U.S. patent application number 11/409170 was filed with the patent office on 2007-05-03 for sirt4 activities.
This patent application is currently assigned to MASSACHUSETTS INSTITUTE OF TECHNOLOGY. Invention is credited to Leonard P. Guarente, Marcia C. Haigis.
Application Number | 20070099830 11/409170 |
Document ID | / |
Family ID | 37997223 |
Filed Date | 2007-05-03 |
United States Patent
Application |
20070099830 |
Kind Code |
A1 |
Guarente; Leonard P. ; et
al. |
May 3, 2007 |
Sirt4 activities
Abstract
It has been discovered that Sirt4 possesses an
ADP-ribosyltransferase activity. Sirt4 is localized to
mitochondria, where it binds to and regulates the activity of
proteins such as glutamate dehydrogenase. The
ADP-ribosyltransferase activity of Sirt4 is important for the
regulation of biological functions such as insulin secretion.
Methods of screening for compounds that modulate the expression or
activity of Sirt4 are provided. Also provided are methods of
modulating insulin secretion, treating metabolic disorders, and
treating neurodegenerative disorders by modulating the expression
or activity of Sirt4.
Inventors: |
Guarente; Leonard P.;
(Newton, MA) ; Haigis; Marcia C.; (Stoneham,
MA) |
Correspondence
Address: |
FISH & RICHARDSON PC
P.O. BOX 1022
MINNEAPOLIS
MN
55440-1022
US
|
Assignee: |
MASSACHUSETTS INSTITUTE OF
TECHNOLOGY
|
Family ID: |
37997223 |
Appl. No.: |
11/409170 |
Filed: |
April 21, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60673565 |
Apr 21, 2005 |
|
|
|
Current U.S.
Class: |
424/94.1 ;
435/15; 514/17.8; 514/18.2; 514/44A; 514/6.7 |
Current CPC
Class: |
C12N 9/80 20130101; C12Q
1/48 20130101; C12N 15/1137 20130101; G01N 2333/91142 20130101 |
Class at
Publication: |
514/012 ;
514/044; 435/015 |
International
Class: |
A61K 48/00 20060101
A61K048/00; A61K 38/17 20060101 A61K038/17; C12Q 1/48 20060101
C12Q001/48 |
Goverment Interests
STATEMENT REGARDING FEDERALLY FUNDED RESEARCH
[0002] At least some of the work described herein was funded, in
part, through grants from the National Institute of Health. The
United States government may, therefore, have certain rights in the
invention.
Claims
1. A method of evaluating Sirt4 activity, the method comprising:
providing a cell-free composition that comprises a Sirt4 protein,
an ADP-ribosyl donor, and a substrate; and evaluating
ADP-ribosylation activity in the composition.
2. The method of claim 1, wherein the ADP-ribosyl donor comprises
NAD.
3. The method of claim 2, wherein the NAD is radio-labeled.
4. The method of claim 1, wherein the substrate comprises a
peptide.
5. The method of claim 1, wherein the substrate comprises GDH,
aldehyde dehydrogenase, or histones, or a fragment of any
thereof.
6. The method of claim 1, wherein the providing comprises combining
a preparation of the Sirt4 protein that is at least 10% pure with
the ADP-ribosyl donor and the substrate.
7. The method of claim 1, wherein the Sirt4 protein comprises a
sequence at least 85% identical to SEQ ID NO:3.
8. The method of claim 1, wherein the Sirt4 protein comprises a
sequence at least 85% identical to SEQ ID NO:1 or amino acid
residues 29-314, 29-308, 30-314, 36-308, 42-308, 42-300, or 56-314
of SEQ ID NO:1.
9. The method of claim 1, further comprising including a test
compound in the cell-free composition.
10. A method of evaluating the effect of a test compound on Sirt4,
the method comprising: a) providing a reaction mixture comprising
Sirt4 and a test compound; and b) evaluating an activity of
Sirt4.
11. The method of claim 10, wherein the activity is
ADP-ribosyltransferase activity.
12. The method of claim 10, wherein the reaction mixture comprises
NAD or an NAD analog.
13. The method of claim 10, wherein the NAD or NAD analog comprises
a radioactive label.
14. The method of claim 10, wherein the reaction mixture comprises
an ADP-ribosylation substrate selected from the group consisting
of: GDH, aldehyde dehydrogenase, and histones.
15. The method of claim 10, wherein the test compound is a small
molecule.
16. The method of claim 10, wherein the method is repeated for each
of a plurality of test compounds from a chemical library.
17. The method of claim 10, wherein the Sirt4 comprises a sequence
at least 85% identical to SEQ ID NO:3.
18. The method of claim 10, wherein the Sirt4 comprises a sequence
at least 85% identical to SEQ ID NO:1 or amino acid residues
29-314, 29-308, 30-314, 36-308, 42-308, 42-300, or 56-314 of SEQ ID
NO: 1.
19. The method of claim 10, further comprising c) comparing the
activity evaluated in (b) with the Sirt4 activity evaluated in the
absence of the test compound.
20. A method of identifying a compound that alters a
Sirt4-associated parameter in a cell, the method comprising: a)
contacting a test compound to a cell that expresses Sirt4; and b)
evaluating a Sirt4-associated parameter associated with the
cell.
21. The method of claim 20, wherein the Sirt4-associated parameter
is ADP-ribosylation activity.
22. The method of claim 20, wherein the test compound is a small
molecule.
23. The method of claim 20, wherein the method is repeated for each
of a plurality of test compounds from a chemical library.
24. The method of claim 20, further comprising c) comparing the
parameter determined in (b) with a Sirt4-associated parameter
evaluated in the absence of the test compound.
25. A method of modulating insulin secretion in response to
glucose, the method comprising modulating the expression or
activity of Sirt4 in an insulin-secreting cell.
26. The method of claim 25, wherein insulin secretion is increased
by decreasing the expression or activity of Sirt4.
27. The method of claim 25, wherein insulin secretion is decreased
by increasing the expression or activity of Sirt4.
28. The method of claim 25, wherein insulin secretion is modulated
in vitro.
29. The method of claim 25, wherein insulin secretion is modulated
in a subject.
30. The method of claim 25, wherein the modulation of Sirt4
expression or activity is effected by administering an agent that
modulates Sirt4 expression or activity.
31. A method of treating or preventing a metabolic disorder, the
method comprising administering, to a subject, an agent that
modulates the expression or activity of Sirt4, in an amount
effective to treat or prevent the metabolic disorder.
32. The method of claim 31, wherein the metabolic disorder is
diabetes, insulin resistance, metabolic syndrome, or
pre-diabetes.
33. The method of claim 31, wherein the levels of Sirt4 are
modulated in an insulin-secreting cell.
34. The method of claim 31, wherein the agent is an antagonistic
nucleic acid that reduces Sirt4 expression.
35. The method of claim 31, wherein the agent comprises an siRNA
that targets Sirt4 mRNA.
36. A method of treating or preventing a symptom of a
neurodegenerative disorder, the method comprising administering to
a subject a compound that increases the expression or activity of
Sirt4 in an amount effective to treat or prevent the
neurodegenerative disorder.
37. The method of claim 36, wherein the neurodegenerative disorder
involves accumulation of .beta.-amyloid peptide.
38. The method of claim 36, wherein the neurodegenerative disorder
is Alzheimer's disease.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. application Ser.
No. 60/673,565, filed on Apr. 21, 2005, the contents of which are
incorporated by reference in their entirety.
SUMMARY
[0003] In one aspect, the disclosure features a method of
evaluating Sirt4 activity. The method includes providing a
composition (e.g., a cell-free composition) that includes one or
more of: a Sirt4 protein, an ADP-ribosyl donor, and a substrate;
and evaluating ADP-ribosylation activity in the composition. The
ADP-ribosyl donor can be NAD or an NAD analog, e.g., a labeled
version of the donor, e.g., radio-labeled versions thereof, e.g.,
.sup.3H, .sup.14C, .sup.32P- or .sup.33P-labeled. ADP-ribosylation
activity can be evaluated by detecting a radiolabel associated with
the substrate, e.g., NAD. ADP-ribosylation activity can be
evaluated by detecting the modification of the substrate or the
ADP-ribosyl donor (e.g., NAD). The substrate can be separated from
the composition prior to evaluation of ADP-ribosylation
activity.
[0004] The substrate can include, e.g., glutamate dehydrogenase
(GDH), aldehyde dehydrogenase (ADH), adenine nucleotide transporter
(ANT) or a homolog thereof, or histones, or a fragment of any of
the above. The substrate can be from a human or other mammal, e.g.,
a mouse, rat, pig, or cow. In one embodiment, the substrate
includes a peptide, e.g., a peptide from a mitochondrial
protein.
[0005] In one embodiment, the composition includes a test
compound.
[0006] In one embodiment, the Sirt4 protein is at least 10% pure,
e.g., 10%, 20%, 30%, 40%, 50%, 75%, 80%, 90%, 95%, or 99% pure. The
Sirt4 protein can be expressed in recombinant cells and isolated
therefrom. The Sirt4 protein can be expressed in, e.g., E. coli
cells, and isolated therefrom. The concentration of the Sirt4
protein in the composition can be between 0.1 pM and 0.1 .mu.M,
e.g., between 0.1 pM and 10 pM, 10 pM and 1 nM, or 1 nM and 0.1
.mu.M.
[0007] In one embodiment, the Sirt4 protein can include the core
domain of Sirt4 or other biologically active portion of full-length
Sirt4. The Sirt4 protein can include the core domain of Sirt4, but
not all sequences of a full-length Sirt4. Alternatively, the Sirt4
protein can include a full-length Sirt4. The Sirt4 protein can
include a sequence that is at least 85% identical, e.g., 85%, 90%,
95%, 98%, 99% identical, to SEQ ID NO:3 (an exemplary fragment of
human Sirt4). The Sirt4 protein can include a sequence at least 85%
identical, e.g., 85%, 90%, 95%, 98%, 99%, or 100% identical, to SEQ
ID NO:1 (full-length human Sirt4) or can include one or more of a
sequence at least 85% identical, e.g., 85%, 90%, 95%, 98%, 99%, or
100% identical, to amino acid residues 29-314, 29-308, 30-314,
36-308, 42-308, 42-300, or 36-314 of SEQ ID NO: 1. The Sirt4
protein can be human. The Sirt4 protein can include an artificial
mutation, e.g., an alanine-scanned mutation.
[0008] In another aspect, this disclosure features a method of
evaluating Sirt4 activity in a cell. The method includes altering
Sirt4 expression in an isolated cell and evaluating
ADP-ribosylation activity associated with the cell. Sirt4
expression can be decreased, e.g., by introducing to the cell a
nucleic acid that decreases expression, e.g., an siRNA. Sirt4
expression can be increased, e.g., by introducing to the cell a
nucleic acid that encodes Sirt4 (e.g., a protein that includes a
Sirt4 core domain) operably linked to a promoter that drives
expression of Sirt4. The isolated cell can include an exogenous
nucleic acid that includes a sequence encoding a Sirt4 protein. The
isolated cell can be modified by introduction of an exogenous
promoter into an endogenous Sirt4 encoding gene. The cell can be,
e.g., a yeast cell or a mammalian cell, e.g., a pancreatic cell,
brain cell, liver cell, adipose cell, muscle cell, skin cell, or
kidney cell.
[0009] In one embodiment, the method further includes comparing the
activity evaluated in the presence of the test compound with the
Sirt4 activity evaluated in the absence of the test compound. In
one embodiment, the method further includes evaluating the test
compound in a cellular or animal model of insulin secretion,
diabetes, or a neurodegenerative disorder, e.g., Alzheimer's
disease.
[0010] In another aspect, this disclosure features a method of
evaluating the effect of a test compound on Sirt4. The method
includes providing a reaction mixture including a Sirt4 protein and
a test compound, and evaluating an activity of Sirt4. In one
embodiment, the activity of Sirt4 is an enzymatic activity, e.g.,
an ADP-ribosyltransferase activity. The reaction mixture can
include NAD or an NAD analog. The NAD or NAD analog can be
radiolabeled, e.g., with .sup.3H, .sup.14C, .sup.32P or .sup.33P.
The reaction mixture can include an ADP-ribosylation substrate,
e.g., GDH, aldehyde dehydrogenase (ADH), an adenine nucleotide
transporter (ANT), or a histone.
[0011] In another embodiment, the activity of Sirt4 is a binding
activity. The binding activity can be binding to the test compound
or binding to a Sirt4 binding partner, e.g., GDH, adenine
nucleotide transporter 1 or 2 (ANT), or insulin-degrading enzyme
(IDE). The reaction mixture can include a Sirt4 binding partner,
e.g., GDH, ANT, or IDE.
[0012] The test compound can be, e.g., a small molecule, a peptide,
a protein, or an antibody. In one embodiment, the method is
repeated for each of a plurality of test compounds from a chemical
library.
[0013] In one embodiment, the Sirt4 protein is at least 10% pure,
e.g., 10%, 20%, 30%, 40%, 50%, 75%, 80%, 90%, 95%, or 99% pure. The
Sirt4 protein can be expressed in recombinant cells and isolated
therefrom. The Sirt4 protein can be expressed in, e.g., E. coli
cells, and isolated therefrom. The concentration of the Sirt4
protein in the composition can be between 0.1 pM and 0.1 .mu.M,
e.g., between 0.1 pM and 10 pM, 10 pM and 1 nM, or 1 nM and 0.1
.mu.M.
[0014] In one embodiment, the Sirt4 protein includes the core
domain of Sirt4. The Sirt4 protein can include the core domain of
Sirt4, but not all sequences of a full-length Sirt4. Alternatively,
the Sirt4 protein can include a full-length Sirt4. The Sirt4
protein can include a sequence that is at least 85% identical,
e.g., 85%, 90%, 95%, 98%, 99% identical, to SEQ ID NO:3 (an
exemplary fragment of human Sirt4). The Sirt4 protein can include a
sequence at least 85% identical, e.g., 85%, 90%, 95%, 98%, 99%
identical, to SEQ ID NO:1 (full-length human Sirt4) or amino acid
residues 29-314, 29-308, 30-314, 36-308, 42-308, 42-300, or 36-314
of SEQ ID NO:1. The Sirt4 protein can be human. The Sirt4 protein
can include an artificial mutation, e.g., an alanine scanned
mutation. The Sirt4 protein can be a processed Sirt4 protein, e.g.,
a Sirt4 protein that lacks at least amino acids 1-14, 1-20, 1-27,
or 1-28 of SEQ ID NO:1.
[0015] In one embodiment, the method further includes comparing the
activity evaluated in the presence of the test compound with the
Sirt4 activity evaluated in the absence of the test compound. In
one embodiment, the method further includes evaluating the test
compound in a cellular or animal model of insulin secretion,
diabetes, or a neurodegenerative disorder, e.g., Alzheimer's
disease.
[0016] In another aspect, this disclosure features a method of
identifying a compound that alters a Sirt4-associated parameter in
a cell. The method includes contacting a test compound to a cell
that expresses Sirt4, and evaluating a Sirt4-associated parameter
associated with the cell. The Sirt4-associated parameter can be
expression of Sirt4, e.g., measured as levels of Sirt4 mRNA or
protein. The Sirt4-associated parameter can be ADP-ribosylation
activity, e.g., measured as ADP-ribosylation of a mitochondrial
protein, e.g., GDH. The Sirt4-associated parameter can be binding
of Sirt4 to a protein, e.g., a mitochondrial protein, e.g., GDH,
ANT, or IDE. The Sirt4-associated parameter can be the subcellular
localization of Sirt4, e.g., measured by immunofluorescence
microscopy. The Sirt4-associated parameter can be a parameter
indicative of mitochondrial function. The Sirt4-associated
parameter can be the proteolytic modification state of Sirt4, e.g.,
an N-terminal proteolytic modification. The Sirt4-associated
parameter can be a level of a primary or secondary metabolite.
[0017] The test compound can be, e.g., a small molecule, a peptide,
a protein, or an antibody. In one embodiment, the method is
repeated for each of a plurality of test compounds from a chemical
library. The cell can be, e.g., a yeast cell or a mammalian cell,
e.g., a pancreatic cell, brain cell, liver cell, adipose cell,
muscle cell, skin cell, or kidney cell. A peptide is generally a
polymer of less than 24 amino acids in length. Exemplary peptides
include peptides between 3-24, 3-20, 3-12, 3-8, or 5-12 amino acids
in length.
[0018] In one embodiment, the method further includes comparing the
parameter evaluated in the presence of the test compound with the
Sirt4-associated parameter evaluated in the absence of the test
compound. In one embodiment, the method further includes evaluating
the test compound in a cellular or animal model of insulin
secretion, diabetes, or a neurodegenerative disorder, e.g.,
Alzheimer's disease, Parkinson's disease, or Huntington's disease
or other neurological disorder.
[0019] In another aspect, this disclosure features a method of
modulating insulin secretion in response to glucose. The method
includes modulating the expression or activity of Sirt4 in an
insulin-secreting cell. Insulin secretion can be increased, e.g.,
by decreasing the expression or activity of Sirt4. Insulin
secretion can be decreased, e.g., by increasing the expression or
activity of Sirt4. Insulin secretion can be modulated in vitro,
e.g., in a cultured cell or tissue explant. Insulin secretion can
be modulated in a subject, e.g., a mammal, e.g., a human, by
administering an agent that modulates Sirt4 expression or
activity.
[0020] In another aspect, this disclosure features an isolated cell
including an RNA (e.g., a dsRNA, anti-sense RNA, or siRNA) that
inhibits the expression of Sirt4. The cell can be a pancreatic
cell, e.g., a pancreatic .beta.-cell. The cell can be an
insulin-secreting cell.
[0021] In another aspect, this disclosure features a method of
treating or preventing diabetes or a diabetes-related disorder
(e.g., pre-diabetes) by administering to a subject an agent that
decreases the expression or activity of Sirt4 in an amount
effective to treat or prevent diabetes or the diabetes-related
disorder. The agent can be an agent identified by any of the
screening methods described herein.
[0022] In another aspect, this disclosure features a method of
treating or preventing a disorder, e.g., a metabolic disorder. The
method includes administering to a subject an agent that modulates
the expression or activity of Sirt4 in an amount effective to treat
or prevent the disorder, e.g., the metabolic disorder. The
metabolic disorder can be, e.g., diabetes, insulin resistance,
metabolic syndrome (syndrome X), obesity, or pre-diabetes. The
expression or activity of Sirt4 can be modulated in an
insulin-secreting cell, e.g., a pancreatic .beta.-cell. In one
embodiment, the expression or activity of Sirt4 is decreased, thus
increasing insulin secretion. In one embodiment, the expression or
activity of Sirt4 is increased, thus decreasing insulin secretion.
The agent can be an antagonistic nucleic acid that reduces Sirt4
expression, e.g., an siRNA that targets Sirt4.
[0023] A related method includes providing a composition that
includes an agent that modulates expression or activity of Sirt4,
evaluating an aliquot of the composition (e.g., using a method
described herein), e.g., for ability of the aliquot to modulate
expression or activity of Sirt4, and administering to a subject an
agent that modulates the expression or activity of Sirt4 in an
amount effective to treat or prevent the disorder, e.g., the
metabolic disorder, or diabetes.
[0024] In another aspect, this disclosure features a method of
treating or preventing a symptom of a neurodegenerative disorder,
e.g., Alzheimer's disease, Parkinson's disease, or Huntington's
disease, or other neurological disorder. The method includes
administering to a subject a compound that increases the expression
or activity of Sirt4 in an amount effective to treat or prevent the
neurodegenerative disorder. The neurodegenerative disorder can be
one, e.g., that involves accumulation of .beta.-amyloid
peptide.
[0025] In another aspect, this disclosure features a method that
includes evaluating a Sirt4-associated parameter in a pancreatic
cell, brain cell, or other Sirt4-expressing cell. The
Sirt4-associated parameter can be an indicator of expression of
Sirt4, levels of Sirt4 mRNA or protein, or ADP-ribosylation
activity by Sirt4 protein. The Sirt4-associated parameter can be an
indicator of ADP-ribosylation of a mitochondrial protein, e.g.,
GDH. The Sirt4-associated parameter can be an indicator of binding
of Sirt4 to a Sirt4 binding partner, e.g., a mitochondrial protein,
e.g., GDH, ANT, or IDE. For example, the Sirt4 binding partner is a
human protein.
[0026] In another aspect, this disclosure features a method that
includes evaluating a Sirt4-associated parameter in mitochondria,
e.g., isolated mitochondria. The Sirt4-associated parameter can be
an indicator of levels of Sirt4 protein, or ADP-ribosylation
activity by Sirt4 protein. The Sirt4-associated parameter can be an
indicator of ADP-ribosylation of a mitochondrial protein, e.g.,
GDH. The Sirt4-associated parameter can be an indicator of binding
of Sirt4 to a Sirt4 binding partner, e.g., GDH, ANT, or IDE. The
Sirt4-associated parameter can be mitochondrial function.
[0027] In another aspect, this disclosure features a method that
includes evaluating the ADP-ribosylation state of glutamate
dehydrogenase in a pancreatic cell, brain cell, or other
Sirt4-expressing cell. The cells can include human cells.
[0028] In another aspect, this disclosure features an antibody that
binds to Sirt4, e.g., human Sirt4, and distinguishes between
mature, processed Sirt4, e.g., human Sirt4, and unprocessed Sirt4,
e.g., human Sirt4. The antibody can bind preferentially to mature,
processed Sirt4, relative to unprocessed Sirt4. For example, the
antibody binds to amino acid residues 29-32 of a Sirt4 whose
N-terminus begins at a residue corresponding to residue 29 of SEQ
ID NO:1. The antibody can bind preferentially to unprocessed Sirt4,
relative to mature, processed Sirt4. For example, the antibody
binds to an epitope within amino acid residues 1-28 of SEQ ID
NO:1.
[0029] In another aspect, this disclosure features a method that
includes processing state of Sirt4, e.g., in the N-terminal region,
e.g., to determine if one or more amino acids from amino acid
residues 1-28 of SEQ ID NO:1 are present or absent. The method can
be used to evaluate a sample that includes Sirt4. The sample can
include, or contain, e.g., a cell, a cell-free extract, or
mitochondria, e.g., isolated mitochondria. The method can include
determining the N-terminal sequence of Sirt4, the sequence of one
or more amino acids in the N-terminal 20% of the protein, or the
size of peptide fragments of Sirt4 (e.g., using mass spectroscopy
and optionally proteolysis). The method can include contacting the
sample to an antibody that binds to Sirt4, e.g., human Sirt4, and
distinguishes between mature, processed Sirt4, e.g., human Sirt4,
and unprocessed Sirt4, e.g., human Sirt4. The sample can include a
pancreatic cell, brain cell, or other Sirt4-expressing cell. The
sample can include human cells.
[0030] In another aspect, this disclosure features a method of
directing expression of a target sequence to an insulin-producing
cell. The method includes providing a pancreatic cell that contains
a nucleic acid that includes regulatory sequences from the SIRT4
gene operably linked to a target sequence for expression, wherein
the target sequence is not a sequence from the Sirt4 gene. The
target sequence can encode a protein, e.g., insulin or another
secreted protein. The target sequence can encode an anti-sense
nucleic acid.
[0031] In another aspect, this disclosure features a method of
isolating a Sirt4 protein. The method includes isolating
mitochondria from a cell that expresses a Sirt4 protein and
separating the Sirt4 from at least one other mitochondrial protein.
The Sirt4 protein can be proteolytically processed. The cell can be
a human cell.
[0032] In another aspect, this disclosure features a method of
isolating a Sirt4 protein. The method includes isolating
mitochondria from a cell that expresses a Sirt4 protein and
separating the Sirt4 from at least one other mitochondrial protein.
The Sirt4 protein can be proteolytically processed.
[0033] In another aspect, this disclosure features a kit including
a Sirt4 protein and an ADP-ribosylation substrate, e.g., GDH. The
kit can further include an ADP-ribosyl donor, e.g., NAD or an NAD
analog.
[0034] In one aspect, the invention features a method that includes
genotyping a human gene that encodes a sirtuin, e.g., SIRT4 or
another SIRT4 and recording information about the genotype in
association with information about a metabolic disorder, e.g.,
diabetes, pre-diabetes, or hyperinsulinemia, or other disorder
described herein (including, e.g., neurological disorders).
[0035] The invention also features a method that includes
genotyping a human gene that encodes a sirtuin, e.g., SIRT4 or
another SIRT4 and recording information about the genotype in
association with information about a metabolic disorder or other
disorder described herein.
[0036] In one aspect, the invention features a method that includes
a) determining the identity of at least one nucleotide in the SIRT4
locus on human chromosome 12q of a subject; and b) creating a
record which includes information about the identity of the
nucleotide and information relating to a metabolic disorder, e.g.,
diabetes, or other disorder described herein, e.g., a
disorder-related parameter of the subject, wherein the a metabolic
disorder (e.g., diabetes, or other disorder described herein) is
other than the genotype of a nucleotide in the 12q region. The
method can be used, e.g., for gathering genetic information.
[0037] In one embodiment, the determining includes evaluating a
sample including human genetic material from the subject.
[0038] Another method includes: a) evaluating a parameter of a
SIRT4 molecule from a mammalian subject; b) evaluating a parameter
associated with a metabolic disorder, e.g., diabetes, or other
disorder described herein of the subject wherein the parameter is
other than a parameter of a SIRT4 molecule; and c) recording
information about the SIRT4 parameter and information about the
parameter, wherein the information about the parameter and
information about the phenotypic trait are associated with each
other in the database. For example, the parameter is a phenotypic
trait of the subject.
[0039] In one embodiment, the SIRT4 molecule is a polypeptide and
the SIRT4 parameter includes information about a SIRT4 polypeptide.
In another embodiment, the SIRT4 molecule is a nucleic acid and the
SIRT4 parameter includes information about identity of a nucleotide
in the SIRT4 gene.
[0040] In an embodiment, the subject is an embryo, blastocyst, or
fetus. In another embodiment, the subject is a post-natal human,
e.g., a child or an adult (e.g., at least 20, 30, 40, 50, 60, 70
years of age).
[0041] In one embodiment, step b) is performed before or concurrent
with step a). In one embodiment, the human genetic material
includes DNA and/or RNA.
[0042] The method can further include comparing the SIRT4 parameter
to reference information, e.g., information about a corresponding
nucleotide from a reference sequence. For example, the reference
sequence is from a reference subject , e.g., a subject who has a
common allele, especially one who is homozygous for a common
allele. In another embodiment, the reference sequence is from a
reference subject that has a metabolic disorder, e.g., diabetes,
e.g., early or late-onset diabetes, or other disorder described
herein.
[0043] In one embodiment, the method further includes comparing the
nucleotide to a corresponding nucleotide from a genetic relative or
family member (e.g., a parent, grandparent, sibling, progeny,
prospective spouse, etc.).
[0044] In one embodiment, the method further includes evaluating
risk or determining diagnosis of a metabolic disorder, e.g.,
diabetes, or other disorder described herein in the subject as a
function of the genotype.
[0045] In one embodiment, the method further includes recording
information about the SIRT4 parameter and parameter, e.g., in a
database. For example, the information is recorded in linked fields
of a database (e.g., SIRT4 parameter is linked to at least one of:
corresponding SIRT4 parameter and/or data regarding comparison with
the reference sequence). The nucleotide can be located in an exon,
intron, or regulatory region of the SIRT4 gene. For example, the
nucleotide is a SNP. The identity of at least one SNP from Table 1
can be evaluated. In one embodiment, a plurality of nucleotides
(e.g., at least 10, 20, 50, 100, 500, or 1000 nucleotides are
evaluated (e.g., consecutive or non-consecutive)) in the SIRT4
locus are evaluated. In another embodiment, a single nucleotide is
evaluated.
[0046] In one embodiment, the method includes one or more of:
evaluating a nucleotide position in the SIRT4 locus on both
chromosomes of the subject; recording the information (e.g., as
phased or unphased information); aligning the genotyped nucleotides
of the sample and the reference sequence; and identifying
nucleotides that differ between the subject nucleotides and the
reference sequence.
[0047] The method can be repeated for a plurality of subjects
(e.g., at least 10, 25, 50, 100, 250, 500 subjects).
[0048] In one embodiment, the method can include comparing the
information of step a) and step b) to information in a database,
and evaluating the association of the genotyped nucleotide(s) with
a metabolic disorder, e.g., diabetes, or other disorder described
herein.
[0049] In another aspect, the disclosure features a protein, e.g.,
an isolated protein, that includes a Sirt4 core domain, but that
does not include all or part of the Sirt4 leader sequence. For
example, the protein can be lacking at least amino acids that
correspond to 1-14, 1-20, 1-26, 1-27, or 1-28 of SEQ ID NO:1, e.g.,
such amino acids of SEQ ID NO:1 itself. The protein may include
another amino acid or another amino acid sequence, e.g., amino
terminal to the remaining region of SIRT4. For example, there may
be a methionine immediately amino terminal to residue 29 of SEQ ID
NO:1, a tag sequence, or a heterologous leader sequence.
[0050] The protein can be recombinantly produced, e.g., from E.
coli. The protein can be provided in a preparation that is
substantially free of mitochondrial proteins. In some embodiments,
the preparation includes one, but not more than five species of
mitochondrial proteins.
[0051] An "isolated" or "purified" polypeptide or protein is
separated from at least some cellular material or other
contaminating proteins from the cell or tissue source from which
the protein is derived, or substantially free from chemical
precursors or other chemicals when chemically synthesized. An
isolated protein can be substantially free of contaminating
materials. "Substantially free" means that the protein of interest
in the preparation is at least 10% pure. In an embodiment, the
preparation of the protein has less than about 30%, 20%, 10% and
more preferably 5% (by dry weight), of a contaminating component
(e.g., a protein not of interest, chemical precursors, and so
forth). When the protein or biologically active portion thereof is
recombinantly produced, it is also preferably separated from
culture medium, e.g., culture medium represents less than about
20%, more preferably less than about 10%, and most preferably less
than about 5% of the volume of the protein preparation. Exemplary
preparations of proteins described herein include isolated or
purified preparations of at least 0.01, 0.1, 1.0, and 10 milligrams
in dry weight.
[0052] A "non-essential" amino acid residue is a residue that can
be altered from the wild-type sequence of protein without
abolishing or substantially altering activity, e.g., the activity
is at least 20%, 40%, 60%, 70%, or 80% of wild-type. An "essential"
amino acid residue is a residue that, when altered from the
wild-type sequence results in abolishing activity such that less
than 20% of the wild-type activity is present. Conserved amino acid
residues are frequently predicted to be particularly unamenable to
alteration.
[0053] A "conservative amino acid substitution" is one in which the
amino acid residue is replaced with an amino acid residue having a
similar side chain. Families of amino acid residues having similar
side chains have been defined in the art. These families include
amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine), nonpolar side
chains (e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a
predicted nonessential amino acid residue in a protein is
preferably replaced with another amino acid residue from the same
side chain family. Alternatively, in another embodiment, mutations
can be introduced randomly along all or part of a coding sequence,
such as by saturation mutagenesis, and the resultant mutants can be
screened for biological activity to identify mutants that retain
activity. Following mutagenesis, the encoded protein can be
expressed recombinantly and the activity of the protein can be
determined.
[0054] As used herein, a "biologically active portion" of a protein
includes a fragment of a protein of interest, e.g., a target
protein, which participates in an interaction, e.g., an
intramolecular or an inter-molecular interaction, e.g., a binding
or catalytic interaction. An inter-molecular interaction can be a
specific binding interaction or an enzymatic interaction (e.g., the
interaction can be transient and a covalent bond is formed or
broken). An inter-molecular interaction can be between the protein
and another protein, between the protein and another compound, or
between a first molecule and a second molecule of the protein
(e.g., a dimerization interaction). Biologically active portions of
a protein include peptides comprising amino acid sequences
sufficiently homologous to or derived from the amino acid sequence
of the protein which include fewer amino acids than the full
length, natural protein, and exhibit at least one activity of the
natural protein.
[0055] Biologically active portions can be identified by a variety
of techniques including truncation analysis, site-directed
mutagenesis, and proteolysis. Mutants or proteolytic fragments can
be assayed for activity by an appropriate biochemical or biological
(e.g., genetic) assay. In some embodiments, a biologically active
portion is folded, e.g., the portion includes one or more folded
domains, e.g., independently folded domains.
[0056] Exemplary biologically active portions can include at least
a minimal enzymatic core domain that has an active site and
detectable enzymatic activity in vitro.
[0057] Exemplary biologically active portions include between
5-100% of a protein, e.g., between 10-99, 10-95, 15-94, 15-90,
20-90, 25-80, 25-70, 25-60, 25-50, 25-40, 5-25, or 75-90% of the
protein, e.g. a target protein. Biologically active portions can
include, e.g., internal deletions, insertions (e.g., of a
heterologous sequence), terminal deletions, and substitutions
(e.g., conservative substitutions). Typically, biologically active
portions comprise a domain or motif with at least one activity of
the protein.
[0058] The terms "modulated" and "differentially regulated" include
increasing (including, for example, activation or stimulation) and
decreasing (including, for example, inhibition or suppression)
relative to a reference level.
[0059] The details of one or more embodiments are set forth in the
accompanying drawings and the description below. Other features,
objects, and advantages of the invention will be apparent from the
description and drawings, and from the claims.
DESCRIPTION OF DRAWINGS
[0060] FIG. 1. Sirt4 is an ADP-ribosyltransferase. FIG. 1A. 293T
cells were transiently transfected with pCMV-FLAG-4A.RTM.
(control), phSirt4FLAG or hSirt4. Protein expression was verified
by anti-FLAG, or anti-hSirt4 antibodies. FIG. 1B. hSirt4-FLAG or
hSirt1-FLAG was immunoprecipitated from 293T cells and dialyzed to
remove FLAG peptide. 50 ng of each protein was assayed for
deacetylase activity after a 1 hour at 37.degree. C. incubation
with the FLUOR DE LYS.TM. substrate (BIOMOL) in the absence (open
bars) or presence (black bars) of 1 mM NAD.sup.+. FIG. 1C.
ADP-ribosyltransferase activity of hSirt4-FLAG, hSirt5-FLAG or
buffer control was assessed. 50 ng of protein was incubated with
[.sup.32P]-NAD.sup.+ in the presence of core histone proteins as
the substrate. Experiments were also performed in the presence of 1
mM nicotinamide. FIG. 1D. Mass spectrometry was used to analyze
histone 2A protein that was incubated for 30 min at 37.degree. C.
in the presence of 1 mM NAD.sup.+with or without Sirt4.
[0061] FIG. 2. Sirt4 localizes to mitochondria in human and mouse
cells. FIG. 2A. HepG2 cells were co-transfected with phSirt4-GFP
(green), pDS-RED-MITO (red) or pGFP alone (FIG. 2B. green), and the
fluorescence was visualized after 48 h using a confocal microscope.
FIG. 2C. MIN6 cells were disrupted by homogenization and the
mitochondrial fraction was isolated by sucrose centrifugation.
Mitochondrial enrichment in the final sample was assessed by
anti-HSP60 antibody. Endogenous Sirt4 was detected by anti-mSirt4
antibody. FIG. 2D. N-terminal sequences for hSirt3 and hSirt4 are
shown, and their potential cleavage sites are indicated by
arrows.
[0062] FIG. 3. Sirt4 is abundant in pancreatic islets. Sections of
mouse pancreas were assessed for Sirt4 expression by anti-mSirt4
antibodies (red), and .beta.-cells were identified by using
antibodies against insulin (green). Samples were visualized at
10.times. (FIG. 3A) or 40.times. (FIG. 3B) magnification.
[0063] FIG. 4. The role of Sirt4 in insulin secretion. FIG. 4A. The
levels of Sirt4 were reduced in MIN6 cells infected with virus that
contained Sirt4 RNAi sequences compared to cells infected with
pSUPER. FIG. 4B. Insulin secretion was measured from control or
RNAi MIN6 cells. Cells were pre-incubated with KRB buffer
containing 3 mM glucose, then cells were shifted to buffer
containing either 3 mM (open bars) or 16.7 mM glucose (filled
bars). Insulin in the buffer was measured by ELISA after a 45 min
incubation at 37.degree. C. FIG. 4C. Insulin remaining inside cells
was measured at the end of the insulin secretion assays by
ELISA.
[0064] FIG. 5. Glutamate dehydrogenase is ADP-ribosylated in vivo
and interacts with Sirt4. FIG. 5A. Mitochondria (50 mg) from 293T
cells were incubated with [.sup.32P]-NAD at 37.degree. C. for 30
min. Proteins were separated by SDS-PAGE, transferred to a
nitrocellulose membrane, and radioactivity was measured by exposure
to film. FIG. 5B. Mitochondrial proteins were ADP-ribosylated as
described for FIG. 5A, and lysis was performed in NP-40 buffer
containing 10 mM DTT and 0.5 mM EDTA. Clarified lysate was
incubated with protein A resin that had been pre-incubated with
antibodies against hSirt4, ANT or glutamate dehydrogenase (GDH).
FIG. 5C. 293T cells were transiently transfected with 10 .mu.g of
pCMV (V) or phSirt4-FLAG (4). Cells were harvested and lysed in
NP-40 buffer containing protease inhibitors and 1 mM DTT. The
clarified lysate was incubated for 2 h at 4.degree. C., while
rotating, with resin that had been pre-incubated with antibodies
for Gal4, FLAG, hSirt4, or GDH. The resin was washed six times with
lysis buffer and protein complexes were subjected to western blot
analysis using antibodies against FLAG or GDH. FIG. 5D. An
endogenous interaction between Sirt4 and glutamate dehydrogenase
was determined in MIN6 cells. 5-10 cm plates of MIN6 cells were
harvested and lysed as described above. Lysates were incubated with
resin pre-incubated with antibodies for mSirt4 or Gal4. Protein
complexes were subjected to western blot analysis using antibodies
for mSirt4 or GDH. FIG. 5E. The effect of Sirt4 overexpression on
glutamate dehydrogenase ADP-ribosylation was investigated in 293T
cells that were transiently transfected with hSirt4-FLAG.
Mitochondria were labeled with [.sup.32P]-NAD, and then proteins
were immunoprecipitated using antibodies against FLAG or GDH.
Proteins were separated by SDS-PAGE, transferred to nitrocellulose
membrane, and the radioactivity was measured by exposure to x-ray
film.
[0065] FIG. 6. Glutamate dehydrogenase inhibition by Sirt4. FIG.
6A. The enzymatic activity of glutamate dehydrogenase (50 mg in 200
ml) was measured before (open bars, T=0) after a 60 min incubation
(filled bars, T=60) at 37.degree. C. with hSirt4-FLAG (50 ng) or a
buffer control in the presence of 1 mM NAD.sup.+. Experiments were
replicated 3-5 times. FIG. 6B. Inhibition of glutamate
dehydrogenase by Sirt4 requires 1 mM NAD.sup.+. Glutamate
dehydrogenase (50 mg) was incubated with Sirt4 at 37.degree. C. in
the presence (pink) or absence (blue) of I mM NAD+. Aliquots (1 ml)
were removed at T=0, 5, 15, 30, 60, or 120 min, and glutamate
dehydrogenase activity was measured for each time point. FIG. 6C.
The activity of glutamate dehydrogenase was measure in
mitochondrial lysates (30 mg) from control or Sirt4 RNAi cells.
Experiments were performed 3-5 times using cell lines from two
separate infections.
[0066] FIG. 7. Elevated glutamate dehydrogenase activity
contributes to increased insulin secretion in Sirt4 RNAi cells.
FIG. 7A. MIN6 cells were co-infected with RNAi for Sirt4 and
glutamate dehydrogenase, and protein levels were compared to
control infections. FIG. 7B. Insulin secretion assays were
performed in double-infected or control cells as described
previously. FIG. 7C. Insulin secretion assays were performed in
control or Sirt4 RNAi treated cells in the presence or absence of
BCH, an activator of glutamate dehydrogenase.
[0067] FIG. 8. ATP and respiration in Sirt4 RNAi treated cells.
FIG. 8A. Total cellular ATP was measured in 20,000 control or Sirt4
RNAi MIN6 cells. ATP was measure after incubation in 3 (open bars)
or 16.7 mM (filled bars) glucose as described for insulin secretion
assays. FIG. 8B. Oxygen consumption was measured using a Clark
electrode in control or Sirt4 RNAi treated cells that were
incubated in 16.7 mM glucose.
[0068] FIG. 9 is a graphical overview of conserved putative
transcription factor binding sites in the region 5 kb upstream of
human and mouse Sirt4 as determined using the rVISTA program. Tick
marks represent conserved binding sites. Predicted transcription
factors are indicated on the left. Percent identity between the
sequences is indicated on the bottom graph.
DETAILED DESCRIPTION
[0069] Sirt4 is a mitochondrial protein. It interacts with
glutamate dehydrogenase, insulin degrading enzyme and an adenine
nucleotide transporter, and can regulate insulin secretion. Agents
that modulate the expression or activity of Sirt4, e.g., agents
described herein or identified by the methods described herein, can
be useful in treating or preventing metabolic disorders, e.g., the
metabolic syndrome, obesity, elevated cholesterol, or diabetes
(e.g., type 1 diabetes mellitus or type 2 diabetes mellitus); and
neurodegenerative disorders, e.g., Alzheimer's disease, as well as
other disorders.
Sirt4 Proteins
[0070] The Sirt4 proteins belong to the sirtuin family, proteins
identified as sharing significant sequence identity to
Saccharomyces cerevisiae SIR2.
[0071] As used herein, the term "Sirt4" or "Sirt4 protein" refers
to proteins, e.g., eukaryotic proteins, e.g., mammalian proteins,
comprising a conserved core domain classified in the Conserved
Domain Database group cd01049 (SIRT4) (Marchler-Bauer et al. (2005)
Nucleic Acids Res. 33: D192-6), functional domains, fragments
(e.g., functional fragments), e.g., fragments of at least 8 amino
acids, e.g., at least 8, 18, 28, 64, 128, 150, 180, 200, 220, 240,
260, or 280 amino acids, and variants thereof. Exemplary Sirt4
proteins include those designated GenBank NP.sub.--036372 (human
Sirt4) and Q8R216 (mouse Sirt4). Homologs of Sirt4 proteins will
share 60%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity to a
known Sirt4 protein and, e.g., feature ADP-ribosyltransferase
activity. Eukaryotic Sirt4 proteins may be localized, e.g., to
mitochondria.
[0072] An exemplary human Sirt4 sequence (NP.sub.--036372) is as
follows: TABLE-US-00001 (SEQ ID NO:1)
MKMSFALTFRSAKGRWIANPSQPCSKASIGLFVPASPPLDPEKVKELQR
FITLSKRLLVMTGAGISTESGIPDYRSEKVGLYARTDRRPIQHGDFVRS
APIRQRYWARNFVGWPQFSSHQPNPAHWALSTWEKLGKLYWLVTQNVDA
LHTKAGSRRLTELHGCMDRVLCLDCGEQTPRGVLQERFQVLNPTWSAEA
HGLAPDGDVFLSEEQVRSFQVPTCVQCGGHLKPDVVFFGDTVNPDKVDF
VHKRVKEADSLLVVGSSLQVYSGYRFILTAWEKKLPIAILNIGPTRSDD
LACLKLNSRCGELLPLIDPC
[0073] An exemplary mouse Sirt4 sequence (Q8R216) is as follows:
TABLE-US-00002 (SEQ ID NO:2)
MSGLTFRPTKGRWITHLSRPRSCGPSGLFVPPSPPLDPEKIKELQRFIS
LSKKLLVMTGAGISTESSIPDYRSEKVGLYARTDRRPIQHIDFVRSAPV
RQRYWARNFVGWPQFSSHQPNPAHWALSNWERLGKLHWLVTQNVDALHS
KAGSQRLTELHGCMHRVLCLNCGEQTARRVLQERFQALNPSWSAEAQGV
APDGDVFLTEEQVRSFQVPCCDRCGGPLKPDVVFFGDTVNPDKVDFVHR
RVKEADSLLVVGSSLQVYSGYRFILTAREQKLPIAILNIGPTRSDDLAC
LKLDSRCGELLPLIDPRRQHSDVQRLEMNFPLSSAAQDP
[0074] Sirt4 is post-translationally processed to form mature Sirt4
by cleavage of the N-terminus after serine 28 of SEQ ID NO:1
(underlined). The N-terminus of SEQ ID NO:2 is predicted to be
similarly cleaved (underlined).
[0075] The conserved Sirt4 core domain of human Sirt4 (SEQ ID NO:3)
includes about amino acids 47-308 of SEQ ID NO:1. That of murine
Sirt4 includes about amino acids 44-305 of SEQ ID NO:2. Other
exemplary fragments of human Sirt4 include: 29-314, 29-308, 30-314,
36-308, 42-308, 42-300, and 56-314. Human and mouse Sirt4 share
about 89% sequence identity within this conserved core domain. It
is also possible to make chimeric proteins that include one or more
segments from human Sirt4 and one or more segments from mouse
Sirt4. Such chimeras, for example, will include a number of
proteins that are between 89% to 100% identical to human or mouse
Sirt4.
[0076] Some simple examples include proteins that have an
N-terminal half from one of the two species and a C-terminal half
from the other. The switch over point can be located at an amino
acid residue between about 5-10, 10-20, 20-30, 30-40, 40-50, 50-60,
60-70, 70-80, 80-90, or 90-95% of the length of human Sirt4.
[0077] A fragment of full length Sirt4 can have at least one
function of Sirt4 protein and/or be folded. Functional fragments
can, for example, have ADP-ribosyltransferase activity and/or
ability to interact with a Sirt4 binding partner, e.g., glutamate
dehydrogenase (GDH), adenine nucleotide transporter 1 or 2 (ANT),
or insulin-degrading enzyme (IDE).
[0078] Variants of Sirt4 proteins can be produced by standard
means, including site-directed and random mutagenesis. Preferably,
amino acid 61 is a threonine, amino acid 70 is a glycine, amino
acid 71 is an isoleucine, amino acid 144 is an asparagine, or amino
acid 146 is an aspartic acid.
Assays
[0079] This disclosure includes methods for evaluating Sirt4,
including Sirt4 enzymatic activity. One general category of methods
including evaluating, directly or indirectly, ADP ribosylation
activity of a Sirt4 protein. The protein can be a full length Sirt4
protein, a fragment thereof, or other variant thereof.
[0080] Detection of ADP ribosylation activity can be used to
evaluate artificial or naturally occurring variants of a Sirt4
protein. In a first exemplary implementation, a Sirt4 genomic
nucleic acid or mRNA (e.g., as cDNA) is amplified from a subject
(e.g., a human subject. Protein encoded by the nucleic acid is
evaluate for ADP ribosylation activity. In a second exemplary
implementation,. a Sirt4 gene is subjected to artificial
mutagenesis to produce varied nucleic acids. One or more Sirt4
proteins encoded by such nucleic acids are evaluated for ADP
ribosylation activity. Examples of artificial mutagenesis include
random point mutagenesis, site-directed mutagenesis (e.g., alanine
scanning), DNA shuffling, and partial exonuclease treatment.
[0081] It is also possible to evaluate Sirt4 activity in the
presence of a test compound or test condition. The method can be
used to screen a collection of compounds, e.g., a chemical library
of small molecules. The method can also be used to evaluate a
single compound, e.g., a quality control step, e.g., for a
pharmaceutical or other product.
[0082] One exemplary method includes providing a reaction mixture
that includes Sirt4 and a test compound, and evaluating an activity
of Sirt4. Another exemplary method includes contacting a test
compound to a cell that expresses Sirt4 and evaluating a Sirt4
parameter, e.g., expression or activity, in the cell. In some
embodiments, in initial rounds of screening, it is possible to use
mixtures (e.g., pools) of different compounds.
[0083] The activity of Sirt4 can be assayed, e.g., in the presence
of a compound.
[0084] Exemplary "Sirt4 activities" include ADP-ribosyltransferase
function (e.g., ability to ADP-ribosylate a substrate, e.g.,
glutamate dehydrogenase, aldehyde dehydrogenase, or a histone); and
interaction with a Sirt4 binding partner, e.g., physical
association with a Sirt4 binding partner such as glutamate
dehydrogenase (GDH), insulin degrading enzyme (IDE), or adenine
nucleotide translocase (ANT), or fragments thereof.
[0085] In one aspect, the effect of a test compound on Sirt4
activity is evaluated by providing a reaction mixture that includes
Sirt4 and the test compound, and evaluating an activity of Sirt4.
The reaction mixture can include nicotinamide adenine nucleotide
(NAD) or an NAD-like compound, e.g., comprising a radioactive
isotope, e.g., .sup.32p, .sup.33P, .sup.14C, .sup.3H. An "NAD-like
compound" refers to a compound (e.g., a synthetic or naturally
occurring chemical, drug, protein, peptide, small organic molecule)
that possesses structural similarity to component groups of NAD
(e.g., adenine, ribose and phosphate groups) or functional
similarity (e.g., oxidation of substrates, supports
ADP-ribosylation of a histone in the presence of Sir2). For
example, NAD-like compounds can be NADH, NADP, NADPH,
3-aminobenzamide or 1,3-dihydroisoquinoline (Vaziri et al. (1997)
EMBO J. 16:6018-6033), nicotinamide, iso-nicotinamide,
non-hydrolyzable NAD, biotinylated NAD, and fluorescent analogs of
NAD (e.g., 1,N6-ethenoNAD).
[0086] A parameter associated with Sirt4 can be evaluated, e.g., in
the presence or absence of a test compound. Exemplary
Sirt4-associated parameters include transcription of Sirt4 mRNA,
levels of Sirt4 mRNA, levels of Sirt4 protein, ADP-ribosylation
activity of Sirt4, levels of ADP-ribosylation of mitochondrial
proteins (e.g., glutamate dehydrogenase), activity of
Sirt4-regulated proteins (e.g., glutamate dehydrogenase activity,
measured by monitoring NADH absorption at 340 nm), binding of Sirt4
to Sirt4-binding partners (e.g., GDH, ANT, and IDE), bound vs.
unbound state of Sirt4-associated proteins, and mitochondrial
function, monitored by measuring respiration and ATP production.
Other parameters that can be evaluated include qualitative or
quantitative measures of insulin secretion, insulin levels,
.beta.-amyloid levels, .beta.-amyloid degradation activity, or
levels of other biomolecules including primary and secondary
metabolites, e.g., small molecules, carbohydrates (e.g., glucose),
peptides, lipids, or lipoproteins (e.g., low density lipoproteins,
high density lipoproteins). The primary and secondary metabolites
assayed can be endogenous, or as a result of the administration of
a compound. Sirt4-associated parameters can be evaluated singly or
in combination, e.g., to form a profile.
[0087] The assays described herein can also be adapted for other
sirtuin proteins, e.g., sirtuins other than Sirt4.
[0088] Proteins described herein can be made by recombinant or
other methods. See, e.g., techniques described in Sambrook &
Russell, Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold
Spring Harbor Laboratory, N.Y. (2001). The proteins can be
purified, e.g., using purification tags and other standard methods.
See, e.g., techniques described in Scopes (1994) Protein
Purification: Principles and Practice, New York:
Springer-Verlag.
ADP-ribosyltransferase Activity
[0089] ADP-ribosylation results in the transfer of one adenosine
diphosphate ribose group from a donor (e.g., NAD) to an amino acid
residue (e.g., cysteine, threonine) of a substrate (e.g., glutamate
dehydrogenase, aldehyde dehydrogenase, or a histone). An
ADP-ribosyltransferase is an enzyme (e.g. a protein or polypeptide)
that can catalyze an ADP-ribosylation reaction.
[0090] For example, it is possible to monitor the addition of ADP
from a [.sup.32P]-NAD to a substrate (e.g., glutamate
dehydrogenase, aldehyde dehydrogenase, or histones). In one
embodiment, the ADP-ribosyltransferase activity is evaluated by
providing a protein to be evaluated (about 0-1 .mu.g) (e.g., a
Sirt4 protein) to a reaction buffer comprising, e.g., 50 mM
Tris-HCl, pH 8.0, 4 mM MgCl.sub.2, 0.2 mM DTT, 1 .mu.M cold or
nonradiolabeled NAD, 0.08 .mu.M [.sup.32P]-NAD, and admixing or
gently vortexing to dilute, resuspend, or mix the protein.
Substrates (e.g., glutamate dehydrogenase, aldehyde dehydrogenase,
or histones, about 0-1 .mu.g) are then added, and the reaction
mixture is incubated at ambient temperature (18-25.degree. C.) for
30-120 minutes. The presence or absence of ADP-ribosylation
products (e.g., ADP-ribosylated proteins) is detected, e.g., using
autoradiography. The amount of ADP-ribosylation, for example, in
the presence and absence of an agent to be tested, can be
determined using suitable techniques, including, but not limited
to, densitometric scanning of autoradiographs or phosphoimaging
techniques of gels. (See Ausubel et al., Current Protocols in
Molecular Biology, John Wiley & Sons (1999).)
[0091] Confirmation of ADP-ribosylation of substrates and, thus,
ADP-ribosyltransferase activity of an agent can be performed, for
example, by adding a suitable amount of snake venom
phosphodiesterase (e.g., 2 mg/ml, specific activity 1.5 U/mg) to
the resulting product of the reaction mixture described above. The
reaction product and phosphodiesterase are incubated at about
37.degree. C. for about an hour. Absence of an autoradiographic
band following phosphodiesterase digestion, as compared with
presence of an autoradiographic band in the absence of digestion,
indicates that the substrate was ADP-ribosylated.
ADP-ribosyltransferase activity can also be verifies by the
addition of one or more specific ADP-ribosylation inhibitors,
including, but not limited to, novobiocin and coumermycin Al, to in
vitro assays described above. The inhibitor(s) can be added before
or after the addition of the substrate. The absence of a band in an
autoradiograph following the addition of a specific
ADP-ribosylation inhibitor indicates that the agent has
ADP-ribosyltransferase activity.
[0092] Exemplary substrates include, e.g., glutamate dehydrogenase
(GDH), aldehyde dehydrogenase (ADH), adenine nucleotide transporter
(ANT) and its homologs, e.g., mitochondrial carrier proteins, or
histones, or a fragment of any of the above. Substrates can be
obtained from any species of organism, including human, murine, or
other mammal, other animals (e.g., nematode, fruit fly), or other
organism, e.g., yeast. In one embodiment, the substrate is obtained
from the same species as the SIRT4 protein.
[0093] The fragment can be conserved, e.g., at or near the ADP
ribosylation site. Exemplary fragments of human GDH include: a
fragment from a region of about 1-50, 50-100, 100-150, 150-200,
250-300, 300-350, 350-400, 400-450, 450-500, or 500-550.
Interaction Assays
[0094] In some embodiments, interaction with (e.g., binding to)
Sirt4 can be assayed, e.g., in vitro or in a cell. The reaction
mixture can include, e.g., a co-factor such as NAD and/or a NAD
analog, a substrate or other binding partner or potentially
interacting fragment thereof. Exemplary binding partners include
GDH, IDE, ANT, or interacting fragments thereof. Preferably the
binding partner is a direct binding partner.
[0095] In other embodiments, the reaction mixture can include a
Sirt4 binding partner, and compounds can be screened, e.g., in an
in vitro assay, to evaluate the ability of a test compound to
modulate interaction between a Sirt4 and a Sirt4 binding partner.
This type of assay can be accomplished, for example, by coupling
one of the components with a radioisotope or enzymatic label such
that binding of the labeled component to the other can be
determined by detecting the labeled compound in a complex. A
component can be labeled with .sup.125I, .sup.35S, .sup.33P,
.sup.32P, .sup.14C, or .sup.3H, either directly or indirectly, and
the radioisotope detected by direct counting of radioemmission or
by scintillation counting. Alternatively, a component 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. Competition assays can also be used to evaluate a
physical interaction between a test compound and a target.
[0096] Cell-free assays involve preparing a reaction mixture of the
target protein (e.g., Sirt4) 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.
[0097] The interaction between two molecules can also be detected,
e.g., using a fluorescence assay in which at least one molecule is
fluorescently labeled. One example of such an assay includes
fluorescence energy transfer (FET or FRET for fluorescence
resonance energy transfer) (see, for example, U.S. Pat. No.
5,631,169; U.S. Pat. No. 4,868,103). A fluorophore label on the
first, `donor` molecule is selected such that its emitted
fluorescent energy will be absorbed by a fluorescent label on a
second, `acceptor` molecule, which in turn is able to fluoresce due
to the absorbed 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 should be maximal. A FET binding event
can be conveniently measured through standard fluorometric
detection means well known in the art (e.g., using a
fluorimeter).
[0098] Another example of a fluorescence assay is fluorescence
polarization (FP). For FP, only one component needs to be labeled.
A binding interaction is detected by a change in molecular size of
the labeled component. The size change alters the tumbling rate of
the component in solution and is detected as a change in FP. See,
e.g., Nasir et al. (1999) Comb Chem HTS 2:177-190; Jameson et al.
(1995) Methods Enzymol 246:283; Seethala et al. (1998) Anal
Biochem. 255:257. Fluorescence polarization can be monitored in
multiwell plates, e.g., using the POLARION.TM. reader (Tecan,
Maennedorf, Switzerland). See, e.g., Parker et al. (2000) J
Biomolecular Screening 5:77-88; and Shoeman, et al. (1999) Biochem
38:16802-16809.
[0099] In another embodiment, evaluating binding of a Sirt4 protein
to a compound can include a real-time monitoring of the binding
interaction, e.g., using 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 which can be used as an indication of real-time
reactions between biological molecules.
[0100] In one embodiment, Sirt4 protein is anchored onto a solid
phase. The Sirt4/test compound complexes anchored on the solid
phase can be detected at the end of the reaction, e.g., the binding
reaction. For example, Sirt4 protein 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.
[0101] It may be desirable to immobilize either Sirt4 protein or a
Sirt4 binding partner to facilitate separation of complexed from
uncomplexed forms of one or both of the proteins, as well as to
accommodate automation of the assay. Binding of a test compound to
Sirt4, or interaction of Sirt4 with a second component 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/mammalian homolog of a SIR2 fusion
proteins or glutathione-S-transferase/target fusion proteins can be
adsorbed onto glutathione SEPHAROSE.RTM. 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 Sirt4, and the mixture
incubated under conditions conducive to 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 Sirt4 binding or activity
determined using standard techniques.
[0102] Other techniques for immobilizing either Sirt4 or a target
molecule on matrices include using conjugation of biotin and
streptavidin. Biotinylated Sirt4 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 Chemical).
[0103] In order 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).
[0104] In one embodiment, this assay is performed utilizing
antibodies reactive with Sirt4 or target molecules, but which do
not interfere with binding of Sirt4 to its target molecule. Such
antibodies can be derivatized to the wells of the plate, and
unbound target or Sirt4 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 Sirt4
or the target molecule, as well as enzyme-linked assays which rely
on detecting an enzymatic activity associated with Sirt4 or the
target molecule.
[0105] 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: differential centrifugation (see, for
example, Rivas, G., and Minton, A. P., (1993) Trends Biochem Sci
18:284-7); chromatography (gel filtration chromatography,
ion-exchange chromatography); electrophoresis (see, e.g., Ausubel,
F. et al., eds. Current Protocols in Molecular Biology 1999, J.
Wiley: New York.); and immunoprecipitation (see, for example,
Ausubel, F. et al., eds. (1999) Current Protocols in Molecular
Biology, J. Wiley: New York). Such resins and chromatographic
techniques are known to one skilled in the art (see, e.g.,
Heegaard, N. H., (1998) J Mol Recognit 11:141-8; Hage, D. S., and
Tweed, S. A. (1997) J Chromatogr B Biomed Sci Appl. 699:499-525).
Further, fluorescence energy transfer may also be conveniently
utilized, as described herein, to detect binding without further
purification of the complex from solution.
[0106] In a preferred embodiment, the assay includes contacting
Sirt4 or a biologically active portion thereof with a known
compound which binds Sirt4 to form an assay mixture, contacting the
assay mixture with a test compound, and determining the ability of
the test compound to interact with Sirt4, wherein determining the
ability of the test compound to interact with Sirt4 includes
determining the ability of the test compound to preferentially bind
to Sirt4 or a biologically active portion thereof, or to modulate
the activity of a target molecule, as compared to the known
compound.
[0107] Sirt4 can, in vivo, interact with one or more cellular
macromolecules, such as proteins. Such cellular macromolecules are
referred to herein as "Sirt4 binding partners." Exemplary Sirt4
binding partners include GDH, ANT, and IDE. Compounds that disrupt
such interactions can be useful in regulating the activity of
Sirt4. Such compounds can include, but are not limited to molecules
such as antibodies, peptides, and small molecules. Compounds that
disrupt binding can themselves interact with Sirt4 protein or with
a Sirt4 binding partner.
[0108] To identify compounds that modulate (e.g., interfere with)
the interaction between the target product and its binding
partner(s), for example, a reaction mixture containing the target
product and the binding partner is prepared, under conditions and
for a time sufficient, to allow the two products to form complex.
In order to test an inhibitory agent, the reaction mixture is
provided in the presence and absence of the test compound. The test
compound can be initially included in the reaction mixture, or can
be added at a time subsequent to the addition of the target and its
cellular or extracellular binding partner. Control reaction
mixtures are incubated without the test compound or with a placebo.
The formation of any complexes between the target product and the
cellular or extracellular binding partner is then detected. The
formation of a complex in the control reaction, but not in the
reaction mixture containing the test compound, indicates that the
compound interferes with the interaction of the target product and
the interactive binding partner. Additionally, complex formation
within reaction mixtures containing the test compound and normal
target product can also be compared to complex formation within
reaction mixtures containing the test compound and mutant target
product. This comparison can be important in those cases wherein it
is desirable to identify compounds that disrupt interactions of
mutant but not normal target products.
[0109] These assays can be conducted in a heterogeneous or
homogeneous format. Heterogeneous assays involve anchoring either
the target product or the binding partner onto a solid phase, and
detecting complexes anchored on the solid phase at the end of the
reaction. In homogeneous assays, the entire reaction is carried out
in a liquid phase. In either approach, the order of addition of
reactants can be varied to obtain different information about the
compounds being tested. For example, test compounds that interfere
with the interaction between the target products and the binding
partners, e.g., by competition, can be identified by conducting the
reaction in the presence of the test substance. Alternatively, test
compounds that disrupt preformed complexes, e.g., compounds with
higher binding constants that displace one of the components from
the complex, can be tested by adding the test compound to the
reaction mixture after complexes have been formed. The various
formats are briefly described below.
[0110] In a heterogeneous assay system, either the target product
or the partner, is anchored onto a solid surface (e.g., a
microtiter plate), while the non-anchored species is labeled,
either directly or indirectly. The anchored species can be
immobilized by non-covalent or covalent attachments. Alternatively,
an immobilized antibody specific for the species to be anchored can
be used to anchor the species to the solid surface.
[0111] In order to conduct the assay, the partner of the
immobilized species is exposed to the coated surface with or
without the test compound. After the reaction is complete,
unreacted components are removed (e.g., by washing) and any
complexes formed will remain immobilized on the solid surface.
Where the non-immobilized species is pre-labeled, the detection of
label immobilized on the surface indicates that complexes were
formed. Where the non-immobilized species 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 initially
non-immobilized species (the antibody, in turn, can be directly
labeled or indirectly labeled with, e.g., a labeled anti-Ig
antibody). Depending upon the order of addition of reaction
components, test compounds that inhibit complex formation or that
disrupt preformed complexes can be detected.
[0112] Alternatively, the reaction can be conducted in a liquid
phase in the presence or absence of the test compound, the reaction
products separated from unreacted components, and complexes
detected; e.g., using an immobilized antibody specific for one of
the binding components to anchor any complexes formed in solution,
and a labeled antibody specific for the other partner to detect
anchored complexes. Again, depending upon the order of addition of
reactants to the liquid phase, test compounds that inhibit complex
or that disrupt preformed complexes can be identified.
[0113] In an alternate embodiment, a homogeneous assay can be used.
For example, a preformed complex of the target product and the
interactive cellular or extracellular binding partner product is
prepared in that either the target 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 product-binding partner interaction
can be identified.
[0114] In yet another aspect, Sirt4 can be used as "bait proteins"
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 Sirt4 binding
proteins that may be involved in Sirt4 activity. In one embodiment,
the two-hybrid assay is used to monitor an interaction between two
components, e.g., Sirt4 and, e.g., GDH, ANT, IDE, or fragments
thereof. The two hybrid assay can also be conducted in the presence
of a test compound, and the assay is used to determine whether the
test compound enhances or diminishes the interaction between the
components.
Cell-Based Assays
[0115] Cell-based assays can be used to evaluate compounds for
their ability to interact with Sirt4 protein, e.g., bind or
modulate the enzymatic activity of a Sirt4 protein. Useful assays
include assays in which a Sirt4-associated parameter is evaluated.
Other parameters that can be evaluated include parameters that
assess insulin production or secretion.
[0116] In addition, it is possible to evaluate the modification
state of a substrate in a Sirt4-expressing cell. For example, one
can evaluate the ADP-ribosylation state of a substrate, such as
glutamate dehydrogenase (GDH), aldehyde dehydrogenase (ADH), or
histones, or a fragment of any of the above, or a Sirt4 binding
partner. Optionally, the substrate can be immunoprecipitated from
an extract made from the Sirt4 expressing cell (e.g., contacted or
not contacted with a test compound). The precipitated substrate can
then be evaluated. In another variation, the modified form of the
substrate is detected using a reagent that discriminates between
the modified and unmodified form. For example, the reagent is an
antibody that specifically recognizes the ADP-ribosylated form.
[0117] Another exemplary cell based assay can include contacting a
cell expressing a Sirt4 protein with a test compound and
determining the ability of the test compound to modulate (e.g.
stimulate or inhibit) an activity of a Sirt4 protein, and/or
determine the ability of the test compound to modulate expression
of Sirt4, e.g., by detecting Sirt4 nucleic acids (e.g., mRNA or
cDNA) or proteins in the cell. Determining the ability of the test
compound to modulate Sirt4 activity can be accomplished, for
example, by determining the ability of a Sirt4 protein or nucleic
acid to bind to or interact with a substrate (e.g., as described
above), to bind or interact with the test molecule, and by
determining the ability of the test molecule to modulate a
parameter, e.g., insulin secretion, insulin levels, or
.beta.-amyloid peptide accumulation.
[0118] Cell-based systems can be used to identify compounds that
decrease expression and/or activity and/or effect of Sirt4. Such
cells can be recombinant or non-recombinant, such as cell lines
that express SIRT4 gene. In some embodiments, the cells can be
recombinant or non-recombinant cells which express a Sirt4 binding
partner. Exemplary systems include mammalian or yeast cells that
express Sirt4, e.g., from a recombinant nucleic acid. In utilizing
such systems, cells are exposed to compounds suspected of
increasing expression and/or activity of Sirt4. After exposure, the
cells are assayed, for example, for Sirt4 expression or
activity.
[0119] Alternatively, the cells may also be assayed for the
activation or inhibition of the ADP-ribosylation function of Sirt4,
or modulation of insulin secretion or .beta.-amyloid peptide
accumulation. In one embodiment, the levels of ADP-ribosylation of
a mitochondrial protein, e.g., glutamate dehydrogenase, are
evaluated, e.g., in isolated mitochondria. In another embodiment,
secreted insulin or .beta.-amyloid peptide can be measured
directly, e.g., with an immunoglobulin, e.g., by ELISA. The cells
can also be assayed for ATP levels or ATP/ADP ratio. ATP and ADP in
sample extracts can be measured using chromatographic methods, as
described herein. ATP levels can also be measured by using cells
transfected with a reporter gene, such as a luciferase expression
construct designed to emit a luminescence signal that is directly
correlated to ATP concentration (Kohler et al. (1998) FEBS Lett 441
:97-102 and Kennedy et al. (1999) J Biol Chem 274:13281-91). ATP
and ADP can also be measured, e.g., using enzymatic methods, e.g.,
using the ENLITEN.RTM. ATP Assay System (Promega, Madison, Wis.) or
see Adra et al. (1987) Gene 60:65-74, U.S. Pat. No. 4,923,796.
[0120] A cell-based assay can be performed using a single cell, or
a collection of at least two or more cells. The cell can be a yeast
cell (e.g., Saccharomyces cerevisiae) or a mammalian cell,
including but not limited to somatic or embryonic cells (e.g.,
pancreatic or brain cells), HepG2 cells, MIN6 cells, INS-1 cells,
Chinese hamster ovary cells, HeLa cells, human 293 cells, and
monkey COS-7 cells. The collection of cells can form a tissue. A
"tissue" refers to a collection of similar cell types (such as
epithelium, connective, muscle, and nerve tissue).
[0121] In another embodiment, modulators of Sirt4 gene expression
are identified. For example, a cell or cell free mixture is
contacted with a candidate compound and the expression of Sirt4
mRNA or protein evaluated relative to the level of expression of
Sirt4 mRNA or protein in the absence of the candidate compound.
When expression of the Sirt4 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 Sirt4 mRNA or
protein expression. Alternatively, when expression of Sirt4 mRNA or
protein is less (statistically significantly less) in the presence
of the candidate compound than in its absence, the candidate
compound is identified as an inhibitor of the Sirt4 mRNA or protein
expression. The level of Sirt4 mRNA or protein expression can be
determined by methods for detecting Sirt4 mRNA or protein, e.g.,
using probes or antibodies, e.g., labeled probes or antibodies.
[0122] In addition to cell-based and in vitro assay systems,
non-human organisms, e.g., transgenic non-human organisms or a
model organism, can also be used. A transgenic organism is one in
which a heterologous DNA sequence is chromosomally integrated into
the germ cells of the animal. A transgenic organism will also have
the transgene integrated into the chromosomes of its somatic cells.
Organisms of any species, including, but not limited to: yeast,
worms, flies, fish, reptiles, birds, mammals (e.g., mice, rats,
rabbits, guinea pigs, pigs, micro-pigs, and goats), and non-human
primates (e.g., baboons, monkeys, chimpanzees) may be used in the
methods described herein.
[0123] A transgenic cell or animal used in the methods disclosed
herein can include a transgene that encodes, e.g., Sirt4. The
transgene can encode a protein that is normally exogenous to the
transgenic cell or animal, including a human protein, e.g., human
Sirt4. The transgene can be linked to a heterologous or a native
promoter. A transgenic animal can also be produced with reduced
expression or activity of, e.g., Sirt4, e.g., a Sirt4 deletion or
mutant. Methods of making transgenic cells and animals are known in
the art.
[0124] Accordingly, in another embodiment, this disclosure features
a method of identifying a compound as a candidate of treatment of a
metabolic disorder, e.g., a disorder characterized by an
insufficiency or excess of insulin, e.g., type 1 diabetes, type 2
diabetes, or hyperinsulinemia. The method includes: providing a
compound which interacts with, e.g., binds to, Sirt4; evaluating
the effect of the compound on insulin secretion; and further
evaluating the effect of the test compound on a subject, e.g., an
animal model, e.g., an animal model for a metabolic disorder, e.g.,
type 1 diabetes or type 2 diabetes. Exemplary animal models are
described below. The interaction between a test compound and Sirt4
can be evaluated by any of the methods described herein, e.g.,
using cell-based assays or cell-free in vitro assays.
[0125] Accordingly, in another embodiment, this disclosure features
a method of identifying a compound as a candidate of treatment of a
.beta.-amyloid disorder, e.g., a disorder characterized by
.beta.-amyloid accumulation, e.g., Alzheimer's disease. The method
includes: providing a compound which interacts with, e.g., binds
to, Sirt4; evaluating the effect of the compound on .beta.-amyloid
accumulation; and further evaluating the effect of the test
compound on a subject, e.g., an animal model, e.g., an animal model
for a .beta.-amyloid disorder, e.g., Alzheimer's disease. Exemplary
animal models are described below. The interaction between a test
compound and Sirt4 can be evaluated by any of the methods described
herein, e.g., using cell-based assays or cell-free in vitro
assays.
Test Compounds
[0126] A "compound" or "test compound" can be any chemical
compound, for example, a macromolecule (e.g., a polypeptide, a
protein complex, or a nucleic acid) or a small molecule (e.g., an
amino acid, a nucleotide, an organic or inorganic compound). The
test compound can have a formula weight of less than about 10 000
grams per mole, less than 5 000 grams per mole, less than 1 000
grams per mole, or less than about 500 grams per mole. The test
compound can be naturally occurring (e.g., a herb or a nature
product), synthetic, or both. Examples of macromolecules are
proteins, protein complexes, and glycoproteins, nucleic acids,
e.g., DNA, RNA (e.g., double stranded RNA or RNAi) and PNA (peptide
nucleic acid). Examples of small molecules are peptides,
peptidomimetics (e.g., peptoids), amino acids, amino acid analogs,
polynucleotides, polynucleotide analogs, nucleotides, nucleotide
analogs, nucleosides, glycosidic compounds, organic or inorganic
compounds e.g., heteroorganic or organometallic compounds. One
exemplary type of protein compound is an antibody or a modified
scaffold domain protein. A test compound can be the only substance
assayed by the method described herein. Alternatively, a collection
of test compounds can be assayed either consecutively or
concurrently by the methods described herein.
[0127] In one preferred embodiment, high throughput screening
methods involve providing a combinatorial chemical or peptide
library containing a large number of potential therapeutic
compounds (potential modulator or ligand compounds). Such
"combinatorial chemical libraries" or "ligand libraries" are then
screened in one or more assays, as described herein, to identify
those library members (particular chemical species or subclasses)
that display a desired characteristic activity. The compounds thus
identified can serve as conventional "lead compounds" or can
themselves be used as potential or actual therapeutics.
[0128] A combinatorial chemical library is a collection of diverse
chemical compounds generated by either chemical synthesis or
biological synthesis, by combining a number of chemical "building
blocks" such as reagents. For example, a linear combinatorial
chemical library such as a polypeptide library is formed by
combining a set of chemical building blocks (amino acids) in every
possible way for a given compound length (i.e., the number of amino
acids in a polypeptide compound). Millions of chemical compounds
can be synthesized through such combinatorial mixing of chemical
building blocks.
[0129] Preparation and screening of combinatorial chemical
libraries is well known to those of skill in the art. Such
combinatorial chemical libraries include, but are not limited to,
peptide libraries (see, e.g., U.S. Pat. 5,010,175; Furka, Int. J.
Pept. Prot. Res. 37:487-493 (1991) and Houghton et al., Nature
354:84-88 (1991)). Other chemistries for generating chemical
diversity libraries can also be used. Such chemistries include, but
are not limited to: peptoids (e.g., PCT Publication No. WO
91/19735), encoded peptides (e.g., PCT Publication No. WO
93/20242), random bio-oligomers (e.g., PCT Publication No. WO
92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514),
diversomers such as hydantoins, benzodiazepines and dipeptides
(Hobbs et al., Proc. Nat. Acad. Sci. USA 90:6909-6913 (1993)),
vinylogous polypeptides (Hagihara et al., J Amer. Chem. Soc.
114:6568 (1992)), nonpeptidal peptidomimetics with glucose
scaffolding (Hirschmann et al., J Amer. Chem. Soc. 114:9217-9218
(1992)), analogous organic syntheses of small compound libraries
(Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)), oligocarbamates
(Cho et al., Science 261:1303 (1993)), and/or peptidyl phosphonates
(Campbell et al., J Org. Chem. 59:658 (1994)), nucleic acid
libraries (see Ausubel, Berger and Sambrook, all supra), peptide
nucleic acid libraries (see, e.g., U.S. Pat. 5,539,083), antibody
libraries (see, e.g., Vaughn et al., Nature Biotechnology,
14(3):309-314 (1996) and PCT/US96/10287), carbohydrate libraries
(see, e.g., Liang et al., Science, 274:1520-1522 (1996) and U.S.
Pat. No. 5,593,853), small organic molecule libraries (see, e.g.,
benzodiazepines, Baum C&EN, Jan 18, page 33 (1993);
isoprenoids, U.S. Pat. No. 5,569,588; thiazolidinones and
metathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat.
Nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No.
5,506,337; benzodiazepines, U.S. Pat. No. 5,288,514, and the like).
Additional 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. U.S.A. 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.
[0130] Some exemplary libraries are used to generate variants from
a particular lead compound. One method includes generating a
combinatorial library in which one or more functional groups of the
lead compound are varied, e.g., by derivatization. Thus, the
combinatorial library can include a class of compounds which have a
common structural feature (e.g., framework).
[0131] Devices for the preparation of combinatorial libraries are
commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem
Tech, Louisville Ky.; SYMPHONY.TM., Rainin, Woburn, Mass.; 433A
Applied Biosystems, Foster City, Calif.; 9050 Plus, Millipore,
Bedford, Mass.). In addition, numerous combinatorial libraries are
themselves commercially available (see, e.g., ComGenex, Princeton,
N.J.; Asinex, Moscow, RU, Tripos, Inc., St. Louis, Mo.; ChemStar,
Ltd, Moscow, RU; 3D Pharmaceuticals, Exton, Pa.; Martek
Biosciences, Columbia, Md.; etc.).
[0132] Test compounds can also be obtained from: 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 which nevertheless
remain bioactive; see, e.g., Zuckermann, R. N. 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 libraries include libraries of nucleic acids and
libraries of proteins. Some nucleic acid libraries encode a diverse
set of proteins (e.g., natural and artificial proteins; others
provide, for example, functional RNA and DNA molecules such as
nucleic acid aptamers or ribozymes. A peptoid library can be made
to include structures similar to a peptide library. (See also Lam
(1997) Anticancer Drug Des. 12:145). A library of proteins may be
produced by an expression library or a display library (e.g., a
phage display library).
[0133] 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).
Antibodies
[0134] Immunoglobulins can be produced that bind to Sirt4 or a
Sirt4 binding partner (e.g., a protein that interacts with Sirt4).
For example, an immunoglobulin can bind to Sirt4 and prevent Sirt4
enzymatic activity or an interaction between Sirt4 and a Sirt4
binding partner (e.g., GDH, IDE, or ANT). In one embodiment, the
immunoglobulin is human, humanized, deimmunized, or otherwise
non-antigenic in the subject.
[0135] In one embodiment, an immunoglobulin can be produced that
can distinguish between mature, processed Sirt4 and unprocessed
Sirt4, e.g., an antibody that binds preferentially to one form
relative to the other. For example, an antibody that binds
preferentially to the mature processed form can be an antibody that
binds to amino acid residues 29-32 of a Sirt4 whose N-terminus
begins at a residue corresponding to residue 29 of SEQ ID NO:1. An
antibody that binds preferentially to an unprocessed Sirt4 can be
an antibody that binds to amino acid residues 1-28 of SEQ ID
NO:1.
[0136] Antibodies that bind specifically to mono-ADP-ribose can be
utilized to distinguish ADP-ribosylated substrates form
non-ADP-ribosylated substrates (see, e.g., Meyer and Hilz (1986)
Eur JBiochem 155:157-65).
[0137] An immunoglobulin can be, for example, an antibody or an
antigen-binding fragment thereof. As used herein, the term
"immunoglobulin" refers to a protein consisting of one or more
polypeptides that include one or more immunoglobulin variable
domain sequences. A typical immunoglobulin includes at least a
heavy chain immunoglobulin variable domain and a light chain
immunoglobulin variable domain. An immunoglobulin protein can be
encoded by immunoglobulin genes. The recognized human
immunoglobulin genes include the kappa, lambda, alpha (IgA1 and
IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon and mu
constant region genes, as well as the myriad immunoglobulin
variable region genes. Full-length immunoglobulin "light chains"
(about 25 kDa or 214 amino acids) are encoded by a variable region
gene at the NH2-terminus (about 110 amino acids) and a kappa or
lambda constant region gene at the COOH-terminus. Full-length
immunoglobulin "heavy chains" (about 50 kDa or 446 amino acids),
are similarly encoded by a variable region gene (about 116 amino
acids) and one of the other aforementioned constant region genes,
e.g., gamma (encoding about 330 amino acids). The term
"antigen-binding fragment" of an antibody (or simply "antibody
portion" or "fragment"), as used herein, refers to one or more
fragments of a full-length antibody that retain the ability to
specifically bind to the antigen. Examples of antigen-binding
fragments include: (i) a Fab fragment, a monovalent fragment
consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2
fragment, a bivalent fragment comprising two Fab fragments linked
by a disulfide bridge at the hinge region; (iii) a Fd fragment
consisting of the VH and CH1 domains; (iv) a Fv fragment consisting
of the VL and VH domains of a single arm of an antibody, (v) a dAb
fragment (Ward et al., (1989) Nature 341:544-546), which consists
of a VH domain; and (vi) an isolated complementarity determining
region (CDR). Furthermore, although the two domains of the Fv
fragment, VL and VH, are coded for by separate genes, they can be
joined, using recombinant methods, by a synthetic linker that
enables them to be made as a single protein chain in which the VL
and VH regions pair to form monovalent molecules (known as single
chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426;
and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).
Such single chain antibodies are also encompassed within the term
"antigen-binding fragment" of an antibody. These antibody fragments
are obtained using conventional techniques, and the fragments are
screened for utility in the same manner as are intact
antibodies.
[0138] In one embodiment, the antibody against Sirt4 or another
protein is a fully human antibody (e.g., an antibody made in a
mouse which has been genetically engineered to produce an antibody
from a human immunoglobulin sequence), or a non-human antibody,
e.g., a rodent (mouse or rat), goat, primate (e.g., monkey).
Preferably, the non-human antibody is a rodent (mouse or rat
antibody). Method of producing rodent antibodies are known in the
art. Non-human antibodies can be modified, e.g., humanized or
deimmunized. Human monoclonal antibodies can be generated using
transgenic mice carrying the human immunoglobulin genes rather than
the mouse system (see, e.g., WO 91/00906 and WO 92/03918). Other
methods for generating immunoglobulin ligands include phage display
(e.g., as described in U.S. Pat. No. 5,223,409 and WO
92/20791).
Sirt4 Modulating Nucleic Acids
[0139] Nucleic acid molecules (e.g., DNA or RNA molecules) can be
used to modulate (e.g., increase or decrease) Sirt4 expression or
activity.
[0140] A Sirt4 modulator can be a nucleic acid molecule designed to
increase expression or activity of Sirt4, e.g., an exogenous copy
of the Sirt4 gene under the control of a promoter, e.g., a targeted
promoter.
[0141] A Sirt4 modulator can be a siRNA, anti-sense RNA, or a
ribozyme, which can decrease the expression of Sirt4. In some
aspects, a cell or subject can be treated with a compound that
modulates the expression of a gene, e.g., a nucleic acid which
modulates, e.g., decreases, expression of a polypeptide which
inhibits Sirt4. Such approaches include oligonucleotide-based
therapies such as RNA interference, antisense, ribozymes, and
triple helices.
[0142] Gene expression can be modified by gene silencing using
double-strand RNA (Sharp (1999) Genes and Development 13: 139-141).
RNAi methods, including double-stranded RNA interference (dsRNAi)
or small interfering RNA (siRNA), have been extensively documented
in a number of organisms, including mammalian cells and the
nematode C. elegans (Fire, A., et al, Nature, 391, 806-811,
1998).
[0143] dsRNA can be delivered to cells or to an organism to
antagonize Sirt4 or another protein described herein. For example,
a dsRNA that is complementary to a Sirt4 nucleic acid can silence
protein expression of the Sirt4. The dsRNA can include a region
that is complementary to a coding region of a Sirt4 nucleic acid,
e.g., a 5' coding region, a region encoding a Sirt4 core domain, a
3' coding region, or a non-coding region, e.g., a 5' or 3'
untranslated region. dsRNA can be produced, e.g., by transcribing a
cassette (in vitro or in vivo) in both directions, for example, by
including a T7 promoter on either side of the cassette. The insert
in the cassette is selected so that it includes a sequence
complementary to the Sirt4 nucleic acid. The sequence need not be
full length, for example, an exon, or between 19-50 nucleotides or
50-200 nucleotides. The sequence can be from the 5' half of the
transcript, e.g., within 1000, 600, 400, or 300 nucleotides of the
ATG. See also, the HISCRIBE.TM. RNAi Transcription Kit (New England
Biolabs, Ma.) and Fire, A. (1999) Trends Genet. 15, 358-363. dsRNA
can be digested into smaller fragments. See, e.g., US Patent
Application 2002-0086356 and 2003-0084471.
[0144] In one embodiment, an siRNA is used. siRNAs are small double
stranded RNAs (dsRNAs) that optionally include overhangs. For
example, the duplex region is about 18 to 25 nucleotides in length,
e.g., about 19, 20, 21, 22, 23, or 24 nucleotides in length.
Typically, the siRNA sequences are exactly complementary to the
target mRNA. It may also be possible to agonize activity of a Sirt4
by using an siRNA to inhibit a negative regulator of the Sirt4.
[0145] Double-stranded inhibitory RNA can also be used to
selectively reduce the expression of one allele of a gene and not
the other, thereby achieving an approximate 50% reduction in the
expression of a Sirt4 antagonist polypeptide. See Garrus et al.
(2001) Cell 107(1):55-65.
[0146] "Ribozymes" are enzymatic RNA molecules which cleave at
specific sites in RNA. Ribozymes that can specifically cleave
nucleic acids that encode or that are required for the expression
of Sirt4 may be designed according to well-known methods.
[0147] A nucleic acid for modulating Sirt4 expression, activity, or
function can be inserted into a variety of DNA constructs and
vectors for the purposes of gene therapy. Vectors include plasmids,
cosmids, artificial chromosomes, viral elements, and RNA vectors
(e.g., based on RNA virus genomes). The vector can be competent to
replicate in a host cell or to integrate into a host DNA. Viral
vectors include, e.g., replication defective retroviruses,
adenoviruses and adeno-associated viruses.
[0148] Examples of vectors include replication defective retroviral
vectors, adenoviral vectors and adeno-associated viral vectors.
Adenoviral vectors suitable for use by the methods disclosed herein
include (Ad.RSV.lacZ), which includes the Rous sarcoma virus
promoter and the lacZ reporter gene as well as (Ad.CMV.lacZ), which
includes the cytomegalovirus promoter and the lacZ reporter gene.
Methods for the preparation and use of viral vectors are described
in WO 96/13597, WO 96/33281, WO 97/15679, and Trapnell et al.,
Curr. Opin. Biotechnol. 5(6):617-625, 1994, the contents of which
are incorporated herein by reference.
[0149] A gene therapy vector is a vector designed for
administration to a subject, e.g., a mammal, such that a cell of
the subject is able to express a therapeutic gene contained in the
vector. The therapeutic gene may encode a protein (e.g., Sirt4).
The therapeutic gene can also be used to provide a non-coding
transcript, e.g., an antisense RNA, a ribozyme, or a dsRNA, that
targets an RNA of a sirtuin gene, e.g., a Sirt4 gene).
[0150] The gene therapy vector can contain regulatory elements,
e.g., a 5' regulatory element, an enhancer, a promoter, a 5'
untranslated region, a signal sequence, a 3' untranslated region, a
polyadenylation site, and a 3' regulatory region. For example, the
5' regulatory element, enhancer or promoter can regulate
transcription of the DNA encoding the therapeutic polypeptide or
other transcript. The regulation can be tissue specific. For
example, the regulation can restrict transcription of the desired
gene, e.g., Sirt4, to pancreas cells, e.g., pancreatic islet
.beta.-cells. For example, the regulation can restrict
transcription of the desired gene, e.g., Sirt4, to nervous tissue
cells, e.g., neuronal or microglial cells. Alternatively,
regulatory elements can be included that respond to an exogenous
drug, e.g., a steroid, tetracycline, or the like. Thus, the level
and timing of expression of the therapeutic nucleic acid can be
controlled.
[0151] Gene therapy vectors can be prepared for delivery as naked
nucleic acid, as a component of a virus, or of an inactivated
virus, or as the contents of a liposome or other delivery vehicle.
See, e.g., US 2003-0143266 and 2002-0150626. In one embodiment, the
nucleic acid is formulated in a lipid-protein-sugar matrix to form
microparticles., e.g., having a diameter between 50 nm to 10
micrometers. The particles may be prepared using any known lipid
(e.g., dipalmitoylphosphatidylcholine, DPPC), protein (e.g.,
albumin), or sugar (e.g., lactose).
[0152] The gene therapy vectors can be delivered using a viral
system. Exemplary viral vectors include vectors from retroviruses,
e.g., Moloney retrovirus, adenoviruses, adeno-associated viruses,
and lentiviruses, e.g., Herpes simplex viruses (HSV). HSV, for
example, is potentially useful for infecting nervous system cells.
See, e.g., US 2003/0147854, 2002/0090716, 2003/0039636,
2002/0068362, and 2003/0104626. The gene delivery agent, e.g., a
viral vector, can be produced from recombinant cells which produce
the gene delivery system.
[0153] A gene therapy vector can be administered to a subject, for
example, by intravenous injection, by local administration (see
U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g.,
Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The
gene therapy agent can be further formulated, for example, to delay
or prolong the release of the agent by means of a slow release
matrix. One method of providing a therapeutic agent, is by
inserting a gene therapy vector into cells harvested from a
subject. The cells are infected, for example, with a retroviral
gene therapy vector, and grown in culture. The subject is then
replenished with the infected culture cells. The subject is
monitored for recovery and for production of the therapeutic
polypeptide or nucleic acid.
[0154] The disclosure also includes vectors, such as gene therapy
vectors, that include a Sirt4 regulatory sequence (e.g., a Sirt4
promoter) for regulating a coding sequence other than Sirt4.
[0155] Cell-based therapeutic methods include introducing a nucleic
acid that provides a therapeutic activity operably linked to a
promoter into a cell in culture. The therapeutic nucleic acid can
provide the desired modulation of Sirt4 activity in a cultured
cell, e.g., an increase or decrease in Sirt4 activity to an
insulin-secreting cell. Further, it is also possible to modify
cells, e.g., stem cells, using nucleic acid recombination, e.g., to
insert a transgene, e.g., a transgene that provides a therapeutic
activity. The modified stem cell can be administered to a subject.
Methods for cultivating stem cells in vitro are described, e.g., in
US Application 2002/0081724. In some examples, the stem cells can
be induced to differentiate in the subject and express the
transgene. For example, the stem cells can be differentiated into
pancreas, liver, adipose, neuronal or skeletal muscle cells. The
stem cells can be derived from a lineage that produces cells of the
desired tissue type, e.g., pancreas, liver, adipose, neuronal, or
skeletal muscle cells.
[0156] Modifications to nucleic acid molecules may be introduced as
a means of increasing intracellular stability and half-life.
Exemplary modifications include the addition of flanking sequences
of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends
of the molecule or the use of phosphorothioate or 2' O-methyl
rather than phosphodiesterase linkages within the
oligodeoxyribonucleotide backbone.
Artificial Transcription Factors
[0157] Artificial transcription factors can also be used to
regulate genes described herein, e.g., genes encoding Sirt4, GDH,
IDE, or ANT.
[0158] The artificial transcription factor can be designed or
selected from a library. The protein can include one or more zinc
finger domains. For example, the protein can be prepared by
selection in vitro (e.g., using phage display, U.S. Pat. No.
6,534,261) or in vivo, or by design based on a recognition code
(see, e.g., WO 00/42219 and U.S. Pat. No. 6,511,808). See, e.g.,
Rebar et al. (1996) Methods Enzymol 267:129; Greisman and Pabo
(1997) Science 275:657; Isalan et al. (2001) Nat. Biotechnol
19:656; and Wu et al. (1995) Proc. Nat. Acad. Sci. USA 92:344 for,
among other things, methods for creating libraries of varied zinc
finger domains.
[0159] Optionally, the artificial transcription factor can be fused
to a transcriptional regulatory domain, e.g., an activation domain
to activate transcription or a repression domain to repress
transcription. The artificial transcription factor can itself be
encoded by a heterologous nucleic acid that is delivered to a cell
(e.g., a vector described herein) or the transcription factor
itself can be delivered to a cell (see, e.g., U.S. Pat. No.
6,534,261). The heterologous nucleic acid that includes a sequence
encoding the transcription factor can be operably linked to an
inducible promoter, e.g., to enable fine control of the level of
the transcription factor in the cell.
Gene Expression and Transcript Analysis
[0160] Different aspects disclosed herein can include evaluating
expression of one or more genes described herein (e.g., genes
encoding Sirt4, insulin, GDH, IDE, and ANT). Expression of a gene
can be evaluated by detecting an mRNA, e.g., the transcript from
the gene of interest or detecting a protein, e.g., the protein
encoded by the gene of interest. Gene expression can also be
measured, e.g., using an indirect method, e.g., using a reporter
construct (e.g., as described below).
[0161] Reporter genes for measuring expression of Sirt4, or a Sirt4
target or binding partner, can be made by operably linking a
regulatory sequence, e.g., a regulatory sequence of the Sirt4 gene,
to a sequence encoding a reporter gene. A number of methods are
available for designing reporter genes. For example, the sequence
encoding the reporter protein can be linked in frame to all or part
of the sequence that is normally regulated by the regulatory
sequence. Such constructs can be referred to as translational
fusions. It is also possible to link the sequence encoding the
reporter protein to only regulatory sequences, e.g., the 5'
untranslated region, TATA box, and/or sequences upstream of the
mRNA start site. Such constructs can be referred to as
transcriptional fusions. Still other reporter genes can be
constructed by inserting one or more copies (e.g., a multimer of
three, four, or six copies) of a regulatory sequence into a neutral
or characterized promoter.
[0162] Reporter constructs can be used to evaluate expression of
any gene, e.g., genes encoding Sirt4, insulin, GDH, ANT, or IDE, or
other gene described herein.
[0163] Exemplary reporter proteins include chloramphenicol
acetyltransferase, green fluorescent protein and other fluorescent
proteins (e.g., artificial variants of GFP), beta-lactamase,
beta-galactosidase, luciferase, and so forth. The reporter protein
can be any protein other than the protein encoded by the endogenous
gene that is subject to analysis. Epitope tags, e.g., flag or his
tags, can also be used.
[0164] Exemplary methods for evaluating mRNAs include northern
analysis, RT-PCR, microarray hybridization, SAGE, differential
display, and monitoring reporter genes. Exemplary methods for
evaluating proteins include immunoassays (e.g., ELISAs,
immunoprecipitations, westerns), 2D-gel electrophoresis, and mass
spectroscopy. It is possible to evaluate fewer than 100, e.g., less
than 20, 10, 5, 4, or 3 different molecular species, e.g., to only
evaluate the expression of the gene of interest, although it is
typically useful to include at least one or two controls (e.g., a
house keeping gene). It is also possible to evaluate multiple
molecular species, e.g., in parallel, e.g., at least 10, 50, 20,
100, or more different species. See, e.g., the usage of
microarrays, e.g. as described below.
[0165] One method for comparing transcripts uses nucleic acid
microarrays that include a plurality of addresses, each address
having a probe specific for a particular transcript, at least one
of which is specific for a gene of interest, e.g., a gene encoding
Sirt4, insulin, GDH, IDE, and ANT. Such arrays can include at least
100, or 1000, or 5000 different probes, so that a substantial
fraction, e.g., at least 10, 25, 50, or 75% of the genes in an
organism are evaluated. mRNA can be isolated from a cell or other
sample of the organism. The mRNA can be reversed transcribed into
labeled cDNA. The labeled cDNAs are hybridized to the nucleic acid
microarrays. The arrays are detected to quantitate the amount of
cDNA that hybridizes to each probe, thus providing information
about the level of each transcript.
[0166] Methods for making and using nucleic acid microarrays are
well known. For example, nucleic acid arrays can be fabricated by a
variety of methods, e.g., photolithographic methods (see, e.g.,
U.S. Pat. Nos. 5,143,854; 5,510,270; and. 5,527,681), mechanical
methods (e.g., directed-flow methods as described in U.S. Pat. No.
5,384,261), pin based methods (e.g., as described in U.S. Pat. No.
5,288,514), and bead based techniques (e.g., as described in PCT
US/93/04145). The probe can be a single-stranded nucleic acid, a
double-stranded nucleic acid (e.g., which is denatured prior to or
during hybridization), or a nucleic acid having a single-stranded
region and a double-stranded region. Preferably, the probe is
single-stranded. The probe can be selected by a variety of
criteria, and preferably is designed by a computer program with
optimization parameters. The probe can be selected to hybridize to
a sequence rich (e.g., non-homopolymeric) region of the nucleic
acid. The T.sub.m of the probe can be optimized by prudent
selection of the complementarity region and length. Ideally, the
T.sub.m of all probes on the array is similar, e.g., within 20, 10,
5, 3, or 2.degree. C. of one another. A database scan of available
sequence information for a species can be used to determine
potential cross-hybridization and specificity problems.
[0167] The isolated mRNA from samples for comparison can be
reversed transcribed and optionally amplified, e.g., by rtPCR,
e.g., as described in (U.S. Pat. No. 4,683,202). The nucleic acid
can be labeled during amplification, e.g., by the incorporation of
a labeled nucleotide. Examples of preferred labels include
fluorescent labels, e.g., red-fluorescent dye Cy5 (Amersham) or
green-fluorescent dye Cy3 (Amersham), and chemiluminescent labels,
e.g., as described in U.S. Pat. No. 4,277,437. Alternatively, the
nucleic acid can be labeled with biotin, and detected after
hybridization with labeled streptavidin, e.g.,
streptavidin-phycoerythrin (Molecular Probes).
[0168] The labeled nucleic acid can be contacted to the array. In
addition, a control nucleic acid or a reference nucleic acid can be
contacted to the same array. The control nucleic acid or reference
nucleic acid can be labeled with a label other than the sample
nucleic acid, e.g., one with a different emission maximum. Labeled
nucleic acids can be contacted to an array under hybridization
conditions. The array can be washed, and then imaged to detect
fluorescence at each address of the array.
[0169] A general scheme for producing and evaluating profiles can
include the following. The extent of hybridization at an address is
represented by a numerical value and stored, e.g., in a vector, a
one-dimensional matrix, or one-dimensional array. The vector x has
a value for each address of the array. For example, a numerical
value for the extent of hybridization at a first address is stored
in variable x.sub.a. The numerical value can be adjusted, e.g., for
local background levels, sample amount, and other variations.
Nucleic acid is also prepared from a reference sample and
hybridized to an array (e.g., the same or a different array), e.g.,
with multiple addresses. The vector y is construct identically to
vector x. The sample expression profile and the reference profile
can be compared, e.g., using a mathematical equation that is a
function of the two vectors. The comparison can be evaluated as a
scalar value, e.g., a score representing similarity of the two
profiles. Either or both vectors can be transformed by a matrix in
order to add weighting values to different nucleic acids detected
by the array.
[0170] The expression data can be stored in a database, e.g., a
relational database such as a SQL database (e.g., Oracle or Sybase
database environments). The database can have multiple tables. For
example, raw expression data can be stored in one table, wherein
each column corresponds to a nucleic acid being assayed (e.g., one
or more of genes encoding Sirt4, insulin, GDH, ANT, or IDE, or
other gene described herein), and each row corresponds to a sample.
A separate table can store identifiers and sample information,
e.g., the batch number of the array used, date, and other quality
control information.
[0171] Other methods for quantitating mRNAs include: quantitative
RT-PCR. In addition, two nucleic acid populations can be compared
at the molecular level, e.g., using subtractive hybridization or
differential display to evaluate differences in mRNA expression,
e.g., between a cell of interest and a reference cell.
Pharmaceutical Compositions
[0172] An agent that modulates activity of Sirt4 can be
incorporated into a pharmaceutical composition, e.g., a composition
that includes a pharmaceutically acceptable carrier.
[0173] As used herein the language "pharmaceutically acceptable
carrier" includes solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like, compatible with pharmaceutical
administration. Supplementary active compounds can also be
incorporated into the compositions.
[0174] A pharmaceutical composition is formulated to be compatible
with its intended route of administration. Examples of routes of
administration include parenteral, e.g., intravenous, intradermal,
subcutaneous, oral (e.g., inhalation), transdermal (topical),
transmucosal, and rectal administration. Solutions or suspensions
used for parenteral, intradermal, or subcutaneous application can
include the following components: a sterile diluent such as water
for injection, saline solution, fixed oils, polyethylene glycols,
glycerine, propylene glycol or other synthetic solvents;
antibacterial agents such as benzyl alcohol or methyl parabens;
antioxidants such as ascorbic acid or sodium bisulfite; chelating
agents such as ethylenediaminetetraacetic acid; buffers such as
acetates, citrates or phosphates and agents for the adjustment of
tonicity such as sodium chloride or dextrose. pH can be adjusted
with acids or bases, such as hydrochloric acid or sodium hydroxide.
The parenteral preparation can be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic.
[0175] Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD.sub.50 (the
dose lethal to 50% of the population) and the ED.sub.50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD.sub.50/ED.sub.50. Compounds
which exhibit high therapeutic indices are preferred. While
compounds that exhibit toxic side effects may be used, care should
be taken to design a delivery system that targets such compounds to
the site of affected tissue in order to minimize potential damage
to uninfected cells and, thereby, reduce side effects.
[0176] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED.sub.50 with
little or no toxicity. The dosage may vary within this range
depending upon the dosage form employed and the route of
administration utilized. For any compound used in the methods
disclosed herein, the therapeutically effective dose can be
estimated initially from cell culture assays. A dose may be
formulated in animal models to achieve a circulating plasma
concentration range that includes the IC.sub.50 (i.e., the
concentration of the test compound which achieves a half-maximal
inhibition of symptoms) as determined in cell culture. Such
information can be used to more accurately determine useful doses
in humans. Levels in plasma may be measured, for example, by high
performance liquid chromatography.
[0177] Examples of modulators of Sirt4 activity include nucleic
acids that encode a Sirt4, or fragments thereof, nucleic acids that
inhibit Sirt4 gene expression, and polypeptides that have a Sirt4
activity, fragments thereof, as well as antibodies that bind to
and/or inhibit a Sirt4. Such modulators can be provided as a
pharmaceutical composition. Other types of modulators include small
molecule inhibitors and activators, e.g., as described herein.
[0178] A therapeutically effective amount of protein or polypeptide
(i.e., an effective dosage) includes ranges, e.g., from about 0.001
to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body
weight. The skilled artisan will appreciate that certain factors
may influence the dosage and timing required to effectively treat a
subject, including but not limited to the severity of the disease
or disorder, previous treatments, the general health and/or age of
the subject, and other diseases present. Moreover, treatment of a
subject with a therapeutically effective amount of a protein,
polypeptide, or antibody can include a single treatment or,
preferably, can include a series of treatments.
[0179] A pharmaceutical composition is formulated to be compatible
with its intended route of administration. Examples of routes of
administration include parenteral, e.g., intravenous, intradermal,
subcutaneous, oral (e.g., inhalation), transdermal (topical),
transmucosal, and rectal administration. Solutions or suspensions
used for parenteral, intradermal, or subcutaneous application can
include the following components: a sterile diluent such as water
for injection, saline solution, fixed oils, polyethylene glycols,
glycerine, propylene glycol or other synthetic solvents;
antibacterial agents such as benzyl alcohol or methyl parabens;
antioxidants such as ascorbic acid or sodium bisulfite; chelating
agents such as ethylenediaminetetraacetic acid; buffers such as
acetates, citrates or phosphates and agents for the adjustment of
tonicity such as sodium chloride or dextrose. pH can be adjusted
with acids or bases, such as hydrochloric acid or sodium hydroxide.
The parenteral preparation can be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic.
[0180] In one embodiment, a composition that includes a modulator
of Sirt4 activity is used to modulate (e.g., increase, decrease)
the amount of insulin secreted by the pancreas.
[0181] In one embodiment, a composition that includes a modulator
of Sirt4 activity is used to decrease the accumulation of
.beta.-amyloid peptide.
Diabetes
[0182] An agent that modulates Sirt4 expression or activity can be
used to treat or prevent diabetes. The agent can be administered to
a subject in an amount effective to treat, prevent, or ameliorate
at least one symptom of diabetes. For example, an agent that
decreases Sirt4 expression or activity can be used to increase
insulin secretion, e.g., in a hypoinsulinemic subject. An agent
that, e.g., increases Sirt4 expression can be used to decrease
insulin secretion, e.g., in a hyperinsulinemic subject.
[0183] Examples of diabetes include insulin dependent diabetes
mellitus and non-insulin dependent diabetes. For example the method
includes administering to a patient having diabetes or at risk of
diabetes a compound described herein.
[0184] For example, a compound described herein can be administered
to a subject in a therapeutically effective amount to increase
insulin secretion in response to glucose, decrease insulin
secretion in response to glucose, decrease gluconeogenesis, improve
glycemic control (i.e., lower fasting blood glucose), or normalize
insulin sensitivity. The compound can be administered to a subject
suffering from or at risk for diabetes or obesity.
[0185] In some instances, a patient can be identified as being at
risk of developing diabetes (pre-diabetes) by having impaired
glucose tolerance (IGT), insulin resistance, obesity, metabolic
syndrome X, misfunction of glucose regulation by liver, fat, brain,
and/or muscle, or fasting hyperglycemia.
[0186] Insulin dependent diabetes mellitus (Type 1 diabetes) is an
autoimmune disease, where insulitis leads to the destruction of
pancreatic .beta.-cells. At the time of clinical onset of type 1
diabetes mellitus, significant number of insulin producing .beta.
cells are destroyed and only 15% to 40% are still capable of
insulin production (McCulloch et al. (1991) Diabetes 40:673-679).
.beta.-cell failure results in a life long dependence on daily
insulin injections and exposure to the acute and late complication
of the disease.
[0187] Type 2 diabetes mellitus is a metabolic disease of impaired
glucose homeostasis characterized by hyperglycemia, or high blood
sugar, as a result of defective insulin action which manifests as
insulin resistance, defective insulin secretion, or both. A patient
with Type 2 diabetes mellitus has abnormal carbohydrate, lipid, and
protein metabolism associated with insulin resistance and/or
impaired insulin secretion. The disease leads to pancreatic beta
cell destruction and eventually absolute insulin deficiency.
Without insulin, high glucose levels remain in the blood. The long
term effects of high blood glucose include blindness, renal
failure, and poor blood circulation to these areas, which can lead
to foot and ankle amputations. Early detection is critical in
preventing patients from reaching this severity. The majority of
patients with diabetes have the non-insulin dependent form of
diabetes, currently referred to as Type 2 diabetes mellitus.
[0188] This disclosure also includes methods of treating disorders
related to or resulting from diabetes, for example end organ
damage, diabetic gastroparesis, diabetic neuropathy, cardiac
dysrythmia, etc.
[0189] Exemplary molecular models of Type II diabetes include: a
transgenic mouse having defective Nkx-2.2 or Nkx-6.1; (U.S. Pat.
No. 6,127,598); Zucker Diabetic Fatty fa/fa (ZDF) rat. (U.S. Pat.
No. 6,569,832); and Rhesus monkeys, which spontaneously develop
obesity and subsequently frequently progress to overt type 2
diabetes (Hotta et al., Diabetes, 50:1126-33 (2001); and a
transgenic mouse with a dominant-negative IGF-1 receptor
(KR-IGF-1R) having Type 2 diabetes-like insulin resistance.
Metabolic Syndrome
[0190] An agent that modulates Sirt4 expression or activity can be
used to treat or prevent metabolic syndrome. The agent can be
administered to a subject in an amount effective to treat,.prevent,
or ameliorate at least one symptom of metabolic syndrome.
[0191] Metabolic syndrome (e.g., Syndrome X) is a syndrome
characterized by a group of metabolic risk factors in one person.
These factors include two or more of (particularly three, four,
five or more, or all of): central obesity (excessive fat tissue in
and around the abdomen), atherogenic dyslipidemia (blood fat
disorders--mainly high triglycerides and low HDL cholesterol--that
foster plaque buildups in artery walls); insulin resistance or
glucose intolerance (the body can't properly use insulin or blood
sugar); prothrombotic state (e.g., high fibrinogen or plasminogen
activator inhibitor-1 (PAI-1) in the blood); raised blood pressure
(i.e., hypertension) (130/85 mmHg or higher); and proinflammatory
state (e.g., elevated high-sensitivity C-reactive protein in the
blood).
[0192] The underlying causes of this syndrome include
overweight/obesity, physical inactivity and genetic factors. People
with metabolic syndrome are at increased risk of coronary heart
disease, other diseases related to plaque buildups in artery walls
(e.g., stroke and peripheral vascular disease), and type 2
diabetes. Metabolic syndrome is closely associated with a
generalized metabolic disorder called insulin resistance, in which
the body fails to utilize insulin efficiently.
Alzheimer's Disease
[0193] An agent that modulates Sirt4 expression or activity,
preferably one that increases Sirt4 expression or activity, can be
used to treat or prevent Alzheimer's Disease (AD). The agent can be
an agent described herein or an agent identified by a method
described herein. The agent can be administered in an amount
effective to treat, prevent, or ameliorate at least one symptom of
AD.
[0194] Alzheimer's Disease (AD) is a complex neurodegenerative
disease that results in the irreversible loss of neurons and is an
example of a neurodegenerative disease that has symptoms caused at
least in part by protein aggregation.
[0195] Clinical hallmarks of Alzheimer's disease include
progressive impairment in memory, judgment, orientation to physical
surroundings, and language. Neuropathological hallmarks of AD
include region-specific neuronal loss, amyloid plaques, and
neurofibrillary tangles.
[0196] Amyloid plaques are extracellular plaques containing the
.beta. amyloid peptide (also known as A.beta., or A.beta.42), which
is a cleavage product of the .beta.-amyloid precursor protein (also
known as APP). Neurofibrillary tangles are insoluble intracellular
aggregates composed of filaments of the abnormally
hyperphosphorylated microtubule-associated protein, tau. Amyloid
plaques and neurofibrillary tangles may contribute to secondary
events that lead to neuronal loss by apoptosis (Clark and
Karlawish, Ann. Intern. Med. 138(5):400-410 (2003)). For example,
.beta.-amyloid induces caspase-2-dependent apoptosis in cultured
neurons (Troy et al. J. Neurosci. 20(4):1386-1392). The deposition
of plaques in vivo may trigger apoptosis of proximal neurons in a
similar manner.
[0197] Mutations in genes encoding APP, presenilin-1, and
presenilin-2 have been implicated in early-onset AD (Lendon et al.
JAMA 227:825 (1997)). Mutations in these proteins have been shown
to enhance proteolytic processing of APP via an intracellular
pathway that produces A.beta.. Aberrant regulation of A.beta.
processing may be central to the formation of amyloid plaques and
the consequent neuronal damage associated with plaques.
[0198] A variety of criteria, including genetic, biochemical,
physiological, and cognitive criteria, can be used to evaluate AD
in a subject. Symptoms and diagnosis of AD are known to medical
practitioners. Some exemplary symptoms and markers of AD are
presented below. Information about these indications and other
indications known to be associated with AD can be used as an
"AD-related parameter." An AD-related parameter can include
qualitative or quantitative information. An example of quantitative
information is a numerical value of one or more dimensions, e.g., a
concentration of a protein or a tomographic map. Qualitative
information can include an assessment, e.g., a physician's comments
or a binary ("yes/no") and so forth. An AD-related parameter
includes information that indicates that the subject is not
diagnosed with AD or does not have a particular indication of AD,
e.g., a cognitive test result that is not typical of AD or a
genetic apolipoprotein E (APOE) polymorphism not associated with
AD.
[0199] Progressive cognitive impairment is a hallmark of AD. This
impairment can present as decline in memory, judgment, decision
making, orientation to physical surroundings, and language
(Nussbaum and Ellis, New Eng. J Med. 348(14):1356-1364 (2003)).
Exclusion of other forms of dementia can assist in making a
diagnosis of AD.
[0200] Neuronal death leads to progressive cerebral atrophy in AD
patients. Imaging techniques (e.g., magnetic resonance imaging, or
computed tomography) can be used to detect AD-associated lesions in
the brain and/or brain atrophy.
[0201] The insulin degrading enzyme (IDE) can degrade
.beta.-amyloid protein in neuronal and microglial cell cultures
(Qiu et al. (1997) J. Biol. Chem. 272:6641-6; Qiu et al. (1998) J.
Biol. Chem. 273:32730-8; Vekrellis et al. (2000) J. Neurosci.
20:1657-65; Sudoh et al. (2002) Biochemistry 41:1091-9), and can
eliminate the neurotoxicity of .beta.-amyloid protein (Mukherjee et
al. (2000) J. Neurosci. 20:8745-9). Furthermore, IDE -/- mice
presented chronic elevation of .beta.-amyloid protein, similar to
that seen in AD patients (Farris et al. (2003) Proc Nat'l Acad Sci
USA 100:4162-7).
[0202] AD patients may exhibit biochemical abnormalities that
result from the pathology of the disease. For example, levels of
tau protein in the cerebrospinal fluid is elevated in AD patients
(Andreasen, N. et al. Arch Neurol. 58:349-350 (2001)). Levels of
amyloid beta 42 (A.beta.42) peptide can be reduced in CSF of AD
patients (Galasko, D., et al. Arch. Neurol. 55:937-945 (1998)).
Levels of A.beta.42 can be increased in the plasma of AD patients
(Ertekein-Taner, N., et al. Science 290:2303-2304 (2000)).
Techniques to detect biochemical abnormalities in a sample from a
subject include cellular, immunological, and other biological
methods known in the art. For general guidance, see, e.g.,
techniques described in Sambrook & Russell, Molecular Cloning:
A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory,
N.Y. (2001), Ausubel et al., Current Protocols in Molecular Biology
(Greene Publishing Associates and Wiley Interscience, N.Y. (1989),
(Harlow, E. and Lane, D. (1988) Antibodies: A Laboratory Manual,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), and
updated editions thereof.
[0203] For example, antibodies, other immunoglobulins, and other
specific binding ligands can be used to detect a biomolecule, e.g.,
a protein or other antigen associated with AD. For example, one or
more specific antibodies can be used to probe a sample. Various
formats are possible, e.g., ELISAs, fluorescence-based assays,
western blots, and protein arrays. Methods of producing polypeptide
arrays are described in the art, e.g., in De Wildt et al. (2000).
Nature Biotech. 18,989-994; Lueking et al. (1999). Anal. Biochem.
270, 103-111; Ge, H. (2000). Nucleic Acids Res. 28, e3, I-VII;
MacBeath, G., and Schreiber, S. L. (2000). Science 289, 1760-1763;
and WO 99/51773A1. Proteins can also be analyzed using mass
spectroscopy, chromatography, electrophoresis, enzyme interaction
or using probes that detect post-translational modification (e.g.,
a phosphorylation, ubiquitination, glycosylation, methylation, or
acetylation).
[0204] Metabolites that are associated with AD can be detected by a
variety of means, including enzyme-coupled assays, using labeled
precursors, and nuclear magnetic resonance (NMR). For example, NMR
can be used to determine the relative concentrations of
phosphate-based compounds in a sample, e.g., creatine levels. Other
metabolic parameters such as redox state, ion concentration (e.g.,
Ca.sup.2+) (e.g., using ion-sensitive dyes), and membrane potential
can also be detected (e.g., using patch-clamp technology).
[0205] In one embodiment, a non-human animal model of AD (e.g., a
mouse model) is used, e.g., to evaluate a compound or a therapeutic
regimen, e.g., of an agent described herein. For example, U.S. Pat.
No. 6,509,515 describes one such model animal which is naturally
able to be used with learning and memory tests. The animal
expresses an amyloid precursor protein (APP) sequence at a level in
brain tissues such that the animal develops a progressive
neurologic disorder within a short period of time from birth,
generally within a year from birth, preferably within 2 to 6
months, from birth. The APP protein sequence is introduced into the
animal, or an ancestor of the animal, at an embryonic stage,
preferably the one cell, or fertilized oocyte, stage, and generally
not later than about the 8-cell stage. The zygote or embryo is then
developed to term in a pseudo-pregnant foster female. The amyloid
precursor protein genes are introduced into an animal embryo so as
to be chromosomally incorporated in a state which results in
super-endogenous expression of the amyloid precursor protein and
the development of a progressive neurologic disease in the
cortico-limbic areas of the brain, areas of the brain which are
prominently affected in progressive neurologic disease states such
as AD. The gliosis and clinical manifestations in affected
transgenic animals model neurologic disease. The progressive
aspects of the neurologic disease are characterized by diminished
exploratory and/or locomotor behavior and diminished 2-deoxyglucose
uptake/utilization and hypertrophic gliosis in the cortico-limbic
regions of the brain. Other animal models are also described in
U.S. Pat. Nos. 5,387,742; 5,877,399; 6,358,752; and 6,187,992.
[0206] Additionally, glutamate is a key neurotransmitter. Sirt4 can
function to regulate glutamate dehydrogenase, and thus glutamate
levels in the brain. Sirt4 can play an important role in monitoring
synapse and neuron function. Mice with an activating mutation in
glutamate dehydrogenase suffer from neurological problems,
Parkinson's Disease
[0207] Parkinson's disease includes neurodegeneration of
dopaminergic neurons in the substantia nigra resulting in the
degeneration of the nigrostriatal dopamine system that regulates
motor function. This pathology, in turn, leads to motor
dysfunctions. (see, e.g., and Lotharius et al., Nat. Rev.
Neurosci., 3:932-42 (2002).) Exemplary motor symptoms include:
akinesia, stooped posture, gait difficulty, postural instability,
catalepsy, muscle rigidity, and tremor. Exemplary non-motor
symptoms include: depression, lack of motivation, passivity,
dementia and gastrointestinal dysfunction (see, e.g., Fahn, Ann.
N.Y. Acad. Sci., 991:1-14 (2003) and Pfeiffer, Lancet Neurol.,
2:107-16 (2003)) Parkinson's has been observed in 0.5 to 1 percent
of persons 65 to 69 years of age and 1 to 3 percent among persons
80 years of age and older. (see, e.g., Nussbaum et al., N. Engl. J.
Med., 348:1356-64 (2003)).
[0208] An agent that modulates Sirt4 activity can be used to
ameliorate at least one symptom of a subject that has Parkinson's
disease.
[0209] Molecular markers of Parkinson's disease include reduction
in aromatic L-amino acid decarboxylase (AADC). (see, e.g., US Appl
20020172664); loss of dopamine content in the nigrostriatal neurons
(see, e.g., Fahn, Ann. N.Y. Acad. Sci., 991:1-14 (2003) and
Lotharius et al., Nat. Rev. Neurosci., 3:932-42 (2002)). In some
familial cases, PD is linked to mutations in single genes encoding
alpha-synuclein and parkin (an E3 ubiquitin ligase) proteins.
(e.g., Riess et al., J. Neurol. 250 Suppl 1:13-10 (2003) and
Nussbaum et al., N. Engl. J. Med., 348:1356-64 (2003)). A missense
mutation in a neuron-specific C-terminal ubiquitin hydrolase gene
is also associated with Parkinson's. (e.g., Nussbaum et al., N.
Engl. J. Med., 348:1356-64 (2003))
[0210] A compound or library of compounds described herein can be
evaluated in a non-human animal model of Parkinson's disease.
Exemplary animal models of Parkinson's disease include primates
rendered parkinsonian by treatment with the dopaminergic neurotoxin
1-methyl-4 phenyl 1,2,3,6-tetrahydropyridine (MPTP) (see, e.g., US
Appl 20030055231 and Wichmann et al., Ann. N.Y. Acad. Sci.,
991:199-213 (2003); 6-hydroxydopamine-lesioned rats (e.g., Lab.
Anim. Sci.,49:363-71 (1999)); and transgenic invertebrate models
(e.g., Lakso et al., J. Neurochem., 86:165-72 (2003) and Link,
Mech. Ageing Dev., 122:1639-49 (2001)).
Evaluating Huntington's Disease
[0211] An agent that modulates the activity of Sirt4 can be used to
ameliorate at least one symptom of Huntington's disease in a
subject.
[0212] A variety of methods are available to evaluate and/or
monitor Huntington's disease. A variety of clinical symptoms and
indicia for the disease are known. Huntington's disease causes a
movement disorder, psychiatric difficulties and cognitive changes.
The degree, age of onset, and manifestation of these symptoms can
vary. The movement disorder can include quick, random, dance-like
movements called chorea.
[0213] One method for evaluating Huntington's disease uses the
Unified Huntington's disease Rating Scale (UNDRS). It is also
possible to use individual tests alone or in combination to
evaluate if at least one symptom of Huntington's disease is
ameliorated. The UNDRS is described in Movement Disorders (vol.
11:136-142,1996) and Marder et al. Neurology (54:452-458, 2000).
The UNDRS quantifies the severity of Huntington's Disease. It is
divided into multiple subsections: motor, cognitive, behavioral,
functional. In one embodiment, a single subsection is used to
evaluate a subject. These scores can be calculated by summing the
various questions of each section. Some sections (such as chorea
and dystonia) can include grading each extremity, face,
bucco-oral-ligual, and trunk separately.
[0214] Exemplary motor evaluations include: ocular pursuit, saccade
initiation, saccade velocity, dysarthria, tongue protrusion, finger
tap ability, pronate/supinate, a fist-hand-palm sequence, rigidity
of arms, bradykinesia, maximal dystonia (trunk, upper and lower
extremities), maximal chorea (e.g., trunk, face, upper and lower
extremities), gait, tandem walking, and retropulsion. An exemplary
treatment can cause a change in the Total Motor Score 4(TMS-4), a
subscale of the UHDRS, e.g., over a one-year period.
[0215] A number of animal model system for Huntington's disease are
available. See, e.g., Brouillet, Functional Neurology 15(4):
239-251 (2000); Ona et al. Nature 399: 263-267 (1999), Bates et al.
Hum Mol Genet. 6(10):1633-7 (1997); Hansson et al. J. of
Neurochemistry 78: 694-703; and Rubinsztein, D. C., Trends in
Genetics, Vol. 18, No. 4, pp. 202-209 (a review on various animal
and non-human models of HD).
Genetic Information
[0216] SIRT4 genetic information can be obtained, e.g., by
evaluating genetic material (e.g., DNA or RNA) from a subject
(e.g., as described below). Genetic information refers to any
indication about nucleic acid sequence content at one or more
nucleotides. Genetic information can include, for example, an
indication about the presence or absence of a particular
polymorphism, e.g., one or more nucleotide variations. Exemplary
polymorphisms include a single nucleotide polymorphism (SNP), a
restriction site or restriction fragment length, an insertion, an
inversion, a deletion, a repeat (e.g., trinucleotide repeat, a
retroviral repeat), and so forth.
[0217] Exemplary SIRT4 SNPs include: rs16950058; rs12425285;
rs12424555; rs12307919; rs12300927; rs11834400; rs11614455;
rs11613753; rs11609118; rs11412750; rs11378799; rs11065078;
rs11065077; rs11065075; rs2905543; rs2701643; rs2522141; rs2522139;
rs2522138; rs2522130; rs2522129; rs2464297; rs2464296; rs2428384;
rs2261612; rs13461273; rs13461272; rs6400038; and rs6399397.
[0218] It is possible to digitally record or communicate genetic
information in a variety of ways. Typical representations include
one or more bits, or a text string. For example, a biallelic marker
can be described using two bits. In one embodiment, the first bit
indicates whether the first allele (e.g., the minor allele) is
present, and the second bit indicates whether the other allele
(e.g., the major allele) is present. For markers that are
multi-allelic, e.g., where greater than two alleles are possible,
additional bits can be used as well as other forms of encoding
(e.g., binary, hexadecimal text, e.g., ASCII or Unicode, and so
forth). In some embodiments, the genetic information describes a
haplotype, e.g., a plurality of polymorphisms on the same
chromosome. However, in many embodiments, the genetic information
is unphased.
Methods of Evaluating Genetic Material
[0219] There are numerous methods for evaluating genetic material
to provide genetic information. These methods can be used to
evaluate a SIRT4 locus as well as other loci.
[0220] Nucleic acid samples can analyzed using biophysical
techniques (e.g., hybridization, electrophoresis, and so forth),
sequencing, enzyme-based techniques, and combinations-thereof. For
example, hybridization of sample nucleic acids to nucleic acid
microarrays can be used to evaluate sequences in an mRNA population
and to evaluate genetic polymorphisms. Other hybridization based
techniques include sequence specific primer binding (e.g., PCR or
LCR); Southern analysis of DNA, e.g., genomic DNA; Northern
analysis of RNA, e.g., mRNA; fluorescent probe based techniques
(see, e.g., Beaudet et al. (2001) Genome Res. 11(4):600-8); and
allele specific amplification. Enzymatic techniques include
restriction enzyme digestion; sequencing; and single base extension
(SBE). These and other techniques are well known to those skilled
in the art.
[0221] Electrophoretic techniques include capillary electrophoresis
and Single-Strand Conformation Polymorphism (SSCP) detection (see,
e.g., Myers et al. (1985) Nature 313:495-8 and Ganguly (2002) Hum
Mutat. 19(4):334-42). Other biophysical methods include denaturing
high pressure liquid chromatography (DHPLC).
[0222] In one embodiment, allele specific amplification technology
that depends on selective PCR amplification may be used to obtain
genetic information. Oligonucleotides used as primers for specific
amplification may carry the mutation of interest in the center of
the molecule (so that amplification depends on differential
hybridization) (Gibbs et al. (1989) Nucleic Acids Res.
17:2437-2448) or at the extreme 3' end of one primer where, under
appropriate conditions, mismatch can prevent, or reduce polymerase
extension (Prossner (1993) Tibtech 11:238). In addition, it is
possible to introduce a restriction site in the region of the
mutation to create cleavage-based detection (Gasparini et al.
(1992) Mol. Cell Probes 6:1). In another embodiment, amplification
can be performed using Taq ligase for amplification (Barany (1991)
Proc. Natl. Acad. Sci USA 88:189). In such cases, ligation will
occur only if there is a perfect match at the 3' end of the 5'
sequence making it possible to detect the presence of a known
mutation at a specific site by looking for the presence or absence
of amplification.
[0223] Enzymatic methods for detecting sequences include
amplification based-methods such as the polymerase chain reaction
(PCR; Saiki, et al. (1985) Science 230, 1350-1354) and ligase chain
reaction (LCR; Wu. et al. (1989) Genomics 4, 560-569; Barringer et
al. (1990), Gene 1989, 117-122; F. Barany. 1991, Proc. Natl. Acad.
Sci. USA 1988, 189-193); transcription-based methods utilize RNA
synthesis by RNA polymerases to amplify nucleic acid (U.S. Pat. No.
6,066,457; U.S. Pat. No. 6,132,997; U.S. Pat. No. 5,716,785; Sarkar
et al., Science (1989) 244:331-34; Stofler et al., Science (1988)
239:491); NASBA (U.S. Pat. Nos. 5,130,238; 5,409,818; and
5,554,517); rolling circle amplification (RCA; U.S. Pat. Nos.
5,854,033 and 6,143,495) and strand displacement amplification
(SDA; U.S. Pat. Nos. 5,455,166 and 5,624,825). Amplification
methods can be used in combination with other techniques.
[0224] Other enzymatic techniques include sequencing using
polymerases, e.g., DNA polymerases and variations thereof such as
single base extension technology. See, e.g., U.S. Pat. No.
6,294,336; U.S. Pat. No. 6,013,431; and U.S. Pat. No.
5,952,174.
[0225] Mass spectroscopy (e.g., MALDI-TOF mass spectroscopy) can be
used to detect nucleic acid polymorphisms. In one embodiment,
(e.g., the MassEXTEND.TM. assay, SEQUENOM, Inc.), selected
nucleotide mixtures, missing at least one dNTP and including a
single ddNTP is used to extend a primer that hybridizes near a
polymorphism. The nucleotide mixture is selected so that the
extension products between the different polymorphisms at the site
create the greatest difference in molecular size. The extension
reaction is placed on a plate for mass spectroscopy analysis.
[0226] Fluorescence based detection can also be used to detect
nucleic acid polymorphisms. For example, different terminator
ddNTPs can be labeled with different fluorescent dyes. A primer can
be annealed near or immediately adjacent to a polymorphism, and the
nucleotide at the polymorphic site can be detected by the type
(e.g., "color") of the fluorescent dye that is incorporated.
[0227] Hybridization to microarrays can also be used to detect
polymorphisms, including SNPs. For example, a set of different
oligonucleotides, with the polymorphic nucleotide at varying
positions with the oligonucleotides can be positioned on a nucleic
acid array. The extent of hybridization as a function of position
and hybridization to oligonucleotides specific for the other allele
can be used to determine whether a particular polymorphism is
present. See, e.g., U.S. Pat. No. 6,066,454.
[0228] In one implementation, hybridization probes can include one
or more additional mismatches to destabilize duplex formation and
sensitize the assay. The mismatch may be directly adjacent to the
query position, or within 10, 7, 5, 4, 3, or 2 nucleotides of the
query position. Hybridization probes can also be selected to have a
particular T.sub.m, e.g., between 45-60.degree. C., 55-65.degree.
C., or 60-75.degree. C. In a multiplex assay, T.sub.m's can be
selected to be within 5, 3, or 2.degree. C. of each other, e.g.,
probes for rs1800591 and rs2866164 can be selected with these
criteria.
[0229] It is also possible to directly sequence the nucleic acid
for a particular genetic locus, e.g., by amplification and
sequencing, or amplification, cloning and sequence. High throughput
automated (e.g., capillary or microchip based) sequencing apparati
can be used. In still other embodiments, the sequence of a protein
of interest is analyzed to infer its genetic sequence. Methods of
analyzing a protein sequence include protein sequencing, mass
spectroscopy, sequence/epitope specific immunoglobulins, and
protease digestion.
[0230] Any combination of the above methods can also be used. The
above methods can be used to evaluate any genetic locus, e.g., in a
method for analyzing genetic information from particular groups of
individuals or in a method for analyzing a polymorphism associated
with a metabolic disorder, e.g., diabetes, or other disorder
described herein, e.g., the SIRT4 locus.
Evaluating Markers of a Metabolic Disorder, e.g., Diabetes, or
Other Disorder Described Herein
[0231] A variety of criteria, including genetic, biochemical,
physiological, and cognitive criteria, can be used to evaluate a
metabolic disorder, e.g., diabetes, or other disorder described
herein in a subject. Symptoms and diagnosis of a metabolic
disorder, e.g., diabetes, or other disorder described herein are
known to medical practitioners. Some exemplary symptoms and markers
of a metabolic disorder, e.g., diabetes, or other disorder
described herein are presented below. Information about these
indications and other indications known to be associated with a
metabolic disorder, e.g., diabetes, or other disorder described
herein can be used as a parameter associated with the disorder.
[0232] A parameter can include qualitative or quantitative
information. An example of quantitative information is a numerical
value of one or more dimensions, e.g., a concentration of a protein
or a tomographic map. Qualitative information can include an
assessment, e.g., a physician's comments or a binary ("yes"/"no")
and so forth. An parameter can include information that indicates
that the subject is not diagnosed with a metabolic disorder, e.g.,
diabetes, or other disorder described herein or does not have a
particular indication of a metabolic disorder, e.g., diabetes, or
other disorder described herein.
[0233] Techniques to detect biochemical abnormalities in a sample
from a subject include cellular, immunological, and other
biological methods known in the art. For general guidance, see,
e.g., techniques described in Sambrook & Russell, Molecular
Cloning: A Laboratory Manual, 3.sup.rd Edition, Cold Spring Harbor
Laboratory, N.Y. (2001), Ausubel et al., Current Protocols in
Molecular Biology (Greene Publishing Associates and Wiley
Interscience, N.Y. (1989), (Harlow, E. and Lane, D. (1988)
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y.), and updated editions thereof.
[0234] For example, antibodies, other immunoglobulins, and other
specific binding ligands can be used to detect a biomolecule, e.g.,
a protein or other antigen associated with a metabolic disorder,
e.g., diabetes, or other disorder described herein. For example,
one or more specific antibodies can be used to probe a sample.
Various formats are possible, e.g., ELISAs, fluorescence-based
assays, Western blots, and protein arrays. Methods of producing
polypeptide arrays are described in the art, e.g., in De Wildt et
al. (2000). Nature Biotech. 18, 989-994; Lueking et al. (1999).
Anal. Biochem. 270, 103-111; Ge, H. (2000). Nucleic Acids Res. 28,
e3, I-VII; MacBeath, G., and Schreiber, S. L. (2000). Science 289,
1760-1763; and WO 99/51773A1.
[0235] Proteins can also be analyzed using mass spectroscopy,
chromatography, electrophoresis, enzyme interaction or using probes
that detect post-translational modification (e.g., a
phosphorylation, ubiquitination, glycosylation, methylation, or
acetylation).
[0236] Nucleic acid expression can be detected in cells from a
subject, e.g., removed by surgery, extraction, post-mortem or other
sampling (e.g., blood, CSF). Expression of one or more genes can be
evaluated, e.g., by hybridization based techniques, e.g., Northern
analysis, RT-PCR, SAGE, and nucleic acid arrays. Nucleic acid
arrays are useful for profiling multiple mRNA species in a sample.
A nucleic acid array can be generated by various methods, e.g., by
photolithographic methods (see, e.g., U.S. Pat. Nos. 5,143,854;
5,510,270; and 5,527,681), mechanical methods (e.g., directed-flow
methods as described in U.S. Pat. No. 5,384,261), pin-based methods
(e.g., as described in U.S. Pat. No. 5,288,514), and bead-based
techniques (e.g., as described in PCT US/93/04145).
[0237] Metabolites that are associated with a metabolic disorder,
e.g., diabetes, or other disorder described herein can be detected
by a variety of means, including enzyme-coupled assays, using
labeled precursors, and nuclear magnetic resonance (NMR). For
example, NMR can be used to determine the relative concentrations
of phosphate-based compounds in a sample, e.g., creatine levels.
Other metabolic parameters such as redox state, ion concentration
(e.g., Ca.sup.2+)(e.g., using ion-sensitive dyes), and membrane
potential can also be detected (e.g., using patch-clamp
technology).
[0238] Information about an a metabolic disorder, e.g., diabetes,
or other disorder described herein-associated marker can be
recorded and/or stored in a computer-readable format. Typically the
information is linked to a reference about the subject and also is
associated (directly or indirectly) with information about the
identity of one or more nucleotides in the subject's SIRT4
genes.
Identifying Relevant Genotypes
[0239] Methods for identifying genotypes associated with a
metabolic disorder, e.g., diabetes, or other disorder described
herein can include comparisons to one or more reference sequences
or an association study among individuals that have a particular
characteristic, e.g., a particular parameter, e.g., associated with
a disorder described herein, or a diagnosis of a metabolic
disorder, e.g., diabetes, or other disorder described herein
diagnosis.
[0240] Multiple sets of reference sequences may be used for
comparison. Exemplary reference sequences include sequences from
subjects at risk for or diagnosed with a metabolic disorder, e.g.,
diabetes, or other disorder described herein and sequences from
subjects that are not at risk for or diagnosed with a metabolic
disorder, e.g., diabetes, or other disorder described herein.
[0241] By evaluating one or more genetic loci, it is possible to
determine an association for each locus or for each allele of each
locus, and a phenotype. One type of test of association is the
G-Test, but other statistical measures can also be used. A high
degree of association, e.g., a high ch-square statistic, can
indicate that a particular locus is associated with a state (e.g.,
a phenotype). This type of associational study can be used to map a
genetic locus that is associated with the state. Associated loci
can be used, e.g., for diagnostic evaluations (e.g., genetic
counseling, risk evaluation, prophylactic care, care management,
and so forth) and for research (e.g., identifying targets for
therapeutics).
[0242] As seen herein, it is also possible to identify genes
associated with disorders by using a method that includes: a)
identifying a plurality of human individuals characterized by a
disorder or having a genetic relationship with an subject
characterized by the disorder; and b) comparing distribution of a
plurality of genetic markers among the subjects of the first
plurality to distribution of markers of the plurality of genetic
markers among subjects of a second plurality of human subjects,
wherein the human subjects of the second plurality have attained at
least 90, 95, 98, or 100 years of age. For example, the plurality
of genetic markers includes at least one, 10, 20, 30 or 50 markers
from each chromosome. The method can further include evaluating a
measure of linkage disequilibrium (e.g., a LOD score). For example,
each subject of the first plurality is suffering or at risk for an
age-associated disorder or each subject of the first plurality is
genetically related to an subject suffering or at risk for an
age-associated disorder.
[0243] In one embodiment, the first plurality includes at least 50,
100, 150, 200, or 300 subjects. In one embodiment, the human
subjects of the second plurality are free of an a metabolic
disorder, e.g., diabetes, or other disorder described herein
diagnosis. For example, the human subjects of the second plurality
are cognitively intact at the age of 85, 90, 95, 98, or 100 and/or
the human subjects of the second plurality are free of a symptom or
diagnosis of the disorder. In one embodiment, the second plurality
includes at least 50, 100, 150, 200, 300, 500 or 800 subjects.
Pharmacogenomics
[0244] Both prophylactic and therapeutic methods of treatment may
be specifically tailored or modified, based on knowledge obtained
from a pharmacogenomics analysis. In particular, a subject can be
treated based on the presence or absence of a genetic polymorphism
associated with a metabolic disorder, e.g., diabetes, or other
disorder described herein, e.g., a polymorphism associated with the
SIRT4 locus.
[0245] Pharmacogenomics allows a clinician or physician to target
prophylactic or therapeutic treatments to patients who will most
benefit from the treatment and to avoid the treatment of patients
who will experience toxic or other undesirable drug-related side
effects. In particular, a diet or drug that affects a metabolic
disorder, e.g., diabetes, or other disorder described herein can be
prescribed as a function of the subject's SIRT4 locus. For example,
if the individual's SIRT4 locus includes an allele that is
predisposed to a metabolic disorder, e.g., diabetes, or other
disorder described herein relative to other alleles, the individual
can be indicated for a prophylactic treatment for a drug that
alleviates a metabolic disorder, e.g., diabetes, or other disorder
described herein. In another example, the individual is placed in a
monitoring program, e.g., to closely monitor for physical
manifestations of a metabolic disorder, e.g., diabetes, or other
disorder described herein onset.
[0246] These and other aspects of the invention are described
further in the following examples, which are illustrative and in no
way limiting.
EXAMPLES
Experimental Procedures
[0247] Plasmids
[0248] EST clones containing human and mouse Sirt4 cDNA were
obtained from the American Type Culture Collection (Manassas, Va.).
Human Sirt4 cDNA was amplified by PCR with the oligonucleotides
MHSirt4-3 (5'-CACCGCGGTGGCGGCCGCATGAAGATGAGCTTTGCGTTGACTTTC-3')
(SEQ ID NO:4) and MHSirt4-4
(5'-CTTGTAATCCTCGAGGCATGGGTCTATCAAAGGCAGC-3') (SEQ ID NO:5). The
human Sirt4 cDNA fragment was digested at sites within these
oligonucleotide primers with NotI and XhoI, and the resulting
fragment was inserted into the vector pCMV-FLAG-4A.TM. (Invitrogen;
Carlsbad, Calif.) that had been digested with the same enzymes. The
resulting plasmid, phSirt4FLAG, directs the overexpression of human
Sirt4 with a C-terminal FLAG in mammalian cells. phSirt4 directs
the overexpression of human Sirt4 in mammalian cells and was
created by amplification of the Sirt4 gene using the primers
MHSirt4-3 and MHSirt4-QC2
(5'.-CTTGTAATCCTCGAGTCAGCATGGGTCTATCAAAGGCAGC-3') (SEQ ID NO:6).
RNAi retroviral plasmids were constructed using pSUPER.retro.TM. as
the backbone plasmid (OligoEngine, Seattle, Wash.). Mouse Sirt4
RNAi-a and -b were constructed by targeting the sequences
5'-CGCTTCCAAGCCCTGAACC-3' (SEQ ID NO:7) and
5'-GGAGAGTTGCTGCCTTTAA-3' (SEQ ID NO:8), respectively.
[0249] Cell Culture
[0250] HEK293T and HEPG2 cells were grown in DMEM medium
supplemented with fetal bovine serum (FBS; 10% v/v), L-glutamine (2
mM), penicillin (100 units/ml), and streptomycin (100 .mu.g/ml).
MIN6 cells were grown in DMEM medium 1640, supplemented with FBS
(15% v/v), L-glutamine (2 mM), sodium bicarbonate (1 mM),
.beta.-mercaptoethanol (2.5 .mu.l/500 ml), penicillin (100
units/ml), and streptomycin (100 .mu.g/ml). Cells were cultured at
37.degree. C. in a humidified incubator containing 5% CO.sub.2. All
studies were performed using asynchronous log-phase cultures, and
MIN6 cells were used between passages 29 and 40.
[0251] Viralproduction and Infections
[0252] The pBp-amphotrophic viruses were produced by cotransfection
of 293T (Phoenix) cells with the pBABE.TM. and pSUPER.TM.
constructs as described (Picard et al. (2004) Nature 429:771-6).
Transfections were carried out using jetPEI.TM. (Qbiogene,
Carlsbad, Calif.). Virus was harvested 48 hours post-transfection
and added to MIN6 cells in the presence of polybrene (10 .mu.g/ml).
Eight hours after viral infection, fresh media was added. MIN6
cells were selected with puromycin (1 .mu.g/mL) 48 h following
infection.
[0253] Immunoprecipitation
[0254] Cells were harvested 48 h post-transfection by scraping into
PBS and centrifugation, and then incubated for 30 minutes in 3 ml
of ice-cold NP40 lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1%
NP40) containing protease inhibitors (EDTA-free COMPLETE.TM.; Roche
Molecular Biochemicals, Indianapolis, Ind.), and dithiothreitol (1
mM DTT; Sigma, St. Louis, Mo.). The lysate was clarified by
centrifugation for 15 min at 4.degree. C. at 14,000 rpm in a
tabletop centrifuge, and the resulting supernatant was incubated at
4.degree. C. for 2 h with Protein A resin (Sigma) that had been
conjugated with anti-FLAG M2 (Sigma) or the appropriate antibody.
Samples were washed four times with 10 ml of NP40 lysis buffer and
the hSirt4-FLAG protein was eluted by the addition of FLAG peptide
(400 .mu.g/ml, in 50 mM Tris pH 8.0, 150 mM NaCl, 10 mM DTT).
Alternatively, protein complexes were eluted from the Protein A
resin by adding protein sample buffer (50 mM Tris-HCl (pH 6.8)
containing 2% .beta.-mercaptoethanol, 2% SDS, 10% glycerol, 0.01%
bromophenol blue), and then analyzed by SDS-PAGE and Western
blotting.
[0255] Deacetylase Assay
[0256] Deacetylase activity of immunoprecipitated and dialyzed
hSirt4-FLAG (50 ng) was assessed by the FLUOR DE LYS.TM. kit
according to the manufacturer's directions (BIOMOL Research
Laboratories Inc., Pa.). hSirt1-FLAG was used as a positive
control. Experiments were performed in the presence and absence of
1 mM NAD.sup.+.
[0257] ADP-ribosylation Assays
[0258] ADP-ribosylation activity was assessed as described in Tanny
et al. (1999) Cell 99:735-45. Briefly, hSirt4-FLAG (50 ng),
hSirt5-FLAG (50 ng), or a buffer control were incubated with 3
.mu.g histones and 3 .mu.Ci of .sup.32P-NAD.sup.+ in 50 mM
Tris-HCl, pH 8.0, 150 mM NaCl, 10 mM DTT. The reaction mixture was
incubated for 1 hour at 37.degree. C., and the reaction stopped by
the addition of 90 .mu.l ice-cold 22% trichloroacetic acid (TCA),
incubated on ice for 15 minutes, and centrifuged at 13,000 g for 15
minutes. The pellets were resuspended and loaded onto 8.5% or 15%
SDS polyacrylamide gels. After electrophoresis, gels were stained
with Coomassie brilliant blue and .sup.32P in the gel detected by
autoradiography.
[0259] Western Blots
[0260] Cells were lysed in protein sample buffer, vortexed, boiled
for 5 minutes, and centrifuged at 17,000.times.g for 5 minutes.
Extracts corresponding to equivalent cell numbers (or 5-15 .mu.g
protein) were subjected to SDS-PAGE (4-15% gradient) according to
the method of Laemmli ((1970) Nature 227:680-685), and transferred
onto nitrocellulose membranes and incubated for 60 minutes with
blocking solution (3% lowfat milk, 0.1% TWEEN.RTM.-20 in PBS).
Membranes were incubated for 1 hour with appropriate primary
antibodies. The anti-mono-ADP-ribose antibody was as described
(Meyer et al. (1986) Eur J Biochem 155:157-65). After 3-10 minute
washes in blocking buffer, membranes were incubated with the
appropriate horseradish peroxidase-conjugated antibody (1:10,000).
The chemilumiiiescent signal was generated by incubation with the
ECL reagent (Amersham Biosciences, Piscataway, N.J.).
[0261] Fluorescence Microscopy
[0262] HepG2 cells were grown on coverslips and transfected with
phSirt4FLAG and pDSRed.TM.2-Mito (BD Biosciences, Palo Alto,
Calif.) 48 hours before the experiment. On the day of the
experiment, cells were washed twice with ice-cold PBS and then
incubated in paraformaldehyde (4% w/v) at room temperature for 10
minutes. The fixed cells were washed three times with PBS and then
incubated in TRITON.RTM. X-100 (0.5% v/v) for 10 min and then
washed with PBS. The coverslips were mounted with VECTASHIELD.RTM.
mounting media containing 4',6-diamidino-2-phenyindole, dilactate
(DAPI; Vector, Burlingame, Calif.). Fluorescence was visualized by
using a confocal microscope (Zeiss LSM510; Zeiss, Thomwood,
N.Y.).
[0263] Immunohistochemistry of Mouse Tissues
[0264] Immunohistochemistry of endogenous Sirt4 in mouse tissue was
performed using a microwave citrate unmasking protocol. Briefly,
mouse organ sections were deparaffinized in xylene and rehydrated
through an ethanol series. Slides were then placed in citrate
buffer (10 mM citric acid and 25 mM NaOH) and microwaved on high
power for 25 minutes. Slides were then cooled in running tap water
and soaked in PBS+0.1% Tween.RTM.-20 for 10 minutes at room
temperature. Slides were incubated in 10% donkey serum for 1 hour
at room temperature to block non-specific antibody binding. After 1
hour, primary antibody (1:100) was added, and slides were incubated
overnight at 4.degree. C. in a humidified chamber. A primary
antibody against mouse Sirt4 was produced in a rabbit against the
peptide LEMNFPLSSAAQDP (SEQ ID NO:9), which is in the C-terminal
region of the protein. Slides were then washed three times in
PBS+0.1% TWEEN.RTM.-20 at room temperature for 10 minutes each.
Secondary antibody (1:200) was added, and the slides were incubated
for 1 hour at 37.degree. C. Slides were then washed three times in
PBS+0.1% TWEEN.RTM.-20 at room temperature, counterstained with
DAPI, and coverslipped. Mouse monoclonal antibody for detection of
insulin was obtained from Zymed Laboratories (San Francisco,
Calif.). Secoridary antibodies were obtained from Molecular Probes
(Eugene, Oreg.): ALEXA FLUOR.RTM.488 donkey .alpha.-mouse IgG and
ALEXA FLUOR.RTM.594 donkey .alpha.-rabbit IgG. Images were acquired
using a SPOT.TM. digital camera (Diagnostic Instruments, Inc,
Sterling Heights, Mich.) mounted on a ECLIPSE E600.TM. fluorescence
microscope (Nikon).
[0265] Purification of Mitochondria
[0266] Mitochondria and other subcellular fractions were purified
by differential centrifugation as described (Schwer et al. (2002) J
Cell Biol 158:647-57). Briefly, cells were collected in SEM, (150
mM NaCl, 10 mM KCl, 10 mM Tris/HCl, pH 6.7), and then disrupted by
a Dounce homogenizer. Samples were centrifuged twice at
1,000.times.g for 5 min to pellet the nuclei and intact cells. To
separate the mitochondrial fraction from the cytosolic fraction, we
centrifuged the supernatant at 5,000.times.g for 10 min. We washed
the resulting pellet with sucrose/Mg.sup.2+ medium (10 mM Tris/HCl,
pH 6.7, 250 mM sucrose, 150 mM MgCl.sub.2,), recentrifuged, and
resuspended the final pellet in mitochondrial suspension medium (10
mM Tris/HCl, pH 7.0, 250 mM sucrose). 10 .mu.g of protein from each
fraction was separated by SDS-PAGE and then analyzed by western
blot.
[0267] Insulin Secretion Assays
[0268] MIN6 cells were plated in the wells of a 12-well plate at
least 24 hours before secretion assays. Cells were washed once with
Krebs-Ringer-HEPES buffer (KRB) containing 128 mMNaCl, 5 mM KCl,
2.7 mM CaCl.sub.2, 1.2 mM MgSO.sub.4, 1 mM Na.sub.2HPO.sub.4, 20 mM
HEPES (pH 7.4). After incubation for 1-3 hours in KRB containing 3
mM glucose, the cells were incubated in KRB containing either 3 or
16.7 mM glucose at 37.degree. C. for one hour. Insulin
concentration was measured by ELISA (Alpco Diagnostics; Windham,
N.H.).
[0269] Glutamate Dehydrogenase Assay
[0270] Glutamate dehydrogenase (50 .mu.g; Ultrapure from Sigma) was
incubated with or without Sirt4 (10 ng) in 200 .mu.g of
ADP-ribosylation buffer (50 mM Tris pH 8.0, 10 mM DTT, 150 mM NaCl)
in the presence or absence of 1 mM NAD or 1 mM nicotinamide at
37.degree. C. for the indicated times. Then, an aliquot of the
reaction was assayed for glutamate dehydrogenase activity. Activity
assays were performed at 25.degree. C. using a BioSpec-1601
spectrophotometer (Shimadzu Scientific Instruments, Inc; Braintree,
Mass.) at A.sub.340 based on the oxidation of NADH as described
previously. Data was collected for 5-10 minutes at 10 second
intervals. At the end of each experiment, less than 10% of
substrates had been depleted.
Example 1
Purification and Enzymatic Activity of Sirt4
[0271] This example demonstrates that Sirt4 possesses an
ADP-ribosyltransferase activity.
[0272] To investigate the enzymatic activity of hSirt4, two
constructs were designed (phSirt4FLAG, phSirt4) to overproduce
human Sirt4 with or without a C-terminal FLAG tag in mammalian
cells. When 293T cells were transfected with phSirt4FLAG, a 34 kDa
band was produced that was recognized by both anti-FLAG and
anti-hSirt4 antibodies (FIG. 1A). The FLAG-tagged protein migrated
more slowly than did the native version of Sirt4.
[0273] The enzymatic activity of Sirt4-FLAG protein
immunoprecipitated from mammalian cells in a nondenaturing buffer
was evaluated. Sirt4 was able to transfer a radioactive ADP-ribosyl
group from NAD to histones (FIG. 1C). This ADP-ribosylation
activity was inhibited by 1 mM nicotinamide (FIG. 1C). Mass
spectroscopy confirmed that Sirt4 transferred an approximately 500
Dalton moiety to Histone 2A. The molecular weight of this moiety
corresponds to the molecular weight of ADP-ribose (FIG. 1D).
Example 2
Subcellular Localization of Sirt4
[0274] This example demonstrates that Sirt4 is localized to
mitochondria.
[0275] Mammalian homologs of Sir2 have been localized to the
nucleus (Luo et al. (2001) Cell 107:137-48; Vaziri et al. (2001)
Cell 107:149-59), cytosol (North et al. (2003) Mol Cell 11:437-44),
and mitochondria (Onyango et al. (2002) Proc Natl Acad Sci USA
99:13653-8; Schwer et al. (2002) J Cell Biol 158:647-57). To
determine the localization of Sirt4, a plasmid was constructed
(phSirt4GFP) that fused the C-terminus of human Sirt4 with GFP
(Sirt4-GFP). When HepG2 cells were transiently transfected with
phSirt4GFP, a punctate staining pattern was observed (FIG. 2A).
Cotransfection experiments were performed with plasmids that target
DSRed 2 to the mitochondria (pDSRed.TM.2-Mito) or peroxisome
(pDSRed.TM.2-Peroxi). Sirt4-GFP colocalized with DSRed.TM.2 that
was targeted to the mitochondria (FIG. 2A), but did not colocalize
with the peroxisomal DSRed.TM. marker. Transfections with GFP alone
(i.e., GFP that is not fused to Sirt4) did not result in a distinct
localization pattern (FIG. 2B).
[0276] The mitochondrial localization of Sirt4 was verified by
subcellular fractionation. Sirt4-FLAG was detected in the cell
extract, whole cell/nucleus, and mitochondrial fractions, but not
in the cytosolic/light fraction (FIG. 2C). HSP60, a mitochondrial
marker, also co-fractionated with Sirt4-FLAG, while catalase could
be detected strongly in the cytosolic/light fraction, but not in
the mitochondrial fraction (FIG. 2C). In separate experiments,
endogenous Sirt4 was detected in the mitochondrial fraction of both
293T and mouse MIN6 cells. These results strongly suggest that
Sirt4 is localized to the mitochondria of human and mouse
cells.
[0277] The N-terminus of Sirt4-FLAG was analyzed by Edman
degradation to determine whether Sirt4 is post-translationally
cleaved during mitochondrial import. As depicted in FIG. 2D, the
first 28 amino acid residues were absent from the N-terminus,
indicating that the protein had been post-translationally
processed. In addition, a Sirt4-FLAG construct lacking the first 28
amino acids was not stable in cells. Thus, Sirt4 is
post-translationally processed, likely by cleavage after serine 28,
upon targeting to the mitochondria.
Example 3
Expression of Sirt4 in Mouse Tissues
[0278] This example demonstrates the distribution of Sirt4 in mouse
tissues.
[0279] To understand the biological function of Sirt4, its
endogenous expression profile in mouse tissues was determined by
immunohistochemistry using antibodies generated against the
C-terminus of mouse Sirt4. High levels of staining were observed in
neurons and pancreatic islets. Moderate staining was observed in
muscle, heart, liver, and kidney, and testes. Sirt4 was expressed
strongly in islets of the pancreas, but not in surrounding goblet
or acinar cells (FIG. 3A and B). The expression of Sirt4 overlapped
with the expression of insulin within islets (FIG. 3), indicating
that Sirt4 may be involved in normal .beta.-cell function.
Example 4
Sirt4 Regulates the Production and Secretion of Insulin
[0280] To test the role of Sirt4 in .beta.-cell biology, insulin
secretion in the mouse insulinoma, MIN6, .beta.-cell line was
studied. Using two separate RNAi constructs, stable MIN6 cells that
had decreased levels of Sirt4 when compared to control cells that
were infected with the empty vector as a control were engineered
(FIG. 4A). To study insulin secretion, these cells were incubated
in KRB containing 3 mM glucose, and then shifted to KRB containing
either 3 (low) or 16.7 mM (high) glucose. Sirt4 RNAi treated cells
secreted 2-fold more insulin when compared to control cells at both
low and high glucose concentrations (FIG. 4B). The Sirt4 RNAi
treated cells maintained a normal ability to respond to glucose, as
the Sirt4 RNAi-expressing cells still responded to 16.7 mM glucose
by secreting proportional insulin. Thus, the magnitude of insulin
secretion differed between normal and Sirt4 RNAi cells, and this
difference was maintained at difference glucose levels. The
intracellular insulin level was determined at low and high glucose
concentrations, and the Sirt4 RNAi treated cells were found to
contain 2-fold higher levels of insulin (FIG. 4C).
Example 5
Sirt4 Interacts With Mitochondrial Proteins
[0281] To identify transient, yet biochemically relevant
interactions, ADP-ribosylation within the mitochondria was
investigated. Isolated mitochondria were incubated with
[.sup.32P]-NAD to identify ADP-ribosylated mitochondrial proteins.
In agreement with previous studies (Ziegler (2000) Eur J Biochem
267:1550-64), three predominant modified proteins were observed
(FIG. 5A), one of which has been identified as glutamate
dehydrogenase (GDH) (Herrero-Yraola et al. (2001) EMBO J
20:2404-12). Anti-glutamate dehydrogenase immunoprecipitated a 55
kDa-radioactive protein (FIG. 5B). By contrast, immunoprecipitation
by antibodies against hSirt4 or the prevalent mitochondrial
protein, ANT1 did not give a significant radioactive band that
migrated at 55 kDa.
[0282] The interaction between Sirt4 and mitochondrial proteins in
293T cells that overexpressed hSirt4-FLAG was investigated.
Immunoprecipitations using .alpha.-FLAG antibodies isolated a
complex between Sirt4-FLAG and glutamate dehydrogenase (GDH),
insulin degrading enzyme (IDE), and adenine nucleotide transporter
(ANT) in Sirt4 overexpressing cells, but not control cells (FIG.
5C). The complex between Sirt4-FLAG and GDH was also be identified
by immunoprecipitation with .alpha.-glutamate dehydrogenase
antibodies (FIG. 5C).
[0283] MIN6 cells were used to investigate the endogenous
interaction between Sirt4 and GDH, IDE, and ANT. Sirt4 was
immunoprecipitated from MIN6 cells using the C-terminal antibody,
and the blots were probed with antibodies against GDH, IDE, and
ANT. Sirt4 was able to form an endogenous complex with glutamate
dehydrogenase (FIG. 5D), insulin degrading enzyme, and adenine
nucleotide transporter in MIN6 cells. These results indicate that
Sirt4 can form a complex with each of GDH, IDE, and ANT under
physiological protein concentrations.
[0284] Mitochondria from 293T control or Sirt4-FLAG overexpressing
cells were incubated with [.sup.32P]-NAD, and the ADP-ribosylation
state of glutamate dehydrogenase was measured by
immunoprecipitation and subsequent autoradiography. Sirt4
overexpression enhanced the ADP-ribosylation of glutamate
dehydrogenase (FIG. 5E). Moreover, a small amount of labeled
glutamate dehydrogenase could be detected in a complex with Sirt4
(FIG. 5E).
[0285] It is believed that the Sirt4 may act in .beta.-cells to
control insulin levels by interactions with all of GDH, ANT, and
IDE.
Example 6
Sirt4 Inhibits Glutamate Dehydrogenase Activity
[0286] Sirt4 or a buffer control was incubated with bovine
recombinant glutamate dehydrogenase for one hour in the presence of
NAD. Then, the glutamate dehydrogenase activity was assessed by
monitoring the disappearance of NADH (A.sub.340). Sirt4, but not
the buffer control, significantly reduced the enzymatic activity of
glutamate dehydrogenase. To verify that this inhibition was
dependent on the availability of NAD.sup.+, we incubated Sirt4 with
or without NAD.sup.+ with glutamate dehydrogenase for various
times. Sirt4 inhibited glutamate dehydrogenase activity only in the
presence of NAD.sup.+ (FIG. 6B), and within 2 hours the activity of
glutamate dehydrogenase was inhibited by 50%. After incubating for
12 hours, the activity of glutamate dehydrogenase was inhibited by
more than 90%.
[0287] MIN6 cells were co-infected with RNAi plasmids targeting
Sirt4 and glutamate dehydrogenase. Co-infected cells contained less
insulin than did Sirt4-RNAi cells (FIG. 7A), indicating that
elevated glutamate dehydrogenase activity could account for the
increase in insulin secreted. To test further whether glutamate
dehydrogenase activity was altered in Sirt4 RNAi cells, insulin
secretion assays were performed in the presence and absence of a
glutamate dehydrogenase activator, BCH, which is known to stimulate
insulin secretion. Control cells that were stimulated with both BCH
and 16.7 mM glucose demonstrated enhanced insulin secretion when
compared to cells stimulated with 16.7 mM glucose alone (FIG. 7B).
Sirt4 RNAi treated cells did not secrete more insulin when
incubated with BCH and 16.7 mM glucose when compared to the 16.7 mM
glucose condition, indicating that glutamate dehydrogenase activity
may be already elevated in these cells. Finally, the activity of
glutamate dehydrogenase from purified mitochondria isolated from
normal or Sirt4 RNAi treated cells was measured. A 20% increase in
the enzymatic activity of glutamate dehydrogenase in the Sirt4 RNAi
treated cells was found (FIG. 7C). Taken together, these data show
that a decrease in the levels of Sirt4 leads to a functional gain
in glutamate dehydrogenase activity.
Example 7
Sirt4 Function in .beta.-cell Energetics
[0288] To explore the mechanism of how Sirt4 and glutamate
dehydrogenase regulate insulin secretion from pancreatic
.beta.-cells, two parameters regulated by the mitochondria and
change upon glucose stimulation were measured: ATP concentration
and oxygen consumption. As expected, control cells showed an
increase in ATP content when shifted from 3 to 16.7 mM glucose
(FIG. 8A). Sirt4 RNAi treated cells demonstrated a significant
increase in ATP content in both 3 and 16.7 mM glucose
concentrations, as compared to control cells. These cells still
produced more ATP when stimulated with 16.7 mM glucose, indicating
that their glucose metabolism was intact.
[0289] Oxygen consumption is another measure of the cell's
metabolic state and will increase with glucose stimulation. The
reduction of Sirt4 levels caused cells to consume 2-fold more
oxygen in 16.7 mM glucose than the control cells (FIG. 8B). As both
ATP level and rate of oxygen consumption increased in Sirt4-RNAi
cells, their metabolic rate is up-regulated in a coupled
process.
Example 8
Determination of Putative Regulatory Sequences for SIRT4
[0290] To determine conserved putative transcription factor binding
sites involved in the regulation of Sirt4, genomic sequences 5 kb
upstream of the start of the first codon of human and mouse Sirt4
were input to the rVISTA.TM. program (Loots et al. (2002) Genome.
Res. 12:832-839). The output of the program is shown in FIG. 9.
[0291] All patents, patent applications, and references cited
herein are hereby incorporated by reference in their entirety.
Other embodiments are within the scope of the following claims.
Sequence CWU 1
1
11 1 314 PRT Homo sapiens 1 Met Lys Met Ser Phe Ala Leu Thr Phe Arg
Ser Ala Lys Gly Arg Trp 1 5 10 15 Ile Ala Asn Pro Ser Gln Pro Cys
Ser Lys Ala Ser Ile Gly Leu Phe 20 25 30 Val Pro Ala Ser Pro Pro
Leu Asp Pro Glu Lys Val Lys Glu Leu Gln 35 40 45 Arg Phe Ile Thr
Leu Ser Lys Arg Leu Leu Val Met Thr Gly Ala Gly 50 55 60 Ile Ser
Thr Glu Ser Gly Ile Pro Asp Tyr Arg Ser Glu Lys Val Gly 65 70 75 80
Leu Tyr Ala Arg Thr Asp Arg Arg Pro Ile Gln His Gly Asp Phe Val 85
90 95 Arg Ser Ala Pro Ile Arg Gln Arg Tyr Trp Ala Arg Asn Phe Val
Gly 100 105 110 Trp Pro Gln Phe Ser Ser His Gln Pro Asn Pro Ala His
Trp Ala Leu 115 120 125 Ser Thr Trp Glu Lys Leu Gly Lys Leu Tyr Trp
Leu Val Thr Gln Asn 130 135 140 Val Asp Ala Leu His Thr Lys Ala Gly
Ser Arg Arg Leu Thr Glu Leu 145 150 155 160 His Gly Cys Met Asp Arg
Val Leu Cys Leu Asp Cys Gly Glu Gln Thr 165 170 175 Pro Arg Gly Val
Leu Gln Glu Arg Phe Gln Val Leu Asn Pro Thr Trp 180 185 190 Ser Ala
Glu Ala His Gly Leu Ala Pro Asp Gly Asp Val Phe Leu Ser 195 200 205
Glu Glu Gln Val Arg Ser Phe Gln Val Pro Thr Cys Val Gln Cys Gly 210
215 220 Gly His Leu Lys Pro Asp Val Val Phe Phe Gly Asp Thr Val Asn
Pro 225 230 235 240 Asp Lys Val Asp Phe Val His Lys Arg Val Lys Glu
Ala Asp Ser Leu 245 250 255 Leu Val Val Gly Ser Ser Leu Gln Val Tyr
Ser Gly Tyr Arg Phe Ile 260 265 270 Leu Thr Ala Trp Glu Lys Lys Leu
Pro Ile Ala Ile Leu Asn Ile Gly 275 280 285 Pro Thr Arg Ser Asp Asp
Leu Ala Cys Leu Lys Leu Asn Ser Arg Cys 290 295 300 Gly Glu Leu Leu
Pro Leu Ile Asp Pro Cys 305 310 2 333 PRT Mus musculus 2 Met Ser
Gly Leu Thr Phe Arg Pro Thr Lys Gly Arg Trp Ile Thr His 1 5 10 15
Leu Ser Arg Pro Arg Ser Cys Gly Pro Ser Gly Leu Phe Val Pro Pro 20
25 30 Ser Pro Pro Leu Asp Pro Glu Lys Ile Lys Glu Leu Gln Arg Phe
Ile 35 40 45 Ser Leu Ser Lys Lys Leu Leu Val Met Thr Gly Ala Gly
Ile Ser Thr 50 55 60 Glu Ser Ser Ile Pro Asp Tyr Arg Ser Glu Lys
Val Gly Leu Tyr Ala 65 70 75 80 Arg Thr Asp Arg Arg Pro Ile Gln His
Ile Asp Phe Val Arg Ser Ala 85 90 95 Pro Val Arg Gln Arg Tyr Trp
Ala Arg Asn Phe Val Gly Trp Pro Gln 100 105 110 Phe Ser Ser His Gln
Pro Asn Pro Ala His Trp Ala Leu Ser Asn Trp 115 120 125 Glu Arg Leu
Gly Lys Leu His Trp Leu Val Thr Gln Asn Val Asp Ala 130 135 140 Leu
His Ser Lys Ala Gly Ser Gln Arg Leu Thr Glu Leu His Gly Cys 145 150
155 160 Met His Arg Val Leu Cys Leu Asn Cys Gly Glu Gln Thr Ala Arg
Arg 165 170 175 Val Leu Gln Glu Arg Phe Gln Ala Leu Asn Pro Ser Trp
Ser Ala Glu 180 185 190 Ala Gln Gly Val Ala Pro Asp Gly Asp Val Phe
Leu Thr Glu Glu Gln 195 200 205 Val Arg Ser Phe Gln Val Pro Cys Cys
Asp Arg Cys Gly Gly Pro Leu 210 215 220 Lys Pro Asp Val Val Phe Phe
Gly Asp Thr Val Asn Pro Asp Lys Val 225 230 235 240 Asp Phe Val His
Arg Arg Val Lys Glu Ala Asp Ser Leu Leu Val Val 245 250 255 Gly Ser
Ser Leu Gln Val Tyr Ser Gly Tyr Arg Phe Ile Leu Thr Ala 260 265 270
Arg Glu Gln Lys Leu Pro Ile Ala Ile Leu Asn Ile Gly Pro Thr Arg 275
280 285 Ser Asp Asp Leu Ala Cys Leu Lys Leu Asp Ser Arg Cys Gly Glu
Leu 290 295 300 Leu Pro Leu Ile Asp Pro Arg Arg Gln His Ser Asp Val
Gln Arg Leu 305 310 315 320 Glu Met Asn Phe Pro Leu Ser Ser Ala Ala
Gln Asp Pro 325 330 3 262 PRT Homo sapiens 3 Leu Gln Arg Phe Ile
Thr Leu Ser Lys Arg Leu Leu Val Met Thr Gly 1 5 10 15 Ala Gly Ile
Ser Thr Glu Ser Gly Ile Pro Asp Tyr Arg Ser Glu Lys 20 25 30 Val
Gly Leu Tyr Ala Arg Thr Asp Arg Arg Pro Ile Gln His Gly Asp 35 40
45 Phe Val Arg Ser Ala Pro Ile Arg Gln Arg Tyr Trp Ala Arg Asn Phe
50 55 60 Val Gly Trp Pro Gln Phe Ser Ser His Gln Pro Asn Pro Ala
His Trp 65 70 75 80 Ala Leu Ser Thr Trp Glu Lys Leu Gly Lys Leu Tyr
Trp Leu Val Thr 85 90 95 Gln Asn Val Asp Ala Leu His Thr Lys Ala
Gly Ser Arg Arg Leu Thr 100 105 110 Glu Leu His Gly Cys Met Asp Arg
Val Leu Cys Leu Asp Cys Gly Glu 115 120 125 Gln Thr Pro Arg Gly Val
Leu Gln Glu Arg Phe Gln Val Leu Asn Pro 130 135 140 Thr Trp Ser Ala
Glu Ala His Gly Leu Ala Pro Asp Gly Asp Val Phe 145 150 155 160 Leu
Ser Glu Glu Gln Val Arg Ser Phe Gln Val Pro Thr Cys Val Gln 165 170
175 Cys Gly Gly His Leu Lys Pro Asp Val Val Phe Phe Gly Asp Thr Val
180 185 190 Asn Pro Asp Lys Val Asp Phe Val His Lys Arg Val Lys Glu
Ala Asp 195 200 205 Ser Leu Leu Val Val Gly Ser Ser Leu Gln Val Tyr
Ser Gly Tyr Arg 210 215 220 Phe Ile Leu Thr Ala Trp Glu Lys Lys Leu
Pro Ile Ala Ile Leu Asn 225 230 235 240 Ile Gly Pro Thr Arg Ser Asp
Asp Leu Ala Cys Leu Lys Leu Asn Ser 245 250 255 Arg Cys Gly Glu Leu
Leu 260 4 45 DNA Artificial Sequence Primer 4 caccgcggtg gcggccgcat
gaagatgagc tttgcgttga ctttc 45 5 37 DNA Artificial Sequence Primer
5 cttgtaatcc tcgaggcatg ggtctatcaa aggcagc 37 6 40 DNA Artificial
Sequence Primer 6 cttgtaatcc tcgagtcagc atgggtctat caaaggcagc 40 7
19 DNA Artificial Sequence Primer 7 cgcttccaag ccctgaacc 19 8 19
DNA Artificial Sequence Primer 8 ggagagttgc tgcctttaa 19 9 14 PRT
Mus musculus 9 Leu Glu Met Asn Phe Pro Leu Ser Ser Ala Ala Gln Asp
Pro 1 5 10 10 30 PRT Homo sapiens 10 Met Ala Phe Trp Gly Trp Arg
Ala Ala Ala Ala Leu Arg Leu Trp Gly 1 5 10 15 Arg Val Val Glu Arg
Val Glu Ala Gly Gly Gly Val Gly Pro 20 25 30 11 32 PRT Homo sapiens
11 Met Lys Met Ser Phe Ala Leu Thr Phe Arg Ser Ala Lys Gly Arg Trp
1 5 10 15 Ile Ala Asn Pro Ser Gln Pro Cys Ser Lys Ala Ser Ile Gly
Leu Phe 20 25 30
* * * * *