U.S. patent application number 15/900698 was filed with the patent office on 2018-07-12 for regulators of nfat.
This patent application is currently assigned to CHILDREN'S MEDICAL CENTER CORPORATION. The applicant listed for this patent is CHILDREN'S MEDICAL CENTER CORPORATION. Invention is credited to Stefan Feske, Yousang Gwack, Patrick Hogan, Anjana Rao.
Application Number | 20180194821 15/900698 |
Document ID | / |
Family ID | 38256924 |
Filed Date | 2018-07-12 |
United States Patent
Application |
20180194821 |
Kind Code |
A1 |
Rao; Anjana ; et
al. |
July 12, 2018 |
REGULATORS OF NFAT
Abstract
Disclosed are methods of identifying an agent that modulates an
NFAT regulator protein. One such method comprises contacting at
least one test agent with a recombinant cell comprising at least
one NFAT regulator protein or fragment or derivative thereof,
assessing the effect of the test agent on an activity, interaction,
expression, or binding to the NFAT regulator protein or fragment or
derivative thereof, and identifying the test agent that has an
effect on an activity, interaction, expression, or binding to the
NFAT regulator protein or fragment or derivative thereof, whereby
the identified test agent is characterized as an agent that
modulates an NFAT regulator protein. Methods and tools for
identifying an agent that modulates intracellular calcium, to
screen for an agent that modulates NFAT regulator function, to
diagnose unexplained immunodeficiency in a subject, and for
identifying an agent for treating or preventing a disease or
disorder associated with a NFAT regulator protein or calcium
signaling are also disclosed.
Inventors: |
Rao; Anjana; (Cambridge,
MA) ; Feske; Stefan; (New York, NY) ; Hogan;
Patrick; (Cambridge, MA) ; Gwack; Yousang;
(Los Angeles, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CHILDREN'S MEDICAL CENTER CORPORATION |
Boston |
MA |
US |
|
|
Assignee: |
CHILDREN'S MEDICAL CENTER
CORPORATION
Boston
MA
|
Family ID: |
38256924 |
Appl. No.: |
15/900698 |
Filed: |
February 20, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14832922 |
Aug 21, 2015 |
9932378 |
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15900698 |
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13161307 |
Jun 15, 2011 |
9163078 |
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14832922 |
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12160030 |
Oct 28, 2008 |
8399185 |
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PCT/US2007/000280 |
Jan 5, 2007 |
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13161307 |
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60756934 |
Jan 5, 2006 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/5041 20130101;
C07H 21/04 20130101; C12N 15/63 20130101; A61K 35/12 20130101; C07K
16/18 20130101; C12N 5/16 20130101; C12Q 1/00 20130101; C12N 15/907
20130101; C12N 2510/00 20130101; C07K 14/4702 20130101 |
International
Class: |
C07K 14/47 20060101
C07K014/47; G01N 33/50 20060101 G01N033/50; C07K 16/18 20060101
C07K016/18 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with Government support under Grant
Nos. RO1 AI40127, HD39685, R21 AI054933, and GM 075256 awarded by
the National Institutes of Health (NIH). The Government has certain
rights in the invention.
Claims
1. A recombinant cell comprising heterologous nucleic acid encoding
at least one mammalian Nuclear Factor of Activated T cells (NFAT)
regulator protein, wherein the heterologous nucleic acid is a
mammalian NFAT regulator gene, wherein the NFAT regulator gene is
ORAI selected from ORAI1, ORAI2, ORAI3 or a homologue or derivative
thereof; and the recombinant cells is an isolated cell.
Description
CROSS-REFERENCED TO RELATED APPLICATIONS
[0001] This Application is a Continuation Application of U.S.
patent application Ser. No. 14/832,922 filed Aug. 21, 2015, which
is a Continuation Application of U.S. Pat. No. 9,163,078, issued on
Oct. 20, 2015, which is a Continuation Application of U.S. Pat. No.
8,399,185, issued on Mar. 19, 2013, which is a 35 U.S.C. .sctn. 371
National Phase Entry Application of International Application No.
PCT/US2007/000280 filed Jan. 5, 2007, which designates the U.S. and
which claims benefit under 35 U.S.C. .sctn. 119(e) to U.S.
Provisional Application 60/756,934, filed, Jan. 5, 2006, the entire
contents of each of which are incorporated by reference herein.
SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Oct. 8, 2015 is named Sequence_Listing_033393-085620-C2 and is
46,928 bytes in size.
FIELD OF THE INVENTION
[0004] The invention relates to the field of regulation of a family
of calcium regulated transcription factors known as NFAT
proteins.
BACKGROUND OF THE INVENTION
[0005] Hyperactivity or inappropriate activity of the immune system
is a serious and widespread medical problem. It contributes to
acute and chronic immune diseases, e.g., allergic and atopic
diseases, e.g., asthma, allergic rhinitis, allergic conjunctivitis
and atopic dermatitis, and to autoimmune diseases, e.g., rheumatoid
arthritis, insulin-dependent diabetes, inflammatory bowel disease,
autoimmune thyroiditis, hemolytic anemia and multiple sclerosis.
Hyperactivity or inappropriate activity of the immune system is
also involved in transplant graft rejections and graft-versus-host
disease.
[0006] A certain family of transcription factors, the NFAT proteins
(nuclear factor of activated T cells), are expressed in immune
cells and play a key role in eliciting immune responses. The NFAT
proteins are activated by an increase in intracellular calcium
levels, e.g., by means of store-operated calcium entry. The
activated NFAT proteins, in turn, induce transcription of cytokine
genes which are required for an immune response. The
immunosuppressive drugs cyclosporin A and FK506 are potent
inhibitors of cytokine gene transcription in activated immune
cells, and have been reported to act by inhibiting calcineurin such
that calcineurin is not able to activate NFAT. These drugs,
however, can display nephrotoxic and neurotoxic effects after long
term usage. Since calcineurin is ubiquitously expressed in many
tissues, the drugs' inhibition of calcineurin activity toward
substrates other than NFAT may contribute to the observed
toxicity.
[0007] There is a need for immunosuppressive agents which
selectively inhibit the store-operated calcium entry activation of
NFAT.
SUMMARY OF THE INVENTION
[0008] The present invention provides a method for identifying an
agent that modulates NFAT activity. In one embodiment, the agent
modulates NFAT activity by means of modulating intracellular
calcium levels. In one preferred embodiment, the agent modulates at
least one component of the CRAC channel, e.g., an ORAI protein,
e.g., proteins encoded by ORAI1 (NM_032790; SEQ ID NO: 1), ORAI2
(BC069270; SEQ ID NO: 2), and/or ORAI3 (NM_152288; SEQ ID NO: 3).
In one embodiment, the agent modulates phosphorylation of NFAT,
e.g., via modulation of a DYRK protein, e.g., proteins encoded by
DYRK1A (NM_001396; SEQ ID NO:4), DYRK1B (NM_004714; SEQ ID NO:5),
DYRK2 (NM_003583; SEQ ID NO:6), DYRK3 (NM_003582; SEQ ID NO:7),
DYRK4 (NM_003845; SEQ ID NO:8) and/or DYRK6 (NM_005734; SEQ ID
NO:9).
[0009] The present invention provides a method of identifying an
agent that modulates an NFAT regulator protein, comprising
contacting at least one test agent with a recombinant cell
comprising at least one NFAT regulator protein or fragment or
derivative thereof; assessing the effect of the test agent on an
activity, interaction, expression, or binding to the NFAT regulator
protein or fragment or derivative thereof; and identifying the test
agent that has an effect on an activity, interaction, expression,
or binding to the NFAT regulator protein or fragment or derivative
thereof, whereby the identified test agent is characterized as an
agent that modulates an NFAT regulator protein.
[0010] In one embodiment, the NFAT regulator protein is encoded by
at least one NFAT regulator selected from the group consisting of
ORAI1 (SEQ ID NO: 1), ORAI2 (SEQ ID NO:2), ORAI3 (SEQ ID NO:3),
DYRK1A (SEQ ID NO:4), DYRK1B (SEQ ID NO:5), DYRK2 (SEQ ID NO:6),
DYRK3 (SEQ ID NO:7), DYRK4 (SEQ ID NO:8) and DYRK6 (SEQ ID NO:9).
In one embodiment, the NFAT regulator protein is encoded by at
least one of the genes listed in Table I.
[0011] In one embodiment, assessing the effect of the test agent
comprises using an antibody which specifically binds to a NFAT
regulator protein encoded by ORAI1 (SEQ ID NO: 1), ORAI2 (SEQ ID
NO: 2), ORAI3 (SEQ ID NO: 3), DYRK1A (SEQ ID NO:4), DYRK1B (SEQ ID
NO:5), DYRK2 (SEQ ID NO:6), DYRK3 (SEQ ID NO:7), DYRK4 (SEQ ID
NO:8), or DYRK6 (SEQ ID NO:9).
[0012] In one embodiment, the method further comprises assessing
the effect of the test agent on electrical current across the
plasma membrane of the cell. In one embodiment, the electrical
current is due to flux of monovalent cations or divalent cations
across the cell. In one embodiment, the method further comprises
assessing the effect of the test agent on intracellular calcium
within the cell. In one embodiment, the method further comprises
identifying the test agent that has an effect on intracellular
calcium within the cell, whereby the identified test agent is
characterized as an agent that modulates intracellular calcium and
an agent that modulates NFAT regulator protein.
[0013] In one embodiment, the cell comprises at least one
heterologous NFAT regulator proteins or a fragment or derivative
thereof. In one embodiment, the cell comprises heterologous nucleic
acid encoding at least one NFAT regulator protein or a fragment or
derivative thereof. In one embodiment, the cell overexpresses, or
underexpresses at least one NFAT regulator protein or fragment or
derivative thereof.
[0014] The present invention further provides a method of
identifying an agent that modulates
intracellular calcium, comprising contacting at least one test
agent with a recombinant cell comprising at least one NFAT
regulator protein or fragment or derivative thereof; assessing the
effect(s) of the test agent on intracellular amounts, or
concentrations, of cations or divalent cations within the cell, or
on ion influx into the cell; and identifying the test agent that
has an effect on intracellular amounts or concentrations of cations
or divalent cations within the cell, or on ion influx into the
cell, whereby the identified test agent is characterized as an
agent that modulates intracellular calcium. In one embodiment, the
intracellular cation is calcium. In one embodiment, assessing the
effect of the test agent comprises monitoring calcium levels in the
cytoplasm, monitoring calcium levels in an intracellular calcium
store, monitoring calcium movement, or monitoring a calcium-entry
mediated event. In one embodiment, the method further comprises
assessing the effect of the test agent on an activity, interaction,
expression, or binding to the NFAT regulator protein or fragment or
derivative thereof. In one embodiment, the NFAT regulator protein
is encoded by at least one NFAT regulator selected from the group
consisting of ORAI1 (SEQ ID NO: 1), ORAI2 (SEQ ID NO: 2), or ORAI3
(SEQ ID NO: 3), DYRK1A (SEQ ID NO:4), DYRK1B (SEQ ID NO:5), DYRK2
(SEQ ID NO:6), DYRK3 (SEQ ID NO:7), DYRK4 (SEQ ID NO:8) or DYRK6
(SEQ ID NO:9). In one embodiment, the agent that modulates
intracellular calcium is further characterized as an agent that
modulates NFAT regulator protein. In one embodiment, the
recombinant cell comprises at least one heterologous NFAT regulator
proteins or a fragment or derivative thereof. In one embodiment,
the recombinant cell comprises a heterologous nucleic acid encoding
at least one NFAT regulator proteins or fragment or derivative
thereof. In one embodiment, the recombinant cell overexpresses at
least one NFAT regulator protein or fragment or derivative thereof.
In one embodiment, the recombinant cell exhibits dyshomeostasis. In
one embodiment, the recombinant cell exhibits calcium
dyshomeostasis
[0015] The present invention further provides a method to screen
for an agent that modulates NFAT regulator function, comprising
administering at least one test agent to a recombinant cell
comprising at least one vector that comprises heterologous nucleic
acid encoding at least one NFAT regulatory domain or a fragment or
derivative thereof, operably linked to a sequence encoding a
reporter protein; and monitoring intracellular localization of at
least one expression product encoded by the vector, whereby a test
agent that has an effect on intracellular localization of the
expression product is characterized as an agent that modulates NFAT
regulator function. In one embodiment, the agent that modulates
NFAT regulator function is associated with cytoplasmic or nuclear
localization of the expression product. In one embodiment, the cell
is under resting conditions. In one embodiment, the cell is
stimulated with a calcium modulating agent. In one embodiment, the
cell is stimulated with thapsigargin or ionomycin. In one
embodiment, the cell is further administered a vector that
comprises a heterologous nucleic acid encoding at least one NFAT
regulator protein, or a fragment or derivative thereof. In one
embodiment, the vector that comprises the heterologous nucleic acid
encoding at least one NFAT regulator protein, or fragment or
derivative thereof, is the same vector that comprises heterologous
nucleic acid encoding at least one NFAT regulatory domain or a
fragment or derivative thereof, operably linked to a sequence
encoding a reporter protein.
[0016] The present invention further provides a method to diagnose
unexplained immunodeficiency in a subject comprising sequencing at
least 25 contiguous nucleotides in a gene from the subject
corresponding to ORAI1 (SEQ ID NO:1), ORAI2 (SEQ ID NO:2), ORAI3
(SEQ ID NO:3), DYRK1A (SEQ ID NO:4), DYRK1B (SEQ ID NO:5), DYRK2
(SEQ ID NO:6), DYRK3 (SEQ ID NO7), DYRK4 (SEQ ID NO:8), DYRK6 (SEQ
ID NO:9), or any of the genes listed in Table I; and comparing the
sequence of the subject's gene to the wild type sequence of the
gene, wherein a variation between the gene from the wild type
sequence indicates the subject's gene is responsible for the
immunodeficiency. In one embodiment, the comparison comprises
obtaining a biological sample from the subject, sequencing the DNA
in the biological sample, and electronically aligning the DNA
sequence obtained from the biological sample to a wild type
sequence. In one embodiment, the variation comprises a nucleotide
mutation from C to T at position 271 of the coding sequence of
ORAI1 (SEQ ID NO: 1). In one embodiment, the unexplained
immunodeficiency is associated with defects in regulation of NFAT
activity. In one embodiment, the variation comprises a mutation in
a splice site. In one embodiment, the variation comprises a
nonsynonymous mutation.
[0017] The present invention further provides a method for
identifying an agent for treating or preventing a disease or
disorder associated with a NFAT regulator protein, comprising
assessing the effects of a test agent on an organism exhibiting a
disease or disorder associated with NFAT regulator protein; and
identifying the test agent as an agent for treating or preventing a
disease or disorder associated with NFAT regulator protein if it
has an effect on a phenotype of the organism associated with the
disease or disorder, wherein the test agent modulates an activity,
interaction, expression, or binding of, at least one NFAT regulator
protein or fragment or derivative thereof. In one embodiment, the
organism comprises one or more cells that exhibit calcium
dyshomeostasis. In one embodiment, the organism exhibits calcium
dyshomeostasis. In one embodiment, the phenotype on which the test
agent has an effect is associated with the disease or disorder.
This method is particularly useful, for diseases or conditions
associated with altered regulation of intracellular calcium. In one
embodiment, the disease or disorder is primarily attributable to
deranged calcium signaling. In one embodiment, the disease or
disorder associated with NFAT regulator protein is rheumatoid
arthritis, inflammatory bowel disease, allogeneic or xenogeneic
transplantation rejection, graft-versus-host disease, aplastic
anemia, psoriasis, lupus erytematosus, inflammatory disease, MS,
type I diabetes, asthma, pulmonary fibrosis, scleroderma,
dermatomyositis, Sjogren's syndrome, postpericardiotomy syndrome,
Kawasaki disease, Hashimoto's thyroiditis, Graves' disease,
myasthenia gravis, pemphigus vulgaris, autoimmune hemolytic anemia,
idiopathic thrombopenia, chronic glomerulonephritis, Goodpasture's
syndrome, Wegner's granulomatosis, multiple sclerosis, cystic
fibrosis, chronic relapsing hepatitis, primary biliary cirrhosis,
uveitis, allergic rhinitis, allergic conjunctivitis, atopic
dermatitis, Crohn's disease, ulcerative colitis,
colitis/inflammatory bowel syndrome, Guilllain-Barre syndrome,
chronic inflammatory demyelinating polyradiculoneuropathy, eczema,
and autoimmune thyroiditis. Transplant graft rejections, acquired
immunodeficiencies, common variable immunodeficiency, myocardial
hypertrophy, severe combined immunodeficiency, dilated
cardiomyopathy, excessive or pathological bone resorption,
excessive adipocyte differentiation, obesity, or reactivation of
latent viruses.
[0018] The present invention further provides an antibody which
specifically binds to a NFAT regulator protein encoded by ORAI1
(SEQ ID NO: 1), ORAI2 (SEQ ID NO: 2), or ORAI3 (SEQ ID NO: 3),
DYRK1A (SEQ ID NO:4), DYRK1B (SEQ ID NO:5), DYRK2 (SEQ ID NO:6),
DYRK3 (SEQ ID NO:7), DYRK4 (SEQ ID NO:8) or DYRK6 (SEQ ID NO:9), or
a homolog thereof.
[0019] The NFAT regulator protein of the invention can be produced
by a variety of means known in the art, e.g. automated peptide
synthesis or culturing a host cell comprising a recombinant vector,
the recombinant vector comprising a nucleic acid sequence, the
nucleic acid sequence comprising/encoding the NFAT regulator or a
fragment or derivative thereof, wherein the host cell is cultured
under conditions suitable for expression of the NFAT regulator.
[0020] The present invention further provides a system comprising
an isolated cell comprising at least one heterologous NFAT
regulator protein or fragment or derivative thereof, and/or at
least one heterologous nucleic acid encoding a NFAT regulator
protein or fragment or derivative thereof; and a monitoring agent
used to monitor, detect, or measure electrical current across the
plasma membrane of the cell. In one embodiment, the monitoring
agent is an apparatus. In one embodiment, the electrical current is
due to flux of cations or divalent ions across the cell. In one
embodiment, the monitoring agent is used to monitor the effect of a
test agent on intracellular calcium within the cell. In one
embodiment, the monitoring agent is used to monitor, detect, or
measure a calcium-entry mediated event.
[0021] The present invention further provides a system comprising a
recombinant cell overexpressing at least one mammalian NFAT
regulator protein or fragment or derivative thereof; and a
monitoring agent used to monitor, detect, or measure a
calcium-entry mediated event. In one embodiment, the NFAT regulator
is encoded by ORAI1 (SEQ ID NO: 1), ORAI2 (SEQ ID NO: 2), or ORAI3
(SEQ ID NO: 3), DYRK1A (SEQ ID NO: 4), DYRK1B (SEQ ID NO: 5), DYRK2
(SEQ ID NO: 6), DYRK3 (SEQ ID NO: 7), DYRK4 (SEQ ID NO: 8) or DYRK6
(SEQ ID NO: 9).
[0022] The present invention further provides a recombinant cell
comprising at least one heterologous NFAT regulator protein or
fragment or derivative thereof, and/or at least one heterologous
nucleic acid encoding a NFAT regulator protein or fragment or
derivative thereof. In one embodiment, the recombinant cell
overexpresses at least one mammalian NFAT regulator protein or
fragment or derivative thereof.
[0023] The present invention further provides a recombinant cell
overexpressing at least on mammalian NFAT regulator protein or
fragment or derivative thereof.
[0024] The present invention further provides a method for
identifying an agent for treating or
[0025] preventing a disease or disorder associated with calcium
signaling. The method comprises assessing the effects of a test
agent on an organism exhibiting the disease or disorder, and
identifying the test agent as an agent for treating or preventing
the disease or disorder if it modulates an activity, interaction,
expression, or binding of at least one NFAT regulator protein or
fragment thereof. In one embodiment, the disease or disorder is
rheumatoid arthritis, inflammatory bowel disease, allogeneic or
xenogeneic transplantation rejection, graft-versus-host disease,
aplastic anemia, psoriasis, lupus erytematosus, inflammatory
disease, MS, type I diabetes, asthma, pulmonary fibrosis,
scleroderma, dermatomyositis, Sjogren's syndrome,
postpericardiotomy syndrome, Kawasaki disease, Hashimoto's
thyroiditis, Graves' disease, myasthenia gravis, pemphigus
vulgaris, autoimmune hemolytic anemia, idiopathic thrombopenia,
chronic glomerulonephritis, Goodpasture's syndrome, Wegner's
granulomatosis, multiple sclerosis, cystic fibrosis, chronic
relapsing hepatitis, primary biliary cirrhosis, uveitis, allergic
rhinitis, allergic conjunctivitis, atopic dermatitis, Crohn's
disease, ulcerative colitis, colitis/inflammatory bowel syndrome,
Guilllain-Barre syndrome, chronic inflammatory demyelinating
polyradiculoneuropathy, eczema, and autoimmune thyroiditis.
Transplant graft rejections, acquired immunodeficiencies, common
variable immunodeficiency, myocardial hypertrophy, severe combined
immunodeficiency, dilated cardiomyopathy, excessive or pathological
bone resorption, excessive adipocyte differentiation, obesity, or
reactivation of latent viruses.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIGS. 1A-1C show gene-dosage effect in store-operated
Ca.sup.2+ entry (SOCE). FIG. 1A shows a pedigree of patients with a
defect in SOCE and CRAC channel function. Two male SCID patients
(subject ID numbers 8 and 11; filled black squares) were born to
consanguineous parents (subject ID numbers 35 and 36). For
functional and genetic analysis, DNA and blood samples were
obtained from all individuals shown in yellow or black. Half black
squares or circles indicate heterozygous disease carriers as
determined by phenotypic analysis. Double horizontal bars indicate
consanguineous marriages, black boxes SCID disease, diagonal bars
death of individuals. FIG. 1B shows reduced SOCE in T cells of both
parents of CRAC deficient SCID patients that defines them as
heterozygous carriers of the disease trait. T cells were stimulated
with thapsigargin (TG) in the absence of extracellular Ca.sup.2+.
The peak (upper panel) and rate (bottom panel) of Ca.sup.2+ influx
were measured after readdition of 0.5 mM extracellular Ca.sup.2+.
FIG. 1C shows reduced SOCE that phenotypically identifies 12/21
family members of the SCID patients as heterozygous disease trait
carriers. Ca.sup.2+ influx was measured as described in B but using
0.2 mM extracellular Ca.sup.2+. Shown are the averages of Ca.sup.2+
influx rates from 4-5 experiments. Individual ID numbers correspond
to those shown in FIG. 1A. Stars indicate heterozygous carriers as
defined by influx rates below 2 nM/s (dotted red line). Co, healthy
control; P, patient.
[0027] FIGS. 2A-2B show that a genome-wide RNAi screen identifies
Drosophila Orai as a protein regulating NFAT translocation and
store-operated Ca.sup.2+ entry. FIG. 2A shows that RNAi of dSTIM or
dOrai inhibits dephosphorylation of NFAT. S2R+ cells stably
transfected with NFAT1(1-460)-GFP were incubated for 4 days with
double-stranded (ds) RNAi against dSTIM, dOrai or an irrelevant DNA
sequence (mock). Cells were left unstimulated (-TG) or stimulated
with thapsigargin (+TG) for 10 min, then lysed after stimulation
with TG, and cell extracts were separated by SDS-PAGE and
immunoblotted with antibodies against NFAT1. Dephosphorylation of
NFAT is evidenced by more rapid migration (lower band) on SDS-PAGE.
FIG. 2B shows that RNAi of either dSTIM or dOrai inhibits Ca.sup.2+
influx in S2R+ cells. Cells were left unstimulated (-TG) or
stimulated with thapsigargin (+TG) for 10 min, then loaded with
Fluo-4 and Fura-Red and analyzed for Ca.sup.2+ influx by flow
cytometry. 1 .mu.M thapsigargin was added at the indicated time.
The top line in each panel shows RNAi for Gfp and the bottom line
RNAi for dSTIM or dOrai. Decreased Ca.sup.2+ influx is indicated by
the much reduced change in emission ratio following addition of
thapsigargin.
[0028] FIGS. 3A-3C show that Orai1 is a transmembrane protein. FIG.
3A shows that Orai1 is highly conserved in eukaryotes. Shown is the
sequence conservation in the first of four putative transmembrane
regions (M1, underlined) of Orai1, which contains the R>W
mutation (bold) found in the SCID patients. FIG. 3B shows membrane
topology of Orai1. Hydropathy plots were calculated from the
full-length amino acid sequence of human Orai1 (301 a.a.,
NP_116179) using the Kyte-Doolittle algorithm with a window size of
19 amino acids. Three transmembrane domains (M2-M4) are predicted
with a score >1.8; M1 has a score of .about.1.3. FIG. 3C shows
schematic representation of the predicted membrane topology of
Orai1, based on the hydropathy plot and immunocytochemistry data.
The site of the R>W mutation in the SCID patients is indicated
by a dark box. FIGS. 4A-4H show that expression of Orai1 restores
CRAC channel function in SCID T cells. FIG. 4A shows activation of
an inward current in an Orai.sup.WT-complemented SCID T cell by
passive store depletion with a pipette solution containing 8 mM
BAPTA. At the indicated times, the 20 mM
Ca.sup.2+.sub..smallcircle. solution was replaced with a divalent
free (DVF) solution. Enhanced current in the absence of divalent
cations is a characteristic of CRAC channels and certain other
Ca.sup.2+-selective channels. FIG. 4B shows the current-voltage
(I-V) relation of currents in 20 mM Ca.sup.2+.sub..smallcircle.
(left) and in DVF solution (right) measured during voltage ramps
from -100 to +100 mV. Data were collected at the times indicated by
the arrows in 4A. Note that the Ca.sup.2+ current I-V relation is
inwardly rectifying with a reversal potential >+90 mV. In DVF
solution, the current reversed at .about.+50 mV. FIG. 4C shows that
SCID T cells expressing mutant Orai1.sup.R>W, inward Ca.sup.2+
and Na.sup.+ currents fail to develop during passive store
depletion by 8 mM BAPTA. FIG. 4D shows noise characteristics of the
depotentiating Na.sup.+ current. Top graph shows the mean current
at a constant holding potential of -100 mV. The dotted line
indicates the zero current level (measured in 20 mM Ca.sup.2++2
.mu.M La.sup.3+). Variance was calculated from 100-ms segments of
the Na.sup.+ current and plotted against mean current in lower
panel. The data are fit by a straight line with a slope of 26 fA,
giving a lower limit to the unitary current. FIG. 4E shows fast
inactivation of the Ca.sup.2+ current in a SCID T cell expressing
Orai.sup.WT. Fast inactivation was measured during 300-ms steps to
-100 mV from a holding potential of +30 mV with 20 mM
Ca.sup.2+.sub..smallcircle.. FIG. 4F shows blockade of the
Ca.sup.2+ current by 2 .mu.M La.sup.3+. After passive induction of
the inward current in a SCID T cell expressing Orai.sup.WT, 2 .mu.M
La.sup.3+ was applied. The dotted line indicates the zero current
level, determined from traces collected at the beginning of the
experiment immediately following whole-cell break-in. FIG. 4G shows
potentiation and blockade of I.sub.CRAC by application,
respectively, of low (5 .mu.M) and high (40 M) doses of 2-APB. FIG.
4H shows the summary of peak current densities in the indicated
cell categories. Peak currents were measured during steps to -100
mV. Reconstitution with wild-type Orai1 thus reconstitutes a
current with the expected characteristics of native CRAC channels.
Cells transduced with Orai1.sup.WT or Orai1R>.sup.W were
visually selected based on GFP-fluorescence; untransduced cells
were GFP-negative.
[0029] FIGS. 5A-5D show that expression of Orai1 in fibroblasts
from SCID patients restores store-operated Ca.sup.2+ influx. FIG.
5A shows inhibition of Ca.sup.2+ influx in Orai1.sup.WT expressing
SCID fibroblasts by 75 .mu.M 2-APB. FIG. 5B shows potentiation of
Ca.sup.2+ influx in Orai1.sup.WT-expressing SCID fibroblasts by 3
.mu.M 2-APB. FIGS. 5C-5D shows inhibition of Ca.sup.2+ influx in
Orai1.sup.WT-expressing SCID fibroblasts by 2 .mu.M La.sup.3+ added
before (FIG. 5C) or after (FIG. 5D) readdition of 20 mM Ca.sup.2+.
For each experiment, .about.15-20 GFP-positive fibroblasts were
analyzed. Experiments were repeated at least three times for each
protocol.
[0030] FIGS. 6A-6C show the NFAT regulatory domain and results of
the genome-wide RNAi screen in Drosophila. FIG. 6A shows a
schematic diagram of the N-terminal regulatory domain of NFAT1,
showing the conserved phosphorylated serine motifs which are
dephosphorylated upon stimulation (circles). Peptides corresponding
to the SRR1, SP2 and SP3 motifs used for in vitro kinase assays are
represented. Serine residues shown underlined have been identified
to be phosphorylated in NFAT1 in vivo, and these are the residues
mutated to alanine in the mutant SP2 and SP3 motifs. FIG. 6B shows
that heterologously expressed NFAT is correctly regulated by
Ca.sup.2+ and calcineurin in Drosophila S2R+ cells. Drosophila S2R+
cells were transfected with NFAT1-GFP expression vector. 48 hrs
later, the cells were left untreated (Untr) or treated with
thapsigargin (TG, 1 .mu.M) for 30 min and lysates from the cells
were analysed by immunoblotting (IB) with anti-NFAT1. P and deP
refer to the migration positions of phosphorylated and
dephosphorylated NFAT-GFP, respectively. FIG. 6C shows the
tabulation of the results of the primary screen.
[0031] FIGS. 7A-7C shows screening of candidate kinases identified
in the Drosophila S2R+ cell RNAi screen, for NFAT phosphorylation
and identification of DYRK as a negative regulator of NFAT. FIG. 7A
shows the ability of overexpressed mammalian homologs of the
candidate kinases to directly phosphorylate the NFAT regulatory
domain. FLAG-tagged mammalian homologues of selected Drosophila
kinases were expressed in HEK293 cells, and immunopurified kinases
were tested using an in vitro kinase assay for phosphorylation of
GST-NFAT1(1-415). Phosphorylation levels were assessed by
autoradiography with either short (top panel) or long (middle
panel) exposures. Expression of each kinase was verified by
immunoblotting (IB) using an anti-FLAG antibody. Kinases tested are
as follows: lane 1, CK1.alpha.; lane 2, CK1.epsilon.; lane 3, Bub1;
lane 4, STK38; lane 5, STK38L; lane 6, CDC42BPA; lane 7, ARAF; lane
8. PRKG1; lane 9, SGK; lanes 10 and 11, CSNKA1 and CSNKA2 (CKII
isoforms); lane 12, SRPK1; lane 13, DYRK2; lane 14, ALS2CR7; lane
15, IRAK4. Bub1 was later dropped from our candidate list because
of >10 predicted off-targets (Example 3). FIG. 7B shows
overexpression of DYRK2 blocks calcineurin-mediated
dephosphorylation of NFAT1. Each kinase was co-transfected with
NFAT-GFP into HEK293 cells; after 18 hrs cells were stimulated with
1 .mu.M ionomycin in the presence of 2 mM CaCl.sub.2. Lysates were
immunoblotted using NFAT1 antibody. Relative expression levels of
the kinases were determined by immunoblot using anti-FLAG antibody,
and were identical to those represented in FIG. 6A (bottom panel).
FIG. 7C shows depletion of endogenous DYRK1A potentiates NFAT
activation. HeLa cells stably expressing Ha-tagged NFAT1-GFP were
transfected with control siRNA or DYRK1A-specific siRNA. After 4
days cells were stimulated with 1 .mu.M thapsigargin (TG) or 1
.mu.M thapsigargin (TG) followed by 20 nM CsA for indicated times;
lysates were immunoblotted for NFAT-GFP using anti-HA antibody
(left). DYRK1A mRNA levels (right) were assessed after 3 and 4 days
by real-time PCR. siControl, scrambled control siRNA; siDYRK1A,
DYRK1A-specific siRNA. Results show the average and standard
deviation of three independent experiments.
[0032] FIGS. 8A-8C show that DYRK2 inhibits NFAT-dependent reporter
activity and endogenous IL-2 expression. FIG. 8A shows that
overexpression of DYRK2 inhibits IL2 promoter-driven luciferase
activity in stimulated Jurkat T cells. (The IL2 promoter is an
example of a cytokine promoter whose activation exhibits a strong
requirement for NFAT.) Exponentially growing Jurkat T cells were
co-transfected with pRLTK (renilla luciferase, internal control),
IL-2-pGL3 (IL-2-promoter driven firefly luciferase, experimental
promoter) and empty vector or increasing amounts of wild type (WT)
or kinase dead (KD) DYRK2 expression plasmids (5, 10, 15 and 20
.mu.g). After 24 h cells untreated or stimulated with PMA and
ionomycin for 6 h were analyzed for IL-2-promoter-driven luciferase
activity. Firefly luciferase was normalized to renilla luciferase
and fold induction calculated relative to IL-2 promoter activity
measured in untreated cells. Results show the average and standard
deviation of three independent experiments. FIG. 8B shows that
overexpression of DYRK2 inhibits endogenous IL-2 expression in
stimulated Jurkat T cells. Exponentially-growing Jurkat T cells
were co-transfected with GFP and empty vector or increasing amounts
of wild type (WT) or kinase dead (KD) DYRK2 expression plasmids
(10, 20 and 30 .mu.g). After 24 h cells untreated or stimulated
with PMA and ionomycin for 6 h were evaluated for IL-2 expression
in GFP+ cells by intracellular cytokine staining and flow
cytometry. FIG. 8C shows quantification of the results shown in 8B.
Results show the average and standard deviation of three
independent experiments.
[0033] FIGS. 9A-9C shows that STIM proteins affect NFAT
localization by altering store-operated Ca.sup.2+ influx. FIG. 9A
shows the percent of cells with nuclear NFAT was quantified in
three independent experiments after mock treatment or treatment
with dsRNAs against dSTIM. Mean and standard deviations are
plotted. 50-100 cells were analyzed for each experiment. FIG. 9B
shows the effect of RNAi-mediated depletion of Drosophila STIM
(dSTIM) on NFAT phosphorylation status. Lysates made from
unstimulated or thapsigargin (TG)-stimulated S2R+ cells were
examined by immunoblotting with antibody against NFAT1. The cells
were mock-treated or treated for 4 days with dsRNAs targeting
dSTIM. FIG. 9C shows intracellular Ca.sup.2+ levels, analyzed by
flow cytometry, in S2R+ cells depleted with dSTIM or novel gene
candidates from the confirmatory screen. GFP dsRNA was used as a
control for non-specific effects caused by dsRNA treatment. After
30 sec of basal [Ca.sup.2+].sub.I measurement, 1 .mu.M thapsigargin
was added (arrow) and [Ca.sup.2+].sub.I measurements were continued
for a further 5 min. Depletion of dSTIM almost completely abolishes
thapsigargin-triggered, that is store-operated, Ca.sup.2+
influx.
[0034] FIGS. 10A-10B shows the phylogenetic relation between
different members of the DYRK family in Drosophila and in humans,
and the expression pattern of human DYRKs in Jurkat T cells. FIG.
10A shows the phylogenetic tree of DYRK family kinases using
distance-based methods (neighbour joining). The left-hand side
figures show the homology relationships between Drosophila CG40478
and human DYRK 2, 3; Drosophila CG4551 (smi35A) and human DYRK 4;
Drosophila CG7826 (mnb) and human DYRK1 A, B (top); as computed by
the program Tcoffee. In the right-hand side figures, the orthologue
bootstrap value for CG40478-DYRK2 is higher than for CG40478-DYRK3
(top). Therefore, DYRK2 is an orthologue of CG40478 (the genes
diverged by a speciation event), while DYRK3 may be a paralogue
(the genes diverged by a duplication event). The calculations of
the ortholog bootstrap values were performed with Orthostrapper.
FIG. 10B shows expression of DYRK family members in Jurkat T cells.
Expression level of mammalian DYRK mRNAsin Jurkat T cells was
estimated by RT-PCR analysis. Primers correspond to:
TABLE-US-00001 DYRK1A sense: (SEQ ID NO: 10)
AGTTCTGGGTATTCCACCTGCTCA DYRK1A anti-sense: (SEQ ID NO: 11)
TGAAGTTTACGGGTTCCTGGTGGT; DYRK2 sense: (SEQ ID NO: 12)
TCCACCTTCTAGCTCAGCTTCCAA, DYRK2 anti-sense: (SEQ ID NO: 13)
TGGCAACACTGTCCTCTGCTGAAT; DYRK1B sense: (SEQ ID NO: 14)
GCCAGCTCCATCTCCAGTTCT, DYRK1B anti-sense: (SEQ ID NO: 15)
CACAATATCGGTTGCTGTAGCGGT; DYRK3 sense: (SEQ ID NO: 16)
TGCAATCCTTCTGAACCACCTCCA, DYRK3 anti-sense: (SEQ ID NO: 17)
GCTGTTCTACCTTCATCTCACCTCCA; DYRK4 sense: (SEQ ID NO: 18)
AGGCTGTCATCACTCGAGCAGAAA, DYRK4 anti-sense: (SEQ ID NO: 19)
AGTCCTGCTGATCACCTGAATGCT; DYRK6 sense: (SEQ ID NO: 20)
GCCGATGAGCATATGGCAAACACA, DYRK6 anti-sense: (SEQ ID NO: 21)
TACCCACTGCAGAAGGCTGGTTTA.
[0035] FIGS. 11A-11I show the nucleotide sequences for NFAT
regulator genes. FIG. 11A shows the nucleotide sequence ORAI1
(NM_032790; SEQ ID NO:1). FIG. 11B shows the nucleotide sequence
for ORAI2 (BC069270; SEQ ID NO:2). FIG. 11C shows the nucleotide
sequence for ORAI3 (NM_152288; SEQ ID NO:3). FIG. 11D shows the
nucleotide sequence for DYRK1A (NM_001396; SEQ ID NO:4). FIG. 11E
shows the nucleotide sequence for DYRK1B (NM_004714; SEQ ID NO:5).
FIG. 11F shows the nucleotide sequence for DYRK2 (NM_003583; SEQ ID
NO:6). FIG. 11G shows the nucleotide sequence for DYRK3 (NM_003582;
SEQ ID NO:7). FIG. 11H shows the nucleotide sequence for DYRK4
(NM_003845; SEQ ID NO:8). FIG. 11I shows the nucleotide sequence
for DYRK6 (NM_005734; SEQ ID NO:9).
DETAILED DESCRIPTION OF THE INVENTION
[0036] Aspects of the present invention relate to the
characterization of genes regulating NFAT activity, for example,
via Store-Operated Calcium Entry (SOCE) or via modulation of NFAT
phosphorylation. In particular, to the discovery of an essential
component of the Ca.sup.2+ release-activated Ca.sup.2+ (CRAC)
channel. Accordingly, aspects of the invention relate to novel
regulators of NFAT activity, particularly with regard to modulation
of NFAT activity in T cells. Aspects of the invention also relate
to methods to screen for novel agents that modulate NFAT activity.
Aspects of the invention further relate to methods to screen for
agents that modulate the activity of the NFAT regulators of the
present invention. The invention further provides methods to screen
for agents that modulate the NFAT regulators of the present
invention by means of modulating intracellular calcium.
NFAT Genes and Proteins
[0037] By NFAT protein (nuclear factor of activated T cells) is
meant a member of a family of transcription factors comprising the
members NFAT1, NFAT2, NFAT3 and NFAT4, with several isoforms. Any
other NFAT protein whose activation is calcineurin dependent is
also meant to be included. NFAT proteins can be, e.g., mammalian
proteins, e.g., human or murine. NFAT1, NFAT2 and NFAT4 are
expressed in immune cells, e.g., T lymphocytes, and play a role in
eliciting immune responses. NFAT proteins are involved in the
transcriptional regulation of cytokine genes, e.g., IL-2, IL-3,
IL-4, TNF-alpha and IFN-gamma, during the immune response.
[0038] The conserved regulatory domain of NFAT is an N-terminal
region of NFAT which is about 300 amino acids in length. The
conserved regulatory domain of murine NFAT1 is a region extending
from amino acid residue 100 through amino acid residue 397, of
human NFAT1 is a region extending from amino acid residue 100
through 395, of human NFAT2 is a region extending from amino acid
residue 106 through 413, of human NFAT2b is a region extending from
amino acid residue 93 through 400, of human NFAT3 is a region
extending from amino acid residue 102 through 404, and of human
NFAT4 is a region extending from amino acid residue 97 through 418.
The conserved regulatory domain is moderately conserved among the
members of the NFAT family, NFAT1, NFAT2, NFAT3 and NFAT4. The
conserved regulatory region binds directly to calcineurin. The
conserved regulatory region is located immediately N-terminal to
the DNA-binding domain (amino acid residues 398 through 680 in
murine NFAT1, amino acid residues 396 through 678 in human NFAT1,
amino acid residues 414 through 696 in human NFAT2, amino acid
residues 401 through 683 in human NFAT2b, amino acid residues 405
through 686 in human NFAT3, and amino acid residues 419 through 700
in human NFAT4).
Store Operated Calcium Entry
[0039] SOCE is one of the main mechanisms to increase intracellular
cytoplasmic free Ca.sup.2+ concentrations ([Ca.sup.2+].sub.I) in
electrically non-excitable cells. Ca.sup.2+ elevations are a
crucial signal transduction mechanism in virtually every cell. The
tight control of intracellular Ca.sup.2+, and its utility as a
second messenger, is emphasized by the fact that [Ca.sup.2+].sub.I
levels are typically 70-100 nM while extracellular Ca.sup.2+ levels
([Ca.sup.2+]ex) are 10.sup.4-fold higher, .about.1-2 mM. The
immediate source of Ca.sup.2+ for cell signaling can be either
intracellular or extracellular (FIG. 1). Intracellular Ca.sup.2+ is
released from ER stores by inositol 1,4,5-triphosphate (IP3), or
other signals, while extracellular Ca.sup.2+ enters the cell
through voltage-gated, ligand-gated, store-operated or second
messenger-gated Ca.sup.2+ channels in the plasma membrane. In
electrically non-excitable cells such as lymphocytes, the major
mechanism for Ca.sup.2+ entry is store-operated Ca.sup.2+ entry, a
process controlled by the filling state of intracellular Ca.sup.2+
stores. Depletion of intracellular Ca.sup.2+ stores triggers
activation of membrane Ca.sup.2+ channels with specific
electrophysiological characteristics, which are referred to as
calcium release-activated Ca.sup.2+ (CRAC) channels (Parekh and
Putney, Jr. 2005, Physiol Rev 85:757).
[0040] Ca.sup.2+ release activated Ca.sup.2+ (CRAC) channels. The
electrophysiological characteristics of CRAC channels have been
studied intensively, but the molecular nature of the channel itself
and the mechanisms of its activation remain unknown. One definition
of CRAC channels holds that depletion of intracellular Ca.sup.2+
stores is both necessary and sufficient for channel activation
without direct need for increases in [Ca.sup.2+]i, inositol
phosphates IP3 or IP4, cGMP or cAMP (Parekh and Penner. 1997,
Physiol Rev. 77:901). Biophysically, CRAC current is defined,
amongst other criteria, by its activation as a result of ER
Ca.sup.2+ store depletion, its high selectivity for Ca.sup.2+ over
monovalent (Cs.sup.+, Na.sup.+) cations, a very low single channel
conductance, a characteristic I-V relationship with pronounced
inward rectification and its susceptibility to pharmacological
blockade for instance by La.sup.3+ and 2-APB (100 .mu.M),
respectively (Parekh and Putney, Jr. 2005, Physiol Rev 85:757;
Lewis, 2001, Annu Rev Immunol 19:497).
[0041] Candidate genes for SOCE and CRAC. The molecular nature of
the CRAC channel remains completely unknown. The most widely
investigated candidate genes for the CRAC channel have been the
>25 mammalian homologues of the Drosophila photoreceptor TRP
(Transient Receptor Potential) gene. But most TRP proteins form
non-specific cation channels and even those that show some
preference for divalent cations do not exhibit all of the key
biophysical hallmarks of the CRAC channel when heterologously
expressed (Clapham, 2003. Nature 426:517). Until recently, TRPV6
was the most promising CRAC channel candidate gene because some of
its biophysical features overlapped with that of CRAC. But while
TRPV6, like CRAC, selectively conducts Ca.sup.2+, it is not
activated by store depletion, a defining characteristic of the CRAC
channel. Knockdown studies using RNAi to suppress TRPV6 expression
and our studies using T cells from TRPV6-/- mice showed no defect
in SOCE or I.sub.CRAC in the absence of TRPV6 (Kahr, et al. 2004. J
Physiol 557:121; Kepplinger, et al. Neither CaT1 nor TRPC3 proteins
contribute to CRAC of T lymphocytes. Manuscript in preparation).
Thus, neither TRPV6 nor any other gene has been confirmed to be
involved in SOCE or CRAC channel activity.
Mechanisms of SOCE and CRAC Channel Activation.
[0042] The mechanism by which CRAC channels are activated is
equally unclear. Depletion of intracellular Ca.sup.2+ stores is
necessary for CRAC activation but how the information about reduced
Ca.sup.2+ concentrations in the ER is conveyed to the CRAC pore is
not known. Three main models have been proposed but no consensus
has been reached (Parekh and Putney, Jr. 2005, Physiol Rev 85:757).
(i) The "conformational coupling model" postulates a conformational
change of a molecule at the surface of the ER which then binds to
the CRAC channel; (ii) The "secretion coupling model" suggests that
(constitutively active) CRAC channels reside in intracytoplasmic
vesicles that fuse to the plasma membrane upon store depletion;
(iii) The "Calcium influx factor (CIF) model" predicts a soluble
small molecule, which activates Ca.sup.2+ influx through CRAC
channels when CIF is released into the cytoplasm of stimulated
cells.
[0043] Stromal interaction molecule 1 (STIM1). Recent evidence
suggests that STIM1 plays an important role in store operated
Ca.sup.2+ entry and CRAC channel function. Three independent RNAi
screens by Roos et al. (2005, J Cell Biol 169:435), Liou et al.
(2005, Curr Biol 15:1235) and by our group (see Example 2 below)
have found that suppression of STIM expression by RNAi impairs
Ca.sup.2+ influx in Drosophila melanogaster S2 cells as well as
mammalian cells (FIG. 5). STIM1 is a type I transmembrane protein
which was initially characterized as a stromal protein promoting
the expansion of pre-B cells and as a putative tumor suppressor
(Oritani, et al. 1996. J Cell Biol 134:771; Sabbioni, et al. 1997.
Cancer Res 57:4493). The human gene for STIM1 is located on
chromosome 11p15.5 which is believed to contain genes associated
with a number of pediatric malignancies, including Wilms tumor
(Parker et al. 1996, Genomics 37:253). STIM1 contains a Ca.sup.2+
binding EF hand motif and a sterile .alpha.-motif (SAM) domain in
its ER/extracellular region, a single membrane-spanning domain, and
two predicted cytoplasmic coiled-coil regions (Manji et al. 2000,
Biochim Biophys Acta 1481:147). Domain structure and genomic
organization are conserved in a related gene called STIM2, which
differs from STIM1 mainly in its C-terminus (Williams et al. 2002,
Biochim Biophys Acta 1596:131). STIM1 is able to homodimerize or
heterodimerize with STIM2 (Williams et al. 2002 supra). Expressed
in the ER, its C-terminal region is located in the cytoplasm
whereas the N-terminus resides in the lumen of the ER, as judged by
glycosylation and phosphorylation studies (Maji et al. 2000 supra;
Williams et al. 2002 supra). A minor fraction of STIM1 is located
in the plasma membrane. Although RNAi mediated suppression of STIM1
expression interferes with SOCE and CRAC channel function, STIM1 is
unlikely to be a Ca.sup.2+ channel itself. Rather it is thought
that STIM1 may sense Ca.sup.2+ levels in the ER via its EF hand
(Putney, Jr. 2005. J Cell Biol 169:381; Marchant, 2005, Curr Biol
15:R493). Consistent with the conformational coupling model of
store-operated Ca.sup.2+ influx, STIM1 could act as a key adapter
protein, which physically bridges the space between ER and plasma
membrane, and thus directly connects sensing of depleted Ca.sup.2+
stores to store-operated Ca.sup.2+ channels in the plasma membrane
(Putney, Jr. 2005. supra; Putney, Jr. 1986, Cell Calcium 7:1).
NFAT Regulators
[0044] As used herein, the term "NFAT regulators" is used to refer
to the proteins (NFAT regulator proteins), and the encoding genes
(NFAT regulator genes) which regulate NFAT activity. The methods of
the present invention are intended to include use of homologues,
analogues, isoforms (e.g. alternative splice variants),
derivatives, and functional fragments of the NFAT regulators
described herein. Preferably, homologues of NFAT regulator proteins
have at least 70%, more preferably, 80%, and more preferably 90%
amino acid identity to those specifically identified herein.
NFAT Regulator Proteins
[0045] In one preferred embodiment, the NFAT regulator proteins of
the present invention are encoded by the ORAI genes. Previous to
the discoveries upon which the present invention is based, the
function of the ORAI genes was unknown. ORAI1 nucleic acid sequence
corresponds to GenBank accession number NM_032790, ORAI2 nucleic
acid sequence corresponds to GenBank accession number BC069270 and
ORAI3 nucleic acid sequence corresponds to GenBank accession number
NM_152288. As used herein, ORAI refers to any one of the ORAI
genes, e.g., ORAI1, ORAI2, ORAI3.
[0046] In one embodiment, the NFAT regulator proteins of the
present invention are encoded by the DYRK genes. Previous to the
discoveries upon which the present invention is based, the DYRK
genes were not known to regulate NFAT activity or function. DYRK1A
is encoded by several nucleic acid isoforms including GenBank
accession numbers NM 001396, NM_101395, NM 130436, NM_130437, and
NM_130438. DYRK1B is encoded by multiple nucleic acid isoforms
including GenBank accession numbers NM_004714, NM_006483, and
NM_006484. DYRK2 is encoded by GenBank accession numbers including
NM_003583 and NM_006482. DYRK3 is encoded by GenBank accession
numbers including NM_001004023 and NM_003582. DYRK4 is encoded by
GenBank accession number NM_003845. DYRK6, also known as HIPK3, is
encoded by GenBank accession number NM 005734.
[0047] In one embodiment, the NFAT regulator proteins of the
present invention are encoded by the genes listed in Table I.
[0048] The term "fragment" or "derivative" when referring to a NFAT
regulator protein means proteins or polypeptides which retain
essentially the same biological function or activity in at least
one assay as the native NFAT regulator proteins. For example, the
NFAT regulator fragments or derivatives of the present invention
maintain at least about 50% of the activity of the native proteins,
preferably at least 75%, more preferably at least about 95% of the
activity of the native proteins, as determined e.g. by a calcium
influx assay described in Example 1.
[0049] Fragments or derivatives as the term is used herein can
include competitors of the native NFAT regulators with respect to a
particular NFAT regulator domain activity. However, the fragment or
derivative shows an overall similarity to NFAT regulators in other
areas as explained herein.
[0050] The term fragment, as used herein, refers to a fragment of
the NFAT regulator protein, or nucleic acid sequence, wherein the
(encoded) protein retains at least one biological activity of the
full length NFAT regulator protein. The term fragment and
functional fragment are used herein interchangeably. A fragment of
a sequence contains less nucleotides or amino acids than the
corresponding full length sequences, wherein the sequences present
are in the same consecutive order as is present in the full length
sequence. As such, a fragment does not contain internal insertions
or deletions of anything (e.g. nucleic acids or amino acids) in to
the portion of the full length sequence represented by the
fragment. This is in contrast to a derivative, which may contain
internal insertions or deletions within the nucleic acids or amino
acids that correspond to the full length sequence, or may have
similarity to full length coding sequences.
[0051] A derivative may comprise the same or different number of
nucleic acids or amino acids as full length sequences. The term
derivative, as used herein with respect to an NFAT regulator
protein, includes NFAT regulator proteins, or fragments thereof,
which contain one or more modified amino acids. e.g. chemically
modified, or modification to the amino acid sequence (substitution,
deletion, or insertion). Such modifications should substantially
preserve at least one biological activity of the NFAT regulator
protein. Such biological activity is readily determined by a number
of assays known in the art, for example, a calcium influx assay
described below in Example 1. By way or nonlimiting example, a
derivative may be prepared by standard modifications of the side
groups of one or more amino acid residues of the NFAT regulator
protein, its analog, or a functional fragment thereof, or by
conjugation of the NFAT regulator protein, its analogs or
fragments, to another molecule e.g. an antibody, enzyme, receptor,
etc., as are well known in the art. Accordingly, "derivatives" as
used herein covers derivatives which may be prepared from the
functional groups which occur as side chains on the residues or the
N- or C-terminal groups, by means known in the art, and are
included in the invention. Derivatives may have chemical moieties
such as carbohydrate or phosphate residues. Such a derivativization
process should preserve at least one biological activity of the
NFAT regulator protein. Derivatives can be made for convenience in
expression, for convenience in a specific assay, to enhance
detection, or for other experimental purposes. Derivatives include
dominant negatives, dominant positives and fusion proteins.
Antibodies
[0052] In one embodiment, the invention provides antibodies to the
NFAT regulators of the present invention. Antibodies can be
prepared that will bind to one or more particular domains of a
peptide of the invention and can be used to modulate NFAT regulator
gene or protein activity.
[0053] Moreover, administration of an antibody against an NFAT
regulator protein or fragment or derivative thereof, preferably
monoclonal or monospecific, to mammalian cells (including human
cells) can reduce or abrogate NFAT induced transcription of immune
system associated genes, thus serving to treat hyperactivity or
inappropriate activity of the immune system. Administration of an
activating antibody against an NFAT regulator protein or fragment
or derivative thereof, e.g. an Orai protein, may serve to treat
hypoactivity of the immune system by activating NFAT and thereby
inducing transcription of immune response associated genes.
Administration of an antibody against an NFAT regulator protein or
fragment or derivative thereof, e.g., a DYRK protein, may serve to
treat hypoactivity of the immune system by activating NFAT and
thereby inducing transcription of immune response associated
genes.
[0054] The present invention also relates to antibodies that bind a
protein or peptide encoded by all or a portion of the NFAT
regulator nucleic acid molecule, as well as antibodies which bind
the protein or peptide encoded by all or a portion of a variant
nucleic acid molecule. For instance, polyclonal and monoclonal
antibodies which bind to the described polypeptide or protein, or
fragments or derivatives thereof, are within the scope of the
invention.
[0055] Antibodies of this invention can be produced using known
methods. An animal, such as a mouse, goat, chicken or rabbit, can
be immunized with an immunogenic form of the protein or peptide (an
antigenic fragment of the protein or peptide which is capable of
eliciting an antibody response). Techniques for conferring
immunogenicity on a protein or peptide include conjugation to
carriers or other techniques well known in the art. The protein or
peptide can be administered in the presence of an adjuvant. The
progress of immunization can be monitored by detection of antibody
titers in plasma or serum. Standard ELISA or other immunoassays can
be used with immunogen as antigen to assess the levels of antibody.
Following immunization, anti-peptide antisera can be obtained, and
if desired, polyclonal antibodies can be isolated from the serum.
Monoclonal antibodies can also be produced by standard techniques
which are well known in the art (Kohler and Milstein, Nature
256:4595-497 (1975); Kozbar et al., Immunology Today 4:72 (1983);
and Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R.
Liss, Inc., pp. 77-96 (1985)). Such antibodies are useful as
diagnostics for the intact or disrupted gene, and also as research
tools for identifying either the intact or disrupted gene.
[0056] As an alternative to preparing monoclonal antibody-secreting
hybridomas, a monoclonal antibody to NFAT regulator proteins may be
identified and isolated by screening a recombinant combinatorial
immunoglobulin library (e.g., an antibody phage display library) to
thereby isolate immunoglobulin library members that bind to NFAT
regulator proteins. Kits for generating and screening phage display
libraries are commercially available from, e.g., Dyax Corp.
(Cambridge, Mass.) and Maxim Biotech (South San Francisco, Calif.).
Additionally, examples of methods and reagents particularly
amenable for use in generating and screening antibody display
libraries can be found in the literature.
[0057] Polyclonal sera and antibodies may be produced by immunizing
a suitable subject, such as a rabbit, with NFAT regulator proteins
(preferably mammalian; more preferably human) or an antigenic
fragment thereof. The antibody titer in the immunized subject may
be monitored over time by standard techniques, such as with ELISA,
using immobilized marker protein. If desired, the antibody
molecules directed against NFAT regulator proteins may be isolated
from the subject or culture media and further purified by
well-known techniques, such as protein A chromatography, to obtain
an IgG fraction.
[0058] Fragments of antibodies to NFAT regulator proteins may be
produced by cleavage of the antibodies in accordance with methods
well known in the art. For example, immunologically active F(ab')
and F(ab').sub.2 fragments may be generated by treating the
antibodies with an enzyme such as pepsin. Additionally, chimeric,
humanized, and single-chain antibodies to NFAT regulator proteins,
comprising both human and nonhuman portions, may be produced using
standard recombinant DNA techniques. Humanized antibodies to NFAT
regulator proteins may also be produced using transgenic mice that
are incapable of expressing endogenous immunoglobulin heavy and
light chain genes, but which can express human heavy and light
chain genes.
NFAT Associated Diseases
[0059] The methods of the present invention can also be utilized to
treat, or identify agents useful in treatment of, conditions and
diseases associated with NFAT disregulation/disfunction and/or
Calcium signaling. Such diseases include, without limitation,
immune system diseases involving hyperactivity or inappropriate
activity of the immune system, e.g., acute immune diseases, chronic
immune diseases and autoimmune diseases Examples of such diseases
include rheumatoid arthritis, inflammatory bowel disease,
allogeneic or xenogeneic transplantation rejection (organ, bone
marrow, stem cells, other cells and tissues), graft-versus-host
disease, aplastic anemia, psoriasis, lupus erytematosus,
inflammatory disease, MS, type I diabetes, asthma, pulmonary
fibrosis, scleroderma, dermatomyositis, Sjogren's syndrome,
postpericardiotomy syndrome, Kawasaki disease, Hashimoto's
thyroiditis, Graves' disease, myasthenia gravis, pemphigus
vulgaris, autoimmune hemolytic anemia, idiopathic thrombopenia,
chronic glomerulonephritis, Goodpasture's syndrome, Wegner's
granulomatosis, multiple sclerosis, cystic fibrosis, chronic
relapsing hepatitis, primary biliary cirrhosis, uveitis, allergic
rhinitis, allergic conjunctivitis, atopic dermatitis, Crohn's
disease, ulcerative colitis, colitis/inflammatory bowel syndrome,
Guilllain-Barre syndrome, chronic inflammatory demyelinating
polyradiculoneuropathy, eczema, and autoimmune thyroiditis.
Transplant graft rejections can result from tissue or organ
transplants. Graft-versus-host disease can result from bone marrow
or stem cell transplantation. Immune system diseases involving
hypoactivity of the immune system include, e.g., immunodeficiency
diseases including acquired immunodeficiencies, such as HIV
disease, and common variable immunodeficiency (CVID).
[0060] The methods of the present invention can also be utilized to
treat or identify agents useful in treatment of conditions and
diseases that are not immune mediated, but which nevertheless
involve the Ca.sup.2+- calcineurin-mediated activation of NFAT,
e.g. a protein-protein interaction between calcineurin and NFAT.
Examples include myocardial hypertrophy, dilated cardiomyopathy,
excessive or pathological bone resorption, excessive adipocyte
differentiation, obesity, and reactivation of latent human
herpesvirus-8 or other viruses. Further, the methods of the present
invention can be utilized to treat, or identify agents useful in
the treatment of, conditions that involve a dysfunction of cellular
Ca.sup.2+ signaling, attributable to altered function of an NFAT
regulator protein, wherein, the dysfunction of Ca.sup.2+ signaling
causes a disease or disorder at least in part through its effects
on other Ca.sup.2+ dependent pathways in addition to the
Ca.sup.2+-calcineurin-NFAT pathway, or wherein the dysfunction of
Ca.sup.2+ signaling acts largely through such other pathways and
the changes in NFAT function are ancillary.
Severe Combined Immunodeficiency
[0061] One NFAT associated disease/disorder is Severe Combined
Immunodeficiency (SCID). SCID is a group of congenital immune
disorders caused by failed or impaired development and/or function
of both T and B lymphocytes. A rare disease with an estimated
prevalence of 1 per 100,000 population, SCID can be caused by
mutations in more than 20 different genes. Mutations in the common
.gamma. chain (c.gamma.) of the interleukin 2 (IL-2), IL-4, -7, -9
and -15 receptors leading to X-linked SCID account for 50% of all
cases. Approximately 10% of all SCID cases are due to a variety of
rare mutations in genes important for T and B cell development or
function, especially signal transduction (CD3.epsilon. and .gamma.,
ZAP-70, p56lck, CD45, JAK3, IL-7R.alpha. chain). Due to the low
incidence of these mutations and small family sizes, classical
positional cloning is usually not possible for most of these SCID
diseases and mutations were often found in known signal transducing
genes by functional analysis of T cells followed by sequencing of
candidate genes. Scientifically, SCID disease has been of
extraordinary value for the elucidation of T cell and B cell
function, highlighting the consequences of gene dysfunction in the
immune system.
[0062] In one embodiment, the invention relates to a method to
diagnose unexplained immunodeficiency in a subject comprising
comparison of a nucleotide sequence corresponding to a gene from
the subject comprising the NFAT regulators of the present invention
to wild type sequence of that gene, wherein alteration of the
nucleotide sequence of the gene from the wild type sequence
indicates that the alteration in the gene is responsible for the
immunodeficiency. In one embodiment, the alteration in the gene is
a mutation in a splice site. In one embodiment, the alteration in
the gene is a nonsynonymous mutation. In one embodiment, the
unexplained immunodeficiency is associated with defects in
regulation of NFAT activity.
[0063] In one embodiment, the comparison is accomplished by way of
obtaining a biological sample from the subject, sequencing the DNA
in the biological sample, and electronically aligning the DNA
sequence obtained from the biological sample to a wild type
sequence.
[0064] In one embodiment, a comparison is accomplished by way of
obtaining a DNA sample, processing the DNA sample such that the DNA
is available for hybridization, combining the DNA with nucleotide
sequences complementary to the nucleotide sequence of a NFAT
regulator of the present invention under conditions appropriate for
hybridization of the probes with complementary nucleotide sequences
in the DNA sample, thereby producing a combination; and detecting
hybridization in the combination, wherein absence of hybridization
in the combination is indicative of alteration in the nucleotide
sequence in the gene.
Method to Screen for Agents that Modulate NFAT Regulator
Function
[0065] In one embodiment, the present invention relates to methods
to screen for agents that alter NFAT regulator expression or
function. In one embodiment, the present invention relates to
methods to screen for agents that alter the function of the NFAT
regulator proteins of the present invention. NFAT regulator
function may be altered as to the modulation of CRAC channel
activation. NFAT regulator function may be altered as to the
modulation of NFAT phosphorylation. NFAT regulator function may be
altered as to modulation of NFAT subcellular localization. NFAT
regulator function may be altered as to modulation of free
intracellular calcium levels. NFAT regulator function may be
altered as to modulation of calcineurin activity. In one
embodiment, alter or modulate refers to upregulation or enhancement
of activity. In one embodiment, alter or modulate refers to
downregulation or inhibition.
[0066] As used herein, the term "NFAT regulator genes" is used to
refer to the genes identified by the methods of the present
invention that regulate NFAT activity, including by way of SOCE, by
way of direct phosphorylation of NFAT or by other means as
described in example 2. The NFAT regulator genes of the present
invention include: ORAI1, ORAI2, ORAI3, the DYRK genes including
DYRK1A, DYRK1B, DYRK2, DYRK3 DYRK4 and DYRK6 and the genes
disclosed in Table I in Example 3. In one preferred embodiment, the
NFAT regulator genes of the present invention are ORAIs, e.g.,
ORAI1, ORAI2, and ORAI3. The NFAT regulator genes and/or their
encoded protein products, modulate the activity of NFAT either
directly or indirectly.
[0067] As used herein, the term "modulates" refers the effect an
agent, including a gene product, has on another agent, including a
second gene product. In one embodiment, an agent that modulates
another agent upregulates or increases the activity of the second
agent. In one embodiment, an agent that modulates another agent
downregulates or decreases the activity of the second agent.
[0068] One example of an NFAT regulator detected through the RNAi
screening described herein is calcineurin. The role of calcineurin
in NFAT signaling was previously known. Specifically, calcineurin
dephosphorylates and activates NFAT, and therefore is a positive
regulator.
[0069] Calcineurin serves to illustrate the relationship between
altered expression of a regulator and altered NFAT signaling:
Overexpression of calcineurin leads to increased activation of NFAT
in standard assays; conversely, diminished expression of
calcineurin, as in the RNAi screen detailed below in Example 1,
leads to a decrease in NFAT activation. Calcineurin also
illustrates that altered activity of a regulator, by an agent, is
reflected in altered NFAT signaling. Thus, cyclosporin A and FK506
are calcineurin inhibitors when complexed with their cytoplasmic
binding proteins (cyclophilin A and FKBP12, respectively), and the
inhibitory action of these compounds on calcineurin can be
detected, for example, by examining the effect of cyclosporin A or
FK506 on NFAT localization in cells stimulated with thapsigargin,
or in T cells stimulated physiologically through the T cell
receptor.
[0070] An assay for an agent that affects an NFAT regulator need
not directly involve NFAT. Thus, a number of agents that alter the
activity of calcineurin, for example, the PVIVIT peptide and its
derivatives, the CsA-cyclophilin A complex, and the FK506-FKBP12
complex, can be assayed by examining their binding to calcineurin;
and the calcineurin autoinhibitory peptide can be assayed by
examining its effect on dephosphorylation of substrates other than
NFAT.
[0071] Positive regulators of NFAT are known to act at other stages
of the Ca.sup.2+-calcineurin-NFAT signaling pathway. For example,
Orai1 and STIM1 contribute to the elevation of cytoplasmic
[Ca.sup.2+], and thereby elicit activation of calcineurin and
subsequently of NFAT. Here again, agents that decrease expression
of Orai1 or STIM1 (e.g., RNAi reagents, as shown herein for both
Orai1 and STIM1; and as shown for dStim and STIM1 in Roos et al
(2005) J Cell Biol 169, 435-445; Liou et al (2005) Current Biology
15, 1235-1241) can be recognized either by their effects on NFAT
activation (e.g., NFAT dephosphorylation or intracellular
localization) or on other parameters diagnostic of the function of
the NFAT regulators in question (e.g., cytoplasmic Ca.sup.2+
levels).
[0072] Agents that inhibit function of the
Ca.sup.2+-calcineurin-NFAT signaling pathway by affecting one or
more NFAT regulator proteins, for example agents that inhibit
Ca.sup.2+ influx through the CRAC channel (e.g., La.sup.3+,
Gd.sup.3+, 2-APB) are likewise readily detected. The inhibitory
agents that are known at present, however, are not entirely
selective, which is the reason that the assays described herein
constitute a valuable tool for the discovery of agents that target
the NFAT modulator proteins of this pathway more selectively.
[0073] The present invention is also inclusive of negative
regulators of Ca.sup.2+-calcineurin-NFAT signaling. These include,
for example, DYRK-family kinases, casein kinase-1 isoforms, and
glycogen synthase kinase (GSK-3). Inhibition of the expression of
these negative regulators, for example by RNAi treatment, or
inhibition of their activity, for example by treatment with an
agent that inhibits enzyme activity (e.g., the casein kinase
inhibitor CKI-7; Li.sup.+ as a GSK-3 inhibitor), in each case can
be detected using an assay that monitors an aspect of NFAT
activation.
[0074] The invention relates to screening methods (also referred to
herein as "assays") for identifying modulators, i.e., candidate
compounds or agents (e.g., proteins, peptides, peptidomimetics,
peptoids, oligonucleotides (such as siRNA or anti-sense RNA), small
non-nucleic acid organic molecules, small inorganic molecules, or
other drugs) that bind to NFAT regulator proteins, or to NFAT, have
an inhibitory (or stimulatory) effect on, for example, NFAT
regulator gene expression or protein activity, NFAT gene expression
or protein activity, or have a stimulatory or inhibitory effect on,
for example, the expression or activity of an NFAT
regulator-interacting protein (e.g. a NFAT regulator substrate) or
a NFAT-interacting protein (e.g. a NFAT substrate). Such
interacting proteins can include Ca.sup.2+ and other subunits of
calcium channels, proteins that interact with one or more Orai
proteins, e.g., additional CRAC channel subunits or CRAC channel
modulatory proteins. Compounds thus identified can be used to
modulate the activity of target gene products (e.g., NFAT regulator
polypeptides, NFAT polypeptides) either directly or indirectly in a
therapeutic protocol, to elaborate the biological function of the
target gene product, or to identify compounds that disrupt the
normal interactions of the target gene or gene product.
Identification of a blocking agent or inhibitor of an NFAT
regulator gene or an encoded product can be carried out using the
screening methods of this invention and other methods known in the
art.
[0075] Compounds that affect NFAT regulator expression or activity
can be identified as described herein or using other methods known
in the art. The modulator compounds can be novel, compounds not
previously identified as having any type of activity as a calcium
channel modulator, or a compound previously known to modulate
calcium channels, but that is used at a concentration not
previously known to be effective for modulating calcium influx. The
modulator can also be a modulator compound for NFAT regulators
other than CRAC channel components.
[0076] The term "agent" or "compound" as used herein and throughout
the specification means any organic or inorganic molecule,
including modified and unmodified nucleic acids such as antisense
nucleic acids, RNAi, such as siRNA or shRNA, peptides,
peptidomimetics, receptors, ligands, and antibodies.
[0077] Compounds that inhibit the activity or expression of an NFAT
regulator are useful in the treatment of disorders involving cells
that express an NFAT regulator. Particularly relevant disorders are
those involving hyperactivity or inappropriate activity of the
immune system or hypoactivity of the immune system, as further
described herein.
[0078] Cells or tissues affected by these disorders can be used in
screening methods, e.g., to test whether an agent that modulates
expression or activity of an NFAT regulator can reduce
proliferation of affected cells, alleviate abnormal SOCE function,
or alleviate abnormal NFAT activity. Other cells useful in the
screening methods of the present invention are cells that exhibit
store-operated calcium entry, which include insect cells, e.g.,
Drosophila cells (e.g., Schneider 2 or S2 cells), human embryonic
kidney (HEK) cells, neuronal or nervous system cells, e.g., SHSY5Y
neuroblastoma cells and PC12 cells, rat basophilic leukemia (RBL)
cells, and immune system cells, e.g., primary T cells from mammals
such as human or mouse, lymphocytes such as T lymphocytes,
including Jurkat cells. Cells derived from the knock out or
transgenic animals described below may be useful. Cells derived
from immunodeficient patients, e.g., patients described in Example
1, including T cells and fibroblasts, may be useful in the methods
of the present invention.
[0079] As used herein, the term "recombinant cell" is used to refer
to a cell with exogenous and/or heterologous nucleic acid
incorporated within, either incorporated stably so as to remain
incorporated in clonal expansion of the cells, or introduced
transiently into a cell (or a population of cells). The nucleic
acid may contain, for example, an NFAT regulator gene or it's mRNA,
or its complementary (antisense) strand, or an shRNA or siRNA, or
any fragment or derivative of the foregoing. The nucleic acid may
comprise genomic DNA of NFAT regulator proteins, fragments, or
derivative thereof. The nucleic acid can comprise corresponding
coding and non-coding mRNA or its complementary (anticoding)
strand, which can be employed to regulate expression of the
corresponding mRNA, e.g. corresponding short nucleotides of shRNA
or siRNA. The nucleic acid can result in altered expression (e.g.
over expression or underexpression) of at least one NFAT regulator
protein or its mRNA or antisense. It may also result in the
expression of a NFAT regulator protein functional fragment or
derivative otherwise not expressed in the recipient cell.
Test Compounds
[0080] The test compounds of the present invention can be obtained
using any of the numerous approaches in combinatorial library
methods known in the art, including: biological libraries; peptoid
libraries (libraries of molecules having the functionalities of
peptides, but with a novel, non-peptide backbone, which are
resistant to enzymatic degradation but that nevertheless remain
bioactive; see, e.g., Zuckermann, et al., 1994 J. Med. Chem. 37:
2678-85); spatially addressable parallel solid phase or solution
phase libraries; synthetic library methods requiring deconvolution;
the `one-bead one-compound` library method; and synthetic library
methods using affinity chromatography selection. The biological
library and peptoid library approaches are limited to peptide
libraries, while the other four approaches are applicable to
peptide, non-peptide oligomer or small molecule libraries of
compounds (Lam (1997) Anticancer Drug Des. 12:145).
[0081] Examples of methods for the synthesis of molecular libraries
can be found in the art, for example in: DeWitt et al., 1993, Proc.
Natl. Acad. Sci. USA. 90:6909; Erb et al., 1994, Proc. Natl. Acad.
Sci. USA 91:11422; Zuckermann et al., 1994, J. Med. Chem. 37:2678;
Cho et al., 1993, Science 261:1303; Carrell et al., 1994, Angew.
Chem. Int. Ed. Engl. 33:2059; Carell et al., 1994, Angew. Chem.
Int. Ed. Engl. 33:2061; and Gallop et al., 1994, J. Med. Chem.
37:1233.
[0082] Libraries of compounds may be presented in solution (e.g.,
Houghten, 1992, Biotechniques 13:412-421), or on beads (Lam (1991)
Nature 354:82-84), chips (Fodor, 1993, Nature 364:555-556),
bacteria (Ladner, U.S. Pat. No. 5,223,409), spores (Ladner, U.S.
Pat. No. 5,223,409), plasmids (Cull et al., 1992, Proc. Natl. Acad.
Sci. USA 89:1865-1869) or on phage (Scott and Smith, 1990, Science
249:386-390; Devlin, 1990, Science 249:404-406; Cwirla et al.,
1990, Proc. Natl. Acad. Sci. 87:6378-6382; Felici, 1991, J. Mol.
Biol. 222:301-310; and Ladner supra.).
[0083] The compounds that can be screened by the methods described
herein include, but are not limited to, any small molecule compound
libraries derived from natural and/or synthetic sources, small
non-nucleic acid organic molecules, small inorganic molecules,
peptides, peptoids, peptidomimetics, oligonucleotides (e.g., siRNA,
antisense RNA, aptamers such as those identified using SELEX), and
oligonucleotides containing synthetic components.
[0084] The test compounds can be administered, for example, by
diluting the compounds into the medium wherein the cell is
maintained, mixing the test compounds with the food or liquid of a
test animal (see below), topically administering the compound in a
pharmaceutically acceptable carrier on the test animal, using
three-dimensional substrates soaked with the test compound such as
slow release beads and the like and embedding such substrates into
the test animal, intracranially administering the compound,
parenterally administering the compound.
[0085] A variety of other reagents may also be included in the
mixture. These include reagents such as salts, buffers, neutral
proteins, e.g. albumin, detergents, etc. which may be used to
facilitate optimal protein-protein and/or protein-nucleic acid
binding and/or reduce non-specific or background interactions, etc.
Also, reagents that otherwise improve the efficiency of the assay,
such as protease inhibitors, nuclease inhibitors, antimicrobial
agents, etc. may be used.
[0086] The language "pharmaceutically acceptable carrier" is
intended to include substances capable of being coadministered with
the compound and which allow the active ingredient to perform its
intended function of preventing, ameliorating, arresting, or
eliminating a disease(s) of the nervous system. Examples of such
carriers include solvents, dispersion media, adjuvants, delay
agents and the like. The use of such media and agents for
pharmaceutically active substances is well known in the art. Any
conventional media and agent compatible with the compound may be
used within this invention.
[0087] The compounds can be formulated according to the selected
route of administration. The addition of gelatin, flavoring agents,
or coating material can be used for oral applications. For
solutions or emulsions in general, carriers may include aqueous or
alcoholic/aqueous solutions, emulsions or suspensions, including
saline and buffered media. Parenteral vehicles can include sodium
chloride, potassium chloride among others. In addition intravenous
vehicles can include fluid and nutrient replenishers, electrolyte
replenishers among others.
[0088] Preservatives and other additives can also be present. For
example, antimicrobial, antioxidant, chelating agents, and inert
gases can be added (see, generally, Remington's Pharmaceutical
Sciences, 16th Edition, Mack, 1980).
Test Assays for Agents that Modulate NFAT Activity
[0089] Another aspect of the invention relates to a method to
screen for regulators of free intracellular Ca.sup.2+ levels,
calcineurin activation and NFAT localization in cells as described
in Examples 1 through 3. In one embodiment, a recombinant vector
encoding a fusion protein comprising the entire NFAT regulatory
domain or a functional fragment or derivative thereof, and an
operably linked reporter protein (for determining subcellular
localization of the regulatory domain, e.g. GFP or an antigenic
epitope) is transfected into cells, i.e. test cells. Test cells
transfected with the vector are contacted with the test agent.
After a period of time, e.g., 48-72 hours, the test cells are
scored for subcellular localization of the NFAT-reporter fusion
protein. Scoring may be accomplished by way of automated
microscopy, as in the examples, or by way of manual microscopy,
e.g., fluorescent microscopy, confocal microscopy. Secondary test
assays include calcium influx detection assays. If the test agent
has an effect on intracellular localization of the expression
product of the recombinant vector, this is indicative that it
modulates NFAT regulator function.
[0090] In one embodiment, the cells also express an exogenous (e.g.
heterologous or homologous) NFAT regulator protein, or fragment or
derivative thereof, and/or exhibit altered expression of a NFAT
regulatory protein or fragment or derivative thereof, achieved with
the tools/methods described herein.
[0091] In one embodiment, the test cells are resting cells wherein
NFAT is normally localized to the cytoplasm. Nuclear localization,
or partial nuclear localization in excess of that observed in
untreated control cells, of the NFAT-reporter fusion protein in the
resting test cell indicates that the test agent successfully
activated NFAT activity.
[0092] In one embodiment, the test cells are stimulated cells,
wherein intracellular Ca.sup.2+ stores are depleted and
store-operated Ca.sup.2+ entry is activated and NFAT is localized
to the nucleus. Ca.sup.2+ store depletion may be accomplished, for
example, by means of contacting the test cells with thapsigargin or
ionomycin. The test cells may be stimulated prior to, concurrently
with or subsequent to contacting the test cells with the test
agent. Cytoplasmic localization, or a reduction in nuclear
localization compared to that observed in control cells, of the
NFAT-reporter fusion protein in the stimulated test cell indicates
that the test agent successfully inhibited NFAT activation.
[0093] A reporter gene which encodes a reporter protein to be
operably linked to nucleotide sequences encoding the NFAT
regulatory domain, any reporter gene for general use is
satisfactory provided that its localization in the cell can be
assessed either directly or indirectly in the context of the fusion
protein. For example, the reporter can be any protein whose
localization can be detected by staining with a labeled antibody,
or a protein epitope such as a hemagglutinin or myc epitope, or
green fluorescent protein (GFP) or one of its variants. In one
preferred embodiment, the reporter protein is GFP. The NFAT protein
in the fusion protein may be full length or may comprise the
regulatory domain, particularly the calcineurin and CK1 docking
sites and the conserved serine rich regions (SRR) and
serine-proline (SP) repeat motifs.
[0094] Another aspect of the invention relates to methods for
identifying an agent for treating or preventing a disease or
disorder associated with calcium signaling. In one embodiment, the
method comprises assessing the effects of a test agent on an
organism that exhibits the disease or disorder, or exhibits at
least one phenotype associated with the disease or disorder. The
test agent is identified as an agent for treating or preventing the
disease or disorder if it modulates an activity, interaction,
expression or binding of at least one NFAT regulator protein,
fragment, or derivative thereof. In one embodiment, the NFAT
regulator protein, fragment, or derivative thereof is expressed
either endogenously or exogenously in cells of the organism.
Appropriate methods of administration of the test agent and
assessment of effects can be determined by the skilled
practitioner.
Test Assays for Agents that Modulate Calcium Levels
[0095] In monitoring the effect of a test agent on intracellular
calcium in any of the screening/identification methods provided
herein, a direct or indirect evaluation or measurement of cellular
(including cytosolic and intracellular organelle or compartment)
calcium and/or movement of ions into, within or out of a cell,
organelle, or portions thereof (e.g., a membrane) can be conducted.
A variety of methods are described herein and/or known in the art
for evaluating calcium levels and ion movements or flux. The
particular method used and the conditions employed can depend on
whether a particular aspect of intracellular calcium is being
monitored. For example, as described herein, reagents and
conditions are known, and can be used, for specifically evaluating
store-operated calcium entry, resting cytosolic calcium levels and
calcium levels and uptake by or release from intracellular
organelles. The effect of test agent on intracellular calcium can
be monitored using, for example, a cell, an intracellular organelle
or storage compartment, a membrane (including, e.g., a detached
membrane patch or a lipid bilayer) or a cell-free assay system.
[0096] Generally, monitoring the effect of a test agent on
intracellular calcium involves contacting a test agent with or
exposing a test agent to (1) a protein (and/or nucleic acid, or
portion(s) thereof, encoding a protein) involved in modulating
intracellular calcium (in particular, a protein provided herein)
and/or (2) a cell, or portion(s) thereof (e.g., a membrane or
intracellular structure or organelle) that may or may not contain a
protein (and/or nucleic acid, or portion(s) thereof, encoding a
protein) involved in modulating intracellular calcium. A cell can
be one that exhibits one or more aspects of intracellular Ca.sup.2+
modulation, such as, for example, store-operated calcium entry.
Before, during and/or after the contacting of test agent, a direct
or indirect assessment of intracellular calcium can be made. An
indirect assessment can be, for example, evaluation or measurement
of current through an ion transport protein (e.g., a store-operated
calcium channel or a Ca.sup.2+-regulated ion channel), or
transcription of a reporter protein operably linked to a
calcium-sensitive promoter. A direct assessment can be, for
example, evaluation or measurement of intracellular (including
cytosolic and intracellular organelle) calcium.
[0097] The assessment of intracellular calcium is made in such a
way as to be able to determine an effect of an agent on
intracellular calcium. Typically, this involves comparison of
intracellular calcium in the presence of a test agent with a
control for intracellular calcium. For example, one control is a
comparison of intracellular calcium in the presence and absence of
the test agent or in the presence of varying amounts of a test
agent. Thus, one method for monitoring an effect on intracellular
calcium involves comparing intracellular calcium before and after
contacting a test agent with a test cell containing a protein that
modulates intracellular calcium, or comparing intracellular calcium
in a test cell that has been contacted with test agent and in a
test cell that has not been contacted with test agent (i.e., a
control cell). Generally, the control cell is substantially
identical to, if not the same as, the control cell, except it is
the cell in the absence of test agent. A difference in
intracellular calcium of a test cell in the presence and absence of
test agent indicates that the agent is one that modulates
intracellular calcium.
[0098] Another method for monitoring an effect on intracellular
calcium involves comparing intracellular calcium of a test cell and
a control cell that is substantially similar to the test cell
(e.g., comparing a cell containing a protein (and/or nucleic acid
encoding a protein) involved in intracellular calcium signaling,
such as the proteins provided herein), and a cell that does not
contain, or that contains lower levels of, the particular protein
involved in modulating intracellular calcium signaling. Thus, for
example, if the test cell containing the protein involved in
intracellular calcium modulation is a recombinant cell generated by
transfer of nucleic acid encoding the protein into a host cell,
then one possible control cell is a host cell that has not been
transfected with nucleic acid encoding the protein or that has been
transfected with vector alone. Such a cell would be substantially
similar to the test cell but would differ from the test cell
essentially only by the absence of the introduced nucleic acid
encoding the protein. Thus, a control cell may contain, e.g.,
endogenously, the particular protein involved in modulating
intracellular calcium, in which case the test cell would contain
higher levels of (or overexpress) the particular protein.
[0099] It may also be useful to experimentally reduce the
endogenous expression or functional levels of a particular protein
(e.g. by inhibition of protein expression or function) to identify
an agent that modulates intracellular calcium by targeting that
particular protein. Expression of an NFAT regulator protein can be
reduced in a cell by known experimental methods such as by
targeting expression at the nucleic acid level, e.g. siRNA or shRNA
treatment, to thereby reduce expression of functional protein.
Systems which comprise such a cell which have reduced, or
completely inhibited, expression of NFAT regulator are included in
this invention. Such systems may further contain an exogenous (e.g.
homologous or heterologous) nucleic acid molecule encoding one or
more mammalian NFAT regulator proteins, or a portion thereof, in
expressible form.
[0100] The type of control comparison described above, where
endogenous expression/functional levels of a particular protein are
reduced in a cell, is particularly useful when trying to identify
an agent that specifically modulates intracellular calcium via an
effect on, or modulation of, a particular protein (and/or nucleic
acid, or portion(s) thereof, encoding a particular protein). Thus,
for example, if there is no detectable or substantial difference in
intracellular calcium in the test (non-modified) versus control
(reduced endogenous expression/function) cells in the presence of
the agent, the agent likely does not mediate its effect on
intracellular calcium via the particular protein (or nucleic acid
encoding the protein). A detectable or substantial difference in
intracellular calcium in the test versus control cells in the
presence of the test agent, indicates the test agent may be a
candidate agent that specifically modulates intracellular calcium
via an effect on or modulation of the particular protein. A
candidate agent can be subjected to further control assays to
compare intracellular calcium in test cells in the presence and
absence of test agent or to compare intracellular calcium in
control cells in the presence and absence of test agent, which can
aid in determination of whether a candidate agent is an agent that
modulates intracellular calcium.
[0101] An assessment of intracellular calcium conducted to monitor
the effect of test compound on intracellular calcium can be made
under a variety of conditions. Conditions can be selected to
evaluate the effect of test compound on a specific aspect of
intracellular calcium. For example, as described herein, reagents
and conditions are known, and can be used, for specifically
evaluating store-operated calcium entry, resting cytosolic calcium
levels and calcium levels of and calcium uptake by or release from
intracellular organelles. For example, as described herein, calcium
levels and/or calcium release from the endoplasmic reticulum can
directly be assessed using mag-fura 2, endoplasmic
reticulum-targeted aequorin or cameleons. One method for indirect
assessment of calcium levels or release is monitoring intracellular
cytoplasmic calcium levels (for example using fluorescence-based
methods) after exposing a cell to an agent that effects calcium
release (actively, e.g., IP3, or passively, e.g., thapsigargin)
from the organelle in the absence of extracellular calcium.
Assessment of the effect of the test agent/compound on
concentrations of cations or divalent cations within the cell, or
of ion influx into the cell, can also be used to identify a test
agent as an agent that modulates intracellular calcium.
[0102] Resting cytosolic calcium levels, intracellular organelle
calcium levels and cation movement may be assessed using any of the
methods described herein or known in the art (see, e.g.,
descriptions herein of calcium-sensitive indicator-based
measurements, such as fluo-3, mag-fura 2 and ER-targeted aequorin,
labeled calcium (such as .sup.45Ca.sup.2+)-based measurements, and
electrophysiological measurements). Particular aspects of ion flux
that may be assessed include, but are not limited to, a reduction
(including elimination) or increase in the amount of ion flux,
altered biophysical properties of the ion current, and altered
sensitivities of the flux to activators or inhibitors of calcium
flux processes, such as, for example, store-operated calcium entry.
Reagents and conditions for use in specifically evaluating
receptor-mediated calcium movement and second messenger-operated
calcium movement are also available.
[0103] In particular embodiments of the methods for screening for
or identifying agents that modulate intracellular calcium, the
methods are conducted under conditions that permit store-operated
calcium entry to occur. Such conditions are described herein and
are known in the art. Test agents can be contacted with a protein
and/or nucleic acid encoding a protein (such as the proteins and
nucleic acids provided herein) involved in modulating intracellular
calcium and/or a cell (or portion thereof) containing such a
protein (or nucleic acid) under these appropriate conditions. For
example, in conducting one method for screening for an agent that
modulates intracellular calcium under conditions selected for
evaluating store-operated calcium entry, intracellular calcium
levels of test cells are monitored over time using a fluorescent
calcium indicator (e.g., FLUO-4). Store-operated calcium entry into
the cells is detected depending on the specific indicator used as,
e.g. an increase in fluorescence, a decrease in fluorescence, or a
change in the ratio of fluorescence intensities elicited by
excitation using light of two different wavelengths. in response to
conditions under which store-operated calcium entry occurs. The
methods for eliciting the fluorescence signal for a specific
calcium indicator and for interpreting its relation to a change in
free calcium concentration are well known in the art. The
conditions include addition of a store-depletion agent, e.g.,
thapsigargin (which inhibits the ER calcium pump and allows
discharge of calcium stores through leakage) to the media of cell
that has been incubated in Ca.sup.2+-free buffer, incubation with
thapsigargin for about 5-15 minutes, addition of test compound (or
vehicle control) to the media and incubation of the cell with test
agent for about 5-15 minutes, followed by addition of external
calcium to the media to a final concentration of about 1.8 mM. By
adding thapsigargin to the cell in the absence of external calcium,
it is possible to delineate the transient increase in intracellular
calcium levels due to calcium release from calcium stores and the
more sustained increase in intracellular calcium levels due to
calcium influx into the cell from the external medium (i.e.,
store-operated calcium entry through the plasma membrane that is
detected when calcium is added to the medium). Because the
fluorescence-based assay allows for essentially continuous
monitoring of intracellular calcium levels during the entire period
from prior to addition of thapsigargin until well after addition of
calcium to the medium, not only can "peak" or maximal calcium
levels resulting from store-operated calcium entry be assessed in
the presence and absence of test agent, a number of other
parameters of the calcium entry process may also be evaluated, as
described herein. For example, the kinetics of store-operated
calcium entry can be assessed by evaluation of the time required to
reach peak intracellular calcium levels, the up slope and rate
constant associated with the increase in calcium levels, and the
decay slope and rate constant associated with the decrease in
calcium levels as store-operated calcium entry discontinues. Any of
these parameters can be evaluated and compared in the presence and
absence of test agent to determine whether the agent has an effect
on store-operated calcium entry, and thus on intracellular calcium.
In other embodiments, store-operated calcium entry can be evaluated
by, for example, assessing a current across a membrane or into a
cell that is characteristic of a store-operated calcium entry
current (e.g., responsiveness to reduction in calcium levels of
intracellular stores) or assessing transcription of a reporter
construct that includes a calcium-sensitive promoter element. In
particular embodiments, a test agent is identified as one that
produces a statistically significant difference. E.g., at least a
30% difference in any aspect or parameter of store-operated calcium
entry relative to control (e.g., absence of compound, i.e., vehicle
only).
[0104] Generally, a test agent is identified as an agent, or
candidate agent, that modulates intracellular calcium if there is a
detectable effect of the agent on intracellular calcium levels
and/or ion movement or flux, such as a detectable difference in
levels or flux in the presence of the test agent. In particular
embodiments, the effect or differences can be substantial or
statistically significant.
Test Assays for Agents that Modulate NFAT Regulator Activity
[0105] In one embodiment, an assay is a cell-based assay in which a
cell that expresses an NFAT regulator protein or biologically
active portion thereof is contacted with a test compound, and the
ability of the test compound to modulate NFAT regulator activity is
determined. Determining the ability of the test compound to
modulate NFAT regulator activity can be accomplished by monitoring,
for example, changes in calcium flux in the cell or by testing
downstream effects of modulating calcium flux such activation or
IL-2 expression. Methods of testing such downstream effects are
known in the art and include modulation of cell proliferation and
cell growth. For example, a compound that decreases the number of
NFAT regulator molecules in a cell or affects the function of an
NFAT regulator channel may decrease cellular proliferation.
Alternatively, transcription of genes requiring NFAT
transactivation may be monitored.
[0106] U.S. Pat. Application No. 20040002117 discloses known gene
targets of NFAT and teaches methods to identify further genes
transcribed due to activity of NFAT. Detection of transcription or
protein expression of NFAT target genes may be useful in the
methods of the present invention. Ablation of induced expression of
NFAT target genes in the presence of a test agent indicates that
the test agent is effective in inhibiting NFAT regulator activity,
where the NFAT regulator is a positive regulator of NFAT.
Conversely, expression of NFAT target genes above basal levels in
the presence of a test agent, in otherwise unstimulated conditions,
indicates that the test agents is effective in inhibiting a
negative regulator of NFAT.
[0107] In some cases, the cell used in such assays does not
normally express the NFAT regulator of interest (e.g. a channel
protein). By way of non-limiting example, a cell such as a Xenopus
oocyte or immune system cell or derivative thereof can be
engineered to expresses a recombinant NFAT regulator protein,
biologically active portion or derivative thereof. In general,
recombinant expression that results in increased expression of the
NFAT regulator compared to a corresponding cell that does not
express recombinant NFAT regulator, is referred to as
"overexpression" of the NFAT regulator. Alternatively, the cell can
be of mammalian origin. The cell can also be a cell that expresses
the NFAT regulator of interest (e.g. a calcium channel) but in
which such NFAT regulator activity can be distinguished from other
NFAT regulator (e.g. calcium channel) activity, for example, by
comparison with controls. The ability of the test compound to
modulate NFAT regulator binding to a compound, e.g., an NFAT
regulator substrate, or to bind to NFAT regulator can also be
evaluated. This can be accomplished, for example, by coupling the
compound, e.g., the substrate, with a radioisotope or enzymatic
label such that binding of the compound, e.g., the substrate, to
NFAT regulator can be determined by detecting the labeled compound,
e.g., substrate, in a complex. Alternatively, NFAT regulator could
be coupled with a radioisotope or enzymatic label to monitor the
ability of a test compound to modulate NFAT regulator binding to an
NFAT regulator substrate in a complex. For example, compounds
(e.g., NFAT regulator substrates) can be labeled with .sup.125I,
.sup.35S, .sup.14C, or .sup.3H, either directly or indirectly, and
the radioisotope detected by direct counting of radioemission or by
scintillation counting. Alternatively, compounds can be
enzymatically labeled with, for example, horseradish peroxidase,
alkaline phosphatase, or luciferase, and the enzymatic label
detected by determination of conversion of an appropriate substrate
to product.
[0108] An example of a screening assay for a compound that
specifically modulates activity of an NFAT regulator polypeptide is
as follows. Incubate a cell that expresses the NFAT regulator
polypeptide of interest (e.g., a Jurkat cell or an HEK293 cell)
with a test compound for a time sufficient for the compound to have
an effect on transcription or activity (e.g., for at least 1
minute, 10 minutes, 1 hour, 3 hours, 5 hours, or 24 or more hours.
Such times can be determined experimentally. The concentration of
the test compound can also be varied (e.g., from 1 nM-100 .mu.M, 10
nM to 10 .mu.M or, 1 nM to 10 .mu.M). Inhibition of calcium influx
in the presence and absence of the test compound is then assayed
using methods known in the art. For example, fura-2, Indo-1,
Fluo-3, or Rho-2 can be used to assay calcium flux. Other methods
can be used as assays of inhibition. For example, a test compound
is considered to have, or suspected of, having a significant impact
on influx if any one or more of the following criteria are met:
[0109] a. there is direct inhibition of increased [Ca.sup.2+]i as
measured by a calcium indicator. [0110] b. there is a direct
inhibition of I.sub.CRAC as measured by patch clamp; [0111] c.
there is inhibition of downstream signaling functions such as
calcineurin activity, NFAT subcellular localization, NFAT
phosphorylation, and/or cytokine, e.g., IL-2, production; or [0112]
d. there are modifications in activation-induced cell
proliferation, differentiation and/or apoptotic signaling
pathways.
[0113] Direct testing of the effect of a test compound on an
activity of a specific NFAT regulator polypeptide can be
accomplished using, e.g., patch clamping to measure I.sub.CRAC.
This method can be used in screening assays as a second step after
testing for general effects on calcium influx or as a second step
after identifying a test compound as affecting expression of an
NFAT regulator mRNA or polypeptide. Alternatively, direct testing
can be used as a first step in a multiple step assay or in single
step assays.
[0114] The ability of a compound (e.g., an NFAT regulator
substrate) to interact with the NFAT regulator with or without the
labeling of any of the interactants can be evaluated. For example,
a microphysiometer can be used to detect the interaction of a
compound with NFAT regulator without the labeling of either the
compound or the NFAT regulator (McConnell et al., 1992, Science
257:1906-1912). As used herein, a "microphysiometer" (e.g.,
Cytosensor) is an analytical instrument that measures the rate at
which a cell acidifies its environment using a light-addressable
potentiometric sensor (LAPS). Changes in this acidification rate
can be used as an indicator of the interaction between a compound
and NFAT regulator polypeptide.
[0115] In yet another embodiment, a cell-free assay is provided in
which a NFAT regulator protein or biologically active portion
thereof is contacted with a test compound and the ability of the
test compound to bind to the NFAT regulator protein or biologically
active portion thereof is evaluated. Preferred biologically active
portions of the NFAT regulator proteins to be used in assays of the
present invention include fragments or derivatives that participate
in interactions with other signaling molecules, or fragments or
derivatives that interact directly with NFAT.
[0116] Cell-free assays involve preparing a reaction mixture of the
target gene protein and the test compound under conditions and for
a time sufficient to allow the two components to interact and bind,
thus forming a complex that can be removed and/or detected.
[0117] The interaction between two molecules can also be detected,
e.g., using fluorescence resonance energy transfer (FRET) (see, for
example, Lakowicz et al., U.S. Pat. No. 5,631,169; Stavrianopoulos
et al., U.S. Pat. No. 4,868,103). A fluorophore label is selected
such that a first `donor` label's emission spectrum overlaps with
the absorption spectrum of a second, `acceptor` molecule, which
then fluoresces on excitation of the donor, if the labels are in
close proximity, due to transfer of energy. Alternately, the
`donor` protein molecule may simply utilize the natural fluorescent
energy of tryptophan residues. Labels are chosen that emit
different wavelengths of light, such that the `acceptor` molecule
label may be differentiated from that of the `donor`. Since the
efficiency of energy transfer between the labels is related to the
distance separating the molecules, the spatial relationship between
the molecules can be assessed. In a situation in which binding
occurs between the molecules, the fluorescent emission of the
`acceptor` molecule label in the assay is increased over the
emission when binding does not occur, or when, e.g., binding is
prevented by the excess of unlabelled competitor protein. A FRET
binding event can be conveniently measured, in comparison to
controls, through standard fluorometric detection means well known
in the art (e.g., using a fluorimeter).
[0118] Assays which monitor assembly of the protein complex in
cells or in cell free assays may also be used.
[0119] In another embodiment, determining the ability of the NFAT
regulator protein to bind to a target molecule can be accomplished
using real-time Biomolecular Interaction Analysis (BIA) (see, e.g.,
Sjolander and Urbaniczky, 1991, Anal. Chem. 63:2338-2345 and Szabo
et al., 1995, Curr. Opin. Struct. Biol. 5:699-705). "Surface
plasmon resonance" or "BIA" detects biospecific interactions in
real time, without labeling any of the interactants (e.g.,
BIAcore). Changes in the mass at the binding surface (indicative of
a binding event) result in alterations of the refractive index of
light near the surface (the optical phenomenon of surface plasmon
resonance (SPR)), resulting in a detectable signal that can be used
as an indication of real-time reactions between biological
molecules.
[0120] In one embodiment, the target gene product, e.g., NFAT
regulator polypeptide or the test substance, is anchored onto a
solid phase. The target gene product/test compound complexes
anchored on the solid phase can be detected at the end of the
reaction. In general, the target gene product can be anchored onto
a solid surface, and the test compound, (which is not anchored),
can be labeled, either directly or indirectly, with detectable
labels discussed herein.
[0121] It may be desirable to immobilize an NFAT regulator, an
anti-NFAT regulator antibody or its target molecule to facilitate
separation of complexed from non-complexed forms of one or both of
the proteins, as well as to accommodate automation of the assay.
Binding of a test compound to an NFAT regulator protein, or
interaction of an NFAT regulator protein with a target molecule in
the presence and absence of a candidate compound, can be
accomplished in any vessel suitable for containing the reactants.
Examples of such vessels include microtiter plates, test tubes, and
micro-centrifuge tubes. In one embodiment, a fusion protein can be
provided which adds a domain that allows one or both of the
proteins to be bound to a matrix. For example,
glutathione-S-transferase/NFAT regulator fusion proteins or
glutathione-S-transferase/target fusion proteins can be adsorbed
onto glutathione Sepharose.TM. beads (Sigma Chemical, St. Louis,
Mo.) or glutathione-derivatized microtiter plates, which are then
combined with the test compound or the test compound and either the
non-adsorbed target protein or NFAT regulator protein, and the
mixture incubated under conditions conducive for complex formation
(e.g., at physiological conditions for salt and pH). Following
incubation, the beads or microtiter plate wells are washed to
remove any unbound components, the matrix immobilized in the case
of beads, complex determined either directly or indirectly, for
example, as described above. Alternatively, the complexes can be
dissociated from the matrix, and the level of NFAT regulator
binding or activity determined using standard techniques.
[0122] Other techniques for immobilizing either NFAT regulator
protein or a target molecule on matrices include using conjugation
of biotin and streptavidin. Biotinylated NFAT regulator protein or
target molecules can be prepared from
biotin-NHS(N-hydroxy-succinimide) using techniques known in the art
(e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and
immobilized in the wells of streptavidin-coated 96 well plates
(Pierce Chemicals).
[0123] To conduct the assay, the non-immobilized component is added
to the coated surface containing the anchored component. After the
reaction is complete, unreacted components are removed (e.g., by
washing) under conditions such that any complexes formed will
remain immobilized on the solid surface. The detection of complexes
anchored on the solid surface can be accomplished in a number of
ways. Where the previously non-immobilized component is
pre-labeled, the detection of label immobilized on the surface
indicates that complexes were formed. Where the previously
non-immobilized component is not pre-labeled, an indirect label can
be used to detect complexes anchored on the surface; e.g., using a
labeled antibody specific for the immobilized component (the
antibody, in turn, can be directly labeled or indirectly labeled
with, e.g., a labeled anti-Ig antibody).
[0124] This assay is performed utilizing antibodies reactive with
NFAT regulator protein or target molecules but which do not
interfere with binding of the NFAT regulator protein to its target
molecule. Such antibodies can be derivatized to the wells of the
plate, and unbound target or NFAT regulator protein trapped in the
wells by antibody conjugation. Methods for detecting such
complexes, in addition to those described above for the
GST-immobilized complexes, include immunodetection of complexes
using antibodies reactive with NFAT regulator protein or target
molecule, as well as enzyme-linked assays which rely on detecting
an enzymatic activity associated with the NFAT regulator protein or
target molecule.
[0125] Alternatively, cell free assays can be conducted in a liquid
phase. In such an assay, the reaction products are separated from
unreacted components, by any of a number of standard techniques,
including, but not limited to: filtration; differential
centrifugation (see, for example, Rivas and Minton, 1993, Trends
Biochem. Sci. 18:284-7); chromatography (gel filtration
chromatography, ion-exchange chromatography); electrophoresis (see,
e.g., Ausubel et al., eds. Current Protocols in Molecular Biology
1999, J. Wiley: New York.); and immunoprecipitation (see, for
example, Ausubel et al., eds. Current Protocols in Molecular
Biology 1999, J. Wiley: New York). Such resins and chromatographic
techniques are known to one skilled in the art (see, e.g.,
Heegaard, 1998, J. Mol. Recognit. 11:141-8; Hage and Tweed, 1997,
J. Chromatogr. B. Biomed. Sci. Appl. 699:499-525). Further,
fluorescence resonance energy transfer may also be conveniently
utilized, as described herein, to detect binding without further
purification of the complex from solution.
[0126] The assay can include contacting the NFAT regulator protein
or biologically active portion thereof with a known compound that
binds NFAT regulator to form an assay mixture, contacting the assay
mixture with a test compound, and determining the ability of the
test compound to interact with an NFAT regulator polypeptide,
wherein determining the ability of the test compound to interact
with an NFAT regulator protein includes determining the ability of
the test compound to preferentially bind to NFAT regulator or
biologically active portion thereof, or to modulate the activity of
a target molecule, as compared to the known compound.
[0127] To the extent that NFAT regulator can, in vivo, interact
with one or more cellular or extracellular macromolecules, such as
proteins, inhibitors of such an interaction are useful. Such
interacting molecules include Ca.sup.2+ and subunits of the calcium
channel complex as well as signaling molecules that directly
interact with the channel, such as kinases, phosphatases and
adapter proteins, can be used to identify inhibitors. For example,
a preformed complex of the target gene product and the interactive
cellular or extracellular binding partner product is prepared such
that either the target gene products or their binding partners are
labeled, but the signal generated by the label is quenched due to
complex formation (see, e.g., U.S. Pat. No. 4,109,496 that utilizes
this approach for immunoassays). The addition of a test substance
that competes with and displaces one of the species from the
preformed complex will result in the generation of a signal above
background. In this way, test substances that disrupt target gene
product-binding partner interaction can be identified.
Alternatively, an NFAT regulator polypeptide can be used as a "bait
protein" in a two-hybrid assay or three-hybrid assay (see, e.g.,
U.S. Pat. No. 5,283,317; Zervos et al., 1993, Cell 72:223-232;
Madura et al., 1993, J. Biol. Chem. 268:12046-12054; Bartel et al.,
1993, Biotechniques 14:920-924; Iwabuchi et al., 1993, Oncogene
8:1693-1696; and Brent WO94/10300), to identify other proteins,
that bind to or interact with NFAT regulator ("NFAT
regulator-binding proteins" or "NFAT regulator-bp") and are
involved in NFAT regulator activity. Such NFAT regulator-bps can be
activators or inhibitors of signals by the NFAT regulator proteins
or NFAT regulator targets as, for example, downstream elements of
an NFAT regulator-mediated signaling pathway, e.g., NFAT target
gene expression or activity.
[0128] Modulators of NFAT regulator expression can also be
identified. For example, a cell or cell free mixture is contacted
with a candidate compound and the expression of an NFAT regulator
mRNA or protein evaluated relative to the level of expression of an
NFAT regulator mRNA or protein in the absence of the candidate
compound. Methods to detect expression or evaluate expression level
are well known to the skilled artisan. When expression of an NFAT
regulator mRNA or protein is greater in the presence of the
candidate compound than in its absence, the candidate compound is
identified as a stimulator of NFAT regulator mRNA or protein
expression. Alternatively, when expression of NFAT regulator mRNA
or protein is less (i.e., statistically significantly less) in the
presence of the candidate compound than in its absence, the
candidate compound is identified as an inhibitor of NFAT regulator
mRNA or protein expression. The level of NFAT regulator mRNA or
protein expression can be determined by methods described herein
for detecting an NFAT regulator mRNA or protein.
[0129] A modulating agent can be identified using a cell-based or a
cell-free assay, and the ability of the agent to modulate the
activity of a NFAT regulator protein can be confirmed in vivo,
e.g., in an animal such as an animal model for a disease (e.g., an
animal with leukemia or autoimmune disease or an animal harboring a
xenograft from an animal (e.g., human) or cells from a cancer
resulting from a leukemia or other lymphocytic disorder, or cells
from a leukemia or other lymphocytic disorder cell line.
[0130] This invention further pertains to novel agents identified
by the above-described screening assays. Accordingly, it is within
the scope of this invention to further use an agent identified as
described herein (e.g., a NFAT regulator-modulating agent, an
antisense NFAT regulator nucleic acid molecule, a NFAT
regulator-specific antibody, or a NFAT regulator-binding partner)
in an appropriate animal model (such as those described above) to
determine the efficacy, toxicity, side effects, or mechanism of
action, of treatment with such an agent. Furthermore, novel agents
identified by the above-described screening assays can be used for
treatments as described herein.
[0131] Animal models that are useful include animal models of
leukemia and autoimmune disorders. Examples of such animal models
are known in the art and can be obtained from commercial sources,
e.g., the Jackson Laboratory (Bar Harbor, Me.) or generated as
described in the relevant literature. Examples of animals useful
for such studies include mice, rats, dogs, cats, sheep, rabbits,
and goats. Other useful animal models include, without limitation,
those for other disorders of Ca.sup.2+-NFAT signaling or of
Ca.sup.2+ signaling, e.g., for myocardial hypertrophy, dilated
cardiomyopathy, excessive or pathological bone resorption,
excessive adipocyte differentiation, obesity, and reactivation of
latent human herpesvirus-8 or other viruses, as discussed elsewhere
in this document.
Systems
[0132] Also provided herein are systems for use in identifying an
agent that modulates one or more of the following: a NFAT protein,
a NFAT regulator protein, and intracellular or cytoplasmic calcium.
Such a system includes a cell, or portion(s) thereof, containing
one or more proteins, e.g., NFAT regulator proteins of the present
invention, or fragments or derivative thereof, e.g., ORAI proteins
or fragments or derivatives thereof. In one embodiment, the
proteins are exogenous (heterologous or homologous) to the cell. In
one embodiment, the cell contains an exogenous (e.g. heterologous
or homologous) nucleic acid encoding a NFAT regulator protein or
fragment or derivative thereof. In one embodiment, the system
further contains a monitoring agent used to monitor, detect or
measure electrical current across the plasma membrane of the cell.
Many such monitoring agents are known in the art. The term
"monitoring agent" is also meant to include any apparatus used for
such monitoring.
[0133] In particular embodiments of the systems, the protein(s)
involved in modulating intracellular calcium are contained in
cells. The cells can be isolated cells or cell cultures that
endogenously express such protein(s) or recombinantly express such
proteins as described above with respect to the methods for
identifying agents, e.g. a recombinant cell overexpressing at least
one NFAT regulator protein or fragment or derivative thereof.
Systems in which the cells recombinantly express the proteins can
be such that the cells are isolated cells or cell cultures or are
contained within an animal,--in particular, a non-human animal,
e.g., a non-human mammal.
[0134] The proteins (and/or nucleic acids encoding proteins) or
cells (or portions thereof) of the system can be contained in a
medium that contains an agent that provides for passive or active
intracellular calcium store reduction or depletion (e.g.,
thapsigargin and other agents described herein or known in the art)
and/or that contains a molecule or molecules that facilitate
monitoring or measurement of intracellular calcium and/or calcium
movement. Such molecules include fluorescent (or otherwise labeled)
calcium indicators, trivalent cations, divalent cations other than
calcium and calcium-buffering agents, e.g., calcium chelators.
Recombinant Cells
[0135] Aspects of the invention further relate to recombinant cells
used in the assays described in the methods discussed herein. In
one aspect, the invention also encompasses any recombinant cells
described herein. In one embodiment, the recombinant cell comprises
at least one exogenous (heterologous or homologous) NFAT regulator
protein or fragment or derivative thereof. The recombinant cell may
also further comprise at least one exogenous (heterologous or
homologous) nucleic acid encoding a NFAT regulator protein or
fragment or derivative thereof. The NFAT regulator protein may be
of mammalian origin. The recombinant cell may over express the NFAT
regulator protein or fragment or derivative thereof. This
overexpression may result from expression of an exogenous
(heterologous or homologous) NFAT regulator protein (e.g. from an
exogenous nucleic acid) or may result from over expression of
native/endogenous NFAT regulator protein.
Transgenic Animals
[0136] The invention provides non-human transgenic animals that are
engineered to overexpress an NFAT regulator, ectopically express an
NFAT regulator, express reduced levels of an NFAT regulator,
express a mutant NFAT regulator, or be knocked out for expression
of an NFAT regulator. Such animals and cell lines derived from such
animals are useful for studying the function and/or activity of an
NFAT regulator protein and for identifying and/or evaluating
modulators of NFAT regulator activity. An animal that overexpresses
an NFAT regulator polypeptide is useful, e.g., for testing the
effects of candidate compounds for modulating the activity of the
NFAT regulator polypeptide and assessing the effect of the compound
in vivo.
[0137] As used herein, a "transgenic animal" is a non-human animal,
in general, a mammal, for example, a rodent such as a rat or mouse,
in which one or more of the cells of the animal include a
transgene. Other examples of transgenic animals include non-human
primates, sheep, dogs, cows, goats, chickens, amphibians, and the
like. A transgene is exogenous DNA or a rearrangement, e.g., a
deletion of endogenous chromosomal DNA, which is in most cases
integrated into or occurs in the genome of the cells of a
transgenic animal. A transgene can direct the expression of an
encoded gene product in one or more cell types or tissues of the
transgenic animal; other transgenes, e.g., a knockout, reduce
expression. Thus, a transgenic animal can be one in which an
endogenous NFAT regulator gene has been altered by, e.g., by
homologous recombination between the endogenous gene and an
exogenous DNA molecule introduced into a cell of the animal, e.g.,
an embryonic cell of the animal, prior to development of the
animal.
[0138] Intronic sequences and polyadenylation signals can also be
included in the transgene to increase the efficiency of expression
of the transgene. A tissue-specific regulatory sequence(s) can be
operably linked to a transgene of the invention to direct
expression of an NFAT regulator protein to particular cells. A
transgenic founder animal can be identified based upon the presence
of an NFAT regulator transgene in its genome and/or expression of
NFAT regulator mRNA in tissues or cells of the animals. A
transgenic founder animal can then be used to breed additional
animals carrying the transgene. Moreover, transgenic animals
carrying a transgene encoding an NFAT regulator protein can further
be bred to other transgenic animals carrying other transgenes.
[0139] NFAT regulator proteins or polypeptides can be expressed in
transgenic animals or plants, e.g., a nucleic acid encoding the
protein or polypeptide can be introduced into the genome of an
animal. In preferred embodiments the nucleic acid is placed under
the control of a tissue specific promoter, e.g., a milk or egg
specific promoter, and recovered from the milk or eggs produced by
the animal. Suitable animals are mice, pigs, cows, goats, and
sheep.
[0140] In one non-limiting example, a mouse is engineered to
express an NFAT regulator polypeptide using a T cell-specific
promoter such as an LCK promoter using methods known in the art
(e.g., Zhang et al., 2002, Nat. Immunol. 3:749-755). In an
alternative example, a mouse is engineered with a tissue-specific
knockdown of an NFAT regulator mRNA and protein, e.g., by Cre-lox
mediated recombination, where expression of the recombinase is
under control of a tissue-specific promoter. Engineered animals can
be identified using known methods of identifying the presence of a
transgene in cells and by assaying a cell sample (e.g., T cells)
for the overexpression or underexpression of the NFAT regulator
(for example, using immunocytochemistry) or by assaying calcium
flux in a cell from the sample. Such transgenic animals are useful,
e.g., for testing compounds for their ability to inhibit NFAT
regulator-mediated cell proliferation.
[0141] The invention also includes a population of cells from a
transgenic animal. Methods of developing primary, secondary, and
immortal cell lines from such animals are known in the art.
Pharmaceutical Compositions
[0142] For therapeutic applications, peptides and nucleic acids of
the invention, the antibodies to the NFAT regulators or the agents
identified by the screening methods of the present invention, e.g.,
small molecules, siRNAs, shRNAs, may be suitably administered to a
subject such as a mammal, particularly a human, alone or as part of
a pharmaceutical composition, comprising the peptide, nucleic acid,
antibody or agent together with one or more acceptable carriers
thereof and optionally other therapeutic ingredients. The
carrier(s) must be "acceptable" in the sense of being compatible
with the other ingredients of the formulation and not deleterious
to the recipient thereof.
[0143] The pharmaceutical compositions of the invention include
those suitable for oral, rectal, nasal, topical, e.g, including
buccal and sublingual, mucosal or parenteral, e.g., including
subcutaneous, intramuscular, intravenous and intradermal
administration. The formulations may conveniently be presented in
unit dosage form, e.g., tablets and sustained release capsules, and
in liposomes, and may be prepared by any methods well know in the
art of pharmacy. See, for example, Remington's Pharmaceutical
Sciences, Mack Publishing Company, Philadelphia, Pa. (17th ed.
1985).
[0144] Such preparative methods include the step of bringing into
association with the molecule to be administered ingredients such
as the carrier which constitutes one or more accessory ingredients.
In general, the compositions are prepared by uniformly and
intimately bringing into association the active ingredients with
liquid carriers, liposomes or finely divided solid carriers or
both, and then if necessary shaping the product.
[0145] Compositions of the present invention suitable for oral
administration may be presented as discrete units such as capsules,
cachets or tablets each containing a predetermined amount of the
active ingredient; as a powder or granules; as a solution or a
suspension in an aqueous liquid or a non-aqueous liquid; or as an
oil-in-water liquid emulsion or a water-in-oil liquid emulsion, or
packed in liposomes and as a bolus, etc.
[0146] A tablet may be made by compression or molding, optionally
with one or more accessory ingredients. Compressed tablets may be
prepared by compressing in a suitable machine the active ingredient
in a free-flowing form such as a powder or granules, optionally
mixed with a binder, lubricant, inert diluent, preservative,
surface-active or dispersing agent. Molded tablets may be made by
molding in a suitable machine a mixture of the powdered compound
moistened with an inert liquid diluent. The tablets optionally may
be coated or scored and may be formulated so as to provide slow or
controlled release of the active ingredient therein.
[0147] Compositions suitable for topical administration include
lozenges comprising the ingredients in a flavored basis, usually
sucrose and acacia or tragacanth; and pastilles comprising the
active ingredient in an inert basis such as gelatin and glycerin,
or sucrose and acacia.
[0148] Compositions suitable for parenteral administration include
aqueous and nonaqueous sterile injection solutions which may
contain anti-oxidants, buffers, bacteriostats and solutes which
render the formulation isotonic with the blood of the intended
recipient; and aqueous and non-aqueous sterile suspensions which
may include suspending agents and thickening agents. The
formulations may be presented in unit-dose or multi-dose
containers, for example, sealed ampules and vials, and may be
stored in a freeze dried (lyophilized) condition requiring only the
addition of the sterile liquid carrier, for example water for
injections, immediately prior to use. Extemporaneous injection
solutions and suspensions may be prepared from sterile powders,
granules and tablets.
[0149] Application of the subject therapeutics often will be local,
so as to be administered at the site of interest. Various
techniques can be used for providing the subject compositions at
the site of interest, such as injection, use of catheters, trocars,
projectiles, pluronic gel, stents, sustained drug release polymers
or other device which provides for internal access. Where an organ
or tissue is accessible because of removal from the patient, such
organ or tissue may be bathed in a medium containing the subject
compositions, the subject compositions may be painted onto the
organ, or may be applied in any convenient way. Systemic
administration of a nucleic acid using lipofection, liposomes with
tissue targeting (e.g. antibody) may also be employed.
[0150] It will be appreciated that actual preferred amounts of a
given peptide or nucleic acid of the invention, or of an antibody
or agent identified by the screening methods of the present
invention, used in a given therapy will vary to the particular
active peptide or nucleic acid or agent being utilized, the
particular compositions formulated, the mode of application, the
particular site of administration, the patient's weight, general
health, sex, etc., the particular indication being treated, etc.
and other such factors that are recognized by those skilled in the
art including the attendant physician or veterinarian. Optimal
administration rates for a given protocol of administration can be
readily determined by those skilled in the art using conventional
dosage determination tests.
[0151] Various embodiments of the invention are further illustrated
in the following examples. All references made to other
publications or disclosures throughout this document are
incorporated by reference herein.
Example 1: Identification of Ca.sup.2+ Release Activated Ca.sup.2+
(CRAC) Channel Gene, ORAI1, in SCID Patients
Materials and Methods:
Case Reports
[0152] Detailed case reports of the two SCID patients investigated
in this study have been described (Feske 1996, 2000).
Cell Lines and Reagents
[0153] T cell lines were established from peripheral blood
lymphocytes of two patients and 21 family members and grown as
described.sup.48. Foreskin fibroblasts from the newborn SCID
patient 2 and a healthy newborn (Hs27 cell line, ATCC, Manassas,
Va.) were immortalized by retroviral transduction with a telomerase
expression plasmid (hTERT, generous gift of S. Lessnick, DFCI,
Boston, Mass.). The macrophage-hemocyte-like Drosophila cell line
S2R.sup.+ was grown in Schneider's medium with 10% fetal calf serum
(Invitrogen) according to standard protocols. Thapsigargin was
purchased from LC Biochemicals (Woburn, Mass.), Charybdotoxin (CTX)
and 2-aminoethoxydiphenylborate (2-APB) from Sigma (St. Louis,
Mo.).
Single Nucleotide Polymorphism (SNP) Array Based Linkage
Analysis
[0154] Genomic DNA of SCID patients and 21 relatives was prepared
from peripheral blood mononuclear cells using genomic DNA Maxi prep
kits (Qiagen). Genotyping was performed at the SNP Genotyping
Center (Broad Institute, Cambridge, Mass.) and the Harvard Partners
Center for Genetics and Genomics (Boston, Mass.), using "GeneChip"
Human Mapping 10K Arrays (Xba 142 2.0, Affymetrix, Santa Clara,
Calif.) with an average SNP heterozygosity of 0.38 and a mean
intermarker density of 258 kb. This platform allowed for
simultaneous genotyping of more than 10,000 SNPs in the human
genome. For parametric linkage analysis, data were converted into
"Linkage" format using "Compare Linkage".sup.49 Mendelian genotype
errors inconsistent with the parental genotypes were detected and
set to missing genotypes. Multipoint parametric linkage analysis
was performed to compute LOD scores at each SNP position using
Allegro.sup.50. To confirm linkage, we reanalyzed the SNP data
using Genehunter 2.1r6.sup.51 and Merlin.sup.52 obtaining very
similar results. For parametric analysis, a disease allele
frequency of 0.001, a penetrance value of 0.99 and a phenocopy of
0.01 were used for all the pedigrees. Parametric linkage analyses
were carried out using recessive and dominant models of
inheritance, respectively. For the "recessive" model, haplotypes
from both patients, their parents, unaffected brother and
grandparents (individuals 8, 11, 35, 36, 37, 38, 39, 63, 64 in FIG.
1A) were analyzed assuming an autosomal recessive mode of
inheritance for the SCID disease with both SCID patients being
homozygous for a common disease-causing mutation. The predicted
maximum log.sub.10 of the odds ratio (LOD) score from this analysis
was .about.1.9 (i.e.
-log.sub.10[0.25.times.0.25.times.0.25.times.0.75]). For the
"dominant" model, 12 family members with reduced store-operated
Ca.sup.2+ entry were defined as "affected", i.e. carriers of a
dominantly acting mutation, and their SNP haplotypes compared to
those of 8 healthy family members with normal store-operated
Ca.sup.2+ entry. The predicted maximum LOD score from this analysis
was .about.3.8 (i.e. -log.sub.10[0.5.sup.12]).
Genomic DNA Sequencing
[0155] Genomic DNA of two patients, 21 family members and three
independent controls was sequenced for mutations in exons 1 and 2
of Orai1 using the following oligonucleotide primers: Orai1ex1for1
5' ACAACAACGCCCACTTCTTGGTGG (SEQ ID NO: 22) (exon 1); Orai1ex1rev1
5' TGCTCACGTCCAGCACCTC (SEQ ID NO: 23) (exon 1); Orai1ex2for1 5'
TCTTGCTTTCTGTAGGGCTTTCTG (SEQ ID NO: 24) (exon 2); Orai1ex2rev1 5'
TCTCAAAGGAGCTGGAAGTGC (SEQ ID NO: 25) (exon 2). DNA was amplified
using AmpliTaq Gold polymerase and separated on 1% agarose gels.
PCR products were gel-purified and sequenced directly using the
following primers: Orai1ex1for2 5' AGCATGCAAAACAGCCCAGG (SEQ ID NO:
26) (exon 1); Orai1ex1rev2 5' ACGGTTTCTCCCAGCTCTTC (SEQ ID NO: 27)
(exon 1); Orai1ex2for2 5' TGACAGGAGGAGAGCTAGG (SEQ ID NO: 28) (exon
2); Orai1ex2rev2 5' AAGAGATCCTCCTGCCTTGG (SEQ ID NO: 29).
Sequencing was done at the DF/HCC DNA Resource Core (DFCI) and DNA
sequences analyzed using Xplorer Lite (dnaTools, Ft. Collins,
Colo.).
Sequenom Analysis of HapMap DNA
[0156] To exclude the possibility that the C>T point mutation at
position 271 in the coding sequence of Orai1 (NM_032790) is a SNP,
we examined DNA from a panel of 270 individuals of diverse
geographical origin assembled for the International HapMap
project.sup.30,31. Genotyping was performed using a high-throughput
primer extension method with detection by mass spectrometry
(MALDI-TOF) on the Sequenom platform as previously described 53. A
detailed description of this method can be found at
http://www.hapmap.org/downloads/genotying_protocols.html under
"Sequenom platform". 89% of samples were genotyped successfully and
all were identified as CC homozygotes.
dsRNA Mediated Knockdown in Drosophila Cells
[0157] PCR fragments (size up to 600 bp) were used as templates for
in vitro transcription reactions, followed by DNase I treatment to
remove the template DNA. After purification, dsRNA (5 .mu.g) was
co-transfected together with the NFAT-GFP expression plasmid into
S2R+ cells in 8-chamber slides (10 .mu.g for 12 well plate). After
72 hrs of incubation, cells were treated with the Ca2+ influx
inducers, 1 .mu.M ionomycin or 1 .mu.M thapsigargin for
localization assays and were trypsinized for the measurement of
[Ca.sup.2+].sub.i levels.
Genome-Wide RNAi Screen
[0158] The RNAi screen was performed essentially as described
(Armknecht S. et al., 2005, Methods Enzymol 392, 55-73; Btros M. et
al. 2004 Science 303, 832-835). The macrophage-hemocyte-like
Drosophila cell line S2R+ was stably transfected with the coding
sequence for the NFAT1 (1-460)-GFP fusion protein subcloned into
the expression plasmid pAc5.1 (Invitrogen). Transfection was
achieved using Effectene (Qiagen) with a 19:1 ratio of the
expression plasmid to pCoHygro (Invitrogen), which encodes a
hygromycin resistance gene under the control of a constitutively
active promoter. The cells were selected for 3-4 weeks with 300
.mu.g/ml hygromycin, and stable clones were selected by visual
inspection. 10.sup.4 S2R.sup.+ cells stably expressing
NFAT1(1-460)-GFP were added onto each well of a 384 well plate
containing 0.25 .mu.g of dsRNAs (in 10 .mu.l of serum-free medium)
against Drosophila mRNAs and incubated for 1 h at 26.degree. C. and
incubated for 48-72 hrs at 26.degree. C. to achieve RNAi. S2R.sup.+
cells were stimulated with 1 .mu.M thapsigargin in Schneider medium
containing 5 mM CaCl.sub.2 at room temperature for 10 min, fixed
and stained with DAPI. Coincident GFP and DAPI images were acquired
by an automated camera from three different locations in each well,
and scored by visual inspection. A total of fifty-eight 384-plates
were analysed, containing a total of 21,884 wells into which
individual dsRNAs had been arrayed. For this study, we note that
the dsRNA amplicons for both dStim and dOrai had no predicted
off-targets with exact matches of 19 nucleotides or greater.
Plasmids and Retroviral Transduction
[0159] Full-length cDNA for Orai1 (BC015369) was purchased from
OpenBiosystems (Huntsville, Ala.) and subcloned into pENTR11
("Gateway" system, Invitrogen, Carlsbad, Calif.) in frame with an
N- or C-terminal terminal sequence encoding the myc epitope. Orai1
was then moved to the bicistronic retroviral expression vector
pMSCV-CITE-eGFP-PGK-Puro (kind gift of Masatsugu Oh-hora), which
allows for simultaneous expression of Orai1, GFP and a puromycin
resistance gene. gp293 packaging cell lines were co-transfected
with plasmids encoding Orai1, gag-pol and env to produce
amphotropic, replication-incompetent retrovirus. Virus containing
supernatant was collected for 24 h, filtered (0.45 microm, low
protein binding) and concentrated by centrifugation at 6000.times.g
for 16 h. T cells and fibroblasts were transduced by addition of
viral supernatant for 4d and 1 d, respectively. Transduction
efficiency was evaluated by GFP expression using flow cytometry and
myc-Orai1 expression using immunoblotting and immunocytochemistry.
In some cases, transduced T cells were further selected with 1
.mu.g/ml puromycin for 3 days.
Bioinformatic Prediction of Membrane Topoplogy
[0160] The hydropathy plot of Orai1 was generated using the
Kyte-Doolittle algorithm.sup.29. Membrane topology was further
evaluated using the Phobius algorithm based on the hidden Markov
model.sup.26. Sequence alignment was performed using MegAlign
(DNAStar, Madison, Wis.).
Confocal Imaging
[0161] Immunocytochemistry for Orai1 was done as described.sup.11.
Briefly, retrovirally transduced T cells and fibroblasts were fixed
with 3% paraformaldehyde, left unpermebealized or permeabilized
with wash buffer containing 0.5% NP-40, incubated with anti-myc
antibodies (9E10) and Cy3-labeled secondary antibodies.
Immunofluorescence was analyzed by confocal imaging using a
Radiance 2000 Laser-scanning confocal system (Bio-Rad Laboratories)
on a BX50BWI Olympus microscope using a 63.times. water immersion
objective.
Single-Cell Ca.sup.2+ Imaging
[0162] T cells were loaded at 1.times.10.sup.6 cells/ml with 1
.mu.M fura-2/AM (Molecular Probes) in loading medium (RPMI+10% FBS)
for 30 min at 22-25.degree. C., resuspended in loading medium and
attached to poly-L-lysine-coated coverslips for 15 min. Fibroblasts
were grown directly on UV-sterilized coverslips and loaded with 3
.mu.M fura-2/AM for 45 min at 22-25.degree. C. For
[Ca.sup.2+].sub.i measurements, cells were mounted in a RC-20
closed-bath flow chamber (Warner Instrument Corp., Hamden, Conn.)
and analyzed on an Axiovert S200 epifluorescence microscope (Zeiss)
with OpenLab imaging software (Improvision). Cells were perfused in
Ca.sup.2+-free Ringer solution and Ca.sup.2+ stores were passively
depleted with 1 .mu.M thapsigargin. Active depletion of stores was
induced by incubation with 10 .mu.g/ml anti-CD3 antibody (OKT3,
eBioscience, San Diego, Calif.) for 10 min at 22-25.degree. C.
Fura-2 emission was detected at 510 nm with excitation at 340 and
380 nm and Fura-2 emission ratios (340/380) were calculated for
each 5-s interval after subtraction of background. For each
experiment, approximately 100 individual cells were analyzed for
340/380 ratios using Igor Pro (Wavemetrics, Lake Oswego, Oreg.)
analysis software. [Ca.sup.2+].sub.i was estimated from the
relation [Ca.sup.2+].sub.i=K*(R-R.sub.min)/(R.sub.max-R). K*,
R.sub.min, and R.sub.max were measured in control human T cells in
situ as previously described.sup.54. Ca.sup.2+ influx rates were
calculated from the maximal rate of rise in Ca.sup.2+
concentrations (d[Ca.sup.2+].sub.i/dt) after readdition of 0.2 mM
extracellular Ca.sup.2+.
[0163] Ca.sup.2+ influx in S2R+ cells was measured by flow
cytometry after detaching cells from the dish with trypsin
(CellGro, Herndon, Va.). Cells were loaded with the Ca.sup.2+
indicator dyes Fluo4-AM and Fura-Red (2 .mu.M each, Molecular
Probes, Eugene, Oreg.) for 45 min at room temperature and then
resuspended in loading medium (Schneider's medium+10% FCS).
Immediately before the flow cytometric Ca.sup.2+ measurements,
cells were resuspended in Ringer solution containing 2 mM Ca.sup.2+
and analyzed on a FACSCalibur (BD Biosciences, San Jose, Calif.).
After 30 sec, thapsigargin (3 .mu.M) in Ca.sup.2+ free Ringer to
deplete intracellular Ca.sup.2+ stores, 4 mM Ca.sup.2+ Ringer
solution was added and cellular Ca.sup.2+ levels were monitored for
300 sec. The ratio of Fluo-4 and Fura-Red emission was analyzed
using FloJo software (Tree Star, Inc., Ashland, Oreg.).
Solutions and Chemicals
[0164] The standard extracellular Ringer's solution contained (in
mM): 155 NaCl, 4.5 KCl, 20 CaCl.sub.2, 1 MgCl.sub.2, 10 D-glucose,
and 5 Na-Hepes (pH 7.4). The standard divalent-free (DVF) Ringer's
solutions contained (in mM): 155 Na, 10 HEDTA, 1 EDTA and 10 Hepes
(pH 7.4). Charybdotoxin (CTX) was included in all external solution
to block Kv1.3 channels to prevent contamination of I.sub.CRAC
recordings in DVF solutions. The standard internal solution
contained (in mM): 150 Cs-aspartate, 8 MgCl.sub.2, 8 BAPTA, and 10
Cs-Hepes (pH 7.2).
[0165] Thapsigargin (LC Biochemicals, Woburn, Mass.) was diluted
from a 1 mM stock in DMSO, CTX (Sigma, St. Louis, Mo.) was diluted
1:1000 from 50 .mu.M stock solution in water.
2-aminoethyoxydiphenylborate (2-APB, Sigma) was diluted from stock
solutions in DMSO. The drugs were diluted to the concentrations
indicated in the legends and applied to the cells using a
multi-barrel local perfusion pipette with a common delivery port.
The time for 90% solution exchange was measured to be <1 s,
based on the rate at which the K.sup.+ current reversal potential
changed when the external [K.sup.+] was switched from 2 mM to 150
mM.
Patch-Clamp Measurements
[0166] Patch-clamp experiments were conducted in the standard
whole-cell recording configuration at 22-25.degree. C. using an
Axopatch 200 amplifier (Axon Instruments, Foster City, Calif.)
interfaced to an ITC-16 input/output board (Instrutech, Port
Washington, N.Y.) and a Macintosh G3 computer. Recording electrodes
were pulled from 100-.mu.l pipettes, coated with Sylgard, and
fire-polished to a final resistance of 2-5 M.OMEGA.. Stimulation
and data acquisition and analysis were performed using in-house
routines developed on the Igor Pro platform (Wavemetrics, Lake
Oswego, Oreg.). The holding potential was +30 mV unless otherwise
indicated. Voltage stimuli usually consisted of a 100-ms step to
-100 mV followed by a 100-ms ramp from -100 to +100 mV, applied
every 1.3 s. Currents were filtered at 2 kHz with a 4-pole Bessel
filter and sampled at 5 kHz. Data are corrected for the liquid
junction potential of the pipette solution relative to Ringer's in
the bath (-10 mV) and for the bath DVF solution relative to
Ringer's in the bath-ground agar bridge (+5 mV). For noise
analysis, 200-ms sweeps were acquired at the rate of 3 Hz at a
holding potential of -100 mV, digitized at 5 kHz, and low-pass
filtered using the Axopatch 200 amplifier's internal Bessel filter
at 2 kHz. The mean and variance were calculated from 100-ms
segments of the digitized data.
Data Analysis
[0167] Unless noted otherwise, all data were corrected for leak
currents collected either with 2 .mu.M La.sup.3+ or with traces
collected prior to I.sub.CRAC induction during passive dialysis
with BAPTA. Permeability ratios (P.sub.Cs/P.sub.Na) was calculated
from the biionic reversal potential using the equation:
P Cs P Na = ( [ Na ] o [ Cs ] i ) e ( E rev F RT ) ##EQU00001##
where R, T, and F have their usual meanings and E.sub.rev, is the
reversal potential.
Introduction
[0168] Ca.sup.2+ is an essential second messenger in almost all
cell types. In particular, sustained Ca.sup.2+ influx across the
plasma membrane is crucial for lymphocyte activation and the
adaptive immune response.sup.1. Antigen recognition by the surface
antigen receptors of T and B lymphocytes triggers an elaborate
signal transduction cascade, involving the activation of multiple
tyrosine kinases and the assembly of large scaffolded complexes
containing diverse adapters and signaling proteins. An early
biochemical consequence is the activation of PLC.gamma., which
releases Ca.sup.2+ from the endoplasmic reticulum (ER) by
generating IP3; the resulting decrease in lumenal ER Ca.sup.2+
opens a class of "store-operated" Ca.sup.2+ channels with very
specific electro-physiological characteristics, which have been
termed Ca.sup.2+ release-activated Ca.sup.2+ (CRAC)
channels.sup.1-3. CRAC channel opening results in sustained influx
of Ca.sup.2+ ions across the plasma membrane, promoting a sustained
elevation of intracellular free Ca.sup.2+ ([Ca.sup.2+].sub.I)
levels and activating diverse Ca.sup.2+/calmodulin-dependent
enzymes including the protein phosphatase calcineurin; an ultimate
consequence is the activation of Ca.sup.2+-dependent
transcriptional pathways required for proliferation and effector
immune function.sup.4,5. One of the major Ca.sup.2+-regulated
transcription factors is NFAT, a family of heavily-phosphorylated
proteins that resides in the cytoplasm of resting cells.sup.5.
Sustained Ca.sup.2+ influx results in the dephosphorylation of NFAT
by calcineurin and promotes its translocation to the nucleus, where
it turns on the expression of a large number of
activation-associated genes.sup.4,6
[0169] A great deal of pharmacological, electrophysiological, and
genetic evidence supports the notion that CRAC channels are the
principal pathway for Ca.sup.2+ influx in both developing and
mature T cells, thus orchestrating essentially all aspects of
lymphocyte development and function.sup.1,7. Analysis of two
families of patients with hereditary severe combined immune
deficiency (SCID), who presented as infants with a marked
propensity to bacterial and viral infections, revealed that the
primary defect is lack of store-operated Ca.sup.2+ entry in the
patients' lymphocytes.sup.8-10. Detailed analysis of T cell lines
derived from one family of patients revealed severe impairment of
NFAT dephosphorylation, nuclear translocation and activation of
NFAT-dependent genes, secondary to a correspondingly severe
impairment of store-operated Ca.sup.2+ influx in cells activated
through the T cell receptor or treated with thapsigargin, an
inhibitor of the SERCA Ca.sup.2+ pump.sup.10. Electrophysiological
analysis of the patients' T cells confirmed an almost complete
absence of CRAC channel function.sup.11. Together these data
highlight the crucial importance of CRAC channels and
store-operated Ca.sup.2+ entry for lymphocyte activation and immune
defense.
[0170] Although the pharmacological and electrophysiological
properties of the CRAC channel have been described in some
detail.sup.1,12,13, its molecular identity has remained elusive to
date. The key biophysical hallmarks of the channel include high
selectivity for Ca.sup.2+ over monovalent cations, low
single-channel conductance (<1 pS), an inwardly rectifying I-V
relationship, a lack of significant voltage-dependent gating, rapid
inactivation by intracellular Ca.sup.2+, extracellular blockade by
submicromolar La.sup.3+, and modulation of channel properties by
2-APB.sup.1,13,14. Several candidate genes belonging to the TRP
family of ion channels have been proposed to encode the CRAC
channel, including TRPC1.sup.15, TRPC3.sup.16, and TRPV6.sup.17,18,
as well as voltage-gated Ca.sup.2+ (Cav) channels.sup.19,20.
However, evidence that TRPs are store-dependent following
heterologous expression in several cell lines is
inconsistent.sup.21,22, and none of the candidates exhibit all of
the biophysical properties of the CRAC channel. Previous sequence
analyses and complementation studies in the SCID patients' cells
had failed to establish a role for several TRP family members
including TRPC3, TRPV5 and TRPV6 in the defect in CRAC channel
function.sup.11. More recently, the type I membrane proteins STIM1
and STIM2 were shown to be essential for store-operated Ca.sup.2+
entry and CRAC channel function.sup.23,24. STIM1 has been suggested
to "sense" the filling state of the ER Ca.sup.2+ stores via its EF
hand domain, thus coupling store depletion to the opening of CRAC
channels. However neither STIM1 nor STIM2 were mutated in the SCID
patients, and expression of STIM1 in SCID T cells did not result in
complementation of the Ca.sup.2+ entry defect.sup.11
[0171] Here we describe the identification of a novel protein
crucial for store-operated Ca.sup.2+ entry and CRAC channel
function. The protein, here termed Orai1, was identified using two
unbiased genetic approaches: a modified linkage analysis to
identify the gene mutated in the SCID patients, and a genome-wide
RNAi screen in Drosophila to identify regulators of store-operated
Ca.sup.2+ entry and NFAT nuclear import. The combination of these
two approaches pinpointed a single candidate gene. We show that
RNAi-mediated depletion of Drosophila Orai abrogates store-operated
Ca.sup.2+ entry as effectively as RNAi against Drosophila Stim. We
further show that a point mutation in Orai1 is responsible for the
Ca.sup.2+ influx defect in the SCID patients, and that
complementation of SCID T cells and fibroblasts with wild type
Orai1 reconstitutes store-operated Ca.sup.2+ influx and CRAC
channel current (I.sub.CRAC). The pharmacological and
electrophysiological properties of the reconstituted currents are
indistinguishable from those of endogenous I.sub.CRAC in control T
cells. The primary sequence of Orai1 predicts four transmembrane
domains, and immunocytochemistry of epitope-tagged Orai1 shows that
the protein is localized at or near the plasma membrane.
Results
Phenotypic Identification of Heterozygous Disease Carriers
[0172] The two SCID patients were born to consanguineous parents,
suggesting an autosomal recessive mode of inheritance as neither
the parents of the SCID patients nor any other members of the SCID
patients' family showed clinical symptoms of immunodeficiency (FIG.
1A). Furthermore, T cells derived from the parents of the SCID
patients showed almost normal store-operated Ca.sup.2+ entry in the
presence of 2 mM extracellular Ca.sup.2+ 10. To unmask a potential
defect in Ca.sup.2+ entry in the parental T cells, we measured the
initial rate of Ca.sup.2+ influx (here defined as the initial rate
of change of intracellular free Ca.sup.2+ concentration,
d[Ca.sup.2+].sub.i/dt) after thapsigargin-mediated store depletion,
but decreased the driving force for Ca.sup.2+ entry by reducing the
extracellular Ca.sup.2+ concentration from 2 mM to 0.2-0.5 mM
CaCl.sub.2. Under these conditions, peak Ca.sup.2+ levels and
Ca.sup.2+ influx rates in T cells from both parents were .about.50%
or less of those observed in wild-type control T cells (FIG. 1B).
We hypothesized that this finding reflected a potential gene-dosage
effect, resulting from the fact that the parents were heterozygous
carriers of the causal mutation in the SCID patients.
[0173] We used this assay to identify other potential heterozygous
carriers of such a mutation in the more extended pedigree. Blood
samples were obtained from 19 additional family members (FIG. 1A),
T cell lines were generated, and Ca.sup.2+ entry phenotype was
evaluated by phenotypic analysis in vitro. Thirteen family members
consistently showed reduced peak Ca.sup.2+ influx and decreased
initial rate of Ca.sup.2+ influx, compared to T cells from 8 other
family members and unrelated controls (FIG. 1C). An arbitrary
cutoff of Ca.sup.2+ influx rate below 2 nM/s was used to
distinguish potential heterozygous disease carriers from unaffected
(homozygous wild-type) individuals (FIG. 1C). With this cutoff, the
distribution of putative heterozygous carriers within the family
appears fully compatible with an autosomal dominant mode of
inheritance (FIG. 1A).
Linkage Mapping by Genome-Wide SNP Array Screen
[0174] Genomic DNA from the 23 members of the SCID family was used
for genotyping using genome-wide SNP arrays. SNP data were
evaluated using two independent linkage analyses. The first
analysis assumed an autosomal recessive mode of inheritance based
on the clinical phenotype, and DNA from the two patients, their
parents, their unaffected brother and their grandparents was
analysed (Pedigree A, indicated by the grey shaded area in FIG.
1A). In contrast, the second analysis utilized the remainder of the
pedigree in a completely independent analysis. Here, an autosomal
dominant mode of inheritance was assumed, based on our ability to
identify heterozygous carriers of the disease mutation by
phenotypic analysis in vitro (Pedigree B, indicated by the green
box in FIG. 1A). Importantly, the first analysis (standard
homozygosity mapping) was performed without consideration of the
heterozygous phenotype status of individuals, and the second
(dominant inheritance) was performed on the large pedigree as two
unrelated halves (the relatives of parent 35 and 36 being treated
independently) such that the results of these two analyses are
fully independent. Thus we can consider the analyses of these two
runs to emerge from three independent pedigrees (one homozygosity
mapping run and two unrelated dominant pedigrees) and can simply
add the parametric LOD scores from these to acquire a statistically
robust combined LOD score (see Materials and Methods).
[0175] Parametric linkage analysis for a recessive trait (Pedigree
A) identified six regions on six chromosomes with LOD scores of
1.5-1.9--while one of these is almost certain to harbor the gene,
it is fully expected that this maximum LOD score would be achieved
several times by chance and thus the homozygosity mapping is not
sufficient alone to map this gene. Satisfyingly, the dominant
analysis identified a unique region on chromosome 12q24, clearly
overlapping with one of the 6 regions identified in the
homozygosity mapping analysis, with a LOD score of .about.3.8. The
combination of these two linkage analyses defines an overlapping
.about.9.8 Mb candidate region with a highly significant cumulative
LOD score of 5.7, representing odds of .about.500,000:1 in favor of
linkage--overwhelmingly likely to contain the true gene. This
region is located between SNP_A-1514003 and SNP_A-1510776 (115.49
Mb-125.27 Mb). In support of this conclusion, no other region in
the genome had a cumulative LOD score exceeding zero. Because
incorrect assignment of heterozygous disease carrier status based
on phenotypic analysis would decrease overall LOD scores rather
than yielding false positives of this magnitude, our novel
combination of recessive and dominant analyses successfully
identifies a genomic region with a very high probability of linkage
to the mutant gene.
[0176] Genomic sequencing of six known genes in this region with a
potential role in Ca.sup.2+ signaling or Ca.sup.2+ binding
(PLA2G1B, CABP1, P2RX7, P2RX4, CAMKK2, PITPNM2) did not reveal any
mutations in exons or immediately adjacent genomic regions. It did
however allow us to narrow down the candidate homozygous region
from .about.9.8 Mb to .about.6.5 Mb, on the basis of several SNPs
in PITPNM2 for which the patients were heterozygous. The .about.6.5
Mb interval contains .about.74 genes, of which 16 were annotated as
"hypothetical proteins" or potential gene loci (Human genome
assembly, NCBI build 35.1). Of these, 2 were predicted to contain
transmembrane domains (KIAA0152 and FLJ14466) using TMHMM and
Phobius algorithms.sup.25,26.
A Genome-Wide RNAi Screen in Drosophila Identifies olf186F (dOrai)
as a Novel Regulator of Store-Operated Ca.sup.2+ Entry
[0177] In parallel with the positional cloning effort, we conducted
a genome-wide RNAi screen for NFAT regulators in Drosophila, as an
independent method of identifying components of the CRAC channel
and the signalling pathway leading to CRAC activation. Drosophila
S2R+ cells, stably-expressing an NFAT-GFP fusion protein, were
incubated for 3 days with arrayed dsRNAs against each of
.about.21,000 Drosophila genes to achieve knockdown of gene
expression. The cells were then stimulated for 10 min with
thapsigargin to deplete Ca.sup.2+ stores, thus activating
store-operated Ca.sup.2+ entry and nuclear translocation of
NFAT-GFP. The cells were then fixed, wells containing the cells
were photographed robotically, and the subcellular distribution of
NFAT-GFP was assessed by visual inspection. Among the positive
candidates whose depletion interfered with NFAT nuclear
translocation were several expected regulators of the
Ca.sup.2+/calcineurin/NFAT signalling pathway, including
Calcineurin B (CanB), Calcineurin A (CanA-14F) and Drosophila
Stim.sup.24,27
[0178] One positive candidate, olf186F, was notable because the
gene encoding one of its three human homologues was located within
the 6.5 Mb homozygous genomic region linked to the SCID mutation at
12q24 (hypothetical protein FLJ14466, NM_032790, NP_116179). For
reasons discussed below, olf186F and its human homologue at 12q24
have been designated Drosophila Orai (dOrai) and human Orai1
respectively; the other two human homologues, C7Orf19 located on
chromosome 7 and MGC13024 located on chromosome 16, have been
designated Orai2 and Orai3 (FIG. 3A). In Drosophila S2R+ cells,
RNAi-mediated depletion of either dStim or dOrai blocked nuclear
translocation and dephosphorylation of NFAT-GFP (FIG. 2B).
Likewise, knockdown of either dSTIM or dOrai completely inhibited
thapsigargin-induced Ca.sup.2+ influx in S2R+ cells (FIG. 2B).
These data confirm previous reports that dSTIM and human STIM1 are
essential for store-operated Ca.sup.2+ entry and CRAC channel
activation in Drosophila and mammalian cells.sup.23,24,28, and
identify dOrai as a second novel regulator of store-operated
Ca.sup.2+ entry in Drosophila cells.
Orai1 is Mutated in the SCID Patients
[0179] Since our data implicated dOrai as a second novel regulator
of store-operated Ca.sup.2+ entry (FIG. 2), and since the gene for
human Orai1 was located in the 12q24 region that is homozygous in
the SCID patients, we asked whether the SCID defect was associated
with a mutation in human Orai1 (FIG. 3). By sequencing genomic DNA
from the 23 individuals (patients and their relatives) shown in
FIG. 1A, we found that both SCID patients were homozygous for a
missense mutation in exon 1 of Orai1. The mutation at position 271
of the coding sequence of Orai1 (position 444 of NM_032790), a
C>T transition, leads to substitution of tryptophan for a
highly-conserved arginine residue at position 91 (R91W) of the
protein (NP_116179, FIG. 3B). The mutated residue is located at the
beginning of the first of four potential transmembrane segments in
Orai1, predicted by calculating the hydrophobicity of Orai1 using
the Kyte-Doolittle method.sup.29 (FIG. 3B, 3C). All 13
phenotypically predicted heterozygous disease carriers (FIG. 1)
were genotypically heterozygous for the mutation (C/T), while
healthy controls and unaffected family members were homozygous for
the wild-type allele (C/C). The mutation at this position is not an
annotated SNP (dbSNP Build 124), rendering it unlikely this is
simply a common polymorphism. To confirm this hypothesis, we typed
this polymorphism in the entire HapMap panel (270 individuals in
total from Utah, Ibadan (Nigeria), Tokyo and Beijing) and did not
find a single copy of the putatively causal "T" allele in this
panel (Materials and Methods, and data not shown).sup.30,31. These
data demonstrate unequivocally that the C>T transition is not a
common sequence variant in the general population; thus the
mutation is likely to have occurred spontaneously in the ancestors
of the SCID patients and is strongly associated with disease.
Expression of Orai1 Restores Store-Operated Ca.sup.2+ Influx in the
SCID T Cells
[0180] We asked whether Orai1 would complement the Ca.sup.2+ influx
defect in the SCID T cells (and fibroblasts) by expressing N- and
C-terminally epitope-tagged versions of wild type and mutant Orai1
in T cells and fibroblasts from the SCID patients. Retroviral
expression of Myc-Orai1.sup.WT in SCID T cells or fibroblasts using
a bicistronic IRES-GFP vector restored Ca.sup.2+ influx in response
to thapsigargin treatment in GFP-positive but not GFP-negative
cells, whereas retroviral expression of mutant R91>W Orai1
(Myc-Orai1.sup.R>W) did not restore Ca.sup.2+ influx. The
inability of Myc-Orai1.sup.R>W to restore Ca.sup.2+ influx in
the SCID T cells and fibroblasts was not due to aberrant expression
of Myc-Orai1.sup.R>W compared to Myc-Orai1.sup.WT, because
mutant and wild-type proteins are present at equivalent levels and
appear to be similarly localized at or near the plasma membrane as
judged by immunoblotting (data not shown) and immunocytochemistry.
We were unable to stain non-permeabilized cells with the anti-myc
antibody, consistent with a topology in which both the N- and
C-termini are cytoplasmically oriented and so inaccessible to the
antibody (FIG. 3C).
[0181] Notably, Ca.sup.2+ influx in SCID T cells (and fibroblasts)
reconstituted with Myc-Orai1.sup.WT did not occur in unstimulated T
cells (or fibroblasts) when 2-20 mM extracellular Ca.sup.2+ was
present but was only observed after store-depletion with
thapsigargin (FIG. 5A-5D). This is an important finding because it
demonstrates that restoration of Ca.sup.2+ influx in
Orai1-expressing cells is dependent on store depletion, a defining
feature of store-operated Ca.sup.2+ entry through CRAC channels,
and is not due to expression or activation of constitutively-open
Ca.sup.2+ channels. Myc-Orai1.sup.WT also restored store-operated
Ca.sup.2+ entry in SCID T cells in response to TCR crosslinking.
The pharmacological characteristics of thapsigargin- and
TCR-induced Ca.sup.2+ entry in SCID T cells and fibroblasts
complemented with Orai1 were exactly those expected for Ca.sup.2+
influx through CRAC channels.sup.12,32. Treatment with 75 .mu.M
2-APB or 2 .mu.M La.sup.3+ inhibited Ca.sup.2+ influx (FIG.
5A,5C,5D), whereas treatment with a low dose of 2-APB (3 .mu.M)
caused a distinct further increase in [Ca.sup.2+].sub.i (FIG. 5B),
although the increase in the Orai1.sup.WT expressing SCID T cells
was slightly lower than that in control T cells (.about.15% vs.
.about.23%). Taken together, these results show clearly that Orai1
is the gene responsible for the Ca.sup.2+ influx defect in the SCID
patients' T cells and fibroblasts.
Expression of Orai1 Restores I.sub.CRAC in the SCID T Cells
[0182] The recovery of Ca.sup.2+ influx seen in the previous
experiments could reflect reconstitution of active CRAC channels in
the patients' cells, or could arise from expression (or activation)
of store-operated, Ca.sup.2+ permeable ion channels distinct from
CRAC. To distinguish between these possibilities, we characterized
in detail the current arising from store-depletion in the SCID
cells reconstituted with wild type or mutant (R91W) Orai1, using
the whole-cell patch-clamp configuration. SCID T cells were
retrovirally transduced with Orai1 in a bicistronic IRES-GFP
vector, and cells expressing Orai1 were identified by GFP
fluorescence as described above. In the experiments shown here,
store depletion was accomplished either by including 8 mM BAPTA in
the patch pipette or by treatment with thapsigargin.
[0183] In SCID cells reconstituted with wild type Orai1, inclusion
of 8 mM BAPTA in the patch pipette caused the slow development of
an inward current in 20 mM Ca.sup.2+.sub..smallcircle., following
whole-cell break-in, reminiscent of the development of I.sub.CRAC
in response to store depletion (FIG. 4A).sup.2,3. By contrast, SCID
T cells expressing the R91W mutant of Orai1 failed to manifest any
inward Ca.sup.2+ currents following store depletion either with
BAPTA (FIG. 4C) or with thapsigargin (data not shown), as expected
from the inability of this mutant protein to reconstitute
store-operated Ca.sup.2+ entry. The current observed in
Orai1-reconstituted SCID T cells displayed many key hallmarks of
the I.sub.CRAC.sup.11,33,34. First, when a divalent-free (DVF)
solution lacking Ca.sup.2+ and Mg.sup.2+, in which the only current
carrier is Na.sup.+, was applied after full development of the
current in 20 mM Ca.sup.2+.sub..smallcircle., an inward Na.sup.+
current was observed that was initially much larger than the
Ca.sup.2+ current but that declined over tens of seconds (FIG. 4A).
This decline of the Na.sup.+ current, known as depotentiation, is
characteristic of CRAC channels in Jurkat T cells, RBL cells and
human T cell lines.sup.11,33,34. Second, both the Ca.sup.2+ and
Na.sup.+ currents showed an inwardly rectifying current-voltage
(I-V) relationship (FIG. 4B). The reversal potential of the inward
current in 20 mM Ca.sup.2+ was >+90 mV, consistent with the
known high selectivity of CRAC channels for Ca.sup.2+, whereas the
reversal potential in divalent-free solution was 49.+-.2 mV (n=4
cells), indicating that the channels are only weakly permeable to
the Cs.sup.+ ions in the patch pipette (P.sub.Cs/P.sub.Na=0.14) and
consistent with the selectivity of CRAC channels for monovalent
ions.sup.33,35. Third, the noise characteristics of the Orai1
complemented current were consistent with those of CRAC channels in
wild-type T cells (FIG. 4D).sup.33. During depotentiation of the
Na.sup.+ current, variance declined linearly with mean current with
an average slope of 29.+-.4 fA (n=4 cells), providing a lower limit
estimate of the unitary current similar to that of previous
measurements of I.sub.CRAC. Furthermore, the Ca.sup.2+ current
resulting from complementation with Orai1 exhibited fast
inactivation in 20 mM Ca.sup.2+.sub..smallcircle. (FIG. 4E); the
extent and time course of inactivation was similar to that
previously reported for CRAC channels in Jurkat T cells (current
inactivates by 54.+-.5% at -100 mV within 200 ms; .tau..sub.fast:
9.+-.2 ms; .tau..sub.slow: 84.+-.12 ms).sup.36. And lastly, the
pharmacological hallmarks of the reconstituted current included
complete block by 2 .mu.M La.sup.3+ (FIG. 4F), inhibition by high
doses of 2-APB (FIG. 4G) and potentiation by low doses of 2-APB
(FIG. 4G); moreover the block observed with high doses of 2-APB was
accompanied by the loss of fast inactivation.sup.32. The
discrepancy between full complementation of CRAC currents by
expression of Orai1 (FIG. 4H) and the partial complementation of
Ca.sup.2+ influx observed by Ca.sup.2+ imaging may be explained by
the fact that for measurements of I.sub.CRAC, we selected T cells
with high GFP/Orai1 levels, whereas for the single-cell Ca.sup.2+
imaging, we averaged responses of all GFP/Orai1-positive cells
(both bright and dim).
[0184] In summary, reconstitution of SCID T cells with Orai1
restores not only store-operated Ca.sup.2+ entry but also a current
that is identical to I.sub.CRAC with regard to store dependence,
ion selectivity and unitary conductance, gating properties, and
pharmacological profile. Thus, we conclude that Orai1 is essential
for CRAC channel function in T cells. The pore properties and
pharmacological characteristics of the channel observed in SCID T
cells complemented with Orai1 are indistinguishable from those of
bonafide CRAC channels.
Discussion
[0185] Here we identify Orai1 as an evolutionarily-conserved
component of store-operated Ca.sup.2+ entry and an essential
contributor to I.sub.CRAC. We show that a point mutation in Orai1
is responsible for the genetic defect in store-operated Ca.sup.2+
entry and I.sub.CRAC function in two patients with a rare form of
severe combined immune deficiency (SCID).sup.10,11. Identification
of Orai1 as the defective gene was accomplished through the
synergistic combination of two independent genetic analyses, both
involving unbiased genome-wide screens.
[0186] Our first screen employed genome-wide SNP analysis to
identify the chromosomal region linked to the SCID disease. Because
only two diseased individuals exist, the theoretically-attainable
LOD score from traditional linkage analysis is .about.1.9,
significantly below the 3.0 value necessary to establish linkage.
Indeed, analysis of a small pedigree including the two SCID
patients, their parents and their grandparents identified 6 regions
on 6 separate chromosomes with maximum LOD scores of 1.9 (Pedigree
A). To extend the amount of genetic information available, we
devised a method of identifying heterozygous carriers of the mutant
allele. This was accomplished through a simple modification of our
in vitro method of measuring store-operated Ca.sup.2+ influx, in
which the driving force for Ca.sup.2+ entry was decreased by
reducing the extracellular Ca.sup.2+ concentration. When this assay
was applied to T cell lines derived from 21 additional family
members of the SCID patients (Pedigree B), 13 members showed a
significantly reduced initial rate of Ca.sup.2+ influx, which we
interpret as reflecting a gene-dosage effect consistent with
heterozygosity for the mutant allele. A second, completely
independent linkage analysis, in which the haplotype of these 13
putatively heterozygous individuals was compared to that of the
remaining 8 homozygous healthy family members, yielded experimental
LOD scores that identified a unique region on 12q24 with a LOD
score of 3.8. This region overlapped with one of the regions
identified by linkage analysis of Pedigree A. Because the
individuals used for each analysis and the phenotypes used to
classify them were distinct, allele sharing and thus linkage
results were completely independent in these analyses; hence we
could combine LOD scores from the two analyses to obtain an
unbiased cumulative and highly significant LOD score of .about.5.7
for an .about.9.8 Md region at 12q24. In principle, this novel and
powerful combination of linkage mapping approaches may be applied
to elucidate the genetic causes of other rare autosomal-recessive
diseases, even if only a very few diseased individuals are
available and conventional homozygosity mapping fails to establish
linkage. Prerequisites are that other family members are available
and that mutation of one allele can be detected as a quantifiable
trait in vitro.
[0187] In the hope of rapidly identifying a gene in the 12q24
region that was involved in store-operated Ca.sup.2+ entry, we
conducted a parallel genome-wide RNAi screen in Drosophila, taking
advantage of the fact that Drosophila S2R cells contain a
store-operated Ca.sup.2+ channel with characteristics very similar
to CRAC.sup.37. Rather than focusing solely on Ca.sup.2+ entry, we
designed the screen to identify evolutionarily-conserved regulators
of the Ca.sup.2+-regulated transcription factor NFAT; although
Ca.sup.2+-regulated NFAT proteins are not themselves represented in
Drosophila, there is strong evolutionary conservation of the
pathways which regulate its nuclear-cytoplasmic shuttling, through
effects on Ca.sup.2+ homeostasis, store-operated Ca.sup.2+ entry,
calcineurin activity and kinase-phosphatase balance.sup.27. The
screen was used to identify candidates whose RNAi-mediated
depletion interfered with nuclear localization of an NFAT-GFP
fusion protein in response to stimulation with thapsigargin. Among
the positive candidates was olf186F (here renamed Drosophila Orai),
which has three human homologues, FLJ14466, C7Orf19 and MGC13024.
Since these are novel proteins without known function, we named
them Orai1-3, respectively. In Greek mythology, the Orai are the
keepers of the gates of heaven: Eunomia (Order or Harmony), Dike
(Justice) and Eirene (Peace).sup.38-40; in Japan, Orai is in part
derived from the sound of "all right" in English and also refers to
comings and goings, communication, streets and traffic in Japanese.
In a satisfying validation of our dual strategy, the gene encoding
Orai1 (hypothetical protein FLJ14466) is located on chromosome
12q24, exactly the region identified by our SNP analysis as linked
genetically to the SCID syndrome. DNA sequencing rapidly revealed
the genetic basis for the SCID defect as a point mutation (C>T)
in exon 1 of Orai1, which resulted in an arginine to tryptophan
substitution at residue 91. This mutation is not a known
polymorphism, as confirmed by sequencing DNA from 270 individuals
of mixed ethnic backgrounds assembled for the international HapMap
project.sup.31. This number of samples is sufficient to find almost
all haplotypes with frequencies of 5% or higher. Although there is
a small chance that the C>T mutation is a SNP confined to a
small ethnic population not represented in the HapMap panel, this
possibility can be ruled out with reasonable certainty based on the
fact that complementation with Orai1 restores store-operated
Ca.sup.2+ entry and I.sub.CRAC in SCID patient cells. Furthermore,
arginine 91 which is mutated in the SCID patients is located in a
putative transmembrane region that is highly conserved across
species (FIG. 3A), highlighting its potential importance in the
function of Orai1.
[0188] The characteristics of Ca.sup.2+ influx and Ca.sup.2+
current in Orai1 1-complemented SCID T cells were indistinguishable
from those observed in control T cells. In particular, both
processes were strictly regulated by store depletion, and the
electrophysiological and pharmacological properties of the restored
current were fully consistent with those of I.sub.CRAC. These
properties include: an extremely high selectivity for Ca.sup.2+
over monovalent cations, inwardly rectifying I-V relation,
depotentiation under divalent-free conditions, current noise
characteristics, rapid Ca2+-dependent inactivation, blockade by low
micromolar La.sup.3 and positive and negative modulation by 2-APB.
We therefore conclude that Orai1 reconstituted I.sub.CRAC in the
SCID patients' T cells, and thus that the C>T transition and
resulting R91W mutation in the Orai1 coding region and protein are
responsible for the SCID defect. While its specific role has not
yet been determined, the available data are consistent with the
possibility that Orai1 encodes a channel subunit or a
closely-associated channel regulator in the plasma membrane. First,
the hydropathy profile of Orai1 predicts a membrane protein with
three, or potentially four, hydrophobic membrane domains (FIG. 3B).
Second, immunocytochemistry of myc-tagged Orai1 is consistent with
localization at the plasma membrane under resting conditions; this
distribution differs from that of STIM1, which is predominantly
located in the ER where it is thought to sense Ca.sup.2+ store
depletion via its luminal EF hand domain (Feske 2005, Liou 2005,
Ref). Notably, both N- and C-terminal epitope tags on Orai1 are
inaccessible to antibody staining in non-permeabilised cells; this
finding is consistent with the prediction of four transmembrane
domains and predicts a topology compatible with a channel subunit,
in which both N- and C-termini are cytoplasmically oriented (FIG.
3C). Further studies will be necessary to determine whether Orai1
is part of the CRAC channel itself, or whether it encodes a
regulator of the channel.
[0189] Orai1 is widely expressed at the mRNA level, potentially
explaining our previous observations that not only T cells but also
B cells and fibroblasts from the SCID patients show a substantial
defect in store-operated Ca.sup.2+ entry. Surprisingly, however,
the clinical phenotype of the SCID patients is predominantly one of
immunodeficiency, associated in the single surviving patient with
ectodermal dysplasia and anhydrosis (EDA) and a mild, congenital,
non-progressive myopathy. EDA is characterized by defective tooth
enamel and hair follicle function, and complete absence of sweat
glands, and many previous studies have linked it to hypoactivation
of NF-.kappa.B.sup.41-45. Ca.sup.2+ mobilization is thought to
contribute to NF.kappa.B activation in T cells and other cell types
under certain conditions of stimulation.sup.46, thus the EDA
syndrome may well reflect defective NF.kappa.B activation, either
during development or acutely in specific cell types. In contrast
the myopathy could potentially be a direct consequence of defective
NFAT activation, given that NFAT has a major role in certain
aspects of skeletal muscle development and function (reviewed
in.sup.7,47).
[0190] In conclusion, our studies establish a critical role for
Orai1 in T cell function and the in vivo immune response. A single
point mutation in Orai1, a novel protein conserved from C. elegans
to humans, disrupts store-operated Ca.sup.2+ entry and CRAC channel
function in patients with an inherited immune deficiency. Future
studies will address the relation between Orai and Stim proteins
and the mechanism by which store depletion couples to CRAC channel
opening.
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Example 2: A Genome-Wide Drosophila RNAi Screen Identifies DYRK as
a Novel Regulator of NFAT
[0245] Materials and Methods the Genome-Wide Primary Screen
[0246] Methods were adapted from refs.sup.12,13. 10.sup.4 S2R.sup.+
cells were added into each well containing 0.25 .mu.g of dsRNAs in
10 .mu.l of serum-free medium and incubated for 1 h at 26.degree.
C. The cells were then transiently transfected with
NFAT1(1-460)-GFP expression plasmid.sup.9,17 (10 ng) in Schneider's
medium (Invitrogen) (30 .mu.l). After incubation for 48-72 hrs at
26.degree. C., the cells were fixed and stained with DAPI, and the
coincident GFP and DAPI images were acquired by an automated camera
from three different locations in each well. A total of fifty-eight
384-plates were analysed, containing a total of 21,884 wells into
which individual dsRNAs had been arrayed.
[0247] Control wells (no dsRNA, dsRNA against GFP, and dsRNA
against a gene (thread-anti-apoptotic) causing cell death) were
present on each plate and served as an internal control for
knockdown efficiency of each plate. All three photographs of GFP
fluorescence in each assay well were manually scored using
MetaMorph 6.1 Software (Universal Imaging Corporation). To identify
even weak effectors of NFAT localization non-stringent criteria
were used in the primary screen, such that wells were scored
positive even if only one cell in each of three fields showed
complete nuclear localization of NFAT-GFP. Since the RNAi library
was constructed before the Drosophila genome was completely
annotated, 39 of the 738 positives did not correspond to known
genes and were eliminated. Another 37 candidates were eliminated
because the dsRNAs used to identify them had more than 10 predicted
"off-targets" with exact matches of 21 nucleotides (nt) (see
Bioinformatics and Classification below).
The Confirmatory Screen
[0248] The confirmatory screening on the 699 potentially positive
candidates from the primary screen was performed essentially as
described for the primary screen, except that S2R+ cells stably
transfected with NFAT1 (1-460)-GFP were used, and candidates were
tested for whether their depletion altered NFAT subcellular
localization in both resting and stimulated S2R+ cells. Wells in
which all cells contained cytoplasmic NFAT-GFP got the lowest score
(0) while wells with >90% of the cells showing nuclear NFAT-GFP
scored the highest (3). The summed scores from all three
experiments are presented in Table I. Note that the highest
possible score is 9, but because we scored conservatively in the
confirmatory screen, the highest actual score obtained by any
candidate is 6. All candidates were also tested for whether they
prevented NFAT nuclear localization in cells treated with
thapsigargin (1 .mu.M, 30 min); only Drosophila STIM (dSTIM) scored
positive in this assay.
[0249] To generate the stably-expressing cell line, the coding
sequence for the NFAT1 (1-460)-GFP fusion protein was subcloned
into the expression plasmid pAc5.1 (Invitrogen), and the
macrophage-hemocyte-like Drosophila cell line S2R+ was transfected
in a 6-well format using Effectene (Qiagen) with a 19:1 ratio of
the expression plasmid to pCoHygro (Invitrogen), which encodes a
hygromycin resistance gene under the control of a constitutively
active promoter. The cells were selected for 3-4 weeks with 300
.mu.g/ml hygromycin, and stable clones were selected by visual
inspection.
Bioinformatics and Classification
[0250] Scores were consolidated and formatted for submission to the
DRSC (Drosophila RNAi Screening Center at Harvard Medical School),
which then provided the identity of the genes assayed (FlyBase
identifier; Drosophila gene name, where known; some Gene Ontology
(GO) identifiers; and some human homologues). Gene Ontology (GO)
annotation was retrieved in two ways. First, we employed Ensembl's
EnsMart tool using the FlyBase identifier for each gene to get the
GO description. Second, we used the GO identifiers provided by the
screening center to get descriptions from the "GO terms and IDs"
file from the Gene Ontology Consortium. Functional categories of
genes were constructed by keyword searches of the positives
followed by manual curation. Positive genes were also examined for
involvement in common pathways using tools such as those at the
KEGG Pathway Database.
[0251] For each candidate that was positive in the primary screen,
the number of off-targets was determined using the off-target
sequence search tool on the DRSC website
(http://www.flyrnai.org/RNAi_primer_design.html). This
bioinformatic tool is based on an algorithm similar to that in
ref.sup.37 except that it does not have a built-in primer design
component (Flockhart et al., submitted). Amplicon (dsRNA) sequences
are searched for predicted off-targets by considering all possible
fragments, of length 16-50 bp with a default value of 21 bp, that
perfectly match sequences in fly transcripts in release 4.0.
Ideally, only 1 match corresponding to the targeted mRNA should be
found, but some amplicons have matches with other mRNAs which are
not the intended target. For the genes in Table I, a default length
of 21 nt was used to compute the number of off-targets for each
positive candidate, and candidates with >10 off-targets were
eliminated. For the genes in Table II (calcineurin) and III
(candidates used for additional experiments), shorter fragments of
19 nt and 20 nt were considered as well. The identity of
off-targets was determined using BLASTN against Drosophila NCBI
RefSeq database. Mammalian orthologues of Drosophila melanogaster
proteins in Table I were retrieved from the NCBI Homologene
database
(http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=homologene). The
human homologues of the fly kinases were obtained by reciprocal
blast method using BLASTP; Altschul, et al. 1990, J. Mol. Biol.
215:403-410), as described.sup.38,39. Phylogenetic analysis was
performed using TCoffee.sup.40, and the reliability of the ortholog
assignments was assessed with the bootstrap method implemented in
Orthostrapper.sup.41.
DsRNA Mediated Knockdown in Drosophila Cells
[0252] PCR fragments (size up to 600 bp) were used as templates for
in vitro transcription reactions, followed by DNase I treatment to
remove the template DNA. After purification, dsRNA (5 .mu.g) was
co-transfected together with the NFAT-GFP expression plasmid into
S2R+ cells in 8-chamber slides (10 .mu.g for 12 well plate). After
72 hrs of incubation, cells were left untreated or were treated
with the Ca.sup.2+ influx inducers, 1 .mu.M ionomycin or 1 .mu.M
thapsigargin for localization assays and were trypsinized for the
measurement of [Ca2+]i levels.
In Vitro Kinase Assays
[0253] FLAG-tagged human kinases were immunoprecipitated from whole
cell lysates of transiently-transfected HEK293 cells using
anti-FLAG antibody-coupled protein G beads (Sigma), and
immunoprecipitates were analysed for phosphorylation of either the
entire NFAT1 regulatory domain (GST-NFAT1 [1-415]) expressed in
bacterial cells, or GST-fused peptides corresponding to the SRR-1
(amino acids 149-183), SP-2 (amino acids 206-237) and SP-3 (amino
acids 264-295) motifs of NFAT1 (both wild-type and Ser.fwdarw.Ala
mutants in serines phosphorylated in vivo).sup.10. Immunocomplexes
were washed twice with lysis buffer (1.0% NP-40, 50 mM HEPES pH
7.4, 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 1 mM dithiothreitol [DTT],
20 mM .beta.-glycerol-phosphate, 10 mM sodium pyrophosphate, 0.1 mM
sodium orthovanadate, 10 mM NaF, 1 mM phenylmethylsulfonyl fluoride
[PMSF], 10 .mu.g/ml aprotinin, 10 .mu.g/ml leupeptin) and twice
with kinase buffer (20 mM HEPES, pH 7.4, 20 mM MgCl.sub.2, 1 mM
DTT, 0.1 mM sodium orthovanadate, 20 mM .beta.-glycerol-phosphate),
and incubated at 30.degree. C. for 20 minutes in a 40 .mu.l final
volume of kinase buffer in the presence of 20 .mu.M ATP, 2 .mu.Ci
[.gamma..sup.32P]-ATP and 10 .mu.g of wild-type or mutant
GST-peptide substrate. Peptides were isolated on
glutathione-sepharose and phosphorylation was assessed by SDS gel
electrophoresis and autoradiography.
[0254] The ability of DYRK1A and DYRK2 to phosphorylate GST-NFAT1
fusion peptides was examined using 20 ng of recombinant protein
kinase (Upstate Biotechnology) in a 40 .mu.l final volume of kinase
buffer in the presence of 20 .mu.M ATP, 2 .mu.Ci [-.sup.32P]-ATP
and 10 .mu.g of GST-peptide substrate. The ability of GSK3 to
phosphorylate NFAT1 was examined by first pre-phosphorylating GST
fusion proteins pre-bound to glutathione sepharose beads using 1 U
of recombinant protein kinase A (PKA) (New England Biolabs [NEB]),
20 ng DYRK1A or DYRK2 in the presence of 1 mM cold ATP for 16 h at
30.degree. C. After cold priming fusion proteins were washed
repeatedly to remove recombinant kinase and ATP. Phosphorylated
fusion proteins were then incubated with 1 U of GSK3 (NEB) in a 40
.mu.l final volume of kinase buffer in the presence of 20 .mu.M
ATP, 2 .mu.Ci [.gamma..sup.32P]-ATP for 45 minutes.
Reporter Assays and IL-2 Expression Assays
[0255] Exponentially growing (10.sup.7) Jurkat T cells stably
expressing HA-tagged full-length NFAT1 in the pOZ vector.sup.42
were transfected by electroporation at 250 V and 960 .mu.F. For
luciferase experiments, cells were transfected with 0.5 .mu.g pRLTK
reporter (Renilla luciferase for internal control), 5.0 .mu.g pGL3
reporter (firefly luciferase, experimental promoter) and expression
plasmids encoding empty vector, wild type or kinase dead DYRK2. At
24 h post transfection cells left untreated or stimulated with PMA
(20 nM), ionomycin (1 M) and 2 mM CaCl.sub.2 for 6 hours were
measured for reporter gene activity using the Dual-Luciferase
Reporter Assay (Promega) as recommended by the manufacturer. For
intracellular cytokine staining, cells were co-transfected with
GFP-encoding plasmid and empty vector plasmids, wild type or
kinase-dead DYRK2. At 24 h post transfection cells left untreated
or stimulated with PMA (20 nM), ionomycin (1 .mu.M) and 2 mM
CaCl.sub.2 for 6 hours in the presence of Brefeldin A (2 .mu.g/mL)
for the last 4 hours were fixed with 4% paraformaldehyde in PBS for
20 min at 25.degree. C., washed twice with PBS, permeabilized in
saponin buffer (PBS, 0.5% saponin [Sigma], 1% BSA and 0.1% sodium
azide) and stained with phycoerythrin-conjugated rat anti-human
IL-2 (PharMingen) for 30 min at 25.degree. C. Cells were washed
twice in PBS and analyzed with a FACSCalibur flow cytometer (Becton
Dickinson) and FlowJo software.
siRNA-Mediated Knockdown of DYRK1A
[0256] 0.5.times.10.sup.6 HeLa cells stably expressing
NFAT1(1-460)-GFP were seeded in 6-well plates and transfected the
next day with siRNAs (Dharmacon, Inc., Lafayette, Colo.)
corresponding to control siRNA or human DYRK1A siRNA using
lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad,
Calif.) according to the manufacturer's protocol. Cells were
reseeded and the transfection procedure was repeated after 24 h to
increase the efficiency of knockdown. Cells were harvested for
immunoblot analysis or immunocytochemistry 4 days post
transfection. DYRK transcript levels were measured by real-time
RT-PCR. Threshold cycles (CT) for DYRK1A were normalized to GAPDH
housekeeping gene expression levels (ACT) and plotted as
0.5.sup..DELTA.Ct.quadrature.*10.sup.4 (arbitrary units). The siRNA
sequences correspond to DYRK1A: AGGUGGAGGUGCAAUAUUA (SEQ ID NO:
31); scrambled control: CUUUAAGCCUCGAGAUAUA (SEQ ID NO: 32). The
RT-PCR primer sequences correspond to DYRK1A sense:
AGTTCTGGGTATTCCACCTGCTCA (SEQ ID NO: 10), DYRK1A anti-sense:
TGAAGTTTACGGGTTCCTGGTGGT (SEQ ID NO: 11).
Intracellular Calcium Measurements by Time-Lapse Video Imaging
[0257] HEK 293T cells were grown directly on UV-sterilized
coverslips, loaded with Ca.sup.2+ indicator dye Fura-2 AM (3 .mu.M,
Molecular Probes, Eugene, Oreg.) for 45 min at room temperature,
washed and resuspended in loading medium (RPMI+10% FCS). For
ratiometric Ca.sup.2+ videoimaging, coverslips were mounted on a
closed bath RC-20 flow chamber (Warner Instrument Corp., Hamden,
Conn.) and perfused in 2 mM Calcium Ringer solution (155 mM NaCl,
4.5 mM KCl, 10 mM D-glucose, 5 mM Hepes (pH 7.4), 1 mM MgCl.sub.2,
2 mM CaCl.sub.2). After switching to Ca.sup.2+ free Ringer solution
(2 mM Ca.sup.2+ replaced with 2 mM MgCl.sub.2), intracellular
Ca.sup.2+ stores were depleted with 1 .mu.M thapisgargin, and
store-operated Ca2+ influx was measured after perfusing cells with
Ringer solution containing 2 mM CaCl2. Single cell video imaging
was performed on a S200 inverted epifluorescence microscope (Zeiss,
Thornwood, N.Y.) using OpenLab imaging software (Improvision,
Lexington, Mass.). Fura-2 emission was detected at 510 nm following
excitation at 340 and 380 nm, respectively, with ratios of 340/380
being calculated for each 5 sec interval after background
subtraction. Calibration values (R.sub.min, R.sub.max, S.sub.f)
were derived from cuvette measurements as previously
described.sup.43. For each experiment, approximately 50-100 cells
were analyzed. For simultaneous measurements of [Ca.sup.2+]i and
DYRK2 expression, Jurkat T cells were cotransfected with DYRK2 cDNA
and eGFP at a ratio of 10:1. 48 hrs post transfection, cells were
used for Ca.sup.2+ imaging as described above. For single cell
analysis of [Ca2+]i, GFP.sup.- (that is, DYRK2.sup.-) and GFP.sup.+
(that is, DYRK.sup.2+) cells were gated and plotted separately.
Intracellular Calcium Measurements by Flow Cvtometry
[0258] S2R+ cells were detached from the dish with trypsin
(CellGro, Herndon, Va.) and loaded with the Ca.sup.2+ indicator dye
Fluo4-AM (2 .mu.M Molecular Probes, Eugene, Oreg.) for 45 min at
room temperature and then resuspended in loading medium (RPMI+10%
FCS). Immediately before the flow cytometric Ca.sup.2+
measurements, cells were resuspended in Ca.sup.2+ free Ringer
solution and analyzed on a FACSCalibur (BD Biosciences, San Jose,
Calif.). 180 sec after addition of thapsigargin (3 .mu.M) in
Ca.sup.2+ free Ringer to deplete intracellular Ca.sup.2+ stores, 4
mM Ca.sup.2+ Ringer solution was added to the cells to achieve a
final concentration of 2 mM Ca.sup.2+. Cellular Ca.sup.2+ levels
were then analyzed using FloJo software (Tree Star, Inc., Ashland,
Oreg.).
Subcloning of Human Orthologues of the Candidate Kinases
[0259] Full-length cDNAs encoding human orthologues of the kinase
candidates were obtained from Flexgene Kinase Repository (Harvard
Institute of Proteomics).sup.36 or the Mammalian Gene Collection
(MGC, Open Biosystems), subcloned into pENTRY. 11 (Invitrogen)
vectors with insertion of Flag-tag at the N-terminus, and then
recombined into pDEST12.2 (Invitrogen). Kinase-dead DYRK2 was
constructed by introducing a K251R point mutation in the ATP
binding pocket of the active site using the PCR-based method
(QuikChange Site-Directed Mutagenesis, Stratagene) and sequenced to
ensure polymerase fidelity.
Introduction and Results
[0260] The subcellular localization of NFAT is determined by a
complex process of signal integration that involves inputs from
diverse signalling pathways.sup.3-5. In resting cells, NFAT
proteins are heavily phosphorylated and reside in the cytoplasm; in
cells exposed to stimuli that raise intracellular free Ca.sup.2+
([Ca.sup.2+].sub.I) levels they are dephosphorylated by the
calmodulin-dependent phosphatase calcineurin and translocate to the
nucleus.sup.3,6. Dephosphorylation of NFAT by calcineurin is
countered by distinct NFAT kinases, among them CK1, GSK3, and
various members of the MAP kinase family.sup.3,7-10. The
transcriptional activity of NFAT is regulated by additional inputs,
including phosphorylation of the N-terminal transactivation domain,
recruitment of co-activators and co-repressors, and choice of
partner proteins in the nucleus.sup.3,9,11.
[0261] We used a strategy, based on genome-wide RNAi screening in
Drosophila S2R+ cells.sup.12-14, to identify regulators of
intracellular free Ca.sup.2+ ([Ca.sup.2+].sub.I) levels,
calcineurin activation and NFAT localization in cells. The strategy
relies on the fact that although Ca.sup.2+-regulated NFAT proteins
are not represented in Drosophila, the pathways of Ca.sup.2+
homeostasis, Ca.sup.2+ influx, and calcineurin activity that
regulate NFAT localization are evolutionarily conserved.sup.15,16.
To validate this point, we used the GFP fusion protein
NFAT1(1-460)-GFP (here termed NFAT-GFP).sup.17. NFAT-GFP contains
the entire regulatory domain of NFAT, including the calcineurin and
CK1 docking sites, the nuclear localization signal (NLS), and the
conserved serine-rich regions (SRR) and serine-proline repeat (SP)
motifs which control NFAT1 subcellular localization and DNA-binding
affinity.sup.3,9,10,17 (FIG. 6A). NFAT-GFP was correctly regulated
in Drosophila S2R+ cells: it was phosphorylated and properly
localized to the cytoplasm under resting conditions and became
dephosphorylated and translocated to the nucleus in response to
Ca.sup.2+ store depletion with the SERCA inhibitor thapsigargin
(FIG. 6B); it was imported into the nucleus with similar kinetics
in S2R+ cells and mammalian HeLa cells and was sensitive to the
calcineurin inhibitor CsA in both cell types. S2R+ cells treated
with limiting amounts of thapsigargin displayed intermediate
phosphorylated forms of NFAT-GFP, most likely reflecting
progressive dephosphorylation of serines within the individual
conserved motifs of the regulatory domain.sup.9,10. Finally,
depletion of the primary NFAT regulator, calcineurin, by RNAi in
S2R+ cells inhibited thapsigargin-dependent dephosphorylation and
nuclear import of NFAT-GFP (Table II). Together these experiments
confirmed that the major pathways regulating NFAT phosphorylation
and subcellular localization--store-operated Ca.sup.2+ influx,
calcineurin activation, and NFAT phosphorylation--are conserved in
Drosophila and appropriately regulate vertebrate NFAT.
[0262] We performed a genome-wide RNAi screen.sup.12,13 on
unstimulated S2R+ cells, and scored visually for aberrant nuclear
localization of NFAT-GFP (see Methods and Example 3). Of 21,884
screened wells, 662 were scored as potentially positive using
non-stringent criteria; in a confirmatory screen, 271/325 (83%)
retested candidates were confirmed as positive, attesting to the
reproducibility of our initial assessment of NFAT nuclear
localization (FIG. 6C). Positive candidates included
Na.sup.+/Ca.sup.2+ exchangers and SERCA Ca.sup.2+ ATP-ases whose
knockdown would be expected to increase basal [Ca.sup.2+].sub.i,
and the scaffold protein Homer which has been linked to Ca.sup.2+
influx and Ca.sup.2+ homeostasis.sup.18,19 (Table I). The screen
also identified Stim, a recently-identified regulator of
store-operated Ca.sup.2+ influx.sup.20-22 as causing nuclear
localization of NFAT-GFP in resting S2R+ cells, possibly because
its depletion resulted in minor dysregulation of NFAT kinases or
small increases in basal [Ca.sup.2+].sub.I levels (FIGS. 9A-9C).
Finally, the screen identified a large number of protein kinases
which could potentially influence basal [Ca.sup.2+].sub.I levels or
calcineurin activity, directly phosphorylate the NFAT regulatory
domain, or indirectly influence the activity of direct NFAT kinases
(Table I).
[0263] We were interested in kinases that directly phosphorylate
the NFAT regulatory domain. In the family member NFAT1, the
regulatory domain bears >14 phosphorylated serines, 13 of which
are dephosphorylated by calcineurin.sup.9 (FIG. 6A). Five of these
serines are located in the SRR-1 motif, which controls exposure of
the NLS and is a target for phosphorylation by CK1.sup.3,10; three
are located in the SP-2 motif, which can be phosphorylated by GSK3
after a priming phosphorylation by protein kinase A (PKA).sup.7,10;
and four are located in the SP-3 motif, for which a relevant kinase
had yet to be identified at the time this study was initiated. The
SP-2 and SP-3 motifs do not directly regulate the subcellular
localization of NFAT1, but their dephosphorylation increases both
the probability of NLS exposure and the affinity of NFAT for
DNA.sup.3,10,23. It was not known how distinct SRR-1, SP-2 and SP-3
kinases acted together to promote the full phosphorylation of NFAT;
nevertheless, we expected that depletion of individual NFAT kinases
in S2R+ cells would result in varying degrees of nuclear
accumulation of NFAT, depending on kinase expression level, the
particular motif phosphorylated, and whether or not other related
kinases were redundantly expressed. We therefore tested at least
one mammalian homologue (where available) of all
constitutively-active kinases identified in the screen, regardless
of their score in the secondary screen. Some inducible kinases were
included, but others (e.g. protein kinases C and D) will be
investigated as part of a separate study.
[0264] FLAG-tagged mammalian homologues of selected Drosophila
kinases were expressed in HEK293 cells, and anti-FLAG
immunoprecipitates were tested in an in vitro kinase assay for
their ability to phosphorylate the GST-NFAT1(1-415) fusion protein
(FIG. 7A). Three novel candidates--PRKG1, DYRK2 and IRAK4--showed
strong activity in this assay (FIG. 7A, lanes 8, 13 and 15; CK1
isoforms CK1.alpha. and CK1.epsilon. were included as positive
controls in lanes 1 and 2). PRKG1 was expressed at equivalent or
higher levels than DYRK2 (FIG. 7A, bottom panel, lanes 8 and 13),
but only DYRK2 could counter the dephosphorylation of NFAT-GFP by
calcineurin (FIG. 7B, lanes 3, 4; 7, 8; 11, 12). IRAK4 was poorly
expressed (FIG. 7A, bottom panel, lane 15); however CD4+Th1 cells
isolated from IRAK4-/- mice showed normal NFAT1 dephosphorylation,
rephosphorylation and nuclear transport compared to control cells.
For these reasons, neither PRKG1 nor IRAK4 were further
investigated.
[0265] We focused on the role of DYRK-family kinases as direct
regulators of NFAT. Overexpression of DYRK2 maintained NFAT-GFP in
its phosphorylated form after ionomycin treatment (FIG. 7B, lanes
5-8); similarly, overexpression of wild type (WT) DYRK2 but not a
kinase-dead (KD) mutant of DYRK2, prevented NFAT nuclear
localization in thapsigargin-treated cells. DYRK overexpression
yielded a slower-migrating form of NFAT (FIG. 7B, lanes 7, 8),
leading to the concern that DYRK (a serine/proline-directed
kinase.sup.24) phosphorylated SPRIEIT (SEQ ID NO: 33), the
calcineurin docking sequence on NFAT1.sup.3,6, preventing
NFAT:calcineurin interaction. However, DYRK2 inhibited the
ionomycin-induced dephosphorylation of NFAT-GFP containing a
SPRIEITPS (SEQ ID NO: 53)>HPVIVITGP (SEQ ID NO: 54) (VIVIT) (SEQ
ID NO: 30) substitution.sup.17, which eliminates the SP and TP
sequences that could be targeted by DYRK. The ability of DYRK to
inhibit dephosphorylation of the VIVIT (SEQ ID NO: 30)-substituted
NFAT-GFP is particularly impressive, given the higher affinity
(.about.40-50-fold) of the VIVIT (SEQ ID NO: 30) docking site for
calcineurin compared to the affinity of the wild type SPRIEIT (SEQ
ID NO: 33) docking site.sup.17. Consistent with direct
phosphorylation of NFAT, Ca.sup.2+ mobilization in response to
thapsigargin was unaffected by depletion of the DYRK-family
candidate CG40478 in S2R+ cells, and only slightly diminished by
DYRK2 overexpression in Jurkat T cells.
[0266] DYRKs constitute an evolutionarily-conserved family of
proline or arginine-directed protein kinases distantly related to
cyclin-dependent kinases (CDK), mitogen-activated protein kinases
(MAPK), glycogen synthetase kinases (GSK), and CDK-like (CLK)
kinases (CMGC kinases.sup.24. The DYRK family has multiple members
(FIG. 11A) which have been designated class I (nuclear, DYRK1A and
DYRK1B) or class II (cytoplasmic, DYRK2-6), depending on their
subcellular localisation.sup.25,26. RT-PCR and western blotting
suggested that DYRK1A and DYRK2 were major representatives of
nuclear and cytoplasmic DYRKs in Jurkat T cells, respectively (FIG.
11B). Depletion of endogenous DYRK1A using DYRK1A-specific siRNA in
HeLa cells stably expressing NFAT-GFP increased the rate and extent
of NFAT1 dephosphorylation and nuclear import while slowing
rephosphorylation and nuclear export, in response to treatment with
thapsigargin for 10 min (to induce dephosphorylation and nuclear
import) followed by CsA addition for 5 to 30 min (to inactivate
calcineurin and permit rephosphorylation by NFAT kinases for
nuclear export) (FIG. 10C left panel). Results obtained using
endogenous DYRK1A depletion, which reflect a knockdown efficiency
of approximately 70% of mRNA levels (FIG. 10C right panel),
indicate that DYRK represent physiological negative regulators of
NFAT activation in cells.
[0267] Further experiments showed that DYRK specifically targeted
the SP-3 motif of NFAT1. FLAG-tagged DYRK2 was expressed in HEK 293
cells, immunoprecipitated with anti-FLAG antibodies, and
phosphorylated peptides corresponding to the conserved SP-3 but not
the SP-2 motif of the NFAT regulatory domain in vitro. To rule out
the possibility that the NFAT kinase was not DYRK itself but rather
a DYRK-associated kinase, we tested bacterially-expressed
recombinant DYRK1A and DYRK2 for in vitro phosphorylation of
peptides corresponding to three conserved serine-rich motifs of
NFAT1 phosphorylated in cells (SRR-1, SP-2 and SP-3 motifs.sup.9).
DYRK2 and DYRK1A both displayed strong and selective kinase
activity towards the SP-3 motif of NFAT1, but neither kinase
phosphorylated an SP-3 peptide with Ser>Ala substitutions in the
specific serine residues known to be phosphorylated in cells.sup.9.
At least 2 serine residues (bold and underlined) in the SP-3 motif
(SPQRSRSPSPQPSPHVAPQDD) (SEQ ID NO: 34) fit the known sequence
preference of DYRK kinases for serine/threonine residues with
arginine at the -2 or -3 position, and proline (or valine) at the
+1 position.sup.27-29, and both are known to be phosphorylated in
cells.sup.9 (see FIG. 6A). Additional studies will be needed to
establish whether the two other phosphorylated serine residues
(underlined) in the SP-3 motif are targets for DYRK or other NFAT
kinases in vivo.
[0268] Phosphorylation at the SP-2 and SP-3 motifs are the primary
determinants for upward mobility shift of phosphorylated NFAT1, and
we have shown here and previously that they are phosphorylated by
GSK3 and DYRK, respectively.sup.9. Because DYRK kinases have been
reported to prime for GSK3-mediated phosphorylation of
protein-synthesis initiation factor eIF2B.epsilon. and the
microtubule-associated protein tau.sup.29, we asked whether DYRK
kinases could similarly prime for GSK3-mediated phosphorylation of
NFAT. The SP2 motif of NFAT1 can be phosphorylated by GSK3.sup.10,
and GSK3 recognition of the target sequence requires a priming
phosphorylation that can be mediated by PKA. In contrast to the
strong priming by PKA, neither DYRK2 nor DYRK1A could efficiently
prime for phosphorylation of the SP-2 motif by GSK3.
[0269] As DYRK2 phosphorylated only the SP-3 motif of NFAT in
vitro, and because it was not a priming kinase for GSK3 at the SP-2
motif, we expected that it would cause only half the expected
mobility shift of NFAT1 when expressed in cells. However,
overexpression of DYRK2 resulted in complete phosphorylation of
NFAT1 (FIG. 7B). To resolve this paradox, we asked whether prior
phosphorylation of the entire NFAT regulatory domain by DYRK would
facilitate further phosphorylation by GSK3. The GST-NFAT1(1-415)
fusion protein was prephosphorylated to completion by PKA or DYRK2
using the recombinant kinases, then washed and incubated briefly
(45 min) in the absence or presence of recombinant GSK3 and
radiolabelled [.gamma.-.sup.32P] ATP. As shown previously, GSK3
does not phosphorylate GST-NFAT1(1-415) without priming, but does
phosphorylate after pre-phosphorylation with either PKA or DYRK2.
Pre-phosphorylation with DYRK2 caused an upward mobility shift of
the GST-NFAT1(1-415) substrate as judged by Coomassie blue
staining, as expected from the fact that DYRK2 phosphorylates the
SP-3 motif; moreover, pre-phosphorylation with DYRK2 yielded a
radioactive GSK3-phosphorylated band of slower mobility compared to
the band observed after pre-phosphorylation with PKA. These results
suggest that while PKA primes for GSK3 by phosphorylating the
fourth serine (bold) in the SP-2 motif (SPRTSPIMSPRTSLAED) (SEQ ID
NO: 35) and permitting processive N-terminal phosphorylation of the
underlined serines by GSK3, while DYRK2 potentiates GSK3-mediated
phosphorylation of the regulatory domain motif by phosphorylating a
separate motif, the SP-3 motif. Indeed, the serine targeted by PKA
in the SP-2 motif is not found phosphorylated in cells.sup.10,
providing further evidence for physiological regulation of NFAT by
DYRK.
[0270] We asked whether DYRK expression regulated the
transcriptional activity of NFAT utilizing the kinase-dead mutant
of DYRK2 as an inhibitor of DYRK activity in cells.sup.30,31.
Jurkat T cells were co-transfected with an IL-2 promoter-driven
luciferase reporter plasmid and increasing amounts of expression
plasmids for either wild type (WT) or kinase-dead (KD) DYRK2; one
day later, the cells were stimulated for 6 h with PMA and ionomycin
and reporter activity was measured. WT DYRK2 strongly diminished
NFAT-dependent activity, while the KD mutant behaved as an
inhibitor by increasing NFAT-dependent luciferase activity at
higher concentrations (FIG. 8A). Similar results were obtained
using luciferase reporters containing tandem copies of the ARRE2
NFAT:AP-1 site of the IL-2 promoter.sup.32 as well as the K3 site
of the TNF.alpha. promoter.sup.33. In related experiments
expression WT DYRK2 also diminished, the production of endogenous
IL-2 by stimulated Jurkat T cells in a dose-dependent manner while
KD DYRK2 again had an inhibitory effect, when expressed at high
concentrations, by increasing IL-2 production under these
conditions (FIGS. 8B, 8C). Furthermore, we detected endogenous
DYRK2 co-immunoprecipitating with HA-NFAT1 stably expressed at low
endogenous levels in a Jurkat cell line; in this respect DYRK may
resemble the SRR-1 kinase CK1, which forms a stable complex with
NFAT under resting conditions but dissociates following
activation.sup.10. A DYRK-NFAT interaction supports the hypothesis
that DYRK is a physiological NFAT kinase: kinase-substrate
interactions of this type are known to be critical in many other
signal transduction pathways, although they are often transient and
difficult to detect at endogenous levels of expression.sup.34.
Discussion
[0271] We have shown that genome-wide RNAi screening in Drosophila
is a valid and powerful strategy for exploring novel aspects of
signal transduction in mammalian cells, provided that key members
of the signaling pathway are evolutionarily conserved and
represented in the Drosophila genome. We have used the method to
identify conserved regulators of the purely vertebrate
transcription factor, NFAT; to our knowledge, this is the first
example of a genome-wide RNAi screen that crosses evolutionary
boundaries in this manner. The strategy was successful because
Drosophila developed an evolutionary niche that was later used by
Ca.sup.2+-regulated NFAT proteins when they emerged in vertebrates.
Using this approach we have identified DYRK as a novel
physiological regulator of NFAT, and the first SP-3 motif-directed
kinase. It is likely that conserved aspects of the regulation of
other mammalian processes will also be successfully defined by
developing assays in Drosophila cells.
[0272] Our data suggest that DYRK regulates NFAT phosphorylation by
a mechanism in which DYRK phosphorylates the NFAT regulatory domain
within the conserved SP-3 motif, and thereby facilitates further
phosphorylation of the NFAT regulatory domain by GSK3. A similar
sequential mechanism may regulate progressive dephosphorylation of
NFAT, whereby dephosphorylation of the SRR-1 motif promotes
dephosphorylation of the SP-2 and SP-3 motifs by increasing their
accessibility to calcineurin.sup.9. It is likely that class II
DYRKs (DYRK2, 3 and 4) which are localized to the cytoplasm.sup.25,
function primarily as "maintenance" kinases that sustain the
phosphorylation status of cytoplasmic NFAT in resting cells,
whereas class I DYRKs (DYRK1A and 1B), which are localized to the
nucleus.sup.25, re-phosphorylate nuclear NFAT and promote its
nuclear export. Notably, DYRK1A and the endogenous calcineurin
regulator RCN/DSCR1/calcipressin-1 are both localized to the Down
Syndrome Critical Region on chromosome 21. Thus overexpression of
these negative regulators of NFAT in Down Syndrome could
contribute, by inhibiting NFAT activation, to the severe
neurological and immune developmental defects associated with
chromosome 21 trisomy.sup.35.
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Example 3
Table I.
[0316] List of candidates that were positive in the secondary
screen, classified into the categories in Table I. The first column
indicates whether or not the candidate was retested in the
confirmatory screen (NT, not tested); if tested, the summed
localization score from 3 separate experiments is shown (see
Methods). Other columns list gene names, Flybase numbers, and human
orthologues as obtained from Homologene (for the kinase category,
the phylogenetic analysis described in Methods was used in
addition), and number of predicted off-targets with exact match of
21-nt, 37 candidates with >10 off-targets are not listed.
Table II.
[0317] Analysis of expression, RNAi phenotype in
thapsigargin-treated cells, and amplicon off-targets for
calcineurin subunits and related proteins. Expression level of the
subunits in S2R+ cells was estimated by RT-PCR analysis, and the
effect of their depletion on NFAT nuclear localization in
thapsigargin (TG)-treated cells was evaluated (+++, strong
inhibition; -, no inhibition). The DRSC amplicons targeting each of
the subunits were analyzed for predicted off-targets with exact
matches of 21-, 20-, or 19-nt as described in Methods. Description
of the off-targets is provided in Table III. Red indicates
off-targets belonging to the same family as the primary
targets.
[0318] Of the three isoforms of calcineurin A, the amplicon for
CanA1 and one amplicon each for Pp2b-14D and CanA-14F show no
predicted off-targets. CanA1 is poorly expressed and its depletion
does not inhibit NFAT nuclear translocation, while Pp2B-14D and
CanA-14F are both expressed and depletion of either isoform results
in strong inhibition of NFAT nuclear translocation.
[0319] Why does depletion of the moderately expressed isoform
CanA-14F give similar inhibition as depletion of the more highly
expressed isoform Pp2B-14D?Different methods have different
sensitivities, and while the eye is able to discern subtle changes
in the nuclear localization of NFAT, such visual estimates are not
as quantitative as (for instance) estimating extent of
dephosphorylation by western blotting.
[0320] Of the three isoforms of calcineurin B, two (CanB and CanB2)
are strongly related to mammalian calcineurin B while CG32812 is
more distantly related, resembling mammalian CHP. RNAi against
either CanB or CanB2 gave equivalent inhibition (.about.70%) of
NFAT nuclear localization, even though CanB is barely expressed
while CanB2 is expressed at high levels. This is most likely due to
the fact that CanB and CanB2 are reciprocal off-targets, with 20 nt
overlap in their respective amplicons DRSC 18449 and DRSC07355.
Table IV.
[0321] Amplicon off-targets for selected candidates that were
evaluated in additional experiments. Scores of the candidates in
the confirmatory screen, evaluating the effects of their
RNAi-mediated depletion on NFAT nuclear accumulation in resting
cells, are shown (taken from Table I). For each candidate with
positive DRSC amplicons, predicted off-targets with exact matches
of 21-, 20-, or 19-nt are listed. Description of the off-targets is
provided below. Red indicates off-targets belonging to the same
family as the primary targets that were positive in the initial
screen.
[0322] The amplicon corresponding to the GSK3 homologue sgg
(DRSC18832) gave the highest score but also has a high number of
off-targets. None of these off-targets corresponds to gskt
(DRSC14056), which gave a low score of 1 in the primary screen.
[0323] The amplicon corresponding to the highest-scoring CK1 family
member gish has no predicted off-targets, indicating that it
represents a bonafide regulator of NFAT. Clear cross-inactivation
exists for amplicons DRSC 16929, DRSC20231 and DRSC19863,
corresponding to the CK1 isoforms dco, CK1alpha/CG2028 and CG2577,
each of which has a positive localization score of 1. Further work
is necessary to determine whether the scores associated with the
other isoforms reflect expression levels of the isoforms,
off-target effects, or both.
[0324] We are fortunate that for the two candidates--DYRK and
STIM--that we focused on for this study, there are no predicted
off-targets for exact matches of either 21, 20 or 19 nt.
TABLE-US-00002 TABLE I Number of Human Score in secondary potential
21nt orthologs (NCBI Description of the human screen Gene FBGN
off-targets Homologene) orthologs (NCBI Gene) PHOSPHATASES 5 pLIP
Fbgn0039111 0 PTPMT1 protein tyrosine phosphatase, mitochondrial 1
3 CanA1 FBgn0010015 0 PPP3CC protein phosphatase 3 (formerly 2B),
catalytic subunit, gamma isoform 3 flw FBgn000711 1 PPP1CB protein
phosphatase 1, catalytic subunit, beta isoform 3 PpD6 FBgn005779 1
3 wdb FBgn0027492 0 PPP2R5E protein phosphatase 2, regulatory
subunit B (B56), epsilon isoform 1 CanB FBgn0010014 0 PPP3R1
protein phosphatase 3 (formerly 2B), regulatory subunit B, 19 kDa,
alpha isoform 1 CanB2 FBgn0015614 0 PPP3R1 protein phosphatase 3
(formerly 2B), regulatory subunit B, 19 kDa, alpha isoform 1
CG32812 FBgn0025642 0 LOC63928 hepatocellular carcinoma antigen
gene 520/related to mammalian CHP 0 Pp2B-14D FBgn0011826 1 PPP3CB
protein phosphatase 3 (formerly 2B), catalytic subunit, beta
isoform PROTEIN KINASES 6 sgg FBgn0003371 3 GSK3B glycogen synthase
kinase 3 beta 5 CG7125 FBgn0038603 0 PRKD protein kinase D 4
CG31640 FBgn0015640 0 DDR 4 gish FBgn0011253 0 CSNK1G casein kinase
1, gamma 4 inaC FBgn0004784 0 PRKCB1 protein kinase C, beta 1 3
CG12147 FBgn0037325 0 CSNK1 casein kinase 1 family 3 CkIIalpha
FBgn0000258 0 CSNK2A1,2 casein kinase 2, alpha 3 pII FBgn0010441 0
IRAK 2 CG2905, FBgn0004661 0 TRRAP transformation/transcription
domain- Nipped-A associated protein 2 aPKC FBgn0022131 0 PRKCI
protein kinase C, iota 2 CG11489 FBgn0025702 0 SRPK1 SFRS protein
kinase 1 2 CG32687 FBgn0052687 0 LOC116064 hypothetical protein
LOC116064 2 CG6498 FBgn0036511 0 MAST2 microtubule associated
serine/threonine kinase 2 2 CG7097 FBgn0034421 0 MAP4K3
mitogen-activated protein kinase kinase kinase kinase 3 2 I(1)G0148
FBgn0028360 0 CDC7 CDC7 cell division cycle 7 2 Pkc53E FBgn0003091
0 PRKCA protein kinase C, alpha 2 Pkcdelta FBgn0030387 0 PRKCD
protein kinase C, delta 2 polo FBgn0003124 0 PLK1 polo-like kinase
1 2 trc FBgn0003744 0 STK38, STK38L serine/threonine kinase 38 like
1 CG40478 FBgn0069975 0 DYRK dual-specificity tyrosine-(Y)-
phosphorylation regulated kinase 1 CG2577 FBgn0030384 3 CSNK1
casein kinase 1 family 1 CG4168 FBgn0028888 0 1 CG5483 FBgn0038816
0 1 CG7094 FBgn0032650 0 CSNK1 casein kinase 1 family 1 CkIalpha
FBgn0015024 3 CSNK1A1 casein kinase 1, alpha 1 1 Cks FBgn0010314 0
CKS1B CDC28 protein kinase regulatory subunit 1B 1 dco FBgn0002413
0 CSNK1D, E casein kinase 1, delta/epsilon 1 for FBgn0000721 2
PRKG1 protein kinase, cGMP-dependent, type I 1 gskt FBgn0046332 0
GSK3A 1 phl FBgn0003079 2 BRAF v-raf murine sarcoma viral oncogene
homolog B1 1 Pk61C FBgn0020386 0 PDPK1 3-phosphoinositide dependent
protein kinase 1 1 Pkc98E FBgn0003093 0 PRKCE protein kinase C,
epsilon 1 Tie FBgn0014073 4 0 CG11533 FBgn0039908 0 0 CG9962
FBgn0031441 0 CSNK1 casein kinase 1 family 0 CG10579 FBgn0005640 0
ALS2CR7, PFTK1 PFTAIRE protein kinase 1 0 png FBgn0000826 0 NT
CG17698 FBgn0040056 0 CAMKK2 calcium/calmodulin-dependent protein
kinase kinase 2, beta NT gek FBgn0023081 0 CDC42BPA, B CDC42
binding protein kinase alpha (DMPK-like) OTHER KINASES/
KINASE-RELATED 1 Pi3K59F FBgn0015277 0 PIK3C3
phosphoinositide-3-kinase, class 3 0 CG8298 FBgn0033673 0 0 Pdk
FBgn0017558 0 PDK3 pyruvate dehydrogenase kinase, isoenzyme 3 NT
CG3809 FBgn0037995 0 NT CG6218 FBgn0038321 0 NAGK
N-acetylglucosamine kinase NT CG6364 FBgn0039179 0 UCK2
uridine-cytidine kinase 2 NT dlg FBgn0001624 8 DLG1 discs, large
homolog 1 MISCELLANEOUS/ CALCIUM- RELATED 5 CG14387 FBgn0038089 0 4
TpnC4 FBgn0033027 0 4 TpnC73F FBgn0010424 0 3 Stim FBgn0045073 0
STIM1 stromal interaction molecule 1 3 Cam FBgn0000253 0 CALM2
calmodulin 2 (phosphorylase kinase, delta) 3 CG11165 FBgn0033238 2
3 CG13898 FBgn0035161 0 2 norpA FBgn0004625 0 PLCB4 phospholipase
C, beta 4 2 TpnC41C FBgn0013348 0 2 TpnC47D FBgn0010423 0 1 CG13526
FBgn0034774 0 1 CG31345 FBgn0051345 0 CAPSL calcyphosine-like 1
CG31650 FBgn0031673 0 RCN2 reticulocalbin 2, EF-hand calcium
binding domain 1 CG31958 FBgn0051958 2 1 CG31960 FBgn0051960 2 1
TpnC25D FBgn0031692 1 MEMBRANE SIGNALLING 5 CG6919 FBgn0038980 0
HTR4 5-hydroxytryptamine (serotonin) receptor 4 4 CG30340
FBgn0050340 0 4 DopR FBgn0011582 0 DRD1 dopamine receptor D1 4
Gr47a FBgn0041242 0 4 Or85d FBgn0037594 0 4 Su(fu) FBgn0005355 0
SUFU suppressor of fused homolog (Drosophila) 3 Ac3 FBgn0023416 0
ADCY3 adenylate cyclase 3 3 Gyc-89Db FBgn0038436 0 3 homer
FBgn0025777 0 HOMER2 homer homolog 2 3 mav FBgn0039914 0 TGFB3
transforming growth factor, beta 3 3 PGRP-LE FBgn0030695 0 PGLYRP3
peptidoglycan recognition protein 3 2 cenB1A FBgn0039056 0 CENTB2
centaurin, beta 2 2 CG10823 FBgn0038880 0 2 CG11319 FBgn0031835 0
DPP10 dipeptidylpeptidase 10 2 CG6989 FBgn0038063 0 2 fz3
FBgn0027343 0 2 N FBgn0004647 0 NOTCH1 Notch homolog 1,
translocation- associated 2 Plc21C FBgn0004611 0 PLCB1
phospholipase C, beta 1 (phosphoinositide-specific 2 pxb
FBgn0053207 1 2 sog FBgn0003463 0 CHRD Chordin 2 spz FBgn0003495 0
1 18w FBgn0004364 0 1 CG16752 FBgn0029768 0 1 CG17262 FBgn0031499 0
1 Crag FBgn0025864 0 MYCPBP c-myc promoter binding protein 1 Grip
FBgn0040917 0 GRIP1 glutamate receptor interacting protein 1 1 nkd
FBgn0002945 0 1 sl FBgn0003416 0 PLCG1 phospholipase C, gamma 1 0
bm FBgn0000221 0 B3GALT2 UDP-Gal:betaGlcNAc beta 1,3-
galactosyltransferase, potypeptide 2 0 CG10747 FBgn0032845 0 PLCXD2
phosphatidylinositol-specific phospholipase C, X domain containing
2 0 CG31350 FBgn0051350 2 0 fz2 FBgn0016797 0 FZD8 frizzled homolog
8 0 Rab-RP1 FBgn0015788 0 RAB32 RAB32, member RAS oncogene family 0
skf FBgn0050021 0 MPP7 membrane protein, palmitoylated 7 NT Alg10
FBgn0052076 0 NT CG30361 FBgn0050361 4 GRM4 glutamate receptor,
metabotropic 4 NT rho-5 FBgn0041723 0 NT Sema-1a FBgn0011259 0
SEMA60 sema domain, transmembrane domain (TM), and cytoplasmic
domain, (semaphorin) 6D NT sif FBgn0019652 0 NT Syx1A FBgn0013343 0
STX1A syntaxin 1A NT tinc FBgn0038554 0 CATION CHANNELS AND
TRANSPORTERS 5 CG13223 FBgn0033599 0 SLC24A6 solute carrier family
24 (sodium/potassium/calcium exchanger), member 6 5 CG14741
FBgn0037989 0 ATP8B2 ATPase, Class I, type 8B, member 2 4 CG10465
FBgn0033017 0 KCTD10 potassium channel tetramerisation domain
containing 10 4 CG6737 FBgn0032294 0 4 Cng FBgn0014462 0 CNGA3
cyclic nucleotide gated channel alpha 3 4 GluRIIA FBgn0004620 0 4
inx6 FBgn0027107 0 4 Irk3 FBgn0032706 0 3 Ca-beta FBgn0015608 4 3
Ca-P60A FBgn0004551 0 ATP2A1 ATPase, Ca++ transporting, cardiac
muscle, fast twitch 1 3 CG11155 FBgn0039927 0 GRIK3 glutamate
receptor, ionotropic, kainate 3 3 CG2165 FBgn0025704 0 ATP2B3
ATPase, Ca++ transporting, plasma membrane 3 3 CG32792 FBgn0052792
0 3 CG3367 FBgn0029871 2 3 CG4450 FBgn0032113 0 3 CG6812
FBgn0036843 0 SFXN2 sideroflexin 2 3 KaiRIA FBgn0028422 1 GRIA4
glutamate receptor, ionotrophic, AMPA 4 3 ppk21 FBgn0039675 0 3 trp
FBgn0003861 0 2 Ca-alpha1D FBgn0001991 0 CACNA1D calcium channel,
voltage-dependent, L type, alpha 1D subunit 2 Calx FBgn0013995 0
SLC8A3 solute carrier family 8 (sodium-calcium exchanger), member 3
2 CG12376 FBgn0033323 0 SLC24A6 solute carrier family 24
(sodium/potassium/calcium exchanger), member 6 2 CG12904
FBgn0033510 0 KCNT2 potassium channel, subfamily T, member 2 2
CG1698 FBgn0033443 1 2 CG31284 FBgn0051284 0 2 CG31729 FBgn0051729
0 ATP9B ATPase, Class II, type 9B 2 CG3822 FBgn0038837 0 GRIK1
glutamate receptor, ionotropic, kainate 1 2 CG4536 FBgn0029904 5 2
CG9361 FBgn0037690 0 KCNK9 potassium channel, subfamily K, member 9
2 elk FBgn0011589 0 KCNH8 potassium voltage-gated channel,
subfamily H (eag-related), member 8 2 GluClalpha FBgn0024963 0
GLRA3 glycine receptor, alpha 3 2 GluRIII FBgn0031293 0 2 Irk2
FBgn0039081 0 KCNJ9 potassium inwardly-rectifying channel,
subfamily J, member 9 2 KCNQ FBgn0033494 3 KCNQ5 potassium
voltage-gated channel, KQT- like subfamily, member 5 2 nAcRalpha-
FBgn0028875 0 CHRNA7 cholinergic receptor, nicotinic, alpha 34E
polypeptide 7 2 nAcRalpha- FBgn0000036 0 CHRNA3 cholinergic
receptor, nicotinic, alpha 96Aa polypeptide 3 2 Nmdar1 FBgn0010399
1 GRIN1 glutamate receptor, ionotropic, N-methyl Daspartate 1 2
Ork1 FBgn0017561 0 KCNK4 potassium channel, subfamily K, member 4 2
sei FBgn0003353 0 KCNH6 potassium voltage-gated channel, subfamily
H (eag-related), member 6 1 Ca-alpha 1T FBgn0029846 0 1 cac
FBgn0005563 0 CACNA1A calcium channel, voltage-dependent, P/Q type,
alpha 1A subunit 1 CG10830 FBgn0038839 0 KCTD12 potassium channel
tetramerisation domain containing 12 1 CG31201 FBgn0051201 1 GRIA4
glutamate receptor, ionotrophic, AMPA 4 1 CG32770 FBgn0052770 0 1
CG33298 FBgn0032120 0 ATP10A ATPase, Class V, type 10A 1 CG40146
FBgn0039941 0 1 CG5621 FBgn0038840 0 1 CG8743 FBgn0036904 0 MCOLN3
mucolipin 3 1 CG9935 FBgn0039916 1 GRIA1 glutamate receptor,
ionotropic, AMPA 1 1 eag FBgn0000535 0 KCNH1 potassium
voltage-gated channel, subfamily H 1 Glu-RIB FBgn0028431 1 GRIA2
glutamate receptor, ionotropic, AMPA 2 1 GluRIIB FBgn0020429 0 1 Ir
FBgn0039061 0 KCNJ5 potassium inwardly-rectifying channel,
subfamily J, member 5 1 I(2)01810 FBgn0010497 0 1 nAcRalpha-
FBgn0000039 1 CHRNA2 cholinergic receptor, nicotinic, alpha 96Ab
polypeptide 2 1 nAcRbeta-64B FBgn0000038 0 CHRNA4 cholinergic
receptor, nicotinic, alpha polypeptide 4 1 nAcRbeta-96A FBgn0004118
0 CHRNB4 cholinergic receptor, nicotinic, beta polypeptide 1 Nmdar2
FBgn0014432 0 GRIN2B glutamate receptor, ionotropic, N-methyl
Daspartate 2B 1 nompC FBgn0016920 0 1 pain FBgn0060296 0 1 Pkd2
FBgn0041195 0 PKD2L1 polycystic kidney disease 2-like 1 1 Shal
FBgn0005564 0 KCND3 potassium voltage-gated channel, Shal- related
subfamily, member 3
1 Sip1 FBgn0010620 0 TFIP11 tuftelin interacting protein 11 1 slo
FBgn0003429 0 KCNMA1 potassium large conductance calcium- activated
channel, subfamily M, alpha member 1 0 Anktm1/TrpA1 FBgn0035934 0
TRPA1 transient receptor potential cation channel, subfamily A,
member 1 0 CG12455 FBgn0028859 0 CACNA2D3 calcium channel,
voltage-dependent, alpha 2/delta 3 subunit 0 CG13762 FBgn0040333 1
PKD2L1 polycystic kidney disease 2-like 1 0 CG14647 FBgn0037244 0
KCTD9 potassium channel tetramerisation domain containing 9 0
CG17922 FBgn0034656 0 CNGB1 cyclic nucleotide gated channel beta 1
0 CG32704 FBgn0052704 0 0 CG32810 FBgn0025394 0 KCTD5 potassium
channel tetramerisation domain containing 5 0 CG4301 FBgn0030747 0
ATP11B ATPase, Class VI, type 11B 0 CG9472 FBgn0036874 0 PKD1L3
polycystic kidney disease 1-like 3 0 clumsy FBgn0026255 0 GRIK2
glutamate receptor, ionotropic, kainate 2 0 cngl FBgn0029090 3 0
Glu-RI FBgn0004619 0 GRIA3 glutamate receptor, ionotrophic, AMPA 3
0 Nckx30C FBgn0028704 0 SLC24A2 solute carrier family 24
(sodium/potassium/calcium exchanger), member 2 0 Rya-r44F
FBgn0011286 0 RYR2 ryanodine receptor 2 (cardiac) 0 Shab
FBgn0003383 0 KCNB1 potassium voltage-gated channel, Shab- related
subfamily, member 1 0 SK FBgn0029761 0 KCNN3 potassium
intermediate/small conductance calcium-activated channel, subfamily
N, member 3 0 trpl FBgn0005614 0 NT CG2196 FBgn0039872 1 NT
nAcRalpha- FBgn0037212 0 80B OTHER TRANSPORTERS 3 ATPsyn-Cf6
FBgn0016119 0 3 CG1599 FBgn0033452 0 SYBL1 synaptobrevin-like 1 3
CG31116 FBgn0051116 0 CLCN2 chloride channel 2 3 CG31158
FBgn0051158 0 3 CG31305 FBgn0051305 0 SLC25A1 solute carrier family
25 (mitochondrial carrier; citrate transporter), member 1 3 CG6901
FBgn0038414 0 3 Mst84Db FBgn00004173 0 2 CG3860 FBgn0034951 0
OSBPL1A oxysterol binding protein-like 1A 2 CG3902 FBgn0036824 1
ACADSB acyl-Coenzyme A dehydrogenase, short/branched chain 2 CG5127
FBgn0039335 0 2 CG7442 FBgn0037140 0 2 CG7578 FBgn0028538 0 ARFGEF1
ADP ribosylation factor guanine nucleotide-exchange factor 1 2
CG9270 FBgn0032908 0 ABCC2 ATP-binding cassette, sub-family C
(CFTR/MRP), member 2 1 CG31731 FBgn0028539 0 1 CG8389 FBgn0034063 0
1 rdgB FBgn0003218 0 PITPNM2 phosphatidylinositol transfer protein,
membrane-associated 2 1 w FBgn0003996 0 0 CG33214 FBgn0053214 0
GLG1 golgi apparatus protein 1 0 CG7458 FBgn0037144 0 NT Beach1
FBgn0043362 0 WDFY3 WD repeat and FYVE domain containing 3 NT
CG12539 FBgn0030586 0 NT CG14482 FBgn0034245 0 NT CG14691
FBgn0037829 0 SV2A synaptic vesicle glycoprotein 2A NT CG17119
FBgn0039045 0 CTNS cystinosis, nephropathic NT CG18324 FBgn0033905
0 SLC25A34 solute carrier family 25, member 34 NT CG3071
FBgn0023527 0 UTP15 UTP15, U3 small nucleolar ribonucleoprotein NT
CG32230 FBgn0052230 0 NT CG6142 FBgn0039415 0 NT CG7181 FBgn0037097
0 NT CG7830 FBgn0032015 0 TUSC3 tumor suppressor candidate 3 NT
CG9990 FBgn0039594 0 NT Cyp49a1 FBgn0033524 0 CYP27A1 cytochrome
P450, family 27, subfamily A, polypeptide 1 NT didum FBgn0015933 0
MYO5A myosin VA (heavy polypeptide 12, myoxin) NT ERp60 FBgn0033663
1 PDIA3 protein disulfide isomerase-associated 3 NT Pbprp2
FBgn0011280 0 NT Syx6 FBgn0037084 3 STX10 syntaxin 10
MISCELLANEOUS/ OTHER 6 Prosalpha7 FBgn0023175 0 PSMA3 proteasome
(prosome, macropain) subunit, alpha type, 3 5 CG3812 FBgn0030421 0
AGPAT1 1-acylglycerol-3-phosphate O- acyltransferase 1
(lysophosphatidic acid acyltransferase, alpha) 4 bif FBgn0014133 3
4 CG11727 FBgn0030299 0 4 CG2781 FBgn0037534 0 ELOVL7 ELOVL family
member 7, elongation of long chain fatty acids 4 CG4960 FBgn0039371
0 CI9orf32 chromosome 19 open reading frame 32 4 CG7304 FBgn0036527
0 GALNT11 UDP-N-acetyl-alpha-D- galactosamine:potypeptide N-
acetylgalactosaminyltransferase 11 (GalNAc-T11) 4 CG8258
FBgn0033342 0 CCT8 chaperonin containing TCP1, subunit 8 (theta) 4
CRMP FBgn0023023 0 DPYS dihydropyrimidinase 4 Eip63F-1 FBgn0004910
0 3 Act57B FBgn0000044 5 ACTB actin, beta 3 CG11299 FBgn0034897 0
SESN3 sestrin 3 3 CG6509 FBgn0032363 0 DLG5 discs, large homolog 5
3 CG9342 FBgn0032904 0 MTP microsomal triglyceride transfer protein
(large polypeptide 88 kDa) 3 CG9467 FBgn0037758 0 KCTD3 potassium
channel tetramerisation domain containing 3 3 eIF-2beta FBgn0004926
0 EIF2S2 eukaryotic translation initiation factor 2, subunit 2
beta, 38 kDa 3 fzo FBgn0011596 0 MFN1 mitofusin 1 3 pros
FBgn0004595 0 PROX1 prospero-related homeobox 1 3 Su(var)3-9
FBgn0003600 0 EIF2S3 eukaryotic translation initiation factor 2,
subunit 3 gamma, 52 kDa 2 14-3- FBgn0020238 0 YWHAE tyrosine
3-monooxygenase/tryptophan 5- 3epsilon monooxygenase activation
protein, epsilon polypeptid 2 ac FBgn0000022 3 ASCL2 achaete-scute
complex-like 2 2 Arp66B FBgn0011744 0 ACTR3 ARP3 actin-related
protein 3 homolog 2 CG10069 FBgn0034611 0 SLC37A2 solute carrier
family 37 (glycerol-3-- phosphate transporter), member 2 2 CG11600
FBgn0038068 1 2 CG11608 FBgn0038069 0 LIPL3 lipase-like,
ab-hydrolase domain containing 3 2 CG14625 FBgn0040358 4 2 CG2678
FBgn0014931 0 2 CG3074 FBgn0034709 0 TINAGL1 tubulointerstitial
nephritis antigen-like 1 2 CG32635 FBgn0052635 1 2 CG4448
FBgn0039067 0 2 CG5278 FBgn0038986 3 2 CG5802 FBgn0038863 0 SLC35B1
solute carrier family 35, member B1 2 CG7140 FBgn0037147 0 2 Rad51D
FBgn0030931 0 XRCC2 X-ray repair complementing defective repair in
Chinese hamster cells 2 1 cer FBgn0034443 0 1 CG6330 FBgn0039464 0
UPP2 uridine phosphorylase 2 1 CG7568 FBgn0039673 0 WDR69 WD repeat
domain 69 1 CG9326 FBgn0032885 0 MPP6 membrane protein,
palmitoylated 6 (MAGUK p55 subfamily member 6) 1 CG9784 FBgn0030761
0 PIB5PA phosphatidylinositol (4,5) bisphosphate 5-phosphatase, A 1
cnc FBgn0000338 0 1 eIF2B-beta FBgn0024996 0 EIF2B2 eukaryotic
translation initiation factor 2B, subunit 2 beta, 39 kDa 1
gammaTub23 C FBgn0004176 0 TUBG1 tubulin, gamma 1 1 Hn FBgn0001208
0 PAH phenylalanine hydroxylase 1 Pgant35A FBgn0001970 0 GALNT11
UDP-N-acetyl-alpha-D- galactosamine:polypeptide N-
acetylgalactosaminyltransferase 11 (GalNAc-T11) 1 pgant4
FBgn0051956 0 1 skpA FBgn0025637 1 LOC401713 organ of Corti protein
2; RNA polymerase II elongation factor-like protein OCP2; cyclin
A/CDK2-associated p19 0 CG15408 FBgn0031523 0 0 CG4500 FBgn0028519
0 ACSBG1 acyl-CoA synthetase bubblegum family member 1 0 CG7348
FBgn0036940 0 0 CG8647 FBgn0035729 0 0 D FBgn0000411 1 0 nahoda
FBgn0034797 0 0 Pde6 FBgn0038237 0 PDE11A phosphodiesterase 11A 0
sdt FBgn0003349 1 MPP5 membrane protein, palmitoylated 5 (MAGUK p55
subfamily member 5) 0 TSG101 FBgn0036666 0 TSG101 tumor
susceptibility gene 101 NT Aats-cys FBgn0027091 0 CARS
cysteinyl-tRNA synthetase NT Aats-met FBgn0027083 0 MARS2
methionine-tRNA synthetase 2 NT Acp70A FBgn0003034 0 NT Act79B
FBgn0000045 5 ACTG2 actin, gamma 2, smooth muscle, enteric NT
Ahcy13 FBgn0014455 0 AHCY s-adenosylhomocysteine hydrolase NT amon
FBgn0023179 0 PCSK2 proprotein convertase subtilisin/kexin type 2
NT asparagine- FBgn0041607 0 synthetase NT ATbp FBgn0039946 5 NT
BEAF-32 FBgn0015602 0 NT beat-Ic FBgn0028644 8 NT beat-Vb
FBgn0038092 0 NT Bin1 FBgn0024491 0 SAP18 sin3-associated
polypeptide, 18 kDa NT BM-40-SPARC FBgn0026562 0 SPARCL1 SPARC-like
1 (mast9, hevin) NT btsz FBgn0010940 0 NT bwa FBgn0045064 0 ASAH3L
N-acylsphingosine amidohydrolase 3-like NT CG10168 FBgn0039087 0 NT
CG11107 FBgn0033160 0 DHX15 DEAH (Asp-Glu-Ala-His) box polypeptide
15 NT CG12162 FBgn0037329 0 POLDIP2 polymerase (DNA-directed),
delta interacting protein 2 NT CG13643 FBgn0040601 0 NT CG13779
FBgn0040954 0 NT CG14869 FBgn0038341 0 NT CG15105 FBgn0034412 0 NT
CG1571 FBgn0029993 0 DNAI2 dynein, axonemal, intermediate
polypeptide 2 NT CG16710 FBgn0039101 0 NT CG16857 FBgn0028482 0 NT
CG17294 FBgn0032032 0 HDHD2 haloacid dehalogenase-like hydrolase
domain containing 2 NT CG17826 FBgn0036227 0 FBN2 fibrillin 2
(congenital contractural arachnodactyly) NT CG18493 FBgn0038701 0
NT CG2051 FBgn0037376 0 HAT1 histone acetyltransferase 1 NT CG3066
FBgn0037515 0 NT CG31115 FBgn0051115 0 MTAP methylthioadenosine
phosphorylase NT CG31159 FBgn0051159 0 GFM2 G elongation factor,
mitochondrial 2 NT CG31224 FBgn0051224 0 NT CG31287 FBgn0051287 0
NT CG31453 FBgn0051453 0 TRIP13 thyroid hormone receptor interactor
13 NT CG31716 FBgn0051716 0 NT CG32284 FBgn0052284 0 NT CG3231
FBgn0027522 0 RBBP6 retinoblastoma binding protein 6 NT CG32557
FBgn0052557 0 NT CG32700 FBgn0052700 0 NT CG32727 FBgn0052727 0
DNAJC15 DnaJ (Hsp40) homolog, subfamily C, member 15 NT CG33100
FBgn0053100 0 EIF4E2 eukaryotic translation initiation factor 4E
member 2 NT CG3356 FBgn0034989 0 UBE3C ubiquitin protein ligase E3C
NT CG3605 FBgn0031493 0 SF3B2 splicing factor 3b, subunit 2, 145
kDa NT CG3654 FBgn0036004 0 NT CG3700 FBgn0034796 1 TMPRSS9
transmembrane protease, serine 9 NT CG3940 FBgn0037788 0 NT CG4017
FBgn0032143 0 CPB1 carboxypeptidase B1 NT CG4030 FBgn0034585 0
RABEP1 rabaptin, RAB GTPase binding effector protein 1 NT CG4090
FBgn0038492 1 NT CG4291 FBgn0031287 0 WBP4 WW domain binding
protein 4 (formin binding protein 21) NT CG4302 FBgn0027073 0
UGT2B10, UDP glucuronosyltransferase 2 family, UGT2B11, polypeptide
B10, B11, B28 UGT2B28 NT CG4653 FBgn0030776 0 NT CG4747 FBgn0043456
0 N-PAC cytokine-like nuclear factor n-pac NT CG4851 FBgn0032358 0
PPT2 palmitoyl-protein thioesterase 2 NT CG4901 FBgn0032194 0 DHX33
DEAH (Asp-Glu-Ala-His) box polypeptide 33 NT CG5103 FBgn0036784 0
TKT transketolase (Wernicke-Korsakoff syndrome) NT CG5122
FBgn0032471 0 NT CG5191 FBgn0038803 0 NT CG5567 FBgn0036760 0
LOC283871 hypothetical protein LOC283871 NT CG5715 FBgn0039180 0 NT
CG6041 FBgn0029826 1 TMPRSS9 transmembrane protease, serine 9 NT
CG6656 FBgn0038912 0 NT CG6717 FBgn0031924 0 SERPINB5 serpin
peptidase inhibitor, clade B (ovalbumin), member 5
NT CG6763 FBgn0039069 1 NT CG6764 FBgn0037899 0 C15orf15 chromosome
15 open reading frame 15 NT CG6841 FBgn0036828 0 C20orf14
chromosome 20 open reading frame 14 NT CG6906 FBgn0036261 0 NT
CG6937 FBgn0038989 0 MKI67IP MKI67 (FHA domain) interacting
nucleolar phosphoprotein NT CG7017 FBgn0036951 0 NT CG7290
FBgn0036949 0 NT CG7928 FBgn0039740 0 NT CG8117 FBgn0030663 0 TCEA2
transcription elongation factor A (SII), 2 NT CG9220 FBgn0030662 0
CHSY1 carbohydrate (chondroitin) synthase 1 NT CG9363 FBgn0037697 0
NT CG9520 FBgn0032078 0 C1GALT1 core 1 synthase, glycoprotein-N-
acetylgalactosamine 3-beta- galactosyltransferase, 1 NT CG9535
FBgn0027501 0 UAP1 UDP-N-acteylglucosamine pyrophosphorylase 1 NT
CG9650 FBgn0029939 2 NT CG9843 FBgn0037237 0 NT CG9947 FBgn0030752
0 TMEM30A transmembrane protein 30A NT comm3 FBgn0053209 0 NT CtBP
FBgn0020496 1 CTBP1 C-terminal binding protein 1 NT dbo FBgn0040230
0 KLHL20 ketch-like 20 (Drosophila) NT Dhfr FBgn0004087 0 DHFR
dihydrofolate reductase NT Dmrt11E FBgn0030477 2 NT drm FBgn0024244
0 NT east FBgn0010110 1 NT ec FBgn0025376 1 NT Ef1alpha 100E
FBgn0000557 1 EEF1A2 eukaryotic translation elongation factor 1
alpha 2 NT faf FBgn0005632 0 USP9X ubiquitin specific peptidase 9,
X-linked (fat facets-like, Drosophila NT fbp FBgn0032820 0 FBP1
fructose-1,6-bisphosphatase 1 NT fred FBgn0051774 0 NT GstD5
FBgn0010041 5 NT GstE2 FBgn0063498 0 NT Hand FBgn0332209 0 HAND2
heart and neural crest derivatives expressed 2 NT HGTX FBgn0040318
0 NKX6-1 NK6 transcription factor related, locus 1 NT Hsp6OB
FBgn0011244 0 NT I(2)k05713 FBgn0022160 0 GPD2 glycerol-3-phosphate
dehydrogenase 2 NT I(3)IX-14 FBgn0002478 0 LMLN leishmanolysin-like
(metallopeptidase M8 family) NT IoIa FBgn0005630 2 LOC441636
similar to submaxillary apomucin NT Map60 FBgn0010342 0 NT Mes-4
FBgn0039559 0 WHSC Wolf-Hirschhorn syndrome candidate 1 NT Mgat2
FBgn0039738 0 MGAT2 mannosyl (alpha-1,6-)-gtycoprotein beta-
1,2-N-acetylglucosaminyltransferase NT mol FBgn0028528 0 NIP
homolog of Drosophila Numb-interacting protein NT mre11 FBgn0020270
0 MRE11A MRE11 meiotic recombination 11 homolog A NT mRpL15
FBgn0036990 1 MRPL15 mitochondrial ribosomal protein L15 NT mRpL2a
FBgn0037833 0 MRPL37 mitochondrial ribosomal protein L37 NT nbs
FBgn0026198 1 NBN nibrin NT NfI FBgn0042696 0 NFIA nuclear factor
I/A NT nos FBgn0002962 2 NOS1 nitric oxide synthase 1 NT Odc1
FBgn0013307 0 ODC1 ornithine decarboxylase 1 NT Peb FBgn0004181 0
PRB1, PRB2 proline rich protein BstNI subfamily 1, proline rich
protein BstNI subfamily 2 NT PH4alphaEFB FBgn0039776 0 P4HA1
procollagen-proline, 2-oxoglutarate 4- dioxygenase (proline
4-hydroxylase), alpha polypeptide I NT Phax FBgn0033380 0 RNUXA RNA
U, small nuclear RNA export adaptor NT ple FBgn0005626 0 TH
tyrosine hydroxylase NT Rb97D FBgn0004903 2 LOC144983 heterogeneous
nuclear ribonucleoprotein A1- like NT Rbp2 FBgn0010256 0 WBSCR1
Williams-Beuren syndrome chromosome region 1 NT Rpl1 FBgn0019938 0
POLR1A polymerase (RNA) I polypeptide A NT RpL10Aa FBgn0038281 0
RPL10A ribosomal protein L10a NT RpS10b FBgn0031035 0 RPS10
ribosomal protein S10 NT Rrp1 FBgn0004584 0 APEX1 APEX nuclease
(multifunctional DNA repair enzyme) 1 NT salr FBgn0000287 0 SALL3
sal-like 3 NT sda FBgn0015541 1 ARTS-1 type 1 tumor necrosis factor
receptor shedding aminopeptidase regulator NT SF1 FBgn0025571 0 SF1
splicing factor 1 NT shn FBgn0003396 0 NT Sirt2 FBgn0038788 0 SIRT2
sirtuin (silent mating type information regulation 2 homolog) 2 NT
snRNP69D FBgn0016940 0 SNRPD1 small nuclear ribonucleoprotein D1
polypeptide 16 kDa NT Spn43Ab FBgn0024293 0 NT Spt3 FBgn0037981 1
NT sqd FBgn0003498 0 NT ST6Gal FBgn0035050 0 ST6GAL2 ST6
beta-galactosamide alpha-2,6- sialyltranferase 2 NT stau
FBgn0003520 0 STAU staufen, RNA binding protein NT stich 1
FBgn0016941 1 NT svr FBgn0004648 0 CPD carboxypeptidase D NT T3dh
FBgn0017482 1 ADHFE1 alcohol dehydrogenase, iron containing, 1 NT
Tdp1 FBgn0051953 0 TDP1 tyrosyl-DNA phosphodiesterase 1 NT Ith
FBgn0030502 5 NT Ugt86Dd FBgn0040256 0 NOVEL 5 CG17142 FBgn0035112
0 4 CG14076 FBgn0036829 0 4 CG14870 FBgn0038342 0 EPPB9 B9 protein
4 CG31145 FBgn0051145 0 FAM20C family with sequence similarity 20,
member C 4 CG31203 FBgn0051203 0 4 CG31288 FBgn0051288 0 4 CG4585
FBgn0025335 0 4 CG7706 FBgn0038640 0 SLC4A1AP solute carrier family
4 (anion exchanger), member 1, adaptor protein 4 Osi10 FBgn0037417
0 3 CG14084 FBgn0036855 0 3 CG14556 FBgn0039413 0 3 CG14744
FBgn0033324 0 SLC24A6 solute carrier family 24
(sodium/potassium/calcium exchanger), member 6 3 CG14945
FBgn0032402 0 3 CG17005 FBgn0032109 0 3 CG1968 FBgn0033401 0 COG6
componen of oligomeric golgi complex 6 3 CG1971 FBgn0039881 0 3
CG3566 FBgn0029854 0 CYB5-M outer mitochondrial membrane cytochrome
b5 3 CG4786 FBgn0037012 0 3 CG8740 FBgn0027585 0 3 CG9264
FBgn0032911 0 3 CG9525 FBgn0032080 0 2 CG10946 FBgn0029974 0 2
CG1113 FBgn0037304 0 2 CG11381 FBgn0029568 3 2 CG12688 FBgn0029707
0 2 CG12958 FBgn0034018 0 2 CG14314 FBgn0038581 0 2 CG14354
FBgn0039376 0 2 CG15897 FBgn0029857 0 WDR4 WD repeat domain 4 2
CG16786 FBgn0034974 0 2 CG30389 FBgn0050389 0 TMEM57 transmembrane
protein 57 2 CG32224 FBgn0036950 0 2 CG3704 FBgn0040346 0 XAB1 XPA
binding protein 1, GTPase 2 CG4098 FBgn0036648 0 NUDT9 nudix
(nucleoside diphosphate linked moiety X)-type motif 9 2 CG4643
FBgn0043010 0 FBX045 F-box protein 45 2 CG5308 FBgn0037908 3 2
CG5348 FBgn0034156 0 SLC24A6 solute carrier family 24
(sodium/potassium/calcium exchanger), member 6 2 CG9205 FBgn0035181
0 2 CG9752 FBgn0034614 0 C9orf64 chromosome 9 open reading frame 64
2 nes FBgn0026630 0 C3F putative protein similar to nessy 1 CG10514
FBgn0039312 0 1 CG13659 FBgn0039319 0 1 CG14160 FBgn0036066 0
SLC2A5 solute carrier family 2 (facilitated glucose/fructose
transporter), member 5 1 CG14515 FBgn0039648 0 1 CG14629
FBgn0040398 1 1 CG14743 FBgn0033326 0 SLC24A6 solute carrier family
24 (sodium/potassium/calcium exchanger), member 6 1 CG18679
FBgn0040663 0 1 CG2879 FBgn0025834 0 LRRC8B leucine rich repeat
containing 8 family, member B 1 CG2921 FBgn0034689 1 1 CG3106
FBgn0030148 0 1 CG31410 FBgn0051410 0 1 CG32159 FBgn0052159 0 1
CG32637 FBgn0052637 0 LGR8 leucine rich repeat containing G
protein- coupled receptor 8 1 CG3634 FBgn0037026 0 ST7 suppression
of tumorigenicity 7 1 CG8858 FBgn0033698 0 KIAA0368 KIAA0368 1 mars
FBgn0033845 0 DLG7 discs, large homolog 7 1 Osi16 FBgn0051561 0 1
sip2 FBgn0031878 0 0 CG10095 FBgn0037993 2 0 CG10183 FBgn0039093 2
0 CG13188 FBgn0033668 8 0 CG14162 FBgn0040823 0 0 CG14471
FBgn0033049 0 0 CG2185 FBgn0037358 0 CHP calcium binding protein
P22 0 CG2656 FBgn0037478 0 ATPBD1C ATP binding domain 1 family,
member C 0 CG31189 FBgn0051189 0 0 CG32432 FBgn0052432 0 0 CG3536
FBgn0050267 0 CNGA1 cyclic nucleotide gated channel alpha 1 0
I(1)G0331 FBgn0029944 8 0 Osi18 FBgn0037428 0 0 ppk13 FBgn0032912 0
NT CG10200 FBgn0033968 0 NT CG10424 FBgn0036848 0 FLJ10769
hypothetical protein FLJ10769 NT CG10589 FBgn0037035 0 NT CG11073
FBgn0034693 0 NT CG11113 FBgn0033165 0 NT CG11310 FBgn0037067 0 NT
CG11576 FBgn0039882 0 C20orf54 chromosome 20 open reading frame 54
NT CG11634 FBgn0032968 0 NT CG11672 FBgn0037563 0 NT CG11699
FBgn0030311 0 NT CG11750 FBgn0030294 0 NT CG11839 FBgn0039271 0
CCDC16 coiled-coil domain containing 16 NT CG11847 FBgn0039281 0
SDCCAG1 serologically defined colon cancer antigen 1 NT CG11875
FBgn0039301 0 NUP37 nucleoporin 37 kDa NT CG11881 FBgn0039638 0 NT
CG11926 FBgn0031640 0 MON1A MON1 homolog A NT CG12508 FBgn0040995 0
NT CG12584 FBgn0037257 0 NT CG12608 FBgn0030630 1 PAK1IP1 PAK1
interacting protein 1 NT CG12672 FBgn0030886 1 NT CG12985
FBgn0030881 0 RDBP RD RNA binding protein NT CG13014 FBgn0030759 1
NT CG13021 FBgn0029669 0 NT CG13075 FBgn0036563 0 NT CG13086
FBgn0032770 0 NT CG13088 FBgn0032047 0 PGDS prostaglandin D2
synthase, hematopoietic NT CG13169 FBgn0033704 0 NT CG13239
FBgn0037197 0 NIT CG13364 FBgn0026879 0 HSPC016 hypothetical
protein HSPC016 NT CG13538 FBgn0034820 0 NT CG13552 FBgn0034864 0
NT CG13599 FBgn0039128 0 NT CG13615 FBgn0039199 2 NT CG13623
FBgn0039205 0 NT CG13654 FBgn0039290 0 NT CG13785 FBgn0031901 0 NT
CG13836 FBgn0039060 0 NT CG1394 FBgn0030277 9 NT CG13984
FBgn0031796 0 NT CG14017 FBgn0031721 0 MGC35043 hypothetical
protein MGC35043 NT CG14047 FBgn0040390 0 NT CG14082 FBgn0036851 1
NT CG14131 FBgn0036205 0 NT CG14252 FBgn0039462 0 NT CG14423
FBgn0029646 3 NT CG14448 FBgn0037191 0 NT CG14453 FBgn0037179 2 NT
CG14550 FBgn0039405 1 DSCR5 Down syndrome critical region gene 5 NT
CG14563 FBgn0037139 0 NT CG14564 FBgn0037131 0 NT CG14565
FBgn0037129 0 NT CG14574 FBgn0037104 0 NT CG14609 FBgn0037483 0
KIAA1212 KIAA1212 NT CG14659 FBgn0037284 0 NT CG14662 FBgn0037291 0
NT CG14843 FBgn0038230 0 NT CG14850 FBgn0038239 0 NT CG14931
FBgn0032374 0 NT CG15059 FBgn0030905 0 NT CG15133 FBgn0032619 0 NT
CG15152 FBgn0032665 0 NT CG15278 FBgn0032554 0 NT CG1529
FBgn0031144 1 ZNF569 zinc finger protein 569 NT CG15366 FBgn0030080
0
NT CG15376 FBgn0029692 5 NT CG15432 FBgn0031603 2 NT CG15471
FBgn0029726 0 NT CG15488 FBgn0032440 0 NT CG15513 FBgn0039705 0
ATG16L ATG16 autophagy related 16-like NT CG15771 FBgn0029801 0
HDHD4 haloacid dehalogenase-like hydrolase domain containing 4 NT
CG15784 FBgn0029766 1 NT CG15888 FBgn0038131 0 NT CG1678
FBgn0031176 0 NT CG16865 FBgn0028919 0 FLJ22965 hypothetical
protein FLJ22965 NT CG16964 FBgn0032385 0 NT CG17261 FBgn0031501 0
NT CG17267 FBgn0038821 0 NT CG17282 FBgn0038857 0 NT CG17382
FBgn0039080 0 NT CG17786 FBgn0039167 1 CNOT6 CCR4-NOT transcription
complex, subunit 6 NT CG17807 FBgn0034748 0 LOC91801 hypothetical
protein 8C015183 NT CG17952 FBgn0034657 0 NT CG18145 FBgn0032189 0
NT CG18275 FBgn0029523 2 NT CG18368 FBgn0033864 0 NT CG18600
FBgn0038601 0 NT CG1896 FBgn0039870 0 NT CG2016 FBgn0037289 0 NT
CG2124 FBgn0030217 0 FLJ13149 hypothetical protein FLJ13149 NT
CG2889 FBgn0030206 0 NT CG30010 FBgn0050010 0 MGC70857 similar to
RIKEN cDNA C030006K11 gene NT CG30101 FBgn0050101 1 NT CG30109
FBgn0050109 0 P53CSV p53-inducible cell-survival factor NT CG30363
FBgn0050363 0 NT CG30419 FBgn0050419 0 NT CG31093 FBgn0051093 0 NT
CG31389 FBgn0051389 0 NT CG31407 FBgn0051407 0 NT CG31825
FBgn0051825 1 NT CG31989 FBgn0051989 0 NT CG31998 FBgn0051998 0 NT
CG32021 FBgn0052021 1 NT CG32345 FBgn0052345 3 NT CG32436
FBgn0052436 0 NT CG32639 FBgn0052639 0 NT CG32783 FBgn0029686 0 NT
CG33109 FBgn0053109 0 NT CG33267 FBgn0053267 2 NT CG3330
FBgn0039511 0 NT CG33301 FBgn0053301 3 NT CG33340 FBgn0053340 0 NT
CG3408 FBgn0036008 0 PRO1855 hypothetical protein PRO1855 NT CG3501
FBgn0034791 0 C14orf122 chromosome 14 open reading frame 122 NT
CG3546 FBgn0029716 4 NT CG3598 FBgn0025645 0 NT CG3713 FBgn0040343
0 NT CG3764 FBgn0036684 0 NT CG3800 FBgn0034802 0 ZNF9 zinc finger
protein 9 (a cellular retroviral nucleic acid binding protein) NT
CG3805 FBgn0031665 0 NT CG3973 FBgn0029881 0 NT CG40402 FBgn0058402
0 NT CG4627 FBgn0033808 0 C16orf51 chromosome 16 open reading frame
51 NT CG4820 FBgn0037876 0 ZNF136 zinc finger protein 136 NT CG5237
FBgn0038693 1 KIAA1409 KIAA1409 NT CG5323 FBgn0034362 0 NT CG5386
FBgn0038945 10 NT CG5467 FBgn0039433 3 NT CG5468 FBgn0039434 0 NT
CG5538 FBgn0038052 0 NT CG5955 FBgn0036997 0 NT CG6018 FBgn0034736
0 NT CG6073 FBgn0039417 0 LOC51236 brain protein 16 NT CG6195
FBgn0038723 1 DRG2 developmentally regulated GTP binding protein 2
NT CG6301 FBgn0034161 0 NT CG6480 FBgn0036964 0 FRG1 FSHD region
gene 1 NT CG6569 FBgn0038909 0 MYH2 myosin, heavy polypeptide 2,
skeletal muscle, adult NT CG6614 FBgn0032369 0 TTC18
tetratricopeptide repeat domain 18 NT CG6631 FBgn0039206 0 NT
CG7053 FBgn0030960 0 FLJ11773 hypothetical protein FLJ11773 NT
CG7200 FBgn0032671 1 JMJD4 jumonji domain containing 4 NT CG7242
FBgn0040494 0 NT CG7381 FBgn0038098 0 NT CG7567 FBgn0039670 0 NT
CG8031 FBgn0038110 0 C2orf4 chromosome 2 open reading frame 4 NT
CG8420 FBgn0037664 0 NT CG8538 FBgn0038223 0 NT CG8852 FBgn0031548
1 LRRTM4 leucine rich repeat transmembrane neuronal 4 NT CG9328
FBgn0032886 0 NT CG9380 FBgn0035094 0 NT CG9773 FBgn0037609 0 NT
CR32205 FBgn0052205 1 NT Edg78E FBgn0000551 0 NT I(1)G0196
FBgn0027279 0 KIAA0433 KIAA0433 protein NT I(1)G0222 FBgn0028343 0
NT Mkm1 FBgn0029152 1 MKRN1 makorin, ring finger protein, 1 NT
msb1I FBgn0027949 0 NT MTA1-like FBgn0027951 4 MTA1 metastasis
associated 1 NT nito FBgn0027548 0 RBM15 RNA binding motif protein
15 NT olf186-M FBgn0015522 0 NT Osi13 FBgn0037422 0 NT Osi17
FBgn0037427 0 NT Osi19 FBgn0037429 0 NT Pcp FBgn0003046 0 NT sano
FBgn0034408 0 NT T48 FBgn0004359 0 NT yellow-d2 FBgn0034856 0
TABLE-US-00003 TABLE II Inhibition of NFAT nuclear # of Identity of
localization potential potential Amplicon In TG- oft-targets
off-targetsof Gene Description CG No. Expression treated cells of
21 nt 21 nt CanA1 Calcineurin CG1455 DRSC 16600 +/- - 0 A1 Pp2B-14D
Protein CG9842 DRSC23315 ++ +++ 0 phosphatase 2B at 14D DRSC20270
+++ 1 CG12238 CanA-14F Calcineurin CG9819 DRSC23296 + +++ 0 A at
14F DRSC20211 +++ 13 not listed CanB Calcineurin CG4209 DRSC 18449
+/- ++ 0 B CanB2 Calcineurin CG11217 DRSC07355 ++ ++ 0 B2 CG32812
CG32812 CG32812 DRSC18478 + - 0 # of Identity of # of Identity of
potential potential potential potential off-targets off-targets
off-targets off-targets Gene of 20 nt of 20 nt of 19 nt of 19 nt
Comments CanA1 0 1 CG7952 Pp2B-14D 0 0 2 CG12238, 3 CG12238,
CG32223 CG32223, CG32025 CanA-14F 0 0 56 not listed 163 not listed
CG9842 (Pp2B-14D) has 18 matches with this amplicon. CanB 1 CG11217
2 CG11217 (CanB2) (CanB2), CG15859 CanB2 1 CG4209 2 CG4209 (CanB)
(CanB), CG5744 CG32812 0 0
TABLE-US-00004 TABLE III Molecule in Potential Suppl Table III
off-target Description of the potential off-target (NCBI Gene) 1.
DIRECT NFAT Shaggy CG13772 neurexin binding; ectoderm development
and neurogenesis; (sgg. CG2621) (neuroligin) CG4771 NA CG12199
peroxidase activity, cell adhesion, defense response; reactive
oxygen species metabolism; transmission of (kek5) nerve impulse;
CG1049 choline-phosphate cytidylyltransferase activity; (cct1)
CG5907 calcium sensitive guanylate cyclase activator activity;
calmodulin binding; neurotransmitter secretion; (frq) synaptic
transmission; CG32538 nicotinic acetylcholine-activated
cation-selective channel activity; muscle contraction; nerve-nerve
(nAcRalpha- synaptic transmission; 18C) CG9176 intracellular cyclic
nucleotide activated cation channel activity; potassium channel
activity; sensory (cngl) perception; signal transduction; CG3427
cAMP-dependent protein kinase regulator activity; small GTPase
mediated signal transduction; (epac) CG33513 N-methyl-D-aspartate
selective glutamate receptor activity; cation transport;
nerve-nerve synaptic (nmdar2) transmission; CG13290 NA CG12708 NA
CG4136 nucleobase, nucleoside, nucleotide and nucleic acid
metabolism regulation of transcription from RNA polymerase II
promoter; ligand-dependent nuclear receptor activity; Gasket
CG12212 transcription factor activity; leading edge cell fate
determination; ectoderm development; photoreceptor cell (gsk. (peb)
morphogenesis; maintenance of tracheal epithelial integrity,
negative regulation of JNK cascade; CG11338, CG12147 CG6205
acyltransferase activity; cell adhesion; regulation of Wnt receptor
signaling pathway; (por) CG14895 receptor signaling protein
serine/threonine kinase activity; MAPKKK cascade; actin filament
organization; (pak3) cell proliferation; cytoskeleton organization
and biogenesis; CG18214 Rho guanyl-nucleotide exchange factor
activity; actin cytoskeleton organization and biogenesis; axon
(trio) guidance; central and peripheral nervous system development;
transmission of nerve impulse. Disc CG2028 receptor signaling
protein serine/threonine kinase activity; Wnt receptor signaling
pathway; negative overgrown regulation of smoothened signaling
pathway; regulation of proteolysis and peptidolysis; CKI alpha
CG2048 receptor signaling protein serine/threonine kinase activity;
Wnt receptor signaling pathway, negative (CG2028) (ckIalpha)
regulation of smoothened signaling pathway, regulation of
proteolysis and peptidolysis; CG2577 receptor signaling protein
serine/threonine kinase activity; casein kinase I activity; CG9102
transcription factor activity; chromatin assembly or disassembly;
eye-antennal disc metamorphosis; sex (bab2) determination; female
gonad development; leg morphogenesis; transmission of nerve
impulse. CG7838 receptor signaling protein serine/threonine kinase
activity; chromosome segregation; mitotic spindle (bub1)
checkpoint; regulation of exit from mitosis. CG7892 receptor
signaling protein serine/threonine kinase activity; anti-apoptosis;
cell proliferation; establishment (nmo) of planar polarity; eye
morphogenesis; wing morphogenesis; negative regulation of Wnt
receptor signaling pathway; negative regulation of frizzled
signaling pathway; CG16973 JUN kinase kinase kinase kinase
activity; small GTPase regulator activity; oogenesis; photoreceptor
cell (msn) morphogenesis; regulation of cell shape; CG2577 CG2048
receptor signaling protein serine/threonine kinase activity; casein
kinase I activity; cell communication; circadian rhythm; imaginal
disc growth; regulation of ecdysteroid secretion; regulation of
protein-nucleus CG2028 receptor signaling protein serine/threonine
kinase activity; Wnt receptor signaling pathway; negative
regulation of smoothened signaling pathway; regulation of
proteolysis and peptidolysis; CG7838 receptor signaling protein
serine/threonine kinase activity; chromosome segregation; mitotic
spindle (bub1) checkpoint; regulation of exit from mitosis CG7236
receptor signaling protein serine/threonine kinase activity;
cytokioesis; regulation of progression through cell cycle; CG3228
ATP-dependent helicase activity; nuclear mRNA splicing, via
spliceosome; proteolysis and peptidolysis. (kurz) CC7094 CG9135
guanyl-nucleotide exchange factor activity; proteolysis and
peptidolysis. CG9962 CG5621 glutamate-gated ion channel activity;
kainate selective glutamate receptor activity; potassium channel
activity; nerve-nerve synaptic transmission. II. OTHER KINASES
CG31640 CG33531 transmembrane receptor protein tyrosine kinase
activity; cell-cell adhesion; ectoderm development; (ddr) mesoderm
development; nervous system development; CG2699 phosphoinositide
3-kinase regulator activity; insulin receptor signaling pathway;
positive regulation of cell size; (Pi3K21B) positive regulation of
growth; regulation of cell proliferation; regulation of cell size;
Pelle CG5263 mRNA 3'-UTR binding; translation repressor activity;
(pll. CG5974) I(1)G0148 CG9463 alpha-mannosidase activity;
hydrolase activity, hydrolyzing N-glycosyl compounds. (CG32742)
Pole hole CG8522 fatty acid biosynthesis; positive regulation of
transcription; transcription from RNA polymerase II promoter; (pbl,
CG2845) (HLH106) CG11073 NA CG3634 NA CG15105 transcription
regulator activity; ubiquitin-protein ligase activity; CG3198
nuclear mRNA splicing, via spliceosome CG17299 receptor signaling
protein serine/threonine kinase activity; defense response; fatty
acid metabolism; regulation of phosphate metabolism; response to
stress CG8465 NA Foraging CG7826 receptor signaling protein
serine/threonine kinase activity; nervous system development;
ectoderm (for. (mnb) development; olfactory learning; cell
proliferation; circadian rhythm; induction of apoptosis; learning
and/or CG32629 NA CG13472 NA CG18389 transcription factor activity;
autophagy; ecdysone-mediated induction of salivary gland cell
death; induction (Eip93F) of apoptosis by hormones; larval midgut
histolysis; CG9310 steroid hormone receptor activity; regulation of
transcription from RNA polymerase II promoter; endoderm (hnf4)
development; mesoderm development; CG16902 steroid hormone receptor
activity; metamorphosis; regulation of transcription from RNA
polymerase II (Hr4) promoter CG4013 corepressor activity;
regulation of transcription from RNA polymerase II promoter. (smr)
CG8949 NA CG 14447 glutamate receptor binding; determination of
muscle attachment site; (grip) CG5683 RNA polymerase II
transcription factor activity; cell proliferation; (Aef1) CG32180
specific RNA polymerase II transcription factor activity;
autophagy; cell death; salivary gland cell death (eip74EF) mesoderm
development; oogenesis; CG32423 mRNA processing; CG3696
ATP-dependent helicase activity; blastoderm segmentation; chromatin
assembly or disassembly; (kis) CG3695 RNA polymerase II
transcription mediator activity; mediator complex; (MED23) CG14023
NA CG13109 transcription coaaivator activity; signal transducer
activity; border follicle cell migration; (tai) CG9381 learning
and/or memory; olfactory learning; (mura) CG5466 NA CG12254 RNA
polymerase II transcription mediator activity; (MED25) CG9354
nucleic acid binding; structural constituent of ribosome; (RpL34b)
CG6575 carbohydrate binding; cell adhesion; heterophilic cell
adhesion; nervous system development. (glec) CG14366 NA CG1161 NA
CG10732 NA CG7368 NA CG12432 NA CG17888 transcription factor
activity; circadian rhythm; mesoderm development; (Pdp1) Pi3K59F
CG3856 octopamine receptor activity; octopamine/tyramine signaling
pathway; ovulation; (CG5373) (Oamb) CG14619 cysteine-type
endopeptidase activity; ubiquitin thiolesterase activity;
ubiquitin-specific protease activity CG10989 NA III. OTHER CG6919
CG18208 G-protein coupled receptor protein signaling pathway;
transmission of nerve impulse. CG31288 CG15415 NA CG32381
neurotransmitter secretion; synaptic vesicle priming. (unc-13-4A)
CanA1 CG7952 (giant) negative regulation of transcription from RNA
polymerise II promoter; posterior head segmentation; (CG1455)
terminal region determination; zygotic determination of
anterior/posterior axis; ring gland development; salivary gland
development; torso signaling pathway. Pp2B-14D CG12238 chromatin
binding; transcription regulator activity; gene silencing;
oogenesis. (CG9842) (I(1)G0084) CG32223 NA CG32025 NA CanA-14F not
listed (CG9819) CanB CG11217 (CanB2) calcium-dependent protein
serine/threonine phosphatase activity; cell homeostasis; (CG4209)
neurotransmitter secretion; vesicle-mediated transport. CG15859 NA
CG11217 CG4209 (CanB) calcium-dependent protein serine/threonine
phosphatase activity; cell homeostasis; (CanB2) neurotransmitter
secretion; vesicle-mediated transport. CG5744 calcium-mediated
signaling; sensory perception; signal transduction; visual
perception.
TABLE-US-00005 TABLE IV # of Score In Description of potential
primary the human Amplicon off-targets screen Gene homologue CG
FBgn No. of 21 nt I. DIRECT NFAT KINASES GSK3 6 shaggy GSK38 CG2621
FBgn0003371 DRSC18832 4 (sgg) 1 gasket GSK3A CG11338, FBgn0046332
DRSC14056 0 (gskt) CG31003 CK1 4 gilgamesh CSNK1G CG6963
FBgn0011253 DRSC16154 0 (glsh) 3 CG12147 CSNK1 CG12147 FBgn0037325
DRSC12192 0 1 discs CSNK1E CG2048 FBgn0002413 DRSC16929 0 overgrown
(dco) 1 CK1alpha CSNK1A1 CG2028 FBgn0015024 DRSC20231 3 1 CG2577
CSNK1 CG2577 FBgn0030384 DRSC19863 3 1 CG7094 CSNK1 CG7094
FBgn00032650 DRSC03005 0 0 CG9962 CSNK1 CG9962 FBgn0031441
DRSC00739 0 DYRK 1 C040478 DYRK2 CG40478 FBgn0069975 DRSC21055 0
II. OTHER KINASES DDR 4 CG31640 DDR CG31640 FBgn0051640 DRSC25O4 0
IRAK 3 pll (pelle) IRAK CG5974 FBgn0010441 DRSC17026 0 CK2 3
CkIIalpha CSNK2A CG17520 FBgn0000258 DRSC11946 0 CDC7 2 I(1)G0148
CDC7 CG32742 FBgn0028360 DRSC18429 0 TRRAP 2 Nipped-A TRRAP CG2905,
FBgn0004661, DRSC4882 0 CG33554 FBgn0053554, FBgn0039989 RAF 1 phl
RAF CG2845 FBgn0003079 DRSC18821 2 (pole hole) PRKG1 1 for PRKG1
CG10033 FBgn0000721 DRSC00195 2 (foraging) P13K 1 Pi3K59F PiK3C3
CG5373 FBgn0015277 DRSC04640 0 III. OTHER HTR 5 CG6919 HTR4 CG6919
FBgn0038980 DRSC16134 0 FAM20 4 CG31145 FAM20C CG31145 FBgn0051145
DRSC14671 0 4 CG31288 CG31288 FBgn0051288 DRSC14667 0 B9 4 CG14870
EPPB9 CG14870 FBgn0038342 DRSC14993 0 4 CG4585 CG4585 FBgn0025335
DRSC4475 0 PGLYRP 3 CG8995 PGLYRP3 CG8995 FBgn0030695 DRSC20137 0
STIM 3 Stim STIM1 CG9126 FBgn0045073 DRSC20158 0 Cathepsln B 2
CG3074 CTSB CG3074 FBgn0034709 DRSC4334 0 Identity of # of Identity
of # of Identity of potential potential potential potential
potential off-targets off-targets off-targets off-targets
off-targets of 21 nt of 20 nt of 20 nt of 19 nt of 19 nt I. DIRECT
NFAT KINASES GSK3 CG5907, 7 CG5907, 12 CG4771, CG13772, CG13772,
CG5907, CG12199, CG12199, CG13772, CG1049 CG1049, CG12199, CG32538,
CG1049, CG9176, CG32538, CG3427 CG9176, CG3427, CG33513, CG13290,
CG12708, CG4138 0 1 CG12212 CK1 0 0 2 CG6205, 3 CG6205, CG14895
CG14895, CG18214 1 CG2028 1 CG2028 CG2048, 4 CG2048, 6 CG2048,
CG2577, CG2577, CG2577, CG7838 CG7838, CG7838, CG16973 CG16973,
CG7892, CG9102 CG2028, 4 CG2028, 5 CG2028, CG2046, CG2048, CG2048,
CG7638 CG7838, CG7838, CG7236, CG7236, CG3228 1 CG9135 1 CG9135 6 1
CG5621 DYRK 0 0 II. OTHER KINASES DDR 2 CG33531, 2 CG33531, CG2699
CG2699 IRAK 1 CG5263 1 CG5263 CK2 0 0 CDC7 1 CG9463 1 CG9463 TRRAP
0 0 RAF CG11073, 4 CG11073, 7 CG3198, CG8522 CG8522, CG11073,
CG3634, CG8522, CG15105 CG3834, CG15105, CG17299, CG8465 PRKG1
CG32629, 4 CG32629, 27 CG18389 CG18389, CG7828, CG9310 P13K 2
CG14619, 3 CG14619, CG10989 CG3856, CG10989 III. OTHER HTR 0 1
CG18208 FAM20 0 0 1 CG15415 2 CG15415, CG32381 B9 0 0 0 0 PGLYRP 0
0 STIM 0 0 Cathepsln B 0 0
Sequence CWU 1
1
5411497DNAHomo sapiens 1agcggcgccg cgggcctgcg tgctggggca gcgggcactt
cttcgacctc gtcctcctcg 60tcctgtgcgg ccggccgggt gaggccgggc ccgcgtaggg
ggcagtcggc ggctgcctcc 120ggcggaggtg cctcgcggcg cccgggccgg
cccgcgcctc ggcggcgtgc tccatgcatc 180cggagcccgc cccgcccccg
agccgcagca gtcccgagct tcccccaagc ggcggcagca 240ccaccagcgg
cagccgccgg agccgccgcc gcagcgggga cggggagccc ccgggggccc
300cgccaccgcc gccgtccgcc gtcacctacc cggactggat cggccagagt
tactccgagg 360tgatgagcct caacgagcac tccatgcagg cgctgtcctg
gcgcaagctc tacttgagcc 420gcgccaagct taaagcctcc agccggacct
cggctctgct ctccggcttc gccatggtgg 480caatggtgga ggtgcagctg
gacgctgacc acgactaccc accggggctg ctcatcgcct 540tcagtgcctg
caccacagtg ctggtggctg tgcacctgtt tgcgctcatg atcagcacct
600gcatcctgcc caacatcgag gcggtgagca acgtgcacaa tctcaactcg
gtcaaggagt 660ccccccatga gcgcatgcac cgccacatcg agctggcctg
ggccttctcc accgtcatcg 720gcacgctgct cttcctagct gaggtggtgc
tgctctgctg ggtcaagttc ttgcccctca 780agaagcagcc aggccagcca
aggcccacca gcaagccccc cgccagtggc gcagcagcca 840acgtcagcac
cagcggcatc accccgggcc aggcagctgc catcgcctcg accaccatca
900tggtgccctt cggcctgatc tttatcgtct tcgccgtcca cttctaccgc
tcactggtta 960gccataagac tgaccgacag ttccaggagc tcaacgagct
ggcggagttt gcccgcttac 1020aggaccagct ggaccacaga ggggaccacc
ccctgacgcc cggcagccac tatgcctagg 1080cccatgtggt ctgggccctt
ccagtgcttt ggccttacgc ccttcccctt gaccttgtcc 1140tgccccagcc
tcacggacag cctgcgcagg gggctgggct tcagcaaggg gcagagcatg
1200gagggaagag gatttttata agagaaattt ctgcactttg aaactgtcct
ctaagagaat 1260aagcatttcc tgttcttcca gctccaggtc cacctcctgt
tgggaggcgg tggggggcca 1320aagtggggcc acacactcgc tgtgtcccct
ctcctcccct gtgccagtgc cacctgggtg 1380cctcctcctg tcctgtccgt
ctcaacctcc ctcccgtcca gcattgagtg tgtacatgtg 1440tgtgtgacac
ataaatatac tcataaggaa aaaaaaaaaa aaaaaaaaaa aaaaaaa
149722495DNAHomo sapiens 2ggagagcctg agttggcatt cgtataaatg
acctgcctgg ctcccaccat gagtgctgag 60cttaacgtgc ctatcgaccc ctctgctcct
gcctgccctg agccaggcca taagggcatg 120gattaccggg actgggtccg
ccgcagctac ctggaactgg tcacctctaa ccaccactcg 180gtacaggccc
tgtcgtggcg gaagctctac ctgagcaggg ccaagctgaa ggcctccagc
240aggacctccg ccctcctctc cggctttgcc atggtggcca tggtggaggt
gcagctggag 300acgcagtacc agtacccgcg gccgctgctg attgccttca
gcgcctgcac cacggtgctg 360gtggccgtgc acctgttcgc cctcctcatc
agcacctgca tcctgcccaa tgtggaggcc 420gtgagcaaca tccacaacct
gaactccatc agcgagtccc cgcatgagcg catgcacccc 480tacatcgagc
tggcctgggg cttctccacc gtgcttggca tcctactctt cctggccgag
540gtggtgctgc tctgctggat caagttcctc cccgtggatg cccggcgcca
gcctggcccc 600ccacctggcc ctgggagtca cacgggctgg caggccgccc
tggtgtccac catcatcatg 660gtgcccgtgg gcctcatctt cgtggtcttc
accatccact tctaccgctc cctggtgcgc 720cacaaaacgg agcgccacaa
ccgcgagatc gaggagctcc acaagctcaa ggtccagctg 780gacgggcatg
agcgcagcct gcaggtcttg tgaggggccg agggccgggg ctgggagcgg
840ccctgtgccc gggagtccgc agaggcgggg atttgtcaga tgcagacatt
ttgcaaggct 900gccgggtagt tcaagaccaa agttttcctc ttgtcttaat
accataagga ctggatgact 960tctcctgaga tagaaccgtt tggttcaatg
agggactgtg ttgctaagag cgttgggggc 1020aaagccaggc tggttccttg
gcctcggggt ttcctgggtc ggggacacgg tgaagaggct 1080ccagcgggac
ctgcccatca gtcctgggcc aggaggggct ccaagcagca cccagcggtc
1140cgggggagtc tcagacccgg catgcgtggc tggcagacct gggagagcca
gggcagggtt 1200ttgcgttcag agaaggattg ccccagagac ccgtggtgga
cttcatgggt gctgagtggc 1260ccgtgtgaca gtgatgacac gaaggcttcg
gcgtttgagt gggtgcaggt gcacgccagg 1320gcttggtgct tccctgcctg
gccctggagg gagctgggtg gcctggcttc aggggaagac 1380aggagccagg
acacacgtca gcccagcagg tgtggggggt gctgcagccc tcggcagtgg
1440ggtcaggccc tgggggatgt ttccaatggt gggcagcctg gccaggccgg
agaagacatg 1500ttcacgggca tctatcagat gcccccttga ggaggctgag
ttatttgagg gctgctgcaa 1560agtacgctag gctcaaattc tcttttccca
gccagagccc tggccacacg gactcagagg 1620ggccaccggg gtggggaaag
gacccctccc cgaccccccg cagccactgg cctccagctc 1680tcggccacag
aatggcctct aaggctgact cagccgctcc cttgggctgt ggcagcagga
1740ggcgggggct ctggctcagg ccccggagcc tgtgcagctt gcccatggcc
ctaggcagcg 1800aggggacagc ctgggggact tcctgcctag gcaaggtcat
tggccgggcc tggcctgtgg 1860atagtggggc caggggccgg cccaggccaa
atgagtgccc tccttgttat gacaccaagt 1920gactacaagg gaggcaagac
ccctccaggc ctctcagccg acactgggtc ccaccacaca 1980cagtgactgt
gccgtgcagt gcaggttctg gccttttcct tgaaggcatc tggtagaccc
2040gaagccacgc tctcgggccg cacatgcacg ccgcagcacc agctgccctg
agctgcttgt 2100acaaccaaac acctttcccc tcttctccag ctgtaacctg
gagagtcagc catgccttgt 2160cttttgttct cataaatagt cactggggcc
gggcgcagtg actcacgcct gtaatcccag 2220cactttggga ggcctaggtg
ggcggatcac ttgaggtcag gagttcgaga ccagcctggc 2280caacatggtg
aaaccctgtc tctactaaaa aaatacagaa aattagctgg gcgtggtggc
2340gggcgcctgt agccccagct acttgggagg ctgaggtggg agaatggcaa
tggcgtgaac 2400ccgggaggca gagcttgcag tgagctgaga tggcgccact
gcactccagc ctgggcgaca 2460gagccagact caatctcaaa aaaaaaaaaa aaaaa
249532239DNAHomo sapiens 3cgctccggct cctggggctc cccgcagacg
ctgcttttct tgctccactg ggggtgcctc 60ttcctgggcg cccgccgcct gcatcctgct
cgccctgtct gggaatgggg ccgcccccgg 120gcttgggccg gcccggctgg
ggcccccgag gcgcttccgc cccgtagtga ccgcctggtg 180ccgccccccc
ccaggatgaa gggcggcgag ggggacgcgg gcgagcaggc cccgctgaac
240cctgagggcg agagccctgc aggctcggcc acgtaccggg agttcgtgca
ccgcggctac 300ctggacctca tgggggccag tcagcactcg ctgcgggcgc
tcagctggcg ccgcctctac 360ctcagccggg ccaagctcaa agcttccagc
cgcacgtctg ccttgctctc gggcttcgcc 420atggtggcca tggtggaggt
gcagctggag agtgaccacg agtacccacc aggcctgctg 480gtggccttca
gtgcctgcac caccgtgctg gtggctgtgc acctctttgc actcatggtc
540tccacgtgtc tgctgcccca cattgaagct gtgagcaaca tccacaacct
caactctgtc 600caccagtcgc cacaccagag actgcaccgc tacgtggagc
tggcctgggg cttctccact 660gccctgggca cctttctctt ccttgctgaa
gttgtcctgg ttggttgggt caagtttgtg 720cccattgggg ctcccttgga
cacaccgacc cccatggtgc ccacatcccg ggtgcccggg 780actctggcac
cagtggctac ctcccttagt ccagcttcca atctcccacg gtcctctgcg
840tctgcagcac cgtcccaggc tgagccagcc tgcccacccc ggcaagcctg
tggtggtggt 900ggggcccatg ggccaggctg gcaagcagcc atggcctcca
cagccatcat ggtacccgtg 960gggctcgtgt ttgtggcctt tgccctgcat
ttctaccgct ccttggtggc acacaagaca 1020gaccgctaca agcaggaact
agaggaactg aatcgcctgc agggggagct gcaggctgtg 1080tgagactggt
gttagccacc gctcactgca agcactgcct ccctccgggg tctgtaagag
1140gccgcagggg cctacagacc tcatcccccc atcccctggc tggagccact
tccagtggcc 1200actctcaggc agagttcaga ttcctgcccg cagggtcctc
tgggctgggc cttggggcag 1260ctcccacatt cccagggatt ttccccatca
gtctgtccct tgggttttgc aagctactct 1320gcacctgggc tggcctcagt
tgaaggatca tgcagtagat agaggggagg cagggagagc 1380ttgtgggacc
ttcagtgctg actttagcca ccatttccat tcctatacag gatgtgaagg
1440tcagaaggca gccaattgtt ggtttaattt tttttttttt tgagacagtc
tgtttcccag 1500gctggagtgt agtgatacag tcacagctca ctgtagcctc
gaccttccag gctcaaaaga 1560tgctcccacc acagcctccc aggtagtgag
tagctggtac tacaggtgtg tgctgccaca 1620cccgactaat ttttttgtag
agacggggtt tcgctgttcc caggctggtc tcaaactcct 1680gggctcaagt
gaacctcccg cctcggcctc ccaaagtgct gggattcctt tctttatttc
1740tgtagaatct attttatggt tggcattttg ggggaagatt tcgatgggtt
ccacattctt 1800gctttagttg ttgtagaggg atttgggtgt ttctacccaa
ggcattggtc tagcttttcc 1860tacaatgaac ctatctttgg aggtttaagc
tccccacctt cccccactgt ggtgacctgt 1920ggccacttgc agaagggatg
gtgcctgacc cactgcccta gccccacgct atgcaccaaa 1980cttgttctcc
ccgtcctggt ccagggctgg ggtctttaga gactgacagc ctctgcccca
2040ggcctgagtc cttagcaagg gttgggtaag gaggttttaa gggagaaggt
ccagtcctta 2100gcccttgaaa tacaaagctc ttctgacact gaatttggat
gcaccttgtt ttatataata 2160aatcgtgttt cacagaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2220aaaaaaaaaa aaaaaaaaa
223945212DNAHomo sapiens 4gttatagttt tgccgctgga ctcttccctc
ccttccccca ccccatcagg atgatatgag 60acttgaaaga agacgatgca tacaggagga
gagacttcag catgcaaacc ttcatctgtt 120cggcttgcac cgtcattttc
attccatgct gctggccttc agatggctgg acagatgccc 180cattcacatc
agtacagtga ccgtcgccag ccaaacataa gtgaccaaca ggtttctgcc
240ttatcatatt ctgaccagat tcagcaacct ctaactaacc aggtgatgcc
tgatattgtc 300atgttacaga ggcggatgcc ccaaaccttc cgtgacccag
caactgctcc cctgagaaaa 360ctttctgttg acttgatcaa aacatacaag
catattaatg aggtttacta tgcaaaaaag 420aagcgaagac accaacaggg
ccagggagac gattctagtc ataagaagga acggaaggtt 480tacaatgatg
gttatgatga tgataactat gattatattg taaaaaacgg agaaaagtgg
540atggatcgtt acgaaattga ctccttgata ggcaaaggtt cctttggaca
ggttgtaaag 600gcatatgatc gtgtggagca agaatgggtt gccattaaaa
taataaagaa caagaaggct 660tttctgaatc aagcacagat agaagtgcga
cttcttgagc tcatgaacaa acatgacact 720gaaatgaaat actacatagt
gcatttgaaa cgccacttta tgtttcgaaa ccatctctgt 780ttagtttttg
aaatgctgtc ctacaacctc tatgacttgc tgagaaacac caatttccga
840ggggtctctt tgaacctaac acgaaagttt gcgcaacaga tgtgcactgc
actgcttttc 900cttgcgactc cagaacttag tatcattcac tgtgatctaa
aacctgaaaa tatccttctt 960tgtaacccca aacgcagtgc aatcaagata
gttgactttg gcagttcttg tcagttgggg 1020cagaggatat accagtatat
tcagagtcgc ttttatcggt ctccagaggt gctactggga 1080atgccttatg
accttgccat tgatatgtgg tccctcgggt gtattttggt tgaaatgcac
1140actggagaac ctctgttcag tggtgccaat gaggtagatc agatgaataa
aatagtggaa 1200gttctgggta ttccacctgc tcatattctt gaccaagcac
caaaagcaag aaagttcttt 1260gagaagttgc cagatggcac ttggaactta
aagaagacca aagatggaaa acgggagtac 1320aaaccaccag gaacccgtaa
acttcataac attcttggag tggaaacagg aggacctggt 1380gggcgacgtg
ctggggagtc aggtcatacg gtcgctgact acttgaagtt caaagacctc
1440attttaagga tgcttgatta tgaccccaaa actcgaattc aaccttatta
tgctctgcag 1500cacagtttct tcaagaaaac agctgatgaa ggtacaaata
caagtaatag tgtatctaca 1560agccccgcca tggagcagtc tcagtcttcg
ggcaccacct ccagtacatc gtcaagctca 1620ggtggctcat cggggacaag
caacagtggg agagcccggt cggatccgac gcaccagcat 1680cggcacagtg
gtgggcactt cacagctgcc gtgcaggcca tggactgcga gacacacagt
1740ccccaggtgc gtcagcaatt tcctgctcct cttggttggt caggcactga
agctcctaca 1800caggtcactg ttgaaactca tcctgttcaa gaaacaacct
ttcatgtagc ccctcaacag 1860aatgcattgc atcatcacca tggtaacagt
tcccatcacc atcaccacca ccaccaccat 1920caccaccacc atggacaaca
agccttgggt aaccggacca ggccaagggt ctacaattct 1980ccaacgaata
gctcctctac ccaagattct atggaggttg gccacagtca ccactccatg
2040acatccctgt cttcctcaac gacttcttcc tcgacatctt cctcctctac
tggtaaccaa 2100ggcaatcagg cctaccagaa tcgcccagtg gctgctaata
ccttggactt tggacagaat 2160ggagctatgg acgttaattt gaccgtctac
tccaatcccc gccaagagac tggcatagct 2220ggacatccaa cataccaatt
ttctgctaat acaggtcctg cacattacat gactgaagga 2280catctgacaa
tgaggcaagg ggctgataga gaagagtccc ccatgacagg agtttgtgtg
2340caacagagtc ctgtagctag ctcgtgacta cattgaaact tgagtttgtt
tcttgtgtgt 2400ttttatagaa gtggtgtttt ttttccaaaa acaaagtgca
aagctgcttg aatcaggagg 2460agattaacac actgaaccgc tacaagaggg
caaagctgat ttttttttta acttgaaaag 2520attgcaaagg gacattgaag
tgtttaaaag agccatgtcc aaacccatct tcatggatag 2580ctcagaggta
tcctcttttt gctcccccat tttaacttgc cacatcccag tcacagtggg
2640gtttttttgt ctttctattc agcaaaagtt aatattcaga tgttggtctt
ggtcatttgc 2700caactaattt taaagtaaaa ggcactgcac ataatttgca
taaagggccc catgagggtg 2760tttttttttt ttctttttgt cccccccatc
cccctttttt tttgttttgt tctgttttgt 2820tttgggtggg agggtgggaa
atttgggttt ttaagtcctc taaacacact tgggcacgga 2880aatgcagtac
tgtaaggaag agggacctcc agcttccaca aacaccatct tcagctgtat
2940gaaagggacg gttgtggtga agtttgtcag gcacagtaag catgctgagt
ggcggggatc 3000agaactctcc tatctgaacc tactgaggag caaagcagca
attacatggg atcctgtggc 3060tctcccgttg cagaggccac aggaagatag
gatggaacgt gactggtctc ctaaccaagg 3120tgcactgaga agcaatcaac
gggtcggtcg tggccagtcc tggggaggtc tgagtggtgg 3180tctttgggat
aacctttggc cttatggatt tggactcgaa attagaagag cctaccattt
3240cagatgcaat cacttttgga catgcttttg cagacagtcc ttaatgctga
aaacacagag 3300aatgggtaat tcaagaggcc tttcttttaa aatagacttt
tgtgacccac taattgtaag 3360gtattgcaag gtcactttgc gtgtgtcata
aagttgactt ccttattggt tgaaggtcac 3420agaagtagtg gtttgctttg
atggaaatag ctacagctgt gtcccttcct gctttttact 3480ttttcttttg
ctttttctcg gcacgtggta tctccaccat ttcttctgca caaagatgtc
3540ttctgttcat cctgaacatt tttaaaaaat gcagaatttt atgtgactgc
ttttttgcct 3600cacaattatg ctgtgaattt tacaaaaatt tattttcttt
tttgataatt tattgtacca 3660aagctgtttt tatagcacat agatgtctgt
aaccaataat gtagcagttc tgcactttga 3720cacaaggtgt aactagacca
tttttaaatg tcagttgaaa attatggctg tactattgct 3780taaacaaaac
tggaactgtt gttgaatcca tagccaatac atttacagca atctgtgtac
3840tgaacatagt agattgacat ctaattcaag attacaacat ctgttacatt
ctaagtgtgt 3900tcaggcttct gaaggtaaag ggacactgga tccagaagct
atggaaccag cagttgattc 3960ttgtattcct gattaaccta cttgtaaact
tgaaagcaag accttgattg caccaacagg 4020tccagagtat gagtgcaagc
aaagcagaac tctcatgcgt gacctgagca gacaggctgg 4080tatttaacag
gtgcctcgtg ttgagattac gctgccttaa tgtaacacag tctggcagtt
4140gctaaatttg tgttcccatt ttaaattgac caattttggg gtgtgacact
tttgagcggt 4200tgaattggga gaatgaagat aagtaattta cctgtccagg
atcaaaagaa gcctagaaaa 4260gaagcagtaa tctacctctg ccgataacct
gtttaagatg actcagcaga acaccgcgtt 4320tcattctatt ggtcaattcc
atgtggctga ctaggtcaat tttttttctg aacaaaagca 4380ggtttttata
tgtaaacagt gagaaaagaa aggctaaaca ctatgtaaat gtgaatggaa
4440acttggaaat actcgttttt ataaactaca aaaacttttt gttgtttatc
aggaaatcca 4500tatttatttt gtaattaact gtcaagcctg tggatgattt
ttttgaactt ggtagttcat 4560aaaggtttac agtgaataaa aggatatcat
cttgagtata gcaatatcaa aaggaattca 4620gtagttactg ctgtttagga
atataaggtt aagatatcat atgggtcagg tcattttttt 4680tttctgtgct
ggttgccaca tcttagcaag caccaaaaaa ctaaagcagt ttttaaaccg
4740atatttacgt aaagaaaatc ataaaatcca atgcttctgc atactgtgtt
atgttacagt 4800ccagttttgt gtgctttact acacagtttg gttacaggac
ttctgtgcat tgtaaacata 4860aacagcatgg aaaaggttaa atacctgtgt
tcagattgta agatctagtc cggacttgct 4920gtgtatattg taacgttaaa
tgaaaaaaga accccccttt gtattatagt catgcggtct 4980tatgtatgat
aaacagttga ataatttgtc ctcagactct ttactatgct tttttaaaaa
5040ttaatttaag aaaaatgtaa acatagtaaa aatcttccta tgcaattaaa
ctggtccagg 5100tctggtaggt atagtatcaa agttgagtta aatgtgtaaa
aaggaaacta tttgagatac 5160attgacatag gcatcagcaa tctctgaaag
taaaaattgg aggtttaaca ga 521252540DNAHomo sapiens 5gggatggggg
cggagtccag ggcgtggggg ggccggtttg ttgtggtcgc cattttgctg 60gttgcattac
tgggtaatcg gggccctggc ttgccgcgtc cgccggatac cctcagccag
120tgggcaggtc tgagctcggg ctccccgagc agtttgagtc cccttgcccg
ctccttcagg 180tctcagcggc ggtggcagcc gaggtgcagg atgcaagaag
gcgccccccg gccgggctcc 240cgctccaggc ctcgctcccc tgcggccctc
tgagcccacc atggccgtcc caccgggcca 300tggtcccttc tctggcttcc
cagggcccca ggagcacacg caggtattgc ctgatgtgcg 360gctactgcct
cggaggctgc ccctggcctt ccgggatgca acctcagccc cgctgcgtaa
420gctctctgtg gacctcatca agacctacaa gcacatcaat gaggtatact
atgcgaagaa 480gaagcggcgg gcccagcagg cgccacccca ggattcgagc
aacaagaagg agaagaaggt 540cctgaaccat ggttatgatg acgacaacca
tgactacatc gtgcgcagtg gcgagcgctg 600gctggagcgc tacgaaattg
actcgctcat tggcaaaggc tcctttggcc aggtggtgaa 660agcctatgat
catcagaccc aggagcttgt ggccatcaag atcatcaaga acaaaaaggc
720tttcctgaac caggcccaga ttgagctgcg gctgctggag ctgatgaacc
agcatgacac 780ggagatgaag tactatatag tacacctgaa gcggcacttc
atgttccgga accacctgtg 840cctggtattt gagctgctgt cctacaacct
gtacgacctc ctgcgcaaca cccacttccg 900cggcgtctcg ctgaacctga
cccggaagct ggcgcagcag ctctgcacgg cactgctctt 960tctggccacg
cctgagctca gcatcattca ctgcgacctc aagcccgaaa acatcttgct
1020gtgcaacccc aagcgcagcg ccatcaagat tgtggacttc ggcagctcct
gccagcttgg 1080ccagaggatc taccagtata tccagagccg cttctaccgc
tcacctgagg tgctcctggg 1140cacaccctac gacctggcca ttgacatgtg
gtccctgggc tgcatccttg tggagatgca 1200caccggagag cccctcttca
gtggctccaa tgaggtcgac cagatgaacc gcattgtgga 1260ggtgctgggc
atcccaccgg ccgccatgct ggaccaggcg cccaaggctc gcaagtactt
1320tgaacggctg cctgggggtg gctggaccct acgaaggacg aaagaactca
ggaaggatta 1380ccagggcccc gggacacggc ggctgcagga ggtgctgggc
gtgcagacgg gcgggcccgg 1440gggccggcgg gcgggggagc cgggccacag
ccccgccgac tacctccgct tccaggacct 1500ggtgctgcgc atgctggagt
atgagcccgc cgcccgcatc agccccctgg gggctctgca 1560gcacggcttc
ttccgccgca cggccgacga ggccaccaac acgggcccgg caggcagcag
1620tgcctccacc tcgcccgcgc ccctcgacac ctgcccctct tccagcaccg
ccagctccat 1680ctccagttct ggaggctcca gtggctcctc cagtgacaac
cggacctacc gctacagcaa 1740ccgatattgt gggggccctg ggccccctat
cacagactgt gagatgaaca gcccccaggt 1800cccaccctcc cagccgctgc
ggccctgggc agggggtgat gtgccccaca agacacatca 1860agcccctgcc
tctgcctcgt cactgcctgg gaccggggcc cagttacccc cccagccccg
1920ataccttggt cgtcccccat caccaacctc accaccaccc ccggagctga
tggatgtgag 1980cctggtgggc ggccctgctg actgctcccc acctcaccca
gcgcctgccc cccagcaccc 2040ggctgcctca gccctccgga ctcggatgac
tggaggtcgt ccacccctcc cgcctcctga 2100tgaccctgcc actctggggc
ctcacctggg cctccgtggt gtaccccaga gcacagcagc 2160cagctcgtga
ccctgccccc tccctggggc ccctcctgaa gccataccct cccccatctg
2220ggggccctgg gctcccatcc tcatctctct ccttgactgg aattgctgct
acccagctgg 2280ggtgggtgag gcctgcactg attggggcct ggggcagggg
ggtcaaggag agggttttgg 2340ccgctccctc cccactaagg actggaccct
tgggcccctc tccccctttt tttctattta 2400ttgtaccaaa gacagtggtg
gtccggtgga gggaagaccc cccctcaccc caggacccta 2460ggagggggtg
ggggcaggta gggggagatg gccttgctcc tcctcgctgt acccccagta
2520aagagctttc tcacaaaaaa 254063466DNAHomo sapiens 6ggactgtgtg
tgtctggctg tagcagacgc gaggcggcga cgaggcgccg gggacccgcg 60cgaggggcgg
ccgggaggcg gcggcggcgg ccgccagaag tagcagcagg accggcggcg
120gcgacggcag ccctgaaatg cattttcctc tccagcggcc atgttaacca
ggaaaccttc 180ggccgccgct cccgccgcct acccgaccga ttggcggcag
taagcacaca atgaatgatc 240acctgcatgt cggcagccac gctcacggac
agatccaggt tcaacagttg tttgaggata 300acagtaacaa gcggacagtg
ctcacgacac aaccaaatgg gcttacaaca gtgggcaaaa 360cgggcttgcc
agtggtgcca gagcggcagc tggacagcat tcatagacgg caggggagct
420ccacctctct aaagtccatg gaaggcatgg ggaaggtgaa agccaccccc
atgacacctg 480aacaagcaat gaagcaatac atgcaaaaac tcacagcctt
cgaacaccat gagattttca 540gctaccctga aatatatttc ttgggtctaa
atgctaagaa gcgccagggc atgacaggtg 600ggcccaacaa tggtggctat
gatgatgacc agggatcata tgtgcaggtg ccccacgatc 660acgtggctta
caggtatgag gtcctcaagg tcattgggaa ggggagcttt gggcaggtgg
720tcaaggccta cgatcacaaa gtccaccagc acgtggccct aaagatggtg
cggaatgaga 780agcgcttcca ccggcaagca gcggaggaga tccgaatcct
ggaacacctg cggaagcagg
840acaaggataa cacaatgaat gtcatccata tgctggagaa tttcaccttc
cgcaaccaca 900tctgcatgac gtttgagctg ctgagcatga acctctatga
gctcatcaag aagaataaat 960tccagggctt cagtctgcct ttggttcgca
agtttgccca ctcgattctg cagtgcttgg 1020atgctttgca caaaaacaga
ataattcact gtgaccttaa gcccgagaac attttgttaa 1080agcagcaggg
tagaagcggt attaaagtaa ttgattttgg ctccagttgt tacgagcatc
1140agcgtgtcta cacgtacatc cagtcgcgtt tttaccgggc tccagaagtg
atccttgggg 1200ccaggtatgg catgcccatt gatatgtgga gcctgggctg
cattttagca gagctcctga 1260cgggttaccc cctcttgcct ggggaagatg
aaggggacca gctggcctgt atgattgaac 1320tgttgggcat gccctcacag
aaactgctgg atgcatccaa acgagccaaa aattttgtga 1380gctccaaggg
ttatccccgt tactgcactg tcacgactct ctcagatggc tctgtggtcc
1440taaacggagg ccgttcccgg agggggaaac tgaggggccc accggagagc
agagagtggg 1500ggaacgcgct gaaggggtgt gatgatcccc ttttccttga
cttcttaaaa cagtgtttag 1560agtgggatcc tgcagtgcgc atgaccccag
gccaggcttt gcggcacccc tggctgagga 1620ggcggttgcc aaagcctccc
accggggaga aaacgtcagt gaaaaggata actgagagca 1680ccggtgctat
cacatctata tccaagttac ctccaccttc tagctcagct tccaaactga
1740ggactaattt ggcgcagatg acagatgcca atgggaatat tcagcagagg
acagtgttgc 1800caaaacttgt tagctgagct cacgtcccct gatgctggta
acctgaaaga tacgacattg 1860ctgagcctta ctgggttgaa aaggagtagc
tcagacctgt ttttatttgc tcaataactc 1920tactcatttg tatcttttca
gcacttaatt ttaatgtaag aaagttgttc attttgtttt 1980tataaaatac
atgaggacaa tgctttaagt ttttatactt tcagaaactt tttgtgttct
2040aaaagtacaa tgagccttac tgtatttagt gtggcagaat aataacatca
gtggcaggcc 2100actgattact tcatgactgc cacgcattta cagattggtg
tcaaagacat tcactatgtt 2160tttatggttc atgttatatc ctccccaggg
tgacagcccc ttaaggccct ccttttccct 2220ccatgctcca ggtccatgca
caggtgtagc atgtcctgct tccgtttttc ataaattaat 2280ctgggtgttg
ggggtagtgg gaggagaacg gtcagaatca aagtgacatt ctaagaaaaa
2340ctgtacctta gagattttcc tctagtgctc aaacaaatac aaaataagat
ccccaaggtt 2400taaactgccc agttagcatt ctgacattct aaaagccggc
aaagcagctt ttagtggata 2460aatgggaatg gaaacgtgtg tgttcctcca
aattttctag tatgatcggt gagctgtttt 2520gtaaagaagc ctcatattac
agagttgctt ttgcacctaa atttagaatt gtattccatg 2580aactgttcct
cccttttctc tgcttttctc ctctctgttc ctcttttaat accacacgtc
2640tgttgcttgc atttagtttg tcttcttcct tcagctgtgt atcccagact
gttaatacag 2700aaaagagaca tttcagctgt gattatgacc attgtttcat
attccaatta aaaaaagaac 2760agcagcctag ctacttaagg tggggatttc
atagttccaa agaagattta gcagattaga 2820gtgagttcac acttttcagg
tgccactgta aggttctctc agcctgggaa actatcaact 2880ctttctttaa
aaagaaagag ggttgaaaat cctctggacg aacagaagtc actttggctg
2940ttcagtaagg ccaatgttaa caacacgttt agaggaggaa aagttcaacc
tcaagttaaa 3000tggtttgact tattcttcgt atcattagaa gaaccccaga
gatagcattc ctctatttta 3060ttttactttc ttttggattg cactgattgt
ttttgtggga atgacacttt atctggcaaa 3120gtaactgaga gtttggtaaa
agaatatttt cttctctgaa taataattat tttcacagtg 3180aaaatttcag
tattttatca ctaatgtatg agcaatgatc tatatcaatt tcaaggcacg
3240tgaaaaaaat tttttagtat gtgcaattta atatagaaag atttctgcct
gtttggacaa 3300taggttttgg gtagtacaga ttaggataag taagcttata
tatgcacaga gattattgta 3360ttacctgtaa attgatttac aagtacttaa
aagcgtggtc cccagtgagg ccaagaaagt 3420ttccggttaa gttctttaat
aataatccta cagtttatct taagaa 346672207DNAHomo sapiens 7acttcccagc
cggggccagt cgggagcgaa agtgcgctga gctgcagtgt ctggtcgaga 60gtacccgtgg
gagcgtcgcg ccgcggaggc agccgtcccg gcgtaggtgg cgtggccgac
120cggaccccca actggcgcct ctccccgcgc ggggtcccga gctaggagat
gggaggcaca 180gctcgtgggc ctgggcggaa ggatgcgggg ccgcctgggg
ccgggctccc gccccagcag 240cggaggttgg gggatggtgt ctatgacacc
ttcatgatga tagatgaaac caaatgtccc 300ccctgttcaa atgtactctg
caatccttct gaaccacctc cacccagaag actaaatatg 360accactgagc
agtttacagg agatcatact cagcactttt tggatggagg tgagatgaag
420gtagaacagc tgtttcaaga atttggcaac agaaaatcca atactattca
gtcagatggc 480atcagtgact ctgaaaaatg ctctcctact gtttctcagg
gtaaaagttc agattgcttg 540aatacagtaa aatccaacag ttcatccaag
gcacccaaag tggtgcctct gactccagaa 600caagccctga agcaatataa
acaccacctc actgcctatg agaaactgga aataattaat 660tatccagaaa
tttactttgt aggtccaaat gccaagaaaa gacatggagt tattggtggt
720cccaataatg gagggtatga tgatgcagat ggggcctata ttcatgtacc
tcgagaccat 780ctagcttatc gatatgaggt gctgaaaatt attggcaagg
ggagttttgg gcaggtggcc 840agggtctatg atcacaaact tcgacagtac
gtggccctaa aaatggtgcg caatgagaag 900cgctttcatc gtcaagcagc
tgaggagatc cggattttgg agcatcttaa gaaacaggat 960aaaactggta
gtatgaacgt tatccacatg ctggaaagtt tcacattccg gaaccatgtt
1020tgcatggcct ttgaattgct gagcatagac ctttatgagc tgattaaaaa
aaataagttt 1080cagggtttta gcgtccagtt ggtacgcaag tttgcccagt
ccatcttgca atctttggat 1140gccctccaca aaaataagat tattcactgc
gatctgaagc cagaaaacat tctcctgaaa 1200caccacgggc gcagttcaac
caaggtcatt gactttgggt ccagctgttt cgagtaccag 1260aagctctaca
catatatcca gtctcggttc tacagagctc cagaaatcat cttaggaagc
1320cgctacagca caccaattga catatggagt tttggctgca tccttgcaga
acttttaaca 1380ggacagcctc tcttccctgg agaggatgaa ggagaccagt
tggcctgcat gatggagctt 1440ctagggatgc caccaccaaa acttctggag
caatccaaac gtgccaagta ctttattaat 1500tccaagggca taccccgcta
ctgctctgtg actacccagg cagatgggag ggttgtgctt 1560gtggggggtc
gctcacgtag gggtaaaaag cggggtcccc caggcagcaa agactggggg
1620acagcactga aagggtgtga tgactacttg tttatagagt tcttgaaaag
gtgtcttcac 1680tgggacccct ctgcccgctt gaccccagct caagcattaa
gacacccttg gattagcaag 1740tctgtcccca gacctctcac caccatagac
aaggtgtcag ggaaacgggt agttaatcct 1800gcaagtgctt tccagggatt
gggttctaag ctgcctccag ttgttggaat agccaataag 1860cttaaagcta
acttaatgtc agaaaccaat ggtagtatac ccctatgcag tgtattgcca
1920aaactgatta gctagtggac agagatatgc ccagagatgc atatgtgtat
atttttatga 1980tcttacaaac ctgcaaatgg aaaaaatgca agcccattgg
tggatgtttt tgttagagta 2040gacttttttt aaacaagaca aaacattttt
atatgattat aaaagaattc ttcaagggct 2100aattacctaa ccagcttgta
ttggccatct ggaatatgca ttaaatgact ttttataggt 2160caatgcaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaa 220781860DNAHomo sapiens
8agtgtgagct gttgaaagcc tgcagctaaa caccagtgtt acttcactcc cctttgtgga
60caccaagggg aagaagaata cggtaagctt cccacacatt agcaagaaag tcctgctgaa
120gtcatccctg ctgtatcagg agaatcaagc tcacaatcag atgccggcct
cagagctcaa 180ggcttcagaa atacctttcc accctagcat taaaacccag
gatcccaagg cagaggagaa 240gtcaccaaag aagcaaaagg tgactctgac
agcggcagag gccctaaagc tttttaagaa 300ccagctgtct ccatatgaac
aaagtgaaat cctgggctac gcggagctgt ggttcctggg 360tcttgaagcc
aagaagctcg acacggctcc tgagaaattt agcaagacga gttttgatga
420tgagcatggc ttctatctga aggttctgca tgatcacatt gcctaccgct
atgaagttct 480ggagacaatc gggaaggggt cctttggaca ggtggccaag
tgcttggatc acaaaaacaa 540tgagctggtg gccctgaaaa tcatcaggaa
caagaagagg tttcaccagc aggccctgat 600ggagctgaag atcctggaag
ctctcagaaa gaaggacaaa gacaacacct acaatgtggt 660gcatatgaag
gactttttct actttcgcaa tcacttctgc atcacctttg agctcctggg
720aatcaacttg tatgagttga tgaagaataa caactttcaa ggcttcagtc
tgtccatagt 780tcggcgcttc actctctctg ttttgaagtg cttgcagatg
ctttcggtag agaaaatcat 840tcactgtgat ctcaagcccg aaaatatagt
gctataccaa aagggccaag cctctgttaa 900agtcattgac tttggatcaa
gctgttatga acaccagaaa gtatacacgt acatccaaag 960ccggttctac
cgatccccag aagtgatcct gggccacccc tacgacgtgg ccattgacat
1020gtggagcctg ggctgcatca cggcggagtt gtacacgggc taccccctgt
tccccgggga 1080gaatgaggtg gagcagctgg cctgcatcat ggaggtgctg
ggtctgccgc cagccggctt 1140cattcagaca gcctccagga gacagacatt
ctttgattcc aaaggttttc ctaaaaatat 1200aaccaacaac agggggaaaa
aaagataccc agattccaag gacctcacga tggtgctgaa 1260aacctatgac
accagcttcc tggactttct cagaaggtgt ttggtatggg aaccttctct
1320tcgcatgacc ccggaccagg ccctcaagca tgcttggatt catcagtctc
ggaacctcaa 1380gccacagccc aggccccaga ccctgaggaa atccaattcc
tttttcccct ctgagacaag 1440gaaggacaag gttcaaggct gtcatcactc
gagcagaaaa gcagatgaga tcaccaaaga 1500gactacagag aaaacaaaag
atagccccac gaagcatgtt cagcattcag gtgatcagca 1560ggactgtctc
cagcacggag ctgacactgt tcagctgcct caactggtag acgctcccaa
1620gaagtcagag gcagctgtcg gggcggaggt gtccatgacc tccccaggac
agagcaaaaa 1680cttctccctc aagaacacaa acgttttacc ccctattgta
tgacctttgc tgagggtatg 1740tcctgctcct ttccaccagt gatttgtatt
aagacagcac ttatattgta caatacttca 1800gactgttttt tttaaataca
taaaacttta tgttaaaaaa ctctaaaaaa aaaaaaaaaa 186094743DNAHomo
sapiens 9ccgggaagga agatgaggga gacgggcccg gcgcttagca gccagagcag
cagcagcagc 60agcagcggtc gggggagggt gtttcgccgt ttcctctcag ccgccaggac
aagatggcag 120cggccgcgga gaggggctga gcccgggctg ggtggtgccg
cctgctgaag cgcctggctc 180ccggtccccg gcacggccct gcgccccacc
ccggacatgc tcagggctgc ggccgcccga 240agaggagaga gcgcgggcct
ctaggaaggt atggcctcac aagtcttggt ctacccacca 300tatgtttatc
aaactcagtc aagtgccttt tgtagtgtga agaaactcaa agtagagcca
360agcagttgtg tattccagga aagaaactat ccacggacct atgtgaatgg
tagaaacttt 420ggaaattctc atcctcccac taagggtagt gcttttcaga
caaagatacc atttaataga 480cctcgaggac acaacttttc attgcagaca
agtgctgttg ttttgaaaaa cactgcaggt 540gctacaaagg tcatagcagc
tcaggcacag caagctcacg tgcaggcacc tcagattggg 600gcgtggcgaa
acagattgca tttcctagaa ggcccccagc gatgtggatt gaagcgcaag
660agtgaggagt tggataatca tagcagcgca atgcagattg tcgatgaatt
gtccatactt 720cctgcaatgt tgcaaaccaa catgggaaat ccagtgacag
ttgtgacagc taccacagga 780tcaaaacaga attgtaccac tggagaaggt
gactatcagt tagtacagca tgaagtctta 840tgctccatga aaaatactta
cgaagtcctt gattttcttg gtcgaggcac gtttggccag 900gtagttaaat
gctggaaaag agggacaaat gaaattgtag caatcaaaat tttgaagaat
960catccttctt atgcccgtca aggtcaaata gaagtgagca tattagcaag
gctcagtact 1020gaaaatgctg atgaatataa ctttgtacga gcttatgaat
gctttcagca ccgtaaccat 1080acttgtttag tctttgagat gctggaacaa
aacttgtatg actttctgaa acaaaataaa 1140tttagtcccc tgccactaaa
agtgattcgg cccattcttc aacaagtggc cactgcactg 1200aaaaaattga
aaagtcttgg tttaattcat gctgatctca agccagagaa tattatgttg
1260gtggatcctg ttcggcagcc ttacagggtt aaagtaatag actttgggtc
ggccagtcat 1320gtatcaaaga ctgtttgttc aacatatcta caatctcggt
actacagagc tccagagatt 1380atattggggt tgccattttg tgaagccata
gacatgtggt cattgggatg tgtgattgca 1440gaattatttc ttggatggcc
gctctaccca ggagccttgg agtatgatca gattcgatac 1500atttctcaga
ctcaaggttt gccaggagaa cagttgttaa atgtgggtac taaatccaca
1560agattttttt gcaaagaaac agatatgtct cattctggtt ggagattaaa
gacattggaa 1620gagcatgagg cagagacagg aatgaagtct aaagaagcca
gaaaatacat tttcaacagt 1680ctggatgatg tagcgcatgt gaacacagtg
atggatttgg aaggaagtga tcttttggct 1740gagaaagctg atagaagaga
atttgttagt ctgttgaaga aaatgttgct gattgatgca 1800gatttaagaa
ttactccagc tgagaccctg aaccatcctt ttgttaatat gaaacatctt
1860ctagatttcc ctcatagcaa ccatgtaaag tcctgttttc atattatgga
tatttgtaag 1920tcccacctaa attcatgtga cacaaataat cacaacaaaa
cttcactttt aagaccagtt 1980gcttcaagca gtactgctac actgactgca
aattttacta aaatcggaac attaagaagt 2040caggcattga ccacatctgc
tcattcagtt gtgcaccatg gaatacctct gcaggcagga 2100actgctcagt
ttggttgtgg tgatgctttt cagcagacat tgattatctg tcccccagct
2160attcaaggta ttcctgcaac acatggtaaa cccaccagtt attcaataag
ggtagataat 2220acagttccac ttgtaactca ggccccagct gtgcagccac
tacagatccg accaggagtt 2280ctttctcaga cgtggtctgg tagaacacag
cagatgctgg tgcctgcctg gcaacaggtg 2340acacccctgg ctcctgctac
tactacacta acttctgaga gtgtggctgg ttcacacagg 2400cttggagact
gggggaagat gatttcatgc agcaatcatt ataactcagt gatgccgcag
2460cctcttctga ccaatcagat aactttatct gcccctcagc cagttagtgt
ggggattgca 2520catgttgtct ggcctcagcc tgccactacc aagaaaaata
aacagtgcca gaacagaggt 2580attttggtaa aactaatgga atgggagcca
ggaagagagg aaataaatgc tttcagttgg 2640agtaattcat tacagaatac
caatatccca cattcagcat ttatttctcc aaagataatt 2700aatgggaaag
atgtcgagga agtaagttgt atagaaacac aggacaatca gaactcagaa
2760ggagaggcaa gaaattgctg tgaaacatct atcagacagg actctgattc
atcagtttca 2820gacaaacagc ggcaaaccat cattattgcc gactccccga
gtcctgcagt gagtgtcatc 2880actatcagca gtgacactga tgaggaagag
acttcccaga gacattcact cagagaatgt 2940aaaggtagtc tagattgtga
agcttgccag agcactttga atattgatcg gatgtgttca 3000ttaagtagtc
ctgatagtac tctgagtacc agctcctcag ggcagtccag cccatccccc
3060tgcaagagac cgaatagtat gtcagatgaa gagcaagaaa gtagttgtga
tacggtggat 3120ggctctccga catctgactc ttccgggcat gacagtccat
ttgcagagag cacttttgtg 3180gaggacactc atgaaaacac agaattggta
tcctctgctg acacagaaac caagccagct 3240gtctgttctg ttgtggtgcc
accagtggaa ctagaaaatg gcttaaatgc cgatgagcat 3300atggcaaaca
cagattctat atgccagcca ttaataaaag gacgatctgc ccctggaaga
3360ttaaaccagc cttctgcagt gggtactcgt cagcaaaaat tgacatcagc
attccagcag 3420cagcatttga acttcagtca ggttcagcac tttggatctg
ggcatcaaga gtggaatgga 3480aactttgggc acagaagaca gcaagcttat
attcctacta gtgttaccag taatccattc 3540actctttctc atggaagtcc
caatcacaca gcagtgcatg cccacctggc tggaaataca 3600cacctcggag
gacagcctac tctacttcca tacccatcat cagccaccct cagtagtgct
3660gcaccagtgg cccacctgtt agcctctccg tgtacctcaa gacctatgtt
acagcatcca 3720acttataata tctcccatcc cagtggcata gttcaccaag
tcccagtggg cttaaatccc 3780cgtctgttac catccccaac cattcatcag
actcagtaca aaccaatctt cccaccacat 3840tcttacattg cagcatcacc
tgcatatact ggatttccac tgagtccaac aaaactcagc 3900cagtatccat
atatgtgaaa aacagtatat tggggaagct caatgataca aacatttgat
3960taaaaataaa aacatggtat ttaatattag ccatggcaca agaaaattat
ttttgaatca 4020tgtagacttg ggtgcaattt aaacaacttt gagctttaaa
aactcacttt tgatgtgttt 4080tgcacatttg gtataacttg tctttggtca
tgttatcttc ttatgtagta actctagaca 4140ggtgacttat gggagcagaa
gtccagtttt gctcctgcta ttttttataa attgccttct 4200aactagtgca
agacacgtct acatttggga agccattctg tgtacagact tagagcaaca
4260gatgcacata tgtcagaatt acagcataca agtgaattgt attatccgtg
tcttagtgta 4320taaatgttgg gtcacttacc taagaaattg agctattgtt
ctttacattt gcatgtgtct 4380tttgcatggg caaaatgttg cctagacttt
gctcttaaat gttgttctaa taatctcagc 4440tgcattgtaa accgttccta
cacatagtgc cttaaatatt tgaggttgtt aatgttatta 4500cctatatata
aatgttgagg actgcagcac ttaaaattca gacctactat ttagtttcct
4560tttgatagcg taatgttcat ttttgttttt gtgtggtatg atttcaggta
gtagctgttt 4620ttttccttat taagagggca gcatgtttgc tatagctgaa
ttctgctgtc tgatttttca 4680gaatgatcta gcttcaagaa aagcaagcag
ttagtagtgc ttaagaaaaa ttgattcagt 4740atc 47431024DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
10agttctgggt attccacctg ctca 241124DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
11tgaagtttac gggttcctgg tggt 241224DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
12tccaccttct agctcagctt ccaa 241324DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
13tggcaacact gtcctctgct gaat 241421DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
14gccagctcca tctccagttc t 211524DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 15cacaatatcg gttgctgtag
cggt 241624DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 16tgcaatcctt ctgaaccacc tcca 241726DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
17gctgttctac cttcatctca cctcca 261824DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
18aggctgtcat cactcgagca gaaa 241924DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
19agtcctgctg atcacctgaa tgct 242024DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
20gccgatgagc atatggcaaa caca 242124DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
21tacccactgc agaaggctgg ttta 242224DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
22acaacaacgc ccacttcttg gtgg 242319DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
23tgctcacgtc cagcacctc 192424DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 24tcttgctttc tgtagggctt tctg
242521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 25tctcaaagga gctggaagtg c 212620DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
26agcatgcaaa acagcccagg 202720DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 27acggtttctc ccagctcttc
202819DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 28tgacaggagg agagctagg 192920DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
29aagagatcct cctgccttgg 20305PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 30Val Ile Val Ile Thr 1 5
3119RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 31agguggaggu gcaauauua
193219RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 32cuuuaagccu cgagauaua 19337PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 33Ser
Pro Arg Ile Glu Ile Thr 1 5 3421PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 34Ser Pro Gln Arg Ser Arg
Ser Pro Ser Pro Gln Pro Ser Pro His Val 1 5 10 15 Ala Pro Gln Asp
Asp 20 3517PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 35Ser Pro Arg Thr Ser Pro Ile Met Ser Pro
Arg Thr Ser Leu Ala Glu 1 5 10 15 Asp 3627PRTHomo sapiens 36Leu Lys
Ala Ser Ser Arg Thr Ser Ala Leu Leu Ser Gly Phe Ala Met 1 5 10 15
Val Ala Met Val Glu Val Gln Leu Asp Ala Asp 20 25 3727PRTMus
musculus 37Leu Lys Ala Ser Ser Arg Thr Ser Ala Leu Leu Ser Gly Phe
Ala Met 1 5 10 15 Val Ala Met Val Glu Val Gln Leu Asp Thr Asp 20 25
3827PRTRattus norvegicus 38Leu Lys Ala Ser Ser Arg Thr Ser Ala Leu
Leu Ser Gly Phe Ala Met 1 5 10 15 Val Ala Met Val Glu Val Gln Leu
Asp Thr Asp 20 25 3927PRTBos taurus 39Leu Lys Ala Ser Ser Arg Thr
Ser Ala Leu Leu Ser Gly Phe Ala Met 1 5 10 15 Val Ala Met Val Glu
Val Gln Leu Asp Ala Asp 20 25 4027PRTCanis familiaris 40Leu Lys Ala
Ser Ser Arg Thr Ser Ala Leu Leu Ser Gly Phe Ala Met 1 5 10 15 Val
Ala Met Val Glu Val Gln Leu Asp Ala Asp 20 25 4127PRTCaenorhabditis
elegans 41Leu Lys Ala Ser Ser Arg Thr Ser Ala Leu Leu Ala Gly Phe
Ala Met 1 5 10 15 Val Cys Leu Val Glu Leu Gln Tyr Asp Gln Ser 20 25
4227PRTGallus gallus 42Leu Lys Ala Ser Ser Arg Thr Ser Ala Leu Leu
Ser Gly Phe Ala Met 1 5 10 15 Val Ala Met Val Glu Val Gln Leu Asp
Ala Glu 20 25 4327PRTTetraodon nigroviridis 43Leu Lys Ala Ser Ser
Arg Thr Ser Ala Leu Leu Ser Gly Phe Ala Met 1 5 10 15 Val Ala Met
Val Glu Val Gln Leu Asp Asn Thr 20 25 4427PRTDanio rerio 44Leu Lys
Ala Ser Ser Arg Thr Ser Ala Leu Leu Ser Gly Phe Ala Met 1 5 10 15
Val Ala Met Val Glu Val Gln Leu Asp Thr Asn 20 25 4527PRTXenopus
tropicalis 45Leu Lys Ala Ser Ser Arg Thr Ser Ala Leu Leu Ser Gly
Phe Ala Met 1 5 10 15 Val Ala Met Val Glu Val Gln Leu Glu Ala Asp
20 25 4627PRTDrosophila melanogaster 46Leu Lys Ala Ser Ser Lys Thr
Ser Ala Leu Leu Ser Gly Phe Ala Met 1 5 10 15 Val Ala Met Val Glu
Val Gln Leu Asp His Asp 20 25 4727PRTStrongylocentrotus purpuratus
47Leu Lys Ala Ser Ser Arg Thr Ser Ala Leu Leu Ala Gly Phe Ala Met 1
5 10 15 Val Ala Met Val Glu Val Gln Leu Ser Ala Thr 20 25
4827PRTHomo sapiens 48Leu Lys Ala Ser Ser Arg Thr Ser Ala Leu Leu
Ser Gly Phe Ala Met 1 5 10 15 Val Ala Met Val Glu Val Gln Leu Glu
Thr Gln 20 25 4927PRTHomo sapiens 49Leu Lys Ala Ser Ser Arg Thr Ser
Ala Leu Leu Ser Gly Phe Ala Met 1 5 10 15 Val Ala Met Val Glu Val
Gln Leu Glu Thr Asp 20 25 5020PRTDrosophila melanogaster 50Gly Tyr
Arg Glu Ser Pro Ala Ser Ser Gly Ser Ser Ala Ser Phe Ile 1 5 10 15
Ser Asp Thr Phe 20 5117PRTDrosophila melanogaster 51Ser Pro Arg Thr
Ser Pro Ile Met Ser Pro Arg Thr Ser Leu Ala Glu 1 5 10 15 Asp
5221PRTDrosophila melanogaster 52Ser Pro Gln Arg Ser Arg Ser Pro
Ser Pro Gln Pro Ser Pro His Val 1 5 10 15 Ala Pro Gln Asp Asp 20
539PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 53Ser Pro Arg Ile Glu Ile Thr Pro Ser 1 5
549PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 54His Pro Val Ile Val Ile Thr Gly Pro 1 5
* * * * *
References