U.S. patent application number 11/196524 was filed with the patent office on 2006-12-28 for novel human uncoupling proteins and polynucleotides encoding the same.
Invention is credited to Brian Mathur, Arthur T. Sands, C. Alexander JR. Turner, Brian Zambrowicz.
Application Number | 20060293512 11/196524 |
Document ID | / |
Family ID | 26817141 |
Filed Date | 2006-12-28 |
United States Patent
Application |
20060293512 |
Kind Code |
A1 |
Turner; C. Alexander JR. ;
et al. |
December 28, 2006 |
Novel human uncoupling proteins and polynucleotides encoding the
same
Abstract
The present invention relates to methods and compositions for
the treatment of biological disorders regulatable by the controlled
expression or inhibition of the described NUCPs.
Inventors: |
Turner; C. Alexander JR.;
(The Woodlands, TX) ; Mathur; Brian; (The
Woodlands, TX) ; Zambrowicz; Brian; (The Woodlands,
TX) ; Sands; Arthur T.; (The Woodlands, TX) |
Correspondence
Address: |
LEXICON GENETICS INCORPORATED
8800 TECHNOLOGY FOREST PLACE
THE WOODLANDS
TX
77381-1160
US
|
Family ID: |
26817141 |
Appl. No.: |
11/196524 |
Filed: |
August 3, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10165813 |
Jun 7, 2002 |
6987178 |
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11196524 |
Aug 3, 2005 |
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09501558 |
Feb 9, 2000 |
6403784 |
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10165813 |
Jun 7, 2002 |
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60119228 |
Feb 9, 1999 |
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60158458 |
Oct 8, 1999 |
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Current U.S.
Class: |
536/23.2 |
Current CPC
Class: |
C07K 14/47 20130101;
A61K 38/00 20130101 |
Class at
Publication: |
536/023.2 |
International
Class: |
C07H 21/04 20060101
C07H021/04 |
Claims
1. (canceled)
2. (canceled)
3. (canceled)
4. (canceled)
5. (canceled)
6. An isolated polypeptide comprising the amino acid sequence of
SEQ ID NO:2 or SEQ ID NO:4.
7. An antibody that selectively binds to the polypeptide sequence
of SEQ ID NO:2 or SEQ ID NO:4.
Description
[0001] The present application claims priority to U.S. application
Ser. No. 60/119,228, filed Feb. 9, 1999, and U.S. application Ser.
No. 60/158,458, filed Oct. 8, 1999, which are herein incorporated
by reference in their entirety.
1. INTRODUCTION
[0002] The present invention relates to the discovery,
identification, and characterization of novel human polynucleotide
sequences and the novel polypeptides encoded thereby. The invention
encompasses the described polynucleotides, host cell expression
systems, the encoded proteins or polypeptides, and fusion proteins
and peptides derived therefrom, antibodies to the encoded proteins
or peptides, and genetically engineered animals that lack
functional forms of the genes encoding the disclosed sequences,
over express the disclosed sequences, as well as antagonists and
agonists of the proteins, along with other compounds that modulate
the expression or activity of the proteins encoded by the disclosed
sequences that can be used for diagnosis, drug screening, clinical
trial monitoring, the treatment of physiological or behavioral
disorders, or otherwise improving the quality of life.
2. BACKGROUND OF THE INVENTION
[0003] Uncoupling proteins (UCPs) are found in the mitochondria,
but are encoded within the nucleus. In the mitochondria, UCPs
uncouple, or regulate, the gradient that drives energy production
in the cell/body. As such, UCPs effectively modulate the efficiency
of energy production in the body, and hence body metabolism. Given
the role of UCPs in the body, they are thought to be important
targets for the study of thermogenesis, obesity, cachexia, and
other metabolically related physiological functions, diseases, and
disorders.
3. SUMMARY OF THE INVENTION
[0004] The present invention relates to the discovery,
identification, and characterization of nucleotides that encode
novel human UCPS, and the corresponding amino acid sequences
encoded by the disclosed sequences. The novel human uncoupling
proteins (NUCPS) described for the first time herein share
structural relatedness with other mammalian uncoupling proteins and
brain mitochondrial carrier proteins. The novel human nucleic acid
sequences described herein encode proteins of 291 and 293 amino
acids in length (see SEQ ID NOS:2 and 4).
[0005] A murine homologue of the described NUCPs has been
identified and a "knockout" ES cell line has been produced using
the methods described in U.S. application Ser. No. 08/942,806,
herein incorporated by reference. Alternatively, such knockout
cells and animals can be produced using conventional methods for
generating genetically engineered animals and cells (see, for
example, PCT Applic. No. PCT/US98/03243, filed Feb. 20, 1998,
herein incorporated by reference). Accordingly, an additional
aspect of the present invention includes knockout cells and animals
having genetically engineered mutations in gene encoding the
presently described NUCPs.
[0006] The invention encompasses the nucleotides presented in the
Sequence Listing, host cells expressing such nucleotides, and the
expression products of such nucleotides, and: (a) nucleotides that
encode mammalian homologs of the described genes, including the
specifically described NUCPs, and the NUCP products; (b)
nucleotides that encode one or more portions of the NUCPs that
correspond to functional domains, and the polypeptide products
specified by such nucleotide sequences, including but not limited
to the novel regions of any active domain(s); (c) isolated
nucleotides that encode mutant versions, engineered or naturally
occurring, of the described NUCPs in which all or a part of at
least one domain is deleted or altered, and the polypeptide
products specified by such nucleotide sequences, including but not
limited to soluble proteins and peptides in which all or a portion
of the signal sequence in deleted; (d) nucleotides that encode
chimeric fusion proteins containing all or a portion of a coding
region of NUCP, or one of its domains (e.g., a transmembrane
domain, accessory protein/self-association domain, etc.) fused to
another peptide or polypeptide.
[0007] The invention also encompasses agonists and antagonists of
NUCPs, including small molecules, large molecules, mutant NUCPs, or
portions thereof that compete with or bind to native NUCPs,
antibodies, and nucleotide sequences that can be used to inhibit
the expression of the described NUCPs (e.g., antisense, ribozyme
molecules, and gene or regulatory sequence replacement constructs)
or to enhance the expression of the described NUCPs (e.g.,
expression constructs that place the described genes under the
control of a strong promoter system), as well as transgenic animals
that express a NUCP transgene, or "knockouts" (which can be
conditional) that do not express functional NUCP.
[0008] Further, the present invention also relates to methods for
using of the described NUCP products for the identification of
compounds that modulate, i.e., act as agonists or antagonists, of
NUCP expression and/or NUCP product activity. Such compounds can be
used as therapeutic agents for the treatment of any of a wide
variety of symptomatic representations of biological disorders or
imbalances.
[0009] An additional embodiment of the present invention includes
therapy and treatments mediated by NUCP gene delivery. Gene
delivery can be to somatic or stem cells, and may be effected using
viral (i.e., retrovirus, adeno-associated virus, etc.) or non-viral
(i.e., cationic lipids, formulations using "naked" DNA, etc.)
methods.
4. DESCRIPTION OF THE SEQUENCE LISTING AND FIGURES
[0010] The Sequence Listing provides the sequences of the NUCP
polynucleotides, and the amino acid sequences encoded thereby.
5. DETAILED DESCRIPTION OF THE INVENTION
[0011] The NUCPs described for the first time herein are novel
proteins that are expressed, inter alia, in gene trapped human
cells, human lymph node or kidney cells, and/or ES cells. The NUCPs
exert biological effect by regulating the efficiency of energy
generation in the body with the result being that excess resources
are converted to heat or are otherwise stored as fat, etc.
Regulating the function of a NUCP product will effect NUCP-mediated
processes with resulting effects on fat production and usage,
superoxide generation and regulation, and all biological properties
and functions that are tied to fatty acid metabolism. Because of
these important roles, UCPs have been the focus of intense
scientific scrutiny (see PCT Application No. PCT/EP98/02645, U.S.
Pat. Nos. 5,853,975, 5,741,666 and 5,702,902 all of which are
herein incorporated by reference in their entirety).
[0012] The present invention encompasses the use of the described
NUCP nucleotides, NUCPs and NUCP peptides therefrom, as well as
antibodies, preferably humanized monoclonal antibodies, or binding
fragments, domains, or fusion proteins thereof, or antiidiotypic
variants derived therefrom, that bind NUCP (which can, for example,
also act as NUCP agonists or antagonists), other antagonists that
inhibit binding activity or expression, or agonists that activate
NUCP receptor activity or increase NUCP expression, in the
diagnosis and/or treatment of disease.
[0013] In particular, the invention described in the subsections
below encompasses NUCP polypeptides or peptides corresponding to
functional domains of NUCPs, mutated, truncated or deleted NUCPs
(e.g., NUCPs missing one or more functional domains or portions
thereof), NUCP fusion proteins (e.g., where NUCP or a functional
domain of NUCP is fused to an unrelated protein or peptide such as
an immunoglobulin constant region, i.e., IgFc), nucleotide
sequences encoding such products, and host cell expression systems
that can produce such NUCP products.
[0014] The invention also encompasses antibodies and anti-idiotypic
antibodies (including Fab fragments), antagonists and agonists of
the NUCP, as well as compounds or nucleotide constructs that
inhibit expression of a NUCP gene (transcription factor inhibitors,
antisense and ribozyme molecules, or gene or regulatory sequence
replacement constructs), or promote expression of a NUCP (e.g.,
expression constructs in which a NUCP coding sequence is
operatively associated with expression control elements such as
promoters, promoter/enhancers, etc.). The invention also relates to
host cells and animals genetically engineered to express a NUCP (or
mutant variants thereof) or to inhibit or "knockout" expression of
an animal homolog of a NUCP gene.
[0015] The NUCPs, NUCP peptides, and NUCP fusion proteins derived
therefrom, NUCP nucleotide sequences, antibodies, antagonists and
agonists can be useful for the detection of mutant NUCPs or
inappropriately expressed NUCPs for the diagnosis of biological
disorders (high blood pressure, obesity, etc.) and disease. The
NUCP products or peptides, NUCP fusion proteins, NUCP nucleotide
sequences, host cell expression systems, antibodies, antagonists,
agonists and genetically engineered cells and animals can also be
used for screening for drugs (or high throughput screening of
combinatorial libraries) effective in the treatment of the
symptomatic or phenotypic manifestations of perturbing the normal
function of NUCP in the body. The use of engineered host cells
and/or animals may offer an advantage in that such systems allow
not only for the identification of compounds that bind to an
endogenous NUCP, but can also identify compounds that facilitate or
inhibit NUCP-mediated uncoupling.
[0016] Of particular interest are genetically engineered nucleotide
constructs, or expression vectors, that encode NUCP products and
derivatives (NUCP peptides, fusions, etc). Nucleotide constructs
encoding such NUCP products and derivatives can be used to
genetically engineer host cells to express such products in vivo;
these genetically engineered cells function as "bioreactors" in the
body delivering a continuous supply of a NUCP product, NUCP
peptide, or NUCP fusion protein to the body. Nucleotide constructs
encoding functional NUCPs, mutant NUCPs, as well as antisense and
ribozyme molecules can also be used in "gene therapy" approaches
for the modulation of NUCP expression. Thus, the invention also
encompasses pharmaceutical formulations and methods for treating
biological disorders.
[0017] Therapeutic gene delivery of the described NUCP nucleotides
can be effected by a variety of methods. For example, methods of
retroviral human gene therapy are described in, inter alia, U.S.
Pat. Nos. 5,399,346 and 5,858,740; adenoviral vectors for gene
therapy/delivery are described in U.S. Pat. No. 5,824,544;
adeno-associated viral vectors are described in U.S. Pat. Nos.
5,843,742, 5,780,280, and 5,846,528; herpes virus vectors are
described in U.S. Pat. No. 5,830,727, and other vectors and methods
of nonvirally (e.g., polynucleotides that are not encapsulated by
viral capsid protein, "naked" DNA, or DNA formulated in lipid or
chemical complexes) introducing foreign genetic material of
recombinant origin into a host mammalian, and preferably human,
cell are described in U.S. Pat. Nos. 5,827,703 and 5,840,710 all of
which are herein incorporated by reference in their entirety. When
the above methods are applied to selectively express or inhibit the
expression of a NUCP in tumor/diseased cells, the described methods
and compositions can be used as chemotherapeutic agents for the
treatment of cancer and other diseases and disorders.
[0018] Various aspects of the invention are described in greater
detail in the subsections below.
[0019] 5.1. The NUCP Polynucleotides
[0020] The cDNA sequences (SEQ ID NOS:1 and 3) and deduced amino
acid sequences (SEQ ID NOS:2 and 4) of the described NUCPs are
presented in the Sequence Listing. The NUCP cDNA sequences were
obtained from human lymph node, kidney, and fetal brain cDNA
libraries (Edge Biosystems, Gaithersburg, Md.) using probes and/or
primers generated from gene trapped sequence tags and a human
homolog of the described NUCPs. RT-PCR analysis indicated that
expression of the described NUCPs can be detected in, inter alia,
human cerebellum, spinal cord, thymus, spleen, lymph node, bone
marrow, trachea, lung, kidney, fetal liver, prostate, testis,
thyroid, salivary gland, stomach, heart, uterus, and mammary gland,
with particularly strong expression in kidney, adrenal gland, and
skeletal muscle. The above expression studies were largely verified
by Northern analysis that also detected particularly strong
expression in human skeletal muscle, heart, adrenal gland, and
kidney.
[0021] The NUCPs of the present invention include: (a) the human
DNA sequences presented in the Sequence Listing and additionally
contemplates any nucleotide sequence encoding a contiguous and
functional NUCP open reading frame (ORF) that hybridizes to a
complement of the DNA sequence presented in the Sequence Listing
under highly stringent conditions, e.g., hybridization to
filter-bound DNA in 0.5 M NaHPO.sub.4, 7% sodium dodecyl sulfate
(SDS), 1 M EDTA at 65.degree. C., and washing in 0.1.times.SSC/0.1%
SDS at 68.degree. C. (Ausubel F. M. et al., eds., 1989, Current
Protocols in Molecular Biology, Vol. I, Green Publishing
Associates, Inc., and John Wiley & sons, Inc., New York, at p.
2.10.3) and encodes a functionally equivalent gene product.
Additionally contemplated are any nucleotide sequences that
hybridize to the complement of the DNA sequence that encode and
express an amino acid sequence presented in the Sequence Listing
under moderately stringent conditions, e.g., washing in
0.2.times.SSC/0.1% SDS at 42.degree. C. (Ausubel et al., 1989,
supra), yet still encode a functionally equivalent NUCP product.
Functional equivalents of a NUCP include-naturally occurring NUCPs
present in other species, and mutant NUCPs whether naturally
occurring or engineered. The invention also includes degenerate
variants of the disclosed sequences.
[0022] The invention also includes nucleic acid molecules,
preferably DNA molecules, that hybridize to, and are therefore the
complements of, the described NUCP nucleotide sequences. Such
hybridization conditions may be highly stringent or less highly
stringent, as described above. In instances wherein the nucleic
acid molecules are deoxyoligonucleotides ("DNA oligos"), such
molecules are particularly about 16 to about 100 bases long, about
20 to about 80, or about 34 to about 45 bases long, or any
variation or combination of sizes represented therein that
incorporate a contiguous region of sequence first disclosed in the
present Sequence Listing. Such oligonucleotides can be used in
conjunction with the polymerase chain reaction (PCR) to screen
libraries, isolate clones, and prepare cloning and sequencing
templates, etc. Alternatively, the NUCP oligonucleotides can be
used as hybridization probes for screening libraries or assessing
gene expression patterns (particularly using a micro array or
high-throughput "chip" format). Chip applications can involve a
series of the described NUCP oligonucleotide sequences, or the
complements thereof, can be used to represent all or a portion of
the described NUCP sequences. The oligonucleotides, typically
between about 16 to about 40 (or any whole number within the stated
range) nucleotides in length may partially overlap each other
and/or the NUCP sequence may be represented using oligonucleotides
that do not overlap. Accordingly, the described NUCP polynucleotide
sequences shall typically comprise at least about two or three
distinct oligonucleotide sequences of at least about 18, and
preferably about 25, nucleotides in length that are each first
disclosed in the described Sequence Listing. Such oligonucleotide
sequences may begin at any nucleotide present within a sequence in
the Sequence Listing and proceed in either a sense (5'-to-3')
orientation vis-a-vis the described sequence or in an antisense
orientation.
[0023] For oligonucleotide probes, highly stringent conditions may
refer, e.g., to washing in 6.times.SSC/0.05% sodium pyrophosphate
at 37.degree. C. (for 14-base oligos), 48.degree. C. (for 17-base
oligos), 55.degree. C. (for 20-base oligos), and 60.degree. C. (for
23-base oligos). These nucleic acid molecules may encode or act as
NUCP gene antisense molecules, useful, for example, in NUCP gene
regulation (for and/or as antisense primers in amplification
reactions of NUCP gene nucleic acid sequences). With respect to
NUCP gene regulation, such techniques can be used to regulate
biological functions. Further, such sequences may be used as part
of ribozyme and/or triple helix sequences that are also useful for
NUCP gene regulation. Additionally, the antisense oligonucleotides
may comprise at least one modified base moiety which is selected
from the group including but not limited to 5-fluorouracil,
5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine,
4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine.
[0024] The antisense oligonucleotide may also comprise at least one
modified sugar moiety selected from the group including but not
limited to arabinose, 2-fluoroarabinose, xylulose, and a
hexose.
[0025] In yet another embodiment, the antisense oligonucleotide
comprises at least one modified phosphate backbone selected from
the group consisting of a phosphorothioate, a phosphorodithioate, a
phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a
methylphosphonate, an alkyl phosphotriester, and a formacetal or
analog thereof.
[0026] In yet another embodiment, the antisense oligonucleotide is
an .alpha.-anomeric oligonucleotide. An .alpha.-anomeric
oligonucleotide forms specific double-stranded hybrids with
complementary RNA in which, contrary to the usual .beta.-units, the
strands run parallel to each other (Gautier et al., 1987, Nucl.
Acids Res. 15:6625-6641). The oligonucleotide is a
2'-O-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.
15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987,
FEBS Lett. 215:327-330).
[0027] Oligonucleotides of the invention may be synthesized by
standard methods known in the art, e.g. by use of an automated DNA
synthesizer (such as are commercially available from Biosearch,
Applied Biosystems, etc.). As examples, phosphorothioate
oligonucleotides may be synthesized by the method of Stein et al.
(1988, Nucl. Acids Res. 16:3209), methylphosphonate
oligonucleotides can be prepared by use of controlled pore glass
polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci. U.S.A.
85:7448-7451), etc.
[0028] Low stringency conditions are well known to those of skill
in the art, and will vary predictably depending on the specific
organisms from which the library and the labeled sequences are
derived. For guidance regarding such conditions see, for example,
Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual (and
periodic updates thereof), Cold Springs Harbor Press, N.Y.; and
Ausubel et al., 1989, Current Protocols in Molecular Biology, Green
Publishing Associates and Wiley Interscience, N.Y.
[0029] Alternatively, suitably labeled NUCP nucleotide probes can
be used to screen a human genomic library using appropriately
stringent conditions or by PCR. The identification and
characterization of human genomic clones is helpful for identifying
polymorphisms, determining the genomic structure of a given
locus/allele, and designing diagnostic tests. For example,
sequences derived from regions adjacent to the intron/exon
boundaries of the human gene can be used to design primers for use
in amplification assays to detect mutations within the exons,
introns, splice sites (e.g., splice acceptor and/or donor sites),
etc., that can be used in diagnostics and pharmacogenomics.
[0030] Further, a NUCP gene homolog can be isolated from nucleic
acid of the organism of interest by performing PCR using two
degenerate or "wobble" oligonucleotide primer pools designed on the
basis of amino acid sequences within the NUCP product disclosed
herein. The template for the reaction may be total RNA, mRNA,
and/or cDNA obtained by reverse transcription of mRNA prepared
from, for example, human or non-human cell lines or tissue, such as
choroid plexus, known or suspected to express a NUCP gene
allele.
[0031] The PCR product may be subcloned and sequenced to ensure
that the amplified sequences represent the sequence of the desired
NUCP gene. The PCR fragment may then be used to isolate a full
length cDNA clone by a variety of methods. For example, the
amplified fragment may be labeled and used to screen a cDNA
library, such as a bacteriophage cDNA library. Alternatively, the
labeled fragment may be used to isolate genomic clones via the
screening of a genomic library.
[0032] PCR technology may also be utilized to isolate full length
cDNA sequences. For example, RNA may be isolated, following
standard procedures, from an appropriate cellular or tissue source
(i.e., one known, or suspected, to express a NUCP gene, such as,
for example, brain tissue). A reverse transcription (RT) reaction
may be performed on the RNA using an oligonucleotide primer
specific for the most 5' end of the amplified fragment for the
priming of first strand synthesis. The resulting RNA/DNA hybrid may
then be "tailed" using a standard terminal transferase reaction,
the hybrid may be digested with RNase H, and second strand
synthesis may then be primed with a complementary primer. Thus,
cDNA sequences upstream of the amplified fragment may easily be
isolated. For a review of cloning strategies which may be used, see
e.g., Sambrook et al., 1989, supra.
[0033] A cDNA of a mutant NUCP gene may be isolated, for example,
by using PCR. In this case, the first cDNA strand may be
synthesized by hybridizing an oligo-dT oligonucleotide to mRNA
isolated from tissue known or suspected to be expressed in an
individual putatively carrying a mutant NUCP allele, and by
extending the new strand with reverse transcriptase. The second
strand of the cDNA is then synthesized using an oligonucleotide
that hybridizes specifically to the 5' end of the normal gene.
Using these two primers, the product is then amplified via PCR,
optionally cloned into a suitable vector, and subjected to DNA
sequence analysis through methods well known to those of skill in
the art. By comparing the DNA sequence of the mutant NUCP allele to
that of the normal NUCP allele, the mutation(s) responsible for the
loss or alteration of function of the mutant NUCP gene product can
be ascertained.
[0034] Alternatively, a genomic library can be constructed using
DNA obtained from an individual suspected of or known to carry the
mutant NUCP allele (e.g., a person manifesting a NUCP-associated
phenotype such as, for example, obesity, high blood pressure,
etc.), or a cDNA library can be constructed using RNA from a tissue
known, or suspected, to express a mutant NUCP allele. The normal
NUCP gene, or any suitable fragment thereof, can then be labeled
and used as a probe to identify the corresponding mutant NUCP
allele in such libraries. Clones containing the mutant NUCP gene
sequences may then be purified and subjected to sequence analysis
according to methods well known to those of skill in the art.
[0035] Additionally, an expression library can be constructed
utilizing cDNA synthesized from, for example, RNA isolated from a
tissue known, or suspected, to express a mutant NUCP allele in an
individual suspected of or known to carry such a mutant allele. In
this manner, gene products made by the putatively mutant tissue may
be expressed and screened using standard antibody screening
techniques in conjunction with antibodies raised against the normal
NUCP product as described below (For screening techniques, see, for
example, Harlow, E. and Lane, eds., 1988, "Antibodies: A Laboratory
Manual", Cold Spring Harbor Press, Cold Spring Harbor.)
[0036] Additionally, screening can be accomplished by screening
with labeled NUCP fusion proteins, such as, for example, AP-NUCP or
NUCP-AP fusion proteins. In cases where a NUCP mutation results in
an expressed gene product with altered function (e.g., as a result
of a missense or a frameshift mutation), a polyclonal set of
antibodies to NUCP are likely to cross-react with the mutant NUCP
gene product. Library clones detected via their reaction with such
labeled. antibodies can be purified and subjected to sequence
analysis according to methods well known in the art.
[0037] The invention also encompasses nucleotide sequences that
encode mutant NUCPs, peptide fragments of NUCPs, truncated NUCPs,
and NUCP fusion proteins. These include, but are not limited to
nucleotide sequences encoding the mutant NUCPs described below;
polypeptides or peptides corresponding to one or more domains of
NUCP or portions of these domains; truncated NUCPs in which one or
more of the domains is deleted, or a truncated nonfunctional NUCPs.
Nucleotides encoding fusion proteins may include, but are not
limited to, full length NUCP sequences, truncated NUCPs, or
nucleotides encoding peptide fragments of a NUCP fused to an
unrelated protein or peptide, such as for example, a NUCP domain
fused to an Ig Fc domain which increases the stability and half
life of the resulting fusion protein (e.g., NUCP-Ig) in the
bloodstream; or an enzyme such as a fluorescent protein or a
luminescent protein which can be used as a marker.
[0038] The invention also encompasses (a) DNA vectors that contain
any of the foregoing NUCP coding sequences and/or their complements
(i.e., antisense); (b) DNA expression vectors that contain any of
the foregoing NUCP coding sequences operatively associated with a
regulatory element that directs the expression of the coding
sequences; (c) genetically engineered host cells that contain any
of the foregoing NUCP coding sequences operatively associated with
a regulatory element that directs the expression of the coding
sequences in the host cell; and (d) genetically engineered host
cells that express an endogenous NUCP gene under the control of an
exogenously introduced regulatory element (i.e., gene activation).
As used herein, regulatory elements include, but are not limited
to, inducible and non-inducible promoters, enhancers, operators and
other elements known to those skilled in the art that drive and
regulate expression. Such regulatory elements include but are not
limited to the cytomegalovirus hCMV immediate early gene,
regulatable, viral (particularly retroviral LTR promoters) the
early or late promoters of SV40 adenovirus, the lac system, the trp
system, the tet system, the TAC system, the TRC system, the major
operator and promoter regions of phage lambda, the control regions
of fd coat protein, the promoter for 3-phosphoglycerate kinase
(PGK), the promoters of acid phosphatase, and the promoters of the
yeast .alpha.-mating factors.
[0039] 5.2. The NUCPS and NUCP Polypeptides and Peptides Derived
Therefrom
[0040] The NUCPs, NUCP polypeptides, NUCP peptide fragments,
mutated, truncated, or deleted forms of a NUCP, and/or NUCP fusion
proteins can be prepared for a variety of uses, including but not
limited to the generation of antibodies, as reagents in diagnostic
assays, the identification of other cellular gene products related
to a NUCP, as reagents in assays for screening for compounds that
can be as pharmaceutical reagents useful in the therapeutic
treatment of mental, biological, or medical disorders and
disease.
[0041] The Sequence Listing discloses the amino acid sequences
encoded by the described NUCP polynucleotides. The NUCP sequences
both display initiator methionines that are present in a DNA
sequence context consistent with a translation initiation site
(Kozak sequence).
[0042] The NUCP sequences of the invention include the nucleotide
and amino acid sequences presented in the Sequence Listing as well
as analogues and derivatives thereof. Further, corresponding NUCP
homologues from other species are encompassed by the invention. In
fact, any NUCP protein encoded by the NUCP nucleotide sequences
described above are within the scope of the invention as are any
novel polynucleotide sequences encoding all or any novel portion of
an amino acid sequence presented in the Sequence Listing. The
degenerate nature of the genetic code is well known, and,
accordingly, each amino acid presented in the Sequence Listing, is
generically representative of the well known nucleic acid "triplet"
codon, or in many cases codons, that can encode the amino acid. As
such, as contemplated herein, the amino acid sequences presented in
the Sequence Listing, when taken together with the genetic code
(see, for example, Table 4-1 at page 109 of "Molecular Cell
Biology", 1986, J. Darnell et al. eds., Scientific American Books,
New York, N.Y., herein incorporated by reference) are generically
representative of all the various permutations and combinations of
nucleic acid sequences that can encode such amino acid
sequences.
[0043] The invention also encompasses proteins that are
functionally equivalent to the NUCP encoded by the presently
described nucleotide sequences, as judged by any of a number of
criteria, including, but not limited to, the ability to partition
into the mitochondria, or other cellular membrane structure, and
effect uncoupling activity, change in cellular metabolism (e.g.,
ion flux, tyrosine phosphorylation, etc.), or change in phenotype
when the NUCP equivalent is expressed at similar levels, or
mutated, in an appropriate cell type (such as the amelioration,
prevention or delay of a biochemical, biophysical, or overt
phenotype). Functional equivalents of a NUCP include naturally
occurring NUCPs present in other species and mutant NUCPs whether
naturally occurring or engineered (by site directed mutagenesis,
gene shuffling, directed evolution as described in, for example,
U.S. Pat. No. 5,837,458). The invention also includes degenerate
nucleic acid variants and splice variant of the disclosed NUCP
polynucleotide sequence.
[0044] Additionally contemplated are polynucleotides encoding NUCP
ORFS, or their functional equivalents, encoded by polynucleotide
sequences that are about 99, 95, 90, or about 85 percent similar or
identical to corresponding regions of the nucleotide sequences of
the Sequence Listing (as measured by BLAST sequence comparison
analysis using, for example, the GCG sequence analysis package
using standard default settings).
[0045] Functionally equivalent NUCP proteins include, but are not
limited to, additions or substitutions of amino acid residues
within the amino acid sequence encoded by the NUCP nucleotide
sequences described above, but which result in a silent change,
thus producing a functionally equivalent gene product. Amino acid
substitutions may be made on the basis of similarity in polarity,
charge, solubility, hydrophobicity, hydrophilicity, and/or the
amphipathic nature of the residues involved. For example, nonpolar
(hydrophobic) amino acids include alanine, leucine, isoleucine,
valine, proline, phenylalanine, tryptophan, and methionine; polar
neutral amino acids include glycine, serine, threonine, cysteine,
tyrosine, aspatagine, and glutamine; positively charged (basic)
amino acids include arginine, lysine, and histidine; and negatively
charged (acidic) amino acids include aspartic acid and glutamic
acid.
[0046] While random mutations can be made to NUCP encoding DNA
(using random mutagenesis techniques well known to those skilled in
the art) and the resulting mutant NUCPs tested for activity,
site-directed mutations of the NUCP coding sequence can be
engineered (using site-directed mutagenesis techniques well known
to those skilled in the art) to generate mutant NUCPs with
increased function, e.g., higher receptor binding affinity,
decreased function, and/or increased physiological half-life, and
increased signal transduction triggering. One starting point for
such analysis is by aligning the disclosed human sequences with
corresponding gene/protein sequences from, for example, other
mammals in order to identify amino acid sequence motifs that are
conserved between different species. Non-conservative changes can
be engineered at variable positions to alter function, signal
transduction capability, or both. Alternatively, where alteration
of function is desired, deletion or non-conservative alterations of
the conserved regions (i.e., identical amino acids) can be
engineered. For example, deletion or non-conservative alterations
(substitutions or insertions) of the various conserved
transmembrane domains.
[0047] Other mutations to a NUCP coding sequence can be made to
generate NUCPs that are better suited for expression, scale up,
etc. in the host cells chosen. For example, cysteine residues can
be deleted or substituted with another amino acid in order to
eliminate disulfide bridges; N-linked glycosylation sites can be
altered or eliminated to achieve, for example, expression of a
homogeneous product that is more easily recovered and purified from
yeast hosts which are known to hyperglycosylate N-linked sites. To
this end, a variety of .amino acid substitutions at one or both of
the first or third amino acid positions of any one or more of the
glycosylation recognition sequences which occur in an ECD (N--X--S
or N--X-T), and/or an amino acid deletion at the second position of
any one or more such recognition sequences in an ECD will prevent
glycosylation of the NUCP at the modified tripeptide sequence.
(See, e.g., Miyajima et al., 1986, EMBO J. 5(6):1193-1197).
[0048] Peptides corresponding to one or more domains of a NUCP,
truncated or deleted NUCPs, as well as fusion proteins in which a
full length NUCP, a NUCP peptide, or a truncated NUCP is fused to
an unrelated protein, are also within the scope of the invention
and can be designed on the basis of the presently disclosed NUCP
gene nucleotide and NUCP amino acid sequences. Such fusion proteins
include, but are not limited to, Ig Fc fusions which stabilize a
NUCP protein, or NUCP peptides, and prolong half-life in vivo; or
fusions to any amino acid sequence that allows the fusion protein
to be anchored to the cell membrane; or fusions to an enzyme,
fluorescent protein, or luminescent protein which provide a marker
function.
[0049] While the NUCPs and NUCP peptides can be chemically
synthesized (e.g., see Creighton, 1983, Proteins: Structures and
Molecular Principles, W. H. Freeman & Co., N.Y.), large
polypeptides derived from a full length NUCP can be advantageously
produced by recombinant DNA technology using techniques well known
in the art for expressing nucleic acids containing NUCP gene
sequences and/or coding sequences. Such methods can be used to
construct expression vectors containing the described NUCP
nucleotide sequences and appropriate transcriptional and
translational control signals. These methods include, for example,
in vitro recombinant DNA techniques, synthetic techniques, and in
vivo genetic recombination. See, for example, the techniques
described in Sambrook et al., 1989, supra, and Ausubel et al.,
1989, supra. Alternatively, RNA corresponding to all or a portion
of a transcript encoded by a NUCP gene sequence can be chemically
synthesized using, for example, synthesizers. See, for example, the
techniques described in "Oligonucleotide Synthesis", 1984, Gait, M.
J. ed., IRL Press, Oxford, which is incorporated by reference
herein in its entirety.
[0050] A variety of host-expression vector systems can be utilized
to express the NUCP-encoding nucleotide sequences of the invention.
Where a NUCP peptide or polypeptide is a soluble derivative (e.g.,
NUCP peptides corresponding to an ECD; truncated or deleted NUCP in
which a TM and/or CD are deleted, etc.) the peptide can be
recovered from the host cell in cases where the NUCP peptide or
polypeptide is not secreted, and from the culture media in cases
where the NUCP peptide or polypeptide is secreted by the cells.
However, such expression systems also encompass engineered host
cells that express a NUCP, or a functional equivalent thereof, in
situ, i.e., anchored in the cell membrane. Purification or
enrichment of a NUCP from such expression systems can be
accomplished using appropriate detergents and lipid micelles and
methods well known to those skilled in the art. However, such
engineered host cells themselves may be used in situations where it
is important not only to retain the structural and functional
characteristics of a NUCP, but to assess biological activity, e.g.,
in drug screening assays.
[0051] The expression systems that can be used for purposes of the
invention include but are not limited to microorganisms such as
bacteria (e.g., E. coli, B. subtilis) transformed with recombinant
bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors
containing NUCP nucleotide sequences; yeast (e.g., Saccharomyces,
Pichia) transformed with recombinant yeast expression vectors
containing NUCP nucleotide sequences; insect cell systems infected
with recombinant virus expression vectors (e.g., baculovirus)
containing NUCP sequences; plant cell systems infected with
recombinant virus expression vectors (e.g., cauliflower mosaic
virus, CaMV; tobacco mosaic virus, TMV) or transformed with
recombinant plasmid expression vectors (e.g., Ti plasmid)
containing NUCP nucleotide sequences; or mammalian cell systems
(e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expression
constructs containing promoters derived from the genome of
mammalian cells (e.g., metallothionein promoter) or from mammalian
viruses (e.g., the adenovirus late promoter; the vaccinia virus
7.5K promoter).
[0052] In bacterial systems, a number of expression vectors may be
advantageously selected depending upon the use intended for the
NUCP product being expressed. For example, when a large quantity of
such a protein is to be produced for the generation of
pharmaceutical compositions of or containing NUCP, or for raising
antibodies to a NUCP, vectors that direct the expression of high
levels of fusion protein products that are readily purified may be
desirable. Such vectors include, but are not limited, to the E.
coli expression vector pUR278 (Ruther et al., 1983, EMBO J.
2:1791), in which a NUCP coding sequence may be ligated
individually into the vector in frame with the lacZ coding region
so that a fusion protein is produced; pIN vectors (Inouye &
Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke &
Schuster, 1989, J. Biol. Chem. 264:5503-5509); and the like. pGEX
vectors may also be used to express foreign polypeptides as fusion
proteins with glutathione S-transferase (GST). In general, such
fusion proteins are soluble and can easily be purified from lysed
cells by adsorption to glutathione-agarose beads followed by
elution in the presence of free glutathione. The PGEX vectors are
designed to include thrombin or factor Xa protease cleavage sites
so that the cloned target gene product can be released from the GST
moiety.
[0053] In an insect system, Autographa Californica nuclear
polyhidrosis virus (AcNPV) is used as a vector to express foreign
genes. The virus grows in Spodoptera Frugiperda cells. The NUCP
gene coding sequence may be cloned individually into non-essential
regions (for example the polyhedrin gene) of the virus and placed
under control of an AcNPV promoter (for example the polyhedrin
promoter). Successful insertion of the NUCP gene coding sequence
will result in inactivation of the polyhedrin gene and production
of non-occluded recombinant virus (i.e., virus lacking the
proteinaceous coat coded for by the polyhedrin gene). These
recombinant viruses are then used to infect Spodoptera frugiperda
cells in which the inserted gene is expressed (e.g., see Smith et
al., 1983, J. Virol. 46: 584; Smith, U.S. Pat. No. 4,215,051).
[0054] In mammalian host cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, the NUCP gene nucleotide sequence of interest
may be ligated to an adenovirus transcription/translation control
complex, e.g., the late promoter and tripartite leader sequence.
This chimeric gene may then be inserted in the adenovirus genome by
in vitro or in vivo recombination. Insertion in a non-essential
region of the viral genome (e.g., region E1 or E3) will result in a
recombinant virus that is viable and capable of expressing NUCP in
infected hosts (e.g., See Logan & Shenk, 1984, Proc. Natl.
Acad. Sci. USA 81:3655-3659). Specific initiation signals may also
be required for efficient translation of NUCP transcripts. These
signals include the ATG initiation codon and adjacent sequences. In
cases where an entire NUCP gene or cDNA, including its own
initiation codon and adjacent sequences, is inserted into the
appropriate expression vector, no additional translational control
signals may be needed (for example an independent ribosome entry
site, or IRES, site). However, in cases where only a portion of a
NUCP coding sequence is inserted, exogenous translational control
signals, including, perhaps, the ATG initiation codon, must be
provided. Furthermore, the initiation codon must be in phase with
the reading frame of the desired coding sequence to ensure
translation of the entire insert. These exogenous translational
control signals and initiation codons can have a variety of
origins, both natural and synthetic. The efficiency of expression
may be enhanced by the inclusion of appropriate transcription
enhancer elements, transcription terminators, etc. (See Bittner et
al., 1987, Methods in Enzymol. 153:516-544).
[0055] In addition, a host cell strain may be chosen that modulates
the expression of the inserted sequences, or modifies and processes
the gene product in the specific fashion desired. Such
modifications (e.g., glycosylation) and processing (e.g., cleavage)
of protein products may be important for the function of the
protein. Different host cells have characteristic and specific
mechanisms for the post-translational processing and modification
of proteins and gene products. Appropriate cell lines or host
systems can be chosen to ensure the correct modification and
processing of the foreign protein expressed. To this end,
eukaryotic host cells which possess the cellular machinery for
proper processing of the primary transcript, glycosylation, and
phosphorylation of the gene product may be used. Such mammalian
host cells include, but are not limited to, CHO, VERO, BHK, HeLa,
COS, MDCK, 293, 3T3, WI38, and in particular, human cell lines.
[0056] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
that stably express the presently described NUCPs can be
engineered. Rather than using expression vectors which contain
viral origins of replication, host cells can be transformed with
DNA controlled by appropriate expression control elements (e.g.,
promoter, enhancer sequences, transcription terminators,
polyadenylation sites, etc.), and a selectable marker. Following
the introduction of the foreign DNA, engineered cells can be
allowed to grow for 1-2 days in an enriched media, and then are
switched to a selective media. The selectable marker in the
recombinant plasmid confers resistance to the selection and allows
cells to stably integrate the plasmid into their chromosomes and
grow to form foci which in turn can be cloned and expanded into
cell lines. This method may advantageously be used to engineer cell
lines that express a NUCP. Such engineered cell lines may be
particularly useful in screening and evaluation of compounds that
affect the endogenous activity of the NUCP product.
[0057] A number of selection systems may be used, including but not
limited to the herpes simplex virus thymidine kinase (wigler, et
al., 1977, Cell 11:223), hypoxanthine-guanine
phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc.
Natl. Acad. Sci. USA 48:2026), and adenine
phosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817) genes
can be employed in tk.sup.-, hgprt.sup.- or aprt.sup.- cells,
respectively. Also, antimetabolite resistance can be used as the
basis of selection for the following genes: dhfr, which confers
resistance to methotrexate (Wigler, et al., 1980, Natl. Acad. Sci.
USA 77:3567; O'Hare, et al., 1981, Proc. Natl. Acad. Sci. USA
78:1527); gpt, which confers resistance to mycophenolic acid
(Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072);
neo, which confers resistance to the aminoglycoside G-418
(Colberre-Garapin, et al., 1981, J. Mol. Biol. 150:1); and hygro,
which confers resistance to hygromycin (Santerre, et al., 1984,
Gene 30:147).
[0058] Alternatively, any fusion protein may be readily purified by
utilizing an antibody specific for the fusion protein being
expressed. For example, a system described by Janknecht et al.
allows for the ready purification of non-denatured fusion proteins
expressed in human cell lines (Janknecht, et al., 1991, Proc. Natl.
Acad. Sci. USA 88: 8972-8976). In this system, the gene of interest
is subcloned into a vaccinia recombination plasmid such that the
gene's open reading frame is translationally fused to an
amino-terminal tag consisting of six histidine residues. Extracts
from cells infected with recombinant vaccinia virus are loaded onto
Ni.sup.2+-nitriloacetic acid-agarose columns and histidine-tagged
proteins are selectively eluted with imidazole-containing
buffers.
[0059] NUCP products can also be expressed in transgenic animals.
Animals of any species, including, but not limited to, worms, mice,
rats, rabbits, guinea pigs, pigs, micro-pigs, birds, goats, and
non-human primates, e.g., baboons, monkeys, and chimpanzees may be
used to generate NUCP transgenic animals.
[0060] Any technique known in the art may be used to introduce a
NUCP transgene into animals to produce the founder lines of
transgenic animals. Such techniques include, but are not limited to
pronuclear microinjection (Hoppe, P. C. and Wagner, T. E., 1989,
U.S. Pat. No. 4,873,191); retrovirus mediated gene transfer into
germ lines (Van der Putten et al., 1985, Proc. Natl. Acad. Sci.,
USA 82:6148-6152); gene targeting in embryonic stem cells (Thompson
et al., 1989, Cell 56:313-321); electroporation of embryos (Lo,
1983, Mol Cell. Biol. 3:1803-1814); and sperm-mediated gene
transfer (Lavitrano et al., 1989, Cell 57:717-723); etc. For a
review of such techniques, see Gordon, 1989, Transgenic Animals,
Intl. Rev. Cytol. 115:171-229, which is incorporated by reference
herein in its entirety.
[0061] The present invention provides for transgenic animals that
carry a NUCP transgene in all their cells, as well as animals which
carry the transgene in some, but not all their cells, i.e., mosaic
animals or somatic cell transgenic animals. The transgene may be
integrated as a single transgene or in concatamers, e.g.,
head-to-head tandems or head-to-tail tandems. The transgene may
also be selectively introduced into and activated in a particular
cell type by following, for example, the teaching of Lasko et al.,
1992, Proc. Natl. Acad. Sci. USA 89:6232-6236. The regulatory
sequences required for such a cell-type specific activation will
depend upon the particular cell type of interest, and will be
apparent to those of skill in the art.
[0062] When it is desired that the NUCP transgene be integrated
into the chromosomal site of the endogenous NUCP gene, gene
targeting is preferred. Briefly, when such a technique is to be
utilized, vectors containing some nucleotide sequences homologous
to the endogenous NUCP gene are designed for the purpose of
integrating, via homologous recombination with chromosomal
sequences, into and disrupting the function of the nucleotide
sequence of the endogenous NUCP gene (i.e., "knockout"
animals).
[0063] The transgene may also be selectively introduced into a
particular cell type, thus inactivating the endogenous NUCP gene in
only that cell type, by following, for example, the teaching of Gu
et al., 1994, Science, 265:103-106. The regulatory sequences
required for such a cell-type specific inactivation will depend
upon the particular cell type of interest, and will be apparent to
those of skill in the art.
[0064] Once trans genic animals have been generated, the expression
of the recombinant NUCP gene may be assayed utilizing standard
techniques. Initial screening may be accomplished by Southern blot
analysis or PCR techniques to analyze animal tissues to assay
whether integration of the transgene has taken place. The level of
mRNA expression of the transgene in the tissues of the transgenic
animals may also be assessed using techniques which include but are
not limited to Northern blot analysis of tissue samples obtained
from the animal, in situ hybridization analysis, and RT-PCR.
Samples of NUCP gene-expressing tissue, may also be evaluated
immunocytochemically using antibodies specific for the NUCP
transgene product.
[0065] 5.3. Antibodies to NUCPS
[0066] Antibodies that specifically recognize one or more epitopes
of a NUCP, or epitopes of conserved variants of a NUCP, or peptide
fragments of a NUCP are also encompassed by the invention. Such
antibodies include but are not limited to polyclonal antibodies,
monoclonal antibodies (mAbs), humanized or chimeric antibodies,
single chain antibodies, Fab fragments, F(ab').sub.2 fragments,
fragments produced by a Fab expression library, anti-idiotypic
(anti-Id) antibodies, and epitope-binding fragments of any of the
above.
[0067] The antibodies of the invention can be used, for example, in
the detection of a NUCP in a biological sample and may, therefore,
be utilized as part of a diagnostic or prognostic technique whereby
patients may be tested for abnormal amounts of a NUCP. Such
antibodies may also be utilized in conjunction with, for example,
compound screening schemes, as described below, for the evaluation
of the effect of test compounds on expression and/or activity of a
NUCP gene product. Additionally, such antibodies can be used in
conjunction gene therapy to, for example, evaluate the normal
and/or engineered NUCP-expressing cells prior to their introduction
into the patient. Such antibodies may additionally be used as a
method for inhibiting abnormally high NUCP activity. Thus, such
antibodies may, therefore, be utilized as part of treatment
methods.
[0068] For the production of antibodies, various host animals may
be immunized by injection with a NUCP, a NUCP peptide (e.g., one
corresponding the a functional domain of a NUCP), truncated NUCP
polypeptides (a NUCP in which one or more domains have been
deleted), functional equivalents of the NUCP or mutants of the
NUCP. Such host animals may include but are not limited to rabbits,
mice, goats, and rats, to name but a few. Various adjuvants may be
used to increase the immunological response, depending on the host
species, including but not limited to Freund's (complete and
incomplete), mineral gels such as aluminum hydroxide, surface
active substances such as lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,
dinitrophenol, and potentially useful human adjuvants such as BCG
(bacille Calmette-Guerin) and Corynebacterium parvum. Polyclonal
antibodies are heterogeneous populations of antibody molecules
derived from the sera of the immunized animals.
[0069] Monoclonal antibodies, which are homogeneous populations of
antibodies to a particular antigen, may be obtained by any
technique which provides for the production of antibody molecules
by continuous cell lines in culture. These include, but are not
limited to, the hybridoma technique of Kohler and Milstein, (1975,
Nature 256:495-497; and U.S. Pat. No. 4,376,110), the human B-cell
hybridoma technique (Kosbor et al., 1983, Immunology Today 4:72;
Cole et al., 1983, Proc. Natl. Acad. Sci. USA 80:2026-2030), and
the EBV-hybridoma technique (Cole et al., 1985, Monoclonal
Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such
antibodies may be of any immunoglobulin class including IgG, IgM,
IgE, IgA, IgD and any subclass thereof. The hybridoma producing the
mAb of this invention may be cultivated in vitro or in vivo.
Production of high titers of mAbs in vivo makes this the presently
preferred method of production.
[0070] In addition, techniques developed for the production of
"chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad.
Sci., 81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608;
Takeda et al., 1985, Nature, 314:452-454) by splicing the genes
from a mouse antibody molecule of appropriate antigen specificity
together with genes from a human antibody molecule of appropriate
biological activity can be used. A chimeric antibody is a molecule
in which different portions are derived from different animal
species, such as those having a variable region derived from a
murine mAb and a human immunoglobulin constant region.
[0071] Alternatively, techniques described for the production of
single chain antibodies (U.S. Pat. No. 4,946,778; Bird, 1988,
Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci.
USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-546) can be
adapted to produce single chain antibodies against NUCP gene
products. Single chain antibodies are formed by linking the heavy
and light chain fragments of the Fv region via an amino acid
bridge, resulting in a single chain polypeptide.
[0072] Antibody fragments that recognize specific epitopes can be
generated using known techniques. For example, such fragments
include, but are not limited to: the F(ab').sub.2 fragments which
can be produced by pepsin digestion of the antibody molecule and
the Fab fragments which can be generated by reducing the disulfide
bridges of the F(ab').sub.2 fragments. Alternatively, Fab
expression libraries may be constructed (Huse et al., 1989,
Science, 246:1275-1281) to allow rapid and easy identification of
monoclonal Fab fragments with the desired specificity.
[0073] Antibodies to a NUCP can, in turn, be utilized to generate
anti-idiotype antibodies that "mimic" a given NUCP, using
techniques well known to those skilled in the art. (See, e.g.,
Greenspan & Bona, 1993, FASEB J 7(5):437-444; and Nissinoff,
1991, J. Immunol. 147(8):2429-2438). For example antibodies that
bind to a NUCP domain and competitively inhibit the binding of a
NUCP to its cognate ligand, chaperonin, or accessory molecule(s)
can be used to generate anti-idiotypes that "mimic" the NUCP and,
therefore, bind and activate or neutralize a receptor. Such
anti-idiotypic antibodies or Fab fragments of such anti-idiotypes
can be used in therapeutic regimens involving a NUCP-mediated
process or pathway.
[0074] 5.4. Diagnosis of Abnormalities Related to a NUCP
[0075] A variety of methods can be employed for the diagnostic and
prognostic evaluation of disorders related to NUCP function, and
for the identification of subjects having a predisposition to such
disorders.
[0076] Such methods may, for example, utilize reagents such as the
NUCP nucleotide sequences described above and the NUCP antibodies
described above. Specifically, such reagents may be used, for
example, for: (1) the detection of the presence of NUCP gene
mutations, or the detection of either over- or under-expression of
NUCP mRNA relative to a given phenotype; (2) the detection of
either an over- or an under-abundance of NUCP gene product relative
to a given phenotype; and (3) the detection of perturbations or
abnormalities in any metabolic, physiologic, or catabolic pathway
mediated by NUCP.
[0077] The methods described herein may be performed, for example,
by utilizing pre-packaged diagnostic kits comprising at least one
specific NUCP nucleotide sequence or NUCP antibody reagent
described herein, which may be conveniently used, e.g., in clinical
settings, to diagnose patients exhibiting, for example, body weight
disorder abnormalities.
[0078] For the detection of NUCP mutations, any nucleated cell can
be used as a starting source for genomic nucleic acid. For the
detection of NUCP gene expression or NUCP gene products, any cell
type or tissue in which the NUCP gene is expressed, such as, for
example, kidney cells, may be utilized.
[0079] Nucleic acid-based detection techniques are described,
below, in Section 5.4.1. Peptide detection techniques are
described, below, in Section 5.4.2.
[0080] 5.4.1. Detention of NUCP Sequences
[0081] Mutations within a NUCP nucleotide sequence can be detected
by utilizing a number of techniques. Nucleic acid from any
nucleated cell can be used as the starting point for such assay
techniques, and can be isolated according to standard nucleic acid
preparation procedures which are well known to those of skill in
the art.
[0082] DNA may be used in hybridization or amplification assays of
biological samples to detect abnormalities involving NUCP gene
structure, including point mutations, insertions, deletions and
chromosomal rearrangements. Such assays may include, but are not
limited to, Southern analyses, single stranded conformational
polymorphism analyses (SSCP), and PCR analyses.
[0083] Such diagnostic methods for the detection of NUCP
gene-specific mutations can involve for example, contacting and
incubating nucleic acids including recombinant DNA molecules,
cloned genes or degenerate variants thereof, obtained from a
sample, e.g., derived from a patient sample or other appropriate
cellular source, with one or more labeled nucleic acid reagents
including recombinant DNA molecules, cloned genes or degenerate
variants thereof, as described above, under conditions favorable
for the specific annealing of these reagents to their complementary
sequences within a NUCP gene. Preferably, the lengths of these
nucleic acid reagents are at least about 15 to about 30
nucleotides. After incubation, all non-annealed nucleic acids are
removed from the nucleic acid:NUCP molecule hybrid. The presence of
nucleic acids which have hybridized, if any such molecules exist,
is then detected. Using such a detection scheme, the nucleic acid
from the cell type or tissue of interest can be immobilized, for
example, to a solid support such as a membrane, or a plastic
surface such as that on a microtiter plate or polystyrene beads. In
this case, after incubation, non-annealed, labeled nucleic acid
reagents of the type described above are easily removed. Detection
of the remaining annealed, labeled NUCP nucleic acid reagents is
accomplished using standard techniques well known to those in the
art. The NUCP encoding nucleotide sequences to which the nucleic
acid reagents have annealed can be compared to the annealing
pattern expected from a normal NUCP gene sequence in order to
determine whether a NUCP gene mutation is present.
[0084] Alternative diagnostic methods for the detection of NUCP
gene specific nucleic acid molecules, in patient samples or other
appropriate cell sources, may involve their amplification, e.g., by
PCR (the experimental embodiment set forth in Mullis, K. B., 1987,
U.S. Pat. No. 4,683,202), followed by the detection of the
amplified molecules using techniques well known to those of skill
in the art. The resulting amplified sequences can be compared to
those which would be expected if the nucleic acid being amplified
contained only normal copies of a NUCP gene in order to determine
whether a NUCP gene mutation exists.
[0085] Additionally, well-known genotyping techniques can be
performed to identify individuals carrying NUCP gene mutations.
Such techniques include, for example, the use of restriction
fragment length polymorphisms (RFLPs), which involve sequence
variations in one of the recognition sites for the specific
restriction enzyme used.
[0086] Additionally, improved methods for analyzing DNA
polymorphisms which can be utilized for the identification of NUCP
gene mutations have been described which capitalize on the presence
of variable numbers of short, tandemly repeated DNA sequences
between the restriction enzyme sites. For example, Weber (U.S. Pat.
No. 5,075,217, which is incorporated herein by reference in its
entirety) describes a DNA marker based on length polymorphisms in
blocks of (dC-dA)n-(dG-dT)n short tandem repeats. The average
separation of (dC-dA)n-(dG-dT)n blocks is estimated to be
30,000-60,000 bp. Markers which are so closely spaced exhibit a
high frequency co-inheritance, and are extremely useful in the
identification of genetic mutations, such as, for example,
mutations within the NUCP gene, and the diagnosis of diseases and
disorders related to NUCP mutations.
[0087] Also, Caskey et al. (U.S. Pat. No. 5,364,759, which is
incorporated herein by reference in its entirety) describe a DNA
profiling assay for detecting short tri and tetra nucleotide repeat
sequences. The process. includes extracting the DNA of interest,
such as the NUCP gene, amplifying the extracted DNA, and labeling
the repeat sequences to form a genotypic map of the individual's
DNA.
[0088] The level of NUCP gene expression can also be assayed by
detecting and measuring NUCP transcription. For example, RNA from a
cell type or tissue known, or suspected to express the NUCP gene,
such as kidney, may be isolated and tested utilizing hybridization
or PCR techniques such as those described above. The isolated cells
can be derived from cell culture or from a patient. The analysis of
cells taken from culture may be a necessary step in the assessment
of cells to be used as part of a cell-based gene therapy technique
or, alternatively, to test the effect of compounds on the
expression of the NUCP gene. Such analyses may reveal both
quantitative and qualitative aspects of the expression pattern of
the NUCP gene, including activation or inactivation of NUCP gene
expression.
[0089] In one embodiment of such a detection scheme, cDNAs are
synthesized from the RNAs of interest (e.g., by reverse
transcription of the RNA molecule into cDNA). A sequence within the
cDNA is then used as the template for a nucleic acid amplification
reaction, such as a PCR amplification reaction, or the like. The
nucleic acid reagents used as synthesis initiation reagents (e.g.,
primers) in the reverse transcription and nucleic acid
amplification steps of this method are chosen from among the NUCP
nucleic acid reagents described above. The preferred lengths of
such nucleic acid reagents are at least 9-30 nucleotides. For
detection of the amplified product, the nucleic acid amplification
may be performed using radioactively or non-radioactively labeled
nucleotides. Alternatively, enough amplified product may be made
such that the product may be visualized by standard ethidium
bromide staining, by utilizing any other suitable nucleic acid
staining method, or by sequencing.
[0090] Additionally, it is possible to perform such NUCP gene
expression assays "in situ", i.e., directly upon tissue sections
(fixed and/or frozen) of patient tissue obtained from biopsies or
resections, such that no nucleic acid purification is necessary.
Nucleic acid reagents such as those described in Section 5.1 may be
used as probes and/or primers for such in situ procedures (See, for
example, Nuovo, G. J., 1992, "PCR In Situ Hybridization: Protocols
And Applications", Raven Press, NY).
[0091] Alternatively, if a sufficient quantity of the appropriate
cells can be obtained, standard Northern analysis can be performed
to determine the level of mRNA expression of the NUCP gene.
[0092] 5.4.2. Detection of NUCP Products
[0093] Antibodies directed against wild type or mutant NUCPs, or
conserved variants or peptide fragments thereof, as discussed
above, can also be used as diagnostics and prognostics, as
described herein. Such diagnostic methods, may be used to detect
abnormalities in the level of NUCP gene expression, or
abnormalities in the structure and/or temporal, tissue, cellular,
or subcellular location of the NUCP (besides mitochondria), and may
be performed in vivo or in vitro, such as, for example, on biopsy
tissue.
[0094] For example, antibodies directed to one or more epitopes of
NUCP can be used in vivo to detect the pattern and level of
expression of NUCP in the body. Such antibodies can be labeled,
e.g., with a radio-opaque or other appropriate compound and
injected into a subject in order to visualize binding to the NUCP
expressed in the body using methods such as X-rays, CAT-scans, or
MRI. Labeled antibody fragments, e.g., the Fab or single chain
antibody comprising the smallest portion of the antigen binding
region, are preferred for this purpose to promote crossing the
blood-brain barrier and permit labeling of NUCP expressed in the
brain.
[0095] Additionally, any NUCP fusion protein or NUCP conjugated
protein whose presence can be detected, can be administered. For
example, NUCP fusion or conjugated proteins labeled with a
radio-opaque or other appropriate compound can be administered and
visualized in vivo, as discussed, above for labeled antibodies.
Further such NUCP fusion proteins (such as AP-NUCP or NUCP-AP) can
be utilized for in vitro diagnostic procedures.
[0096] Alternatively, immunoassays or fusion protein detection
assays, as described above, can be utilized oh biopsy and autopsy
samples in vitro to permit assessment of the expression pattern of
the NUCP. Such assays are not confined to the use of antibodies
that define a NUCP domain, but can include the use of antibodies
directed to epitopes of any domain of a NUCP. The use of each or
all of these labeled antibodies will yield useful information
regarding translation and intracellular transport of the NUCP to
the cell surface and can identify defects in processing.
[0097] The tissue or cell type to be analyzed will generally
include those which are known, or suspected, to express the NUCP
gene, such as, for example, epithelial cells, kidney cells, adipose
tissue, brain cells, etc. The protein isolation methods employed
herein may, for example, be such as those described in Harlow and
Lane (Harlow, E. and Lane, D., 1988, "Antibodies: A Laboratory
Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y.), which is incorporated herein by reference in its entirety.
The isolated cells can be derived from cell culture or from a
patient. The analysis of cells taken from culture may be a
necessary step in the assessment of cells that could be used as
part of a cell-based gene therapy technique or, alternatively, to
test the effect of compounds on the expression of the NUCP
gene.
[0098] For example, antibodies, or fragments of antibodies, such as
those described above useful in the present invention may be used
to quantitatively or qualitatively detect the presence of a NUCP,
or conserved variants or peptide fragments thereof. This can be
accomplished, for example, by immunofluorescence techniques
employing a fluorescently labeled antibody (see below, this
Section) coupled with light microscopic, flow cytometric, or
fluorimetric detection. Such techniques are especially preferred if
such NUCP products can be found, at least transiently, on the cell
surface.
[0099] The antibodies (or fragments thereof) or NUCP fusion or
conjugated proteins useful in the present invention may
additionally be employed histologically, as in immunofluorescence,
immunoelectron microscopy or non-immuno assays, for in situ
detection of NUCP gene products or conserved variants or peptide
fragments thereof, or to assay NUCP binding (in the case of labeled
NUCP-fusion protein).
[0100] In situ detection may be accomplished by removing a
histological specimen from a patient, and applying thereto a
labeled antibody or fusion protein of the present invention. The
antibody (or fragment) or fusion protein is preferably applied by
overlaying the labeled antibody (or fragment) onto a biological
sample. Through the use of such a procedure, it is possible to
determine not only the presence of the NUCP product, or conserved
variants or peptide fragments, or NUCP binding, but also its
distribution in the examined tissue. Using the present invention,
those of ordinary skill will readily perceive that any of a wide
variety of histological methods (such as staining procedures) can
be modified in order to achieve such in situ detection.
[0101] Immunoassays and non-immunoassays for a NUCP, or conserved
variants or peptide fragments thereof, will typically comprise
incubating a sample, such as a biological fluid, a tissue extract,
freshly harvested cells, or lysates of cells which have been
incubated in cell culture, in the presence of a detectably labeled
antibody capable of identifying NUCP products or conserved variants
or peptide fragments thereof, and detecting the bound antibody by
any of a number of techniques well-known in the art. Alternatively,
the labeled antibody can be directed against an antigenic tag that
has been directly or indirectly attached to a NUCP.
[0102] The biological sample may be brought in contact with and
immobilized onto a solid phase support or carrier such as
nitrocellulose, or other solid support which is capable of
immobilizing cells, cell particles or soluble proteins. The support
may then be washed with suitable buffers followed by treatment with
the detectably labeled NUCP antibody or NUCP ligand/accessory
molecule fusion protein. The solid phase support may then be washed
with the buffer a second time to remove unbound antibody or fusion
protein. The amount of bound label on solid support may then be
detected by conventional means.
[0103] By "solid phase support or carrier" is intended any support
capable of binding an antigen or an antibody. Well-known supports
or carriers include glass, polystyrene, polypropylene,
polyethylene, dextran, nylon, amylases, natural and modified
celluloses, polyacrylamides, gabbros, and magnetite. The nature of
the carrier can be either soluble to some extent or insoluble for
the purposes of the present invention. The support material may
have virtually any possible structural configuration so long as the
coupled molecule is capable of binding to an antigen or antibody.
Thus, the support configuration may be spherical, as in a bead, or
cylindrical, as in the inside surface of a test tube, or the
external surface of a rod. Alternatively, the surface may be flat
such as a sheet, test strip, etc. Preferred supports include
polystyrene beads. Those skilled in the art will know many other
suitable carriers for binding antibody or antigen, or will be able
to ascertain the same by use of routine experimentation.
[0104] The binding activity of a given lot of NUCP antibody or NUCP
ligand fusion protein may be determined according to well known
methods. Those skilled in the art will be able to determine
operative and optimal assay conditions for each determination by
employing routine experimentation.
[0105] With respect to antibodies, one of the ways in which the
NUCP antibody can be detectably labeled is by linking the same to
an enzyme and use in an enzyme immunoassay (EIA) (Voller, A., "The
Enzyme Linked Immunosorbent Assay (ELISA)", 1978, Diagnostic
Horizons 2:1-7, Microbiological Associates Quarterly Publication,
Walkersville, Md.); Voller, A. et al., 1978, J. Clin. Pathol.
31:507-520; Butler, J. E., 1981, Meth. Enzymol. 73:482-523; Maggio,
E. (ed.), 1980, Enzyme Immunoassay, CRC Press, Boca-Raton, Fla.,;
Ishikawa, E. et al., (eds.), 1981, Enzyme Immunoassay, Kgaku Shoin,
Tokyo). The enzyme that is bound to the antibody will react with an
appropriate substrate, preferably a chromogenic substrate, in such
a manner as to produce a chemical moiety which can be detected, for
example, by spectrophotometric, fluorimetric or by visual means.
Enzymes which can be used to detectably label the antibody include,
but are not limited to, malate dehydrogenase, staphylococcal
nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase,
alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase,
horseradish peroxidase, alkaline phosphatase, asparaginase, glucose
oxidase, beta-galactosidase, ribonuclease, urease, catalase,
glucose-6-phosphate -dehydrogenase, glucoamylase and
acetylcholinesterase. The detection can be accomplished by
colorimetric methods which employ a chromogenic substrate for the
enzyme. Detection may also be accomplished by visual comparison of
the extent of enzymatic reaction of a substrate in comparison with
similarly prepared standards.
[0106] Detection may also be accomplished using any of a variety of
other immunoassays. For example, by radioactively labeling the
antibodies or antibody fragments, it is possible to detect NUCP
through the use of a radioimmunoassay (RIA) (see, for example,
Weintraub, B., Principles of Radioimmunoassays, Seventh Training
Course on Radioligand Assay Techniques, The Endocrine Society,
March, 1986, which is incorporated by reference herein). The
radioactive isotope can be detected by such means as the use of a
gamma counter or a scintillation counter or by autoradiography.
[0107] It is also possible to label the antibody with a fluorescent
compound. When the fluorescently labeled antibody is exposed to
light of the proper wave length, its presence can then be detected
due to fluorescence. Among the most commonly used fluorescent
labeling compounds are fluorescein isothiocyanate, rhodamine,
phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and
fluorescamine.
[0108] The antibody can also be detectably labeled using
fluorescence emitting metals such as .sup.152Eu, or others of the
lanthanide series. These metals can be attached to the antibody
using such metal chelating groups as diethylenetriaminepentacetic
acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
[0109] The antibody also can be detectably labeled by coupling it
to a chemiluminescent compound. The presence of the
chemiluminescent-tagged antibody is then determined by detecting
the presence of luminescence that arises during the course of a
chemical reaction. Examples of particularly useful chemiluminescent
labeling compounds are luminol, isoluminol, theromatic acridinium
ester, imidazole, acridinium salt and oxalate ester.
[0110] Likewise, a bioluminescent compound may be used to label the
antibody of the present invention. Bioluminescence is a type of
chemiluminescence found in biological systems in, which a catalytic
protein increases the efficiency of the chemiluminescent reaction.
The presence of a bioluminescent protein is determined by detecting
the presence of luminescence. Important bioluminescent compounds
for purposes of labeling are luciferin, luciferase and
aequorin.
[0111] 5.5. Screening Assays for Compounds that Modulate NUCP
Expression of Activity
[0112] The following assays are designed to identify compounds that
interact with (e.g., bind to) a NUCP, compounds that interfere with
the interaction of a NUCP with any ligand or accessory molecules,
compounds that modulate NUCP gene expression (i.e., modulate the
level of NUCP activity by regulating gene expression) or otherwise
modulate the levels of a NUCP in the body. Assays may additionally
be utilized which identify compounds that bind to NUCP gene
regulatory sequences (e.g., promoter sequences) and, consequently,
may modulate NUCP gene expression. See e.g., Platt, K. A., 1994, J.
Biol. Chem. 269:28558-28562, which is incorporated herein by
reference in its entirety.
[0113] The compounds which can be screened in accordance with the
invention include but are not limited to peptides, antibodies and
fragments thereof, and other organic compounds (e.g.,
peptidomimetics) that bind to NUCP and either mimic the activity of
the natural product (i.e., agonists) or inhibit the activity of the
natural ligand/accessory molecule (i.e., antagonists); as well as
peptides, antibodies or fragments thereof, and other organic
compounds that mimic the NUCP (or a portion thereof) and bind to
and "inactivate" or "neutralize" the NUCP ligand/accessory
protein.
[0114] Such compounds may include, but are not limited to, peptides
such as, for example, soluble peptides, including but not limited
to members of random peptide libraries; (see, e.g., Lam, K. S. et
al., 1991, Nature 354:82-84; Houghten, R. et al., 1991, Nature
354:84-86), and combinatorial chemistry-derived molecular library
made of D- and/or L-configuration amino acids, phosphopeptides
(including, but not limited to members of random or partially
degenerate, directed phosphopeptide libraries; see, e.g., Songyang,
Z. et al., 1993, Cell 72:767-778), antibodies (including, but not
limited to, polyclonal, monoclonal, humanized, anti-idiotypic,
chimeric or single chain antibodies, and FAb, F(ab').sub.2 and FAb
expression library fragments, and epitope-binding fragments
thereof), and small organic or inorganic molecules.
[0115] Other compounds that can be screened in accordance with .the
invention include but are not limited to small organic molecules
that are able to cross the blood-brain barrier, gain entry into an
appropriate cell (e.g., in the choroid plexus, pituitary, the
hypothalamus, etc.) and affect the expression of a NUCP gene or
some other gene involved in a NUCP mediated pathway (e.g., by
interacting with the regulatory region or transcription factors
involved in gene expression); or such compounds that affect or
substitute for the activity of the NUCP or the activity of some
other intracellular factor involved in a NUCP-mediated catabolic,
or metabolic pathway.
[0116] Computer modeling and searching technologies permit
identification of compounds, or the improvement of already
identified compounds, that can modulate NUCP expression or
activity. Having identified such a compound or composition the
active sites or regions are identified. Such active sites might
typically be ligand binding sites. The active site can be
identified using methods known in the art including, for example,
from the amino acid sequences of peptides, from the nucleotide
sequences of nucleic acids, or from study of complexes of the
relevant compound or composition with its natural ligand. In the
latter case, chemical or X-ray crystallographic methods can be used
to find the active site by finding where on the factor the
complexed ligand is found.
[0117] Next, the three dimensional geometric structure of the
active site is determined. This can be done by known methods,
including X-ray crystallography, which can determine a complete
molecular structure. On the other hand, solid or liquid phase NMR
can be used to determine certain intra-molecular distances. Any
other experimental method of structure determination can be used to
obtain partial or complete geometric structures. The geometric
structures may be measured with a complexed ligand, natural or
artificial, which may increase the accuracy of the active site
structure determined.
[0118] If an incomplete or insufficiently accurate structure is
determined, the methods of computer based numerical modeling can be
used to complete the structure or improve its accuracy. Any
recognized modeling method may be used, including parameterized
models specific to particular biopolymers such as proteins or
nucleic acids, molecular dynamics models based on computing
molecular motions, statistical mechanics models based on thermal
ensembles, or combined models. For most types of models, standard
molecular force fields, representing the forces between constituent
atoms and groups, are necessary, and can be selected from force
fields known in physical chemistry. The incomplete or less accurate
experimental structures can serve as constraints on the complete
and more accurate structures computed by these modeling
methods.
[0119] Finally, having determined the structure of the active site
(or binding site), either experimentally, by modeling, or by a
combination, candidate modulating compounds can be identified by
searching databases containing compounds along with information on
their molecular structure. Such a search seeks compounds having
structures that match the determined active site structure and that
interact with the groups defining the active site. Such a search
can be manual, but is preferably computer assisted. These compounds
found from this search are potential NUCP modulating compounds.
[0120] Alternatively, these methods can be used to identify
improved modulating compounds from an already known modulating
compound or ligand. The composition of the known compound can be
modified and the structural effects of modification can be
determined using the experimental and computer modeling methods
described above applied to the new composition. The altered
structure is then compared to the active site structure of the
compound to determine if an improved fit or interaction results. In
this manner systematic variations in composition, such as by
varying side groups, can be quickly evaluated to obtain modified
modulating compounds or ligands of improved specificity or
activity.
[0121] Further experimental and computer modeling methods useful to
identify modulating compounds based upon identification of the
active sites (or binding sites) of NUCP, and related transduction
and transcription factors will be apparent to those of skill in the
art.
[0122] Examples of molecular modeling systems are the CHARMm and
QUANTA programs (Polygen Corporation, Waltham, Mass.). CHARMm
performs the energy minimization and molecular dynamics functions.
QUANTA performs the construction, graphic modeling and analysis of
molecular structure. QUANTA allows interactive construction,
modification, visualization, and analysis of the behavior of
molecules with each other.
[0123] A number of articles review computer modeling of drugs
interactive with specific proteins, such as Rotivinen, et al.,
1988, Acta Pharmaceutical Fennica 97:159-166; Ripka, New Scientist
54-57 (Jun. 16, 1988); McKinaly and Rossmann, 1989, Annu. Rev.
Pharmacol. Toxiciol. 29:111-122; Perry and Davies, OSAR:
Quantitative Structure-Activity Relationships in Drug Design pp.
189-193 (Alan R. Liss, Inc. 1989); Lewis and Dean, 1989 Proc. R.
Soc. Lond. 236:125-140 and 141-162; and, with respect to a model
receptor for nucleic acid components, Askew, et al., 1989, J. Am.
Chem. Soc. 111:1082-1090. Other computer programs that screen and
graphically depict chemicals are available from companies such as
BioDesign, Inc. (Pasadena, Calif.), Allelix, Inc. (Mississauga,
Ontario, Canada), and Hypercube, Inc. (Cambridge, Ontario).
Although these are primarily designed for application to drugs
specific to particular proteins, they can be adapted to design of
drugs specific to regions of DNA or RNA, once that region is
identified.
[0124] Although described above with reference to design and
generation of compounds which could alter binding, one could also
screen libraries of known compounds, including natural products or
synthetic chemicals, and biologically active materials, including
proteins, for compounds which are inhibitors or activators.
[0125] Cell-based systems can also be used to identify compounds
that bind (or mimic). NUCP as well as assess the altered activity
associated with such binding in living cells. One tool of
particular interest for such assays is green fluorescent protein
which is described, inter alia, in U.S. Pat. No. 5,625,048, herein
incorporated by reference. Cells that may be used in such cellular
assays include, but are not limited to, leukocytes, or cell lines
derived from leukocytes, lymphocytes, stem cells, including
embryonic stem cells, and the like. In addition, expression host
cells (e.g., B95 cells, COS cells, CHO cells, OMK cells,
fibroblasts, Sf9 cells) genetically engineered to express a
functional NUCP of interest and to respond to activation by the
test, or natural, ligand, as measured by a chemical or phenotypic
change, or induction of another host cell gene, can be used as an
end point in the assay.
[0126] Compounds identified via assays such as those described
herein may be useful, for example, in elucidating the biological
function of NUCP. Such compounds can be administered to a patient
at therapeutically effective doses to treat any of a variety of
physiological or mental disorders. A therapeutically effective dose
refers to that amount of the compound sufficient to result in any
amelioration, impediment, prevention, or alteration of any
biological symptom.
[0127] 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
that exhibit large 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.
[0128] 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 method of the
invention, 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.
[0129] Pharmaceutical compositions for use in accordance with the
present invention may be formulated in conventional manner using
one or more physiologically acceptable carriers or excipients.
Thus, the compounds and their physiologically acceptable salts and
solvates may be formulated for administration by inhalation or
insufflation (either through the mouth or the nose) or oral,
buccal, parenteral, intracranial, intrathecal, or rectal
administration.
[0130] For oral administration, the pharmaceutical compositions may
take the form of, for example, tablets or capsules prepared by
conventional means with pharmaceutically acceptable excipients such
as binding agents (e.g., pregelatinised maize starch,
polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers
(e.g., lactose, microcrystalline cellulose or calcium hydrogen
phosphate); lubricants (e.g., magnesium stearate, talc or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g., sodium lauryl sulphate). The tablets may be
coated by methods well known in the art. Liquid preparations for
oral administration may take the form of, for example, solutions,
syrups or suspensions, or they may be presented as a dry product
for constitution with water or other suitable vehicle before use.
Such liquid preparations may be prepared by conventional means with
pharmaceutically acceptable additives such as suspending agents
(e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible
fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous
vehicles (e.g., almond oil, oily esters, ethyl alcohol or
fractionated vegetable oils); and preservatives (e.g., methyl or
propyl-p-hydroxybenzoates or sorbic acid). The preparations may
also contain buffer salts, flavoring, coloring and sweetening
agents as appropriate.
[0131] Preparations for oral administration may be suitably
formulated to give controlled release of the active compound.
[0132] For buccal administration the compositions may take the form
of tablets or lozenges formulated in conventional manner.
[0133] For administration by inhalation, the compounds for use
according to the present invention are conveniently delivered in
the form of an aerosol spray presentation from pressurized packs or
a nebulizer, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit may be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of e.g. gelatin for use in an inhaler or insufflator may
be formulated containing a powder mix of the compound and a
suitable powder base such as lactose or starch.
[0134] The compounds may be formulated for parenteral
administration by injection, e.g., by bolus injection or continuous
infusion. Formulations for injection may be presented in unit
dosage form, e.g., in ampoules or in multi-dose containers, with an
added preservative. The compositions may take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents. Alternatively, the active ingredient may
be in powder form for constitution with a suitable vehicle, e.g.,
sterile pyrogen-free water, before use.
[0135] The compounds may also be formulated in rectal compositions
such as suppositories or retention enemas, e.g., containing
conventional suppository bases such as cocoa butter or other
glycerides.
[0136] In addition to the formulations described previously, the
compounds may also be formulated as a depot preparation. Such long
acting formulations may be administered by implantation (for
example subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compounds may be formulated with
suitable polymeric or hydrophobic materials (for example as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives, for example, as a sparingly soluble
salt.
[0137] The compositions may, if desired, be presented in a pack or
dispenser device which may contain one or more unit dosage forms
containing the active ingredient. The pack may for example comprise
metal or plastic foil, such as a blister pack. The pack or
dispenser device may be accompanied by instructions for
administration.
[0138] 5.5.1. In Vitro Screening Assays for Compounds that Bind to
a NUCP
[0139] In vitro systems may be designed to identify compounds
capable of interacting with (e.g., binding to) or mimicking a NUCP.
The compounds identified can be useful, for example, in modulating
the activity of wild type and/or mutant NUCP; can be useful in
elaborating the biological function of NUCP; can be utilized in
screens for identifying compounds that disrupt normal NUCP
interactions; or may themselves disrupt or activate such
interactions.
[0140] The principle of the assays used to identify compounds that
bind to a NUCP, or NUCP ligands/accessory molecules, involves
preparing a reaction mixture of NUCP and the test compound under
conditions and for a time sufficient to allow the two components to
interact and bind, thus forming a complex which can be removed
and/or detected in the reaction mixture. The NUCP species used can
vary depending upon the goal of the screening assay. For example,
where agonists of a natural NUCP accessory molecule or ligand are
desired, a full length NUCP, or a soluble truncated NUCP, a NUCP
peptide, or NUCP fusion protein containing one or more NUCP domains
fused to a protein or polypeptide that affords advantages in the
assay system (e.g., labeling, isolation of the resulting complex,
etc.) can be utilized. Where compounds that directly interact with
a NUCP are sought, peptides corresponding to NUCP and fusion
proteins containing a NUCP, or a portion thereof, can be used.
[0141] The screening assays can be conducted in a variety of ways.
For example, one method to conduct such an assay would involve
anchoring a NUCP, NUCP polypeptide, NUCP peptide, or fusion protein
thereof, or the test substance onto a solid phase and detecting
NUCP/test compound complexes anchored on the solid phase at the end
of the reaction. In one embodiment of such a method, the NUCP
reactant may be anchored onto a solid surface, and the test
compound, which is not anchored, may be labeled, either directly or
indirectly.
[0142] In practice, microtiter plates may conveniently be utilized
as the solid phase. The anchored component may be immobilized by
non-covalent or covalent attachments. Non-covalent attachment may
be accomplished by simply coating the solid surface with a solution
of the protein and drying. Alternatively, an immobilized antibody,
preferably a monoclonal antibody, specific for the protein to be
immobilized may be used to anchor the protein to the solid surface.
The surfaces may be prepared in advance and stored.
[0143] In order to conduct the assay, the nonimmobilized 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 nonimmobilized component is
pre-labeled, the detection of label immobilized on the surface
indicates that complexes were formed. Where the previously
nonimmobilized 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 previously nonimmobilized
component (the antibody, in turn, may be directly labeled or
indirectly labeled with a labeled anti-Ig antibody).
[0144] Alternatively, a reaction can be conducted in a liquid
phase, the reaction products separated from unreacted components,
and complexes detected; e.g., using an immobilized antibody
specific for a NUCP, NUCP polypeptide, peptide or fusion protein,
or the test compound to anchor any complexes formed in solution,
and a labeled antibody specific for the other component of the
possible complex to detect anchored complexes.
[0145] Alternatively, cell-based assays can be used to identify
compounds that interact with a NUCP. To this end, cell lines that
express a NUCP, or cell lines (e.g., COS cells, CHO cells,
fibroblasts, etc.) that have been genetically engineered to express
a NUCP or a NUCP ligand/accessory molecule (e.g., by transfection
or transduction of NUCP DNA, etc.) can be used. Interaction of the
test compound with, for example, NUCP ligand expressed by the host
cell can be determined by comparison or competition with native
NUCP.
[0146] 5.5.2. Assays for Compounds that Interfere with NUCP
Receptor/Intracellular or NUCP/Transmembrane Macromolecule
Interaction
[0147] Macromolecules that interact with a NUCP are referred to,
for purposes of this discussion, as "binding partners". These
binding partners are likely to be involved in NUCP mediated
biological pathways. Therefore, it is desirable to identify
compounds that interfere with or disrupt the interaction of such
binding partners which may be useful in regulating or augmenting
NUCP activity in the body and/or controlling disorders associated
with NUCP activity (or a deficiency thereof).
[0148] The basic principle of the assay systems used to identify
compounds that interfere with the interaction between NUCP, or NUCP
polypeptides, peptides or fusion proteins as described above
(collectively, the NUCP moiety), and its binding partner or
partners involves preparing a reaction mixture containing the NUCP
moiety and the binding partner under conditions and for a time
sufficient to allow the two to interact and bind, thus forming a
complex. In order to test a compound for inhibitory activity, the
reaction mixture is prepared in the presence and absence of the
test compound. The test compound may be initially included in the
reaction mixture, or may be added at a time subsequent to the
addition of the NUCP moiety and its binding partner. Control
reaction mixtures are incubated without the test compound or with a
placebo. The formation of any complexes between the NUCP moiety and
the 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 NUCP moiety and the interactive binding partner.
Additionally, complex formation within reaction mixtures containing
the test compound and normal NUCP may also be compared to complex
formation within reaction mixtures containing the test compound and
a mutant NUCP. This comparison may be important in those cases
wherein it is desirable to identify compounds that specifically
disrupt interactions of mutant, or mutated, NUCPs but not normal
NUCPs.
[0149] The assay for compounds that interfere with the interaction
of the NUCP moiety and its binding partners can be conducted in a
heterogeneous or homogeneous format. Heterogeneous assays involve
anchoring either the NUCP moiety 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 by competition can be identified by
conducting the reaction in the presence of the test substance;
i.e., by adding the test substance to the reaction mixture prior
to, or simultaneously with, the NUCP moiety and interactive binding
partner. 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 described briefly
below.
[0150] In a heterogeneous assay system, either the NUCP moiety or
an interactive binding partner, is anchored onto a solid surface,
while the non-anchored species is labeled, either directly or
indirectly. In practice, microtiter plates are conveniently
utilized. The anchored species may be immobilized by non-covalent
or covalent attachments. Non-covalent attachment may be
accomplished simply by coating the solid surface with a solution of
the NUCP moiety or binding partner and drying. Alternatively, an
immobilized antibody specific for the species to be anchored may be
used to anchor the species to the solid surface. The surfaces may
be prepared in advance and stored.
[0151] 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. The
detection of complexes anchored on the solid surface can be
accomplished in a number of ways. 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, may be directly labeled or indirectly labeled with a
labeled anti-Ig antibody). Depending upon the order of addition of
reaction components, test compounds which inhibit complex formation
or which disrupt preformed complexes can be detected.
[0152] 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 which inhibit complex
or which disrupt preformed complexes can be identified.
[0153] In an alternate embodiment of the invention, a homogeneous
assay can be used. In this approach, a preformed complex of the
NUCP moiety and an interactive binding partner is prepared in which
either the NUCP moiety or its binding partners is labeled, but the
signal generated by the label is quenched due to formation of the
complex (see, e.g., U.S. Pat. No. 4,190,496 by Rubenstein which
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 which disrupt
NUCP/intracellular binding partner interaction can be
identified.
[0154] In a particular embodiment, a NUCP fusion can be prepared
for immobilization. For example, NUCP or a peptide fragment can be
fused to a glutathione-S-transferase (GST) gene using a fusion
vector, such as pGEX-5X-1, in such a manner that its binding
activity is maintained in the resulting fusion protein. The
interactive binding partner can be purified and used to raise a
monoclonal antibody, using methods routinely practiced in the art
and/or described above. This antibody can be labeled with the
radioactive isotope .sup.125I, for example, by methods routinely
practiced in the art. In a heterogeneous assay, e.g., the GST-NUCP
fusion protein can be anchored to glutathione-agarose beads. The
interactive binding partner can then be added in the presence or
absence of the test compound in a manner that allows interaction
and binding to occur. At the end of the reaction period, unbound
material can be washed away, and the labeled monoclonal antibody
can be added to the system and allowed to bind to the complexed
components. The interaction between the NUCP moiety and the
interactive binding partner can be detected by measuring the amount
of radioactivity that remains associated with the
glutathione-agarose beads. A successful inhibition of the
interaction by the test compound will result in a decrease in
measured radioactivity.
[0155] Alternatively, the GST-NUCP moiety fusion protein and the
interactive binding partner can be mixed together in liquid in the
absence of the solid glutathione-agarose beads. The test compound
can be added either during or after the species are allowed to
interact. This mixture can then be added to the glutathione-agarose
beads and unbound material is washed away. Again the extent of
inhibition of the NUCP moiety/binding partner interaction can be
detected by adding the labeled antibody and measuring the
radioactivity associated with the beads.
[0156] In another embodiment of the invention, these same
techniques can be employed using peptide fragments that correspond
to the binding domain(s) of the NUCP moiety and/or the interactive
or binding partner (in cases where the binding partner is a
protein), in place of one or both of the full length proteins. Any
number of methods routinely practiced in the art can be used to
identify and isolate the binding sites. These methods include, but
are not limited to, mutagenesis of the gene encoding one of the
proteins and screening for disruption of binding in a
co-immunoprecipitation assay. Compensatory mutations in the gene
encoding the second species in the complex can then be selected.
Sequence analysis of the genes encoding the respective proteins
will reveal the mutations that correspond to the region of the
protein involved in interactive binding. Alternatively, one protein
can be anchored to a solid surface using methods described above,
and allowed to interact with and bind to its labeled binding
partner, which has been treated with a proteolytic enzyme, such as
trypsin. After washing, a relatively short, labeled peptide
comprising the binding domain may remain associated with the solid
material, which can be isolated and identified by amino acid
sequencing. Also, once the gene coding for the intracellular
binding partner is obtained, short gene segments can be engineered
to express peptide fragments of the protein, which can then be
tested for binding activity and purified or synthesized.
[0157] For example, and not by way of limitation, the NUCP moiety
can be anchored to a solid material as described, above, by making
a GST-NUCP moiety fusion protein and allowing it to bind to
glutathione agarose beads. The interactive binding partner can be
labeled with a radioactive isotope, such as .sup.35S, and cleaved
with a proteolytic enzyme such as trypsin. Cleavage products can
then be added to the anchored GST-NUCP moiety fusion protein and
allowed to bind. After washing away unbound peptides, labeled bound
material, representing the intracellular binding partner binding
domain, can be eluted, purified, and analyzed for amino acid
sequence by well-known methods. Peptides so identified can be
produced synthetically or fused to appropriate facilitative
proteins using recombinant DNA technology.
[0158] The present invention is not to be limited in scope by the
specific embodiments described herein, which are intended as single
illustrations of individual aspects of the invention, and
functionally equivalent methods and components are within the scope
of the invention. Indeed, various modifications of the invention,
in addition to those shown and described herein will become
apparent to those skilled in the art from the foregoing description
and accompanying drawings. Such modifications are intended to fall
within the scope of the appended claims. All patents, patent
applications, and publications cited herein are hereby incorporated
by reference.
Sequence CWU 1
1
4 1 876 DNA Homo sapiens 1 atgtcagccc tcaactggaa gccgtttgtg
tacggggggc tggcctccat cactgctgag 60 tgcggtacat ttccaattga
tttaaccaag acacggctcc agattcaagg ccagacgaat 120 gatgcaaaat
ttaaggaaat tagataccga ggaatgttgc acgcattagt gaggataggc 180
agagaagaag ggctgaaagc actctactcg gggattgccc ccgcgatgtt acgccaggca
240 tcctatggca ccatcaagat aggcacttac cagagcttga agcgactatt
cattgaacgc 300 ccagaagatg aaactctacc gataaatgtg atatgtggaa
ttctgtctgg agtcatatct 360 tcaaccattg ctaatccaac tgatgttttg
aaaattcgga tgcaagcgca aagcaacacc 420 attcaaggag gaatgatagg
caacttcatg aacatttacc agcaagaggg gacaagagga 480 ctgtggaagg
gtgtgtccct tactgcgcag agggctgcta ttgttgttgg tgtggagctg 540
ccggtctatg acatcaccaa gaagcatctt attctctcag gcctgatggg agacactgtg
600 tatacccact tcctctcaag cttcacctgt ggtctggcag gggccctggc
ctcaaaccct 660 gttgatgttg tgaggacacg tatgatgaat cagagagtgc
ttcgagatgg cagatgttct 720 ggctacacag gaaccctgga ttgcttgtta
cagacatgga agaatgaagg gttttttgct 780 ctctataaag gcttttggcc
aaattggttg agacttggtc cttggaatat cattttcttt 840 gtgacatacg
agcagttgaa gaaattggat ttgtga 876 2 291 PRT Homo sapiens 2 Met Ser
Ala Leu Asn Trp Lys Pro Phe Val Tyr Gly Gly Leu Ala Ser 1 5 10 15
Ile Thr Ala Glu Cys Gly Thr Phe Pro Ile Asp Leu Thr Lys Thr Arg 20
25 30 Leu Gln Ile Gln Gly Gln Thr Asn Asp Ala Lys Phe Lys Glu Ile
Arg 35 40 45 Tyr Arg Gly Met Leu His Ala Leu Val Arg Ile Gly Arg
Glu Glu Gly 50 55 60 Leu Lys Ala Leu Tyr Ser Gly Ile Ala Pro Ala
Met Leu Arg Gln Ala 65 70 75 80 Ser Tyr Gly Thr Ile Lys Ile Gly Thr
Tyr Gln Ser Leu Lys Arg Leu 85 90 95 Phe Ile Glu Arg Pro Glu Asp
Glu Thr Leu Pro Ile Asn Val Ile Cys 100 105 110 Gly Ile Leu Ser Gly
Val Ile Ser Ser Thr Ile Ala Asn Pro Thr Asp 115 120 125 Val Leu Lys
Ile Arg Met Gln Ala Gln Ser Asn Thr Ile Gln Gly Gly 130 135 140 Met
Ile Gly Asn Phe Met Asn Ile Tyr Gln Gln Glu Gly Thr Arg Gly 145 150
155 160 Leu Trp Lys Gly Val Ser Leu Thr Ala Gln Arg Ala Ala Ile Val
Val 165 170 175 Gly Val Glu Leu Pro Val Tyr Asp Ile Thr Lys Lys His
Leu Ile Leu 180 185 190 Ser Gly Leu Met Gly Asp Thr Val Tyr Thr His
Phe Leu Ser Ser Phe 195 200 205 Thr Cys Gly Leu Ala Gly Ala Leu Ala
Ser Asn Pro Val Asp Val Val 210 215 220 Arg Thr Arg Met Met Asn Gln
Arg Val Leu Arg Asp Gly Arg Cys Ser 225 230 235 240 Gly Tyr Thr Gly
Thr Leu Asp Cys Leu Leu Gln Thr Trp Lys Asn Glu 245 250 255 Gly Phe
Phe Ala Leu Tyr Lys Gly Phe Trp Pro Asn Trp Leu Arg Leu 260 265 270
Gly Pro Trp Asn Ile Ile Phe Phe Val Thr Tyr Glu Gln Leu Lys Lys 275
280 285 Leu Asp Leu 290 3 882 DNA Homo sapiens 3 atgtcagccc
tcaactggaa gccgtttgtg tacggggggc tggcctccat cactgctgag 60
tgcggtacat ttccaattga tttaaccaag acacggctcc agattcaagg ccagacgaat
120 gatgcaaaat ttaaggaaat tagataccga ggaatgttgc acgcattagt
gaggataggc 180 agagaagaag ggctgaaagc actctactcg gggattgccc
ccgcgatgtt acgccaggca 240 tcctatggca ccatcaagat aggcacttac
cagagcttga agcgactatt cattgaacgc 300 ccagaagatg aaactctacc
gataaatgtg atatgtggaa ttctgtctgg agtcatatct 360 tcaaccattg
ctaatccaac tgatgttttg aaaattcgga tgcaagcgca aagcaacacc 420
attcaaggag gaatgatagg caacttcatg aacatttacc agcaagaggg gacaagagga
480 ctgtggaagg gtgtgtccct tactgcgcag agggctgcta ttgttgttgg
tgtggagctg 540 ccggtctatg acatcaccaa gaagcatctt attctctcag
gcctgatggg agacactgtg 600 tatacccact tcctctcaag cttcacctgt
ggtctggcag gggccctggc ctcaaaccct 660 gttgatgttg tgaggacacg
tatgatgaat cagagagtgc ttcgagatgg cagatgttct 720 ggctacacag
gaaccctgga ttgcttgtta cagcttacag tgctggaaag tttttccacc 780
acagcaaagc cacaaaagct tatcagcgta gatgccatct cagaagaggc tgataccagg
840 ggatttacat atctcagctg tgatctttct gctccaagct ga 882 4 293 PRT
Homo sapiens 4 Met Ser Ala Leu Asn Trp Lys Pro Phe Val Tyr Gly Gly
Leu Ala Ser 1 5 10 15 Ile Thr Ala Glu Cys Gly Thr Phe Pro Ile Asp
Leu Thr Lys Thr Arg 20 25 30 Leu Gln Ile Gln Gly Gln Thr Asn Asp
Ala Lys Phe Lys Glu Ile Arg 35 40 45 Tyr Arg Gly Met Leu His Ala
Leu Val Arg Ile Gly Arg Glu Glu Gly 50 55 60 Leu Lys Ala Leu Tyr
Ser Gly Ile Ala Pro Ala Met Leu Arg Gln Ala 65 70 75 80 Ser Tyr Gly
Thr Ile Lys Ile Gly Thr Tyr Gln Ser Leu Lys Arg Leu 85 90 95 Phe
Ile Glu Arg Pro Glu Asp Glu Thr Leu Pro Ile Asn Val Ile Cys 100 105
110 Gly Ile Leu Ser Gly Val Ile Ser Ser Thr Ile Ala Asn Pro Thr Asp
115 120 125 Val Leu Lys Ile Arg Met Gln Ala Gln Ser Asn Thr Ile Gln
Gly Gly 130 135 140 Met Ile Gly Asn Phe Met Asn Ile Tyr Gln Gln Glu
Gly Thr Arg Gly 145 150 155 160 Leu Trp Lys Gly Val Ser Leu Thr Ala
Gln Arg Ala Ala Ile Val Val 165 170 175 Gly Val Glu Leu Pro Val Tyr
Asp Ile Thr Lys Lys His Leu Ile Leu 180 185 190 Ser Gly Leu Met Gly
Asp Thr Val Tyr Thr His Phe Leu Ser Ser Phe 195 200 205 Thr Cys Gly
Leu Ala Gly Ala Leu Ala Ser Asn Pro Val Asp Val Val 210 215 220 Arg
Thr Arg Met Met Asn Gln Arg Val Leu Arg Asp Gly Arg Cys Ser 225 230
235 240 Gly Tyr Thr Gly Thr Leu Asp Cys Leu Leu Gln Leu Thr Val Leu
Glu 245 250 255 Ser Phe Ser Thr Thr Ala Lys Pro Gln Lys Leu Ile Ser
Val Asp Ala 260 265 270 Ile Ser Glu Glu Ala Asp Thr Arg Gly Phe Thr
Tyr Leu Ser Cys Asp 275 280 285 Leu Ser Ala Pro Ser 290
* * * * *