U.S. patent application number 10/320778 was filed with the patent office on 2003-08-21 for peptides and other small molecules derived from regions of interacting proteins and uses thereof.
This patent application is currently assigned to Mount Sinai School of Medicine of the City University of New York. Invention is credited to Buck, Elizabeth, Chen, Yibang, Iyengar, Srinivas Ravi V., Weinstein, Harel, Weng, Gezhi.
Application Number | 20030157644 10/320778 |
Document ID | / |
Family ID | 26754861 |
Filed Date | 2003-08-21 |
United States Patent
Application |
20030157644 |
Kind Code |
A1 |
Iyengar, Srinivas Ravi V. ;
et al. |
August 21, 2003 |
Peptides and other small molecules derived from regions of
interacting proteins and uses thereof
Abstract
The present invention relates generally to the field of peptides
and other small molecules (i.e. peptide mimetics) as pharmaceutical
and/or therapeutic agents, and to methods for identification and
design of peptides and peptide mimetics having desired functional
activities. Specifically, peptides and other small molecules
derived from regions of interacting intracellular signaling
proteins are provided. More specifically, peptides and other small
molecules derived from regions of the G.beta. subunit of
heterotrimeric GTP binding proteins are provided. Such molecules
include specific agonists and antagonists of G.beta. downstream
effectors, including adenylyl cyclase and phospholipase C. Such
molecules are targeted to predicted regions of interaction between
intracellular signaling proteins and tested for activity in
functional assays using methods of the invention. One major
advantage of the invention is the incorporation of
three-dimensional structural information in models used for
predicting interaction surfaces between intracellular proteins.
Another major advantage is the ability to distinguish, within a
predicted interaction surface, a signal transfer region from a
general binding domain. Resolution of such signal transfer regions
from general binding domains is useful for prediction and
validation of pharmacologic and therapeutic agonists and
antagonists.
Inventors: |
Iyengar, Srinivas Ravi V.;
(Mohegan Lake, NY) ; Weng, Gezhi; (New York,
NY) ; Chen, Yibang; (Woodside, NY) ;
Weinstein, Harel; (New York, NY) ; Buck,
Elizabeth; (Hadlyme, CT) |
Correspondence
Address: |
PENNIE AND EDMONDS
1155 AVENUE OF THE AMERICAS
NEW YORK
NY
100362711
|
Assignee: |
Mount Sinai School of Medicine of
the City University of New York
|
Family ID: |
26754861 |
Appl. No.: |
10/320778 |
Filed: |
December 16, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10320778 |
Dec 16, 2002 |
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09245039 |
Feb 5, 1999 |
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6555522 |
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60073765 |
Feb 5, 1998 |
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Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/325; 530/324; 530/388.1; 536/23.5 |
Current CPC
Class: |
C07K 14/4722 20130101;
A61K 38/00 20130101 |
Class at
Publication: |
435/69.1 ;
435/320.1; 435/325; 530/324; 530/388.1; 536/23.5 |
International
Class: |
C12P 021/02; C12N
005/06; C07K 014/47; C07H 021/04; C07K 016/18 |
Claims
We claim:
1. An isolated peptide or derivative thereof selected from the
group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID
NO:8, SEQ ID NO:9 and SEQ ID NO:10.
2. The peptide or derivative of claim 1, which is capable of
immunospecific binding to an anti-peptide antibody.
3. A chimeric peptide comprising the peptide or derivative of claim
1 fused by a covalent bond to a second peptide.
4. The chimeric peptide of claim 3, wherein the peptide is capable
of immunospecific binding to an anti-peptide antibody.
5. A purified antibody or an antigen-binding derivative thereof
capable of immunospecific binding to the peptide or derivative
thereof of claim 1 and not to a protein from which the peptide was
derived.
6. The antibody of claim 5 which is polyclonal.
7. The antibody of claim 5 which is monoclonal.
8. A method of producing a recombinant peptide comprising: (a)
growing a recombinant cell containing a nucleic acid comprising a
nucleotide sequence encoding an amino acid sequence selected from
the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ
ID NO:8, SEQ ID NO:9 and SEQ ID NO:10 such that the encoded peptide
is expressed by the cell, and (b) recovering the expressed
recombinant peptide.
9. A purified recombinant peptide produced by the method of claim
8.
10. A pharmaceutical composition comprising: (a) a peptide or
derivative thereof selected from the group consisting of SEQ ID
NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID
NO:10; and (b) a pharmaceutically acceptable carrier.
11. A method of identifying a peptide or derivative thereof having
a biological activity of interest comprising: (a) providing a
molecular model of an intracellular protein-protein interaction
based on three dimensional structure information; (b) predicting a
candidate interaction surface of the intracellular protein-protein
interaction from the molecular model provided; and (c) measuring an
activity in a functional assay of a peptide encoded by at least a
portion of the candidate interaction surface predicted.
12. The method of claim 11, wherein the functional assay is
selected from the group consisting of an adenylyl cyclase assay, a
phospholipase C.beta. assay, a potassium channel assay and a
calcium channel assay.
13. A method of identifying one or more molecules that specifically
bind to a peptide or derivative thereof selected from the group
consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,
SEQ ID NO:9 and SEQ ID NO:10 comprising: (a) contacting the peptide
or derivative thereof with a plurality of molecules under
conditions conducive to binding between the peptide or derivative
thereof and the plurality of molecules; and (b) identifying any
molecules from within the plurality of molecules contacted in step
(a) that specifically bind to the peptide or fragment thereof.
14. The method of claim 13, wherein the peptide or derivative
thereof is labeled with a detectable label.
15. A method for identification of a peptide mimetic comprising:
predicting an interaction surface between a first interacting
protein and a second interacting protein; identifying a portion of
the interaction surface predicted which comprises a core signal
transfer region or a general binding domain by measuring a
functional activity of a peptide comprising at least three residues
of the portion in a functional assay; designing a molecule having a
three dimensional conformation of the peptide as folded in the
portion of the interaction surface identified; and testing whether
the molecule designed has the functional activity of the peptide in
the functional assay, wherein the molecule is identified as the
peptide mimetic if the functional activity of the molecule is at
least 0.1 times the functional activity of the peptide when tested
at the same molar concentration.
16. The method of claim 15, wherein the core signal transfer region
is identified by presence of agonist activity.
17. The method of claim 15, wherein the general binding domain is
identified by absence of agonist activity and presence of
antagonist activity.
18. The method of claim 15, wherein the interaction surface between
the first interacting protein and the second interacting protein is
predicted using three-dimensional structure information.
19. The method of claim 15, wherein the functional activity of the
molecule is greater than 0.1 times the functional activity of the
peptide in the functional assay.
20. The method of claim 15, wherein the functional activity of the
molecule is selected from the group consisting of from 0.1 to
10,000,000, from 0.1 to 100,000, from 0.1 to 1,000 and from 0.1 to
10 times the functional activity of the peptide in the functional
assay.
21. The method of claim 15, wherein the first interacting protein
is a G.beta. protein and the core signal transfer region of the
G.beta. protein comprises at least six contiguous residues of SEQ
ID NO:5.
22. The method of claim 15, wherein the first interacting protein
is a G.beta. protein and the general binding domain of the G.beta.
protein comprises at least six contiguous residues of SEQ ID
NO:6.
23. The method of claim 15, wherein the second interacting protein
is selected from the group consisting of an adenylyl cyclase
protein and a phospholipase C protein.
24. A method for mapping a surface of an intracellular interacting
protein comprising: modeling a surface representation of the
interacting protein with one or more other proteins; predicting an
interaction from the surface representation modeled; and testing
the interaction predicted in a functional assay by measuring a
functional activity of a peptide comprising at least six amino acid
residues of the surface representation.
Description
[0001] This application claims priority of U.S. Provisional Patent
Application No. 60/073,765, filed Feb. 5, 1998, which is
incorporated by reference herein in its entirety. This invention
was made with United States government support under grant numbers
DK-38761, GM-54508, DK-07645, DA-00060, GM-15599, and DA-07135, all
from the National Institutes of Health. Accordingly, the United
States has certain rights in the invention.
TABLE OF CONTENTS
PEPTIDES AND OTHER SMALL MOLECULES DERIVED FROM REGIONS OF
INTERACTING PROTEINS AND USES THEREOF
[0002] 1. FIELD OF THE INVENTION . . . -1-
[0003] 2. BACKGROUND OF THE INVENTION . . . -2-
[0004] 3. SUMMARY OF THE INVENTION . . . -4-
[0005] 4. BRIEF DESCRIPTION OF THE DRAWINGS . . . -8-
[0006] 5. DETAILED DESCRIPTION OF THE INVENTION . . . -11-
[0007] 5.1. PEPTIDES DERIVED FROM REGIONS OF G.beta. PROTEINS . . .
-15-
[0008] 5.1.1. ADENYLYL CYCLASE EFFECTOR PATHWAY . . . - 15-
[0009] 5.1.2. PHOSPHOLIPASE C EFFECTOR PATHWAY . . . -15-
[0010] 5.1.3. OTHER EFFECTOR PATHWAYS . . . - 15-
[0011] 5.2. TROUBLESHOOTING . . . -17-
[0012] 5.3. METHODS OF USE WITH THE INVENTION . . . -18-
[0013] 5.3.1. NUCLEIC ACID CLONING METHODS . . . -18-
[0014] 5.3.2. NUCLEIC ACID HYBRIDIZATION . . . -21-
[0015] 5.3.3. NUCLEIC ACID AMPLIFICATION . . . -23-
[0016] 5.4. DISEASES, DISORDERS AND CONDITIONS . . . -24-
[0017] 5.5. PHARMACEUTICAL COMPOSITIONS . . . -25-
[0018] 5.6. PEPTIDE DERIVATIVES . . . -31-
[0019] 5.7. ANTIBODIES . . . -39-
[0020] 5.8. STRUCTURE OF PEPTIDES AND NUCLEIC ACIDS . . . -44-
[0021] 5.8.1. PEPTIDE STRUCTURAL ANALYSIS . . . -44-
[0022] 5.8.2. NUCLEIC ACID STRUCTURAL ANALYSIS . . . -45-
[0023] 5.9. EXPRESSION OF RECOMBINANT PEPTIDES . . . -46-
[0024] 5.10. IDENTIFICATION OF MOLECULES HAVING BINDING CAPACITY .
. . -50-
[0025] 6. EXAMPLES . . . -66-
[0026] 6.1. A SURFACE ON THE G PROTEIN .beta. SUBUNIT INVOLVED IN
INTERACTIONS WITH ADENYLYL CYCLASES . . . -66-
[0027] 6.1.1. INTRODUCTION . . . -67-
[0028] 6.1.2. MATERIALS AND METHODS . . . -68-
[0029] 6.1.3. RESULTS . . . -70-
[0030] 6.1.4. DISCUSSION . . . -72-
[0031] 6.2. RESOLUTION OF A SIGNAL TRANSFER REGION FROM A GENERAL
BINDING DOMAIN IN G.beta. FOR STIMULATION OF PHOSPHOLIPASE
C-.beta.2 . . . -73-
[0032] 6.2.1. INTRODUCTION . . . -74-
[0033] 6.2.2. METHODS . . . -75-
[0034] 6.2.3. RESULTS AND DISCUSSION . . . -77-
1. FIELD OF THE INVENTION
[0035] The present invention relates generally to the field of
peptides and other small molecules (i.e. peptide mimetics) as
pharmaceutical and/or therapeutic agents, and to methods for
identification and design of peptides and peptide mimetics having
desired functional activities. Specifically, peptides and other
small molecules derived from regions of interacting intracellular
signaling proteins are provided. More specifically, peptides and
other small molecules derived from regions of the G.beta. subunit
of heterotrimeric GTP binding proteins are provided. Such molecules
include specific agonists and antagonists of G.beta. downstream
effectors, including adenylyl cyclase and phospholipase C. Such
molecules are targeted to predicted regions of interaction between
intracellular signaling proteins and tested for activity in
functional assays using methods of the invention. One major
advantage of the invention is the incorporation of
three-dimensional structural information in models used for
predicting interaction surfaces between intracellular proteins.
Another major advantage is the ability to distinguish, within a
predicted interaction surface, a signal transfer region from a
general binding domain. Resolution of such signal transfer regions
from general binding domains is useful for prediction and
validation of pharmacologic and therapeutic agonists and
antagonists.
2. BACKGROUND OF THE INVENTION
[0036] The ability to target a desired drug intervention to a
specific site in a biological system underlies the rational design
of safe and effective drugs. Past drug design efforts have often
focused on development of molecules believed to interact with cell
surface receptors. For example, high-throughput assays have been
used to screen synthetic organic compounds to identify molecules
interacting with an extracellular domain of a cell surface receptor
(Tian et al., 1998, A small, nonpeptidyl mimic of
granulocyte-colony-stimulating factor, Science 281, 257-259).
Further, methods have been developed for determining whether a
candidate compound is an agonist of a peptide hormone receptor (see
Kopin et al., U.S. Pat. No. 5,750,353, issued May 12, 1998, Assay
for non-peptide agonists to peptide hormone receptors). Peptides
and mimetics have also been developed based on the transmembrane
domains of G-protein-coupled receptors (Bouvier et al., Jan. 8,
1998, Peptides and peptidomimetic compounds affecting the activity
of G-protein-coupled-receptors by altering receptor
oligomerization, International Publication No. WO 98/00538).
Examples of other extracellular ligands for which peptide mimetics
have been developed include erythropoietin and TNF.alpha. (Wrighton
et al., 1997, Increased potency of an erythropoietin peptide
mimetic through covalent dimerization, Nature Biotechnology 15,
1261-1265; Takasaki et al., 1997, Structure-based design and
characterization of exocyclic peptidomimetics that inhibit
TNF.alpha. binding to its receptor, Nature Biotechnology 15,
1266-1270). Finally, distinct regions of peptide hormones have even
been considered for design of receptor antagonists (Portoghese et
al., 1990, Design of peptidomimetic .delta. opioid receptor
antagonists using the message-address concept, J. Med. Chem. 33,
1714-1720).
[0037] Heterotrimeric GTP-binding proteins (G proteins) consisting
of G.alpha..beta..gamma. subunits are ubiquitous signal
transduction proteins that play essential roles in intracellular
communication (see e.g. DeVivo and Iyengar, 1994, G protein
pathways: signal processing by effectors, Molec. Cell. Endocrinol.
100, 65-70). For example, the enzymatic production of cyclic AMP
(cAMP) via adenylyl cyclases is regulated by G proteins (Smit and
Iyengar, 1998, Mammalian adenylyl cyclases, Adv. Sec. Mess.
Phosphoprot. Res. 32, 1-21; Iyengar, 1993; Multiple families of
Gs-regulated adenylyl cyclases, Adv. Sec. Mess. Phosphoprot. Res.
28, 27-36; Pieroni et al., 1993, Signal recognition and integration
by Gs-stimulated adenylyl cyclases, Curr. Opin. Neurobiol. 3,
345-351; Weng et al., 1996, G beta subunit interacts with a peptide
encoding region 956-982 of adenylyl cyclase 2, cross-linking of the
peptide to free G beta gamma but not the heterotrimer, J. Biol.
Chem. 271, 26445-264488; Harr et al., 1997, Differential regulation
of adenylyl cyclases by G alphas, J. Biol. Chem. 272, 19017-19021).
G proteins provide a versatile system for investigation of
intracellular protein-protein interactions by virtue of their
interactions with multiple downstream effectors. For example, G
protein .beta..gamma. subunits regulate the activity of not on
adenylyl cyclase but also phospholipase C-.beta.2, calcium
channels, potassium channels, and .beta.-adrenergic receptor kinase
(see e.g. Ford et al., 1998, Molecular basis for interactions of G
protein .beta..gamma. subunits with effectors, Science 280,
1271-1274).
[0038] Drug intervention beyond the cell surface, i.e. at
intracellular protein-protein interaction sites, would broaden the
array of potential targets for achieving a desired therapeutic
effect. Intracellular targets may also. provide intervention points
having enhanced specificity compared to drugs targeted strictly at
cell surface receptors. The ability to use intracellular
interacting proteins as therapeutic targets for drug design has
been less clearly established, however. One reason may be that an
intracellular protein-protein interaction, unlike a typical cell
surface hormone-receptor interaction, will often involve a
multiplicity of proteins. Thus, resolution of specific interactions
among three or more proteins will often be necessary to carry out
design of safe and effective drugs. Accordingly, a need exists for
a generally-applicable approach for identification of peptides and
mimetics thereof having selective activity at a chosen
intracellular site of action.
3. SUMMARY OF THE INVENTION
[0039] This invention provides peptides and other small molecules
derived from regions of intracellular interacting proteins and
methods for identification of such molecules. More specifically,
the present invention provide peptides and other small molecules
derived from regions of G.beta. proteins which function as agonists
or antagonists of adenylyl cyclase or phospholipase C-.beta.2. The
invention is based, at least in part, on the discovery of the
inventors that it is possible to resolve, within a given
intracellular signal transduction protein, a signal transfer region
from a general binding domain. Such resolution provides a rational
basis for design of agonists and antagonists of virtually any
desired intracellular protein-protein interaction. The drug design
methods of the invention utilize three-dimensional structural
information for prediction of protein-protein interactions followed
by evaluation of predictions in functional assays.
[0040] The present invention relates generally to the field of
peptides and peptide mimetics as pharmaceutical and/or therapeutic
agents. More particularly, the present invention relates to
peptides and other small molecules (e.g. peptide mimetics) derived
from regions of G.beta. proteins and their use as pharmaceutical
and/or therapeutic agents. For example, peptides and derivatives
thereof for modulating adenylyl cyclase and phospholipase C-.beta.2
activities are provided. Still further, methods for identification
of peptides and derivatives thereof useful for modulating a chosen
effector-of-interest among various effectors are provided. One
advantage of the methods of the invention is the use of structural
modeling information to predict and validate pharmacologic and
therapeutic agents.
[0041] Predictions about effector interactions of G.beta. proteins
have been made using a combination of molecular modeling and
experimental validation in which the predictions of the model are
tested. Through an iterative process involving cycles of structural
modeling followed by experimental testing, precise definition of
individual effector domains within a G.beta. signal protein has
been achieved. The validated procedure has general applicability
for drug design targeted at other intracellular protein-protein
interactions in virtually any intracellular signal transduction
pathway.
[0042] This invention provides an isolated G.beta. peptide or
derivative thereof. This invention provides a peptide having an
amino acid sequence selected from the group consisting of SEQ ID
NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID
NO:10. In one embodiment, a derivative of a peptide is capable of
immunospecific binding to an anti-peptide antibody. In a preferred
embodiment, a peptide or a derivative thereof displays only one
functional activity of an intracellular signaling protein from
which it is derived. This invention provides a purified fragment of
a peptide, which fragment displays one or more functional
activities of an intracellular signaling protein. this invention
provides a purified fragment of a peptide comprising a region of
the peptide selected from the group consisting of an adenylyl
cyclase interaction region and a phospholipase C interaction
region. This invention provides a purified molecule comprising the
fragment. This invention provides a chimeric peptide comprising the
fragment, which fragment consists of at least 6 amino acids fused
by a covalent bond to an amino acid sequence of a second
peptide.
[0043] This invention provides a purified antibody or an
antigen-binding derivative thereof capable of immunospecific
binding to a peptide selected from the group consisting of SEQ ID
NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID
NO:10 and not to a protein from which the peptide was derived. In
one embodiment, the antibody is polyclonal. In another embodiment,
the antibody is monoclonal.
[0044] This invention provides a method of making a recombinant
protein comprising: (a) growing a recombinant cell containing a
nucleic acid comprising a nucleotide sequence encoding an amino
acid sequence selected from the group consisting of SEQ ID NO:5,
SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10
such that the recombinant protein is expressed by the cell; and (b)
recovering the expressed recombinant protein. Further, this
invention provides a purified recombinant protein produced by said
method. Any method known in the art may be used for growing the
recombinant cell (see e.g. Freshney, 1994, Culture of animal cells,
A manual of basic technique, 3d ed., Wiley-Liss, Inc., New York).
Any method known in the art may be used for recovering the
recombinant protein, such as routine size exclusion chromatography,
molecular tagging with histidine and purification on a nickel
column, etc.
[0045] This invention provides a pharmaceutical composition
comprising: (a) a peptide or derivative thereof selected from the
group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID
NO:8, SEQ ID NO:9 and SEQ ID NO:10; and (b) a pharmaceutically
acceptable carrier. The pharmaceutically acceptable carrier can be
any carrier known to one skilled in the art.
[0046] This invention provides a method of identifying a peptide or
derivative thereof having a biological activity of interest
comprising: (a) providing a molecular model of an intracellular
protein-protein interaction, which model predicts one or more
interaction surfaces among a plurality of interacting proteins
from-three-dimensional structure information; and (b) testing a
candidate interaction surface predicted by the molecular model by
determining whether a peptide encoding at least a portion of the
surface has a functional activity in a functional assay. In one
embodiment, the functional activity is an agonist activity. In
another embodiment, the functional activity is an antagonist
activity.
[0047] This invention provides a method of identifying a functional
activity of a G.beta. peptide comprising: (a) expressing a protein
comprising a peptide selected from the group consisting of SEQ ID
NO:9 and SEQ ID NO:10 in a biological system; and (b) measuring an
effect of expression in a biological assay. In one embodiment, the
biological system is selected from the group consisting of an
animal cell culture and an experimental animal. In another
embodiment, the experimental animal is selected from the group
consisting of a fly (e.g. D. melanogaster), a worm (e.g. C.
elegans), a fish (e.g. zebrafish), a rat, a mouse and a guinea pig.
In yet another embodiment, the biological assay is selected from
the group consisting of an adenylyl cyclase assay, a phospholipase
C assay, a potassium channel assay, a calcium channel assay and a
.beta.-adrenergic receptor kinase assay.
[0048] This invention provides a method of detecting an effect of
expression of a recombinant protein comprising a peptide selected
from the group consisting of SEQ ID NO:9 and SEQ ID NO:10 on a
signal transduction pathway, the method comprising: (a) expressing
the recombinant protein in a cell culture or experimental animal
already having a mutation in the signal transduction pathway; and
(b) detecting the effect of expression in a biological assay. In
one embodiment, the biological assay is selected from the group
consisting of an adenylyl cyclase assay, a phospholipase C.beta.
assay, a potassium channel assay and a calcium channel assay. In
another embodiment, the mutation in the signal transduction pathway
is in a gene selected from the group consisting of an adenylyl
cyclase gene, a phospholipase C gene, a potassium channel gene and
a calcium channel gene.
[0049] This invention provides a method of identifying a molecule
that specifically binds to a peptide or derivative thereof selected
from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,
SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, the method comprising:
(a) contacting the peptide or derivative thereof with a plurality
of molecules under conditions conducive to binding; and (b)
identifying a molecule from the plurality of molecules that
specifically binds to the peptide or derivative thereof.
4. BRIEF DESCRIPTION OF THE DRAWINGS
[0050] The file of this patent contains at least one drawing
executed in color. Copies of this patent with color drawings will
be provided by the Patent and Trademark Office upon request and
payment of the necessary fee.
[0051] FIG. 1. Regions of G.beta. involved in contacts with the AC2
956-982 peptide. (FIG. 1A) Ribbon diagram of the G.beta. backbone
from the crystal structure of G.beta..gamma. (Sondek et al., 1996,
Nature 379, 369-374; Lambright et al., 1996, Nature 379, 311-319);
the residues in contact with the AC2 peptide are shown in pink
(Weng et al., 1996, J. Biol. Chem. 271, 26445-26448). (FIG. 1B)
Predicted core contacts between the AC2 956-982 peptide and
G.beta.. The AC2 peptide residues are in the blue boxes. The AC2
peptide residues are numbered 1-27 from the N terminus. G.beta.1
residues are in green boxes. The G.beta.1 residues are shown in the
spatial sequence in which they are predicted to interact with the
AC2 peptide.
[0052] FIG. 2. Effects of the G.beta.86-105 peptides on AC2 and AC1
activities. (FIG. 2A) Ribbon diagram of the G.beta. backbone with
residues 86-105 in yellow. Other residues in contact with the AC2
peptide are shown in pink. (FIG. 2B) Effect of the G.beta.86-105
peptide (TTN) and the M101N G.beta.86-105 mutant peptide (m-TTN) on
basal, .alpha..sub.s* (2 nM), and various concentrations of TTN
peptide on G.beta..gamma.-stimulated AC2 activity in the presence
of .alpha..sub.s* (2 nM) plus G.beta..gamma. (50 nM) stimulated AC2
activities. (FIG. 2C) Effect of various concentrations of TTN
peptide on G.beta..gamma.-stimulated AC2 activity in the presence
of .alpha..sub.s* (2 nM). (FIG. 2D) Effect of TTN and m-TTN
peptides on basal and CaM (100 nM) plus G.beta..gamma. (30 nM)
regulated AC1 activities. (FIG. 2E) Effect of TTN and m-TTN
peptides on basal and CaM (100 nM) stimulated AC1 activities.
[0053] FIG. 3. Effects of the G.beta. 115-135 peptide on AC2 and
AC1 activities. (FIG. 3A) Ribbon diagram of the G.beta.1 backbone
with residues 115-135 in yellow. Other residues in contact with the
AC2 peptide are shown in pink. (FIG. 3B) Effect of the
G.beta.115-135 peptide (GGL) and the Y124V G.beta.115-135 mutant
peptide (m-GGL) on basal, .alpha..sub.s* (2 nM), and .alpha..sub.s*
(2 NM) plus G.beta..gamma. (50 nM) stimulated AC2 activities. (FIG.
3C) Effect of GGL and m-GGL peptides on basal, CaM (100 nM), or CaM
(100 nM) plus G.beta..gamma. (30 nM) regulated AC1 activities.
[0054] FIG. 4. Schematic representation of the regions of G.beta.
involved in interactions with G.alpha. (outlined in green) and some
regions that may interact with adenylyl cyclases 1 and 2 (outlined
in red). The space-filling model of G.beta. was obtained from tfhe
crystallographic coordinates; G.alpha. contact regions are those
identified by Sigler and coworkers (Sondek et al., 1996, Nature
379, 369-374; Lambright et al., 1996, Nature 379, 311-319) from the
crystal structure of the heterotrimer. The AC2 peptide interaction
region was deduced from molecular modeling studies (Weng et al.,
1996, J. Biol. Chem. 271, 26445-26448) and the functional data in
FIG. 2 and FIG. 3 indicate that these regions may be involved in
interactions with AC1 and AC2.
[0055] FIG. 5. Effects of varying concentrations of G.beta. 86-105
peptide on PLC-.beta.2 activity. Upper Panel: Effects of G.beta.
86-105 peptide on basal and G.beta..gamma. (100 nM) stimulated
PLC-.beta.2 activity. Lower Panel: Effects of G.beta. 86-105
peptide and M101N G.beta. 86-105 peptide on PLC-.beta.2 basal
activity.
[0056] FIG. 6. Effects of varying concentrations of G.beta. 86-105
peptide and (FIG. 6A) K89A, H91A, and R96A substituted peptides on
PLC-.beta.2 activity (FIG. 6B) K89A, H91A, and R96A triple
substituted peptide on basal (Upper Panel) and G.beta..gamma. (100
nM) (Lower Panel) stimulated PLC-.beta.2 activity. (FIG. 6C)
Effects of varying concentrations of G.beta. 86-105 peptide and
FLLT peptide on PLC-.beta.2 activity. (FIG. 6D) Effects of 100 nM
G.beta..gamma. and varying concentrations of G.beta. 86-105 peptide
on PLC-.beta.2 and PLCX.beta. activity.
[0057] FIG. 7. Effects of varying concentrations of G.beta. 86-105
peptide and (Upper Panel) S97A G.beta. 86-105 peptide, (Middle
Panel) S97,98R G.beta. 86-105 peptide, and (Lower Panel) S97,98D
and S97,98C peptides on PLC-.beta.2 activity.
[0058] FIG. 8. Effects of shorter peptides from G.beta. 86-105
region on PLC-.beta.2 activity. (FIG. 8A) Effects of 100 .mu.M
G.beta. 96-98, G.beta. 96-101, and G.beta. 89-101 peptides on
PLC-.beta.2 activity. (FIG. 8B) Effects of varying concentrations
of G.beta. 96-101 peptide and S97, 98R (Upper Panel) and S97, 98D
(Lower Panel) G.beta. 96-101 peptides on PLC-.beta.2 basal
activity. Values for (FIG. 8A) are given as mean.+-.SEM of three
experiments.
[0059] FIG. 9. Effects of G.beta. 115-135 peptide on PLC-.beta.2
activity. (FIG. 9A) Effects of 30 nM G.beta. 115-135 peptide and
Y124V G.beta. 115-135 peptide on basal and G.beta..gamma. (100 nM)
stimulated PLC-.beta.2 activity. (FIG. 9B) Effect of varying
concentrations of G.beta. 115-135 peptide on G.beta..gamma. (100
nM) stimulated PLC-.beta.2 activity. Values for (FIG. 9A) are given
as mean.+-.SEM of three experiments.
[0060] FIG. 10. Ribbon diagram of G.beta..gamma.. G.beta. is shown
in khaki. G.gamma. is shown in grey. Residues 96-101 of G.beta. are
shown in pink, and residues 115-135 of G.beta. are shown in
aqua.
5. DETAILED DESCRIPTION OF THE INVENTION
[0061] The present invention relates to peptides and other small
molecules (e.g. peptide mimetics) derived from regions of
intracellular interacting proteins (e.g. signal transduction
proteins) and to their use as pharmaceutics. The present invention
also relates to methods for identifying peptides and derivatives
thereof as candidate pharmaceutics. Such methods combine molecular
modeling of surface interactions between two or more intracellular
proteins with experimental validation of model predictions. More
specifically, modeling of surface interactions is based on
three-dimensional structure information and validation of model
predictions is based on measuring activities of peptides or
derivatives thereof encoding at least a portion of a predicted
interaction surface in a functional assay. The invention further
relates to fragments and analogs of identified peptides. Nucleic
acids encoding such peptides are also within the scope of the
invention. Production of peptides and derivatives thereof, e.g., by
recombinant or chemical synthetic methods, is provided. Antibodies
specifically immunoreactive with identified peptides and
derivatives are additionally provided.
[0062] The invention is illustrated by way of Examples set forth in
Section 6 below which disclose, inter alia, the identification and
characterization of peptides derived from a G.beta. protein, human
G.beta.1, which have specific interactions with adenylyl cyclase
and phospholipase C-.beta.2. The complete G.beta.1 protein amino
acid sequence, which is identical in humans, dogs, cows and mice,
is set forth in SEQ ID NO:1 (Codina et al., 1986, Beta-subunits of
the human liver Gs/Gi signal-transducing proteins and those of
bovine retinal rod cell transducin are identical, FEBS Lett. 207,
187-192).
[0063] Any functional assay known to one skilled in the art may be
used to measure a functional activity of a peptide of the
invention. For example, an adenylyl cyclase activity or a
phospholipase C-.beta.2 activity may be measured. Such enzyme
activities may be measured in in vivo or in vitro experimental
systems. Functional assays used to determine an activity of a
peptide may employ any cloned, recombinant enzyme available. Many
such enzymes are known in the art. Examples include but are not
limited to: the bovine adenylyl cyclase 1 (AC1) amino acid sequence
set forth in SEQ ID NO:2 (Krupinski et al., 1989, Science 244,
1558-1564; the rat adenylyl cyclase 2 (AC2) amino acid sequence is
set forth in SEQ ID NO:3 (Feinstein et al., 1991, Proc. Natl. Acad.
Sci. U.S.A. 88, 10173-10177); and the human phospholipase C-.beta.2
(PLC-.beta.2) amino acid sequence is set forth in SEQ ID NO:4 (Park
et al., 1992, J. Biol. Chem. 267, 16048-16055).
[0064] In particular aspects, the invention provides amino acid
sequences of peptides, fragments and derivatives thereof, and other
small molecules, and fragments and derivatives thereof, which
comprise an antigenic determinant (i.e., can be recognized by an
antibody) or which are otherwise functionally active. In the case
of peptides, nucleic acid sequences encoding them are also
provided. "Functionally active" material as used herein refers to
material displaying one or more functional activities associated
with an identified peptide or other small molecule of the
invention, e.g., activation or inhibition of a downstream effector
(e.g., adenylyl cyclase 1 or 2, phospholipase C-.beta.2, etc.) or
binding to another protein binding partner, antigenicity (binding
to an antibody of the invention), immunogenicity, etc.
[0065] In specific embodiments, the invention provides fragments of
a peptide or derivative thereof consisting of at least 3 amino
acids, 6 amino acids, 10 amino acids, 15 amino acids, 20 amino
acids, 30 amino acids, or 50 amino acids. Nucleic acids encoding
the foregoing are also provided.
[0066] Once a peptide of the invention is identified, it may be
isolated and purified by any number of standard methods including
but not limited to chromatography (e.g., ion exchange, affinity,
and sizing column chromatography), centrifugation, differential
solubility, etc. The functional properties of an identified peptide
of interest may be evaluated using any functional assay known in
the art. In preferred embodiments, assays for evaluating downstream
effector functions in intracellular signal transduction pathways
are used (see Examples in Section 6).
[0067] In other specific embodiments, a peptide, fragment, analog,
or derivative may be expressed as a fusion, or chimeric protein
product (comprising the peptide, fragment, analog, or derivative
joined via a peptide bond to a heterologous protein sequence of a
different protein). Such a chimeric product can be made by ligating
the appropriate nucleic acid sequences encoding the desired amino
acid sequences to each other by methods known in the art, in the
proper reading frame, and expressing the chimeric product by
methods known in the art. Such exemplary but not limiting methods
are described below. Alternatively, a chimeric product may be made
by protein synthetic techniques, e.g., by use of a peptide
synthesizer. Standard chemical methods for peptide synthesis are
also well known in the art (see e.g. Hunkapiller et al., 1984,
Nature 310, 105-111). The terms "peptide", "polypeptide" and
"protein" are used synonymously herein.
[0068] This invention provides methods for identification of
peptides and peptide mimetics. In a preferred embodiment, the
methods of the invention provide for identification of peptides
(and/or fragments, analogs, derivatives, and mimetics thereof, i.e.
other small molecules) by first modeling an interaction surface
from three-dimensional structural information of one or more
interacting proteins. In a preferred embodiment, interactions of a
heterotrimeric G protein .beta. subunit with one or more downstream
effecteors is modeled to predict one or more interaction regions.
Predicted interaction regions are next evaluated using synthetic or
recombinant peptides (or other small molecules) in functional
assays. Through an iterative process which may involve, for
example, changing one or more residues of a given peptide, the
method can be used to identify peptides having very specific
functional effects. For example, peptide agonists or antagonists of
a specific pathway are identified by activation or inhibition,
respectively, of the functional pathway with a given peptide or
derivative thereof. For an intracellular protein having more than
one interaction partner, interaction regions specific for each
interaction partner may be identified. In a preferred embodiment,
the methods of the invention are used to resolve a specific signal
transfer region from a general binding domain within an
intracellular signaling protein. Such resolution permits design of
selective agonists and antagonists of the identified
interactions.
[0069] 5.1. Peptides Derived from Regions of G.beta. Proteins
[0070] The peptides of the invention described herein which have
been derived from regions of G.beta. proteins include but are not
limited to peptides having amino acid sequences as set forth in SEQ
ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ
ID NO:10.
[0071] 5.1.1. Adenylyl Cyclase Effector Pathway
[0072] Peptides which modulate the adenylyl cyclase effector
pathway include but are not limited to peptides having amino acid
sequences as set forth in SEQ ID NO:5 and SEQ ID NO:6.
[0073] 5.1.2. Phospholipase C Effector Pathway
[0074] Peptides which modulate the phospholipase C effector pathway
include but are not limited to peptides having amino acid sequences
as set forth in SEQ ID NO:9 and SEQ ID NO:10.
[0075] 5.1.3. Other Effector Pathways
[0076] The methods of the invention may be applied to virtually any
intracellular signal transduction pathway. For example, many
cancers have been linked to perturbations in regulation of the cell
cycle. Cell cycle gene products are amenable to the methods of the
invention for identification of peptides and other small molecules
which may act as agonists or antagonists. Such molecules are of
potentially great benefit for cancer treatment.
[0077] Briefly, the cell cycle consists of four stages: G1 (for
Gap1) phase, the resting stage prior to DNA synthesis; S (for
synthesis) phase, in which DNA synthesis occurs; G2 (for Gap2)
phase, the resting stage after DNA synthesis and prior to mitosis;
and M phase, mitosis, in which cell division occurs. For a review
of the cell cycle, including a list of genes encoding intracellular
interacting proteins of the cell cycle, see Murray and Hunt ("The
Cell Cycle, An Introduction", 1993, Oxford University Press, New
York, pp. 1-251, incorporated by reference herein in its
entirety).
[0078] Progression of a cell through the cell cycle is driven by a
group of cyclin-dependent kinases (CDKs) (see e.g. Elledge, 1996,
Science 274, 1664-1672; Nasmyth, 1996, Science 274, 1643-1645). The
kinase activities of CDKs require their positive subunits, the
cyclins. Further, the activities of specific CDK/cyclin complexes
are in turn positively and negatively regulated by phosphorylation
events and CDK inhibitors (CKIs) (see Hunter and Pines, 1995, Cell
80, 225-236; Morgan, 1995, Nature 374, 131-134). While specific
CDKs (CDK2, CDK4 and CDK6) and cyclins D and E regulate the
progression from G1 into S phase, cdc2 and cyclins A and B regulate
the cell cycle progression from G1 into mitosis (see Hunter and
Pines, 1995, Cell 80, 225-236).
[0079] Human tumor suppressor genes often act as negative
regulators of the cell cycle, and several tumor suppressors are
known to influence activities of CDK/cyclin complexes. For example,
p53 activates transcription of the p21 CDK inhibitor
(p21.sup.WAF1/CIP1) in response to DNA damage signals, and p21 in
turn binds and inactivates the CDK4 and CDK6 cyclin D complexes
(Gartel et al., 1996, Proc. Soc. Exp. Biol. Med. 213, 138-149).
Another CDK inhibitor, p16, is itself a potent tumor suppressor
(Biggs and Kraft, 1995, J. Mol. Med. 73, 509-514).
[0080] By systematically applying the methods of the invention to
intracellular protein-protein interactors such as the cyclins and
CDKs, it is possible to identify peptides and derivatives thereof
having functional activity in disease states such as cancer. In
this way, application of the methods of the invention may identify
important pharmacologic and therapeutic cancer drugs.
[0081] 5.2. Troubleshooting
[0082] If any given signal transduction protein or pathway is
initially resistant to the above-described approaches for
identifying peptides and other small molecules therefrom for use as
pharmaceutics, the following troubleshooting discussion may be
helpful. A resistant intracellular signal transduction protein may
be indicated by the identification of no peptide or other small
molecule capable of modulating a downstream effector in a specific
fashion. Consider a case where an initial molecular model of a
given effector interaction does not identify a peptide or other
small molecule when tested experimentally using functional assays
for cyclins, CDKs, or such as those described in the Examples set
forth in Section 6. In this instance, careful attention should be
paid to refining the molecular model.
[0083] For example, a synergistic effect between two or more
domains of a given signal transduction protein, or two or more
domains of more than one signal transduction protein, may be
required to elicit an experimental manifestation of an effector
interaction using a peptide or derivative thereof of the invention.
In this instance, it is desirable to identify and enumerate in a
systematic fashion any and all protein-protein interaction domains
which may have an influence in the downstream effector pathway. In
this way, an accounting is made for the possibility of multiple
molecular determinants in any given effector pathway.
[0084] In this regard, a current review of the literature is often
warranted in an effort to determine whether all possible signal
transduction proteins (and other biologic signaling agents) have
been considered in the design of prospective peptides and peptide
mimetics to be experimentally evaluated. This is particularly so in
the present post-genomic era where vast catalogs of genes encoding
predicted proteins having known or predicted functions are publicly
available in computer databases.
[0085] An effective literature review generally involves reviewing
the relevant chemical, biological, and medical literature
(including clinical data) in connection with a signal transduction
pathway or other biological event of interest. In this regard,
reference to a variety of frequently-updated computer databases is
often the best course to follow (e.g. Medline.RTM., GenBank.RTM.,
etc.).
[0086] 5.3. Methods of Use with the Invention
[0087] Any method known to one of ordinary skill in the art may be
used together with the peptides, derivatives, and methods of the
invention. Set forth below are well known methods for nucleic acid
cloning, hybridization, and amplification which are of general use
together with the invention. These methods enable the production
of, e.g., synthetic and recombinant peptides and derivatives
thereof, including fusion proteins.
[0088] 5.3.1. Nucleic Acid Cloning Methods
[0089] Methods for cloning nucleic acids are very well known in the
art. Several examples of use with the invention are set forth
below. These methods shall not be construed to limit the invention
in any way. The following description sets forth methods by which
clones of any desired nucleic acid may be obtained.
[0090] Any prokaryotic or eukaryotic cell may serve as the nucleic
acid source for molecular cloning. For example, the nucleic acid
sequences encoding proteins and fragments thereof may be isolated
from vertebrate, mammalian, human, porcine, bovine, feline, avian,
equine, canine, as well as additional primate sources, insects
(e.g., Drosophila), invertebrates (e.g., C. elegans), plants, etc.
The DNA may be obtained by standard procedures known in the art
from cloned DNA (e.g., a DNA "library"), by chemical synthesis, by
cDNA cloning, or by the cloning of genomic DNA, or fragments
thereof, purified from the desired cell (see e.g., Sambrook et al.,
1989, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y.; see also Glover,
ed., 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd.,
Oxford, U.K. Vol. I, II.). Clones derived from genomic DNA may
contain regulatory and intron DNA regions in addition to coding
regions; clones derived from cDNA will contain only exon
sequences.
[0091] Once nucleic acid fragments are generated, identification of
the specific nucleic acid fragment of interest may be accomplished
in a number of ways. For example, if a portion of a nucleic acid is
available and can be purified and labeled, the generated nucleic
acid fragments may be screened by hybridization to the labeled
probe (Benton and Davis, 1977, Science 196, 180; Grunstein and
Hogness, 1975, Proc. Natl. Acad. Sci. U.S.A. 72, 3961). Those
fragments with substantial homology to the probe will hybridize. It
is also possible to identify the appropriate fragment by
restriction enzyme digestion(s) and comparison of fragment sizes
with those expected according to a known restriction map if such is
available.
[0092] Alternatively, the presence of the desired nucleic acid may
be detected by assays based on the physical, chemical, or
immunological properties of any expressed product. For example,
cDNA clones, or DNA clones which hybrid-select the cognate mRNAs,
can be selected and expressed to produce a protein that has, e.g.,
similar or identical electrophoretic migration, isoelectric
focusing behavior, proteolytic digestion maps, hormonal activity,
binding activity, or antigenic properties as known for a protein of
interest. Using an antibody to a known protein, other proteins may
be identified by binding of the labeled antibody to expressed
putative proteins, e.g., in an ELISA (enzyme-linked immunosorbent
assay)-type procedure. Further, using a binding protein specific to
a known protein, other proteins may be identified by binding to
such a protein (see e.g., Clemmons, 1993, "IGF binding proteins and
their functions," Mol. Reprod. Dev. 35, 368-374; Loddick et al.,
1998, "Displacement of growth factors from their binding proteins
as a potential treatment for stroke," Proc. Natl. Acad. Sci. U.S.A.
95, 1894-1898).
[0093] An identified and isolated nucleic acid may be inserted into
an appropriate cloning vector. Any of a large number of vector-host
systems known in the art may be used. Possible vectors include, but
are not limited to, plasmids or modified viruses, but the vector
system must be compatible with the host cell used. Such vectors
include, but are not limited to, bacteriophages such as lambda
derivatives, or plasmids such as PBR322 or pUC plasmid derivatives
or the Bluescript vector (Stratagene). The insertion into a cloning
vector can, for example, be accomplished by ligating the DNA
fragment into a cloning vector which has complementary cohesive
termini. However, if the complementary restriction sites used to
fragment the DNA are not present in the cloning vector, the ends of
the DNA molecules may be enzymatically modified. Alternatively, any
site desired may be produced by ligating nucleotide sequences
(linkers) onto the DNA termini; these ligated linkers may comprise
specific chemically synthesized oligonucleotides encoding
restriction endonuclease recognition sequences. In an alternative
method, the cleaved vector and an gene may be modified by
homopolymeric tailing. Recombinant molecules can be introduced into
host cells via transformation, transfection, infection,
electroporation, etc., so that many copies of the desired sequence
are generated.
[0094] In specific embodiments, transformation of host cells with
recombinant DNA molecules that incorporate an isolated nucleic acid
sequence enables generation of multiple copies of the nucleic acid.
Thus, the nucleic acid may be obtained in large quantities by
growing transformants, isolating the recombinant DNA molecules from
the transformants and, when necessary, retrieving the inserted
nucleic acid from the isolated recombinant DNA (e.g. by restriction
digestion or PCR).
[0095] 5.3.2. Nucleic Acid Hybridization
[0096] Nucleic acid hybridization under various stringency
conditions (e.g. low, moderate, or high stringency conditions) is
quite well known to one skilled in the art. Guidelines for nucleic
acid hybridization are widely available, including detailed
protocols for determination and use of an appropriate stringency
(see e.g., Sambrook et al., 1989, Molecular Cloning, A Laboratory
Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.; see also, Ausubel et al., eds., in the Current
Protocols in Molecular Biology series of laboratory technique
manuals, .COPYRGT.1987-1994 Current Protocols, .COPYRGT.1994-1997
John Wiley and Sons, Inc.; see especially, Dyson, 1991,
Immobilization of nucleic acids and hybridization analysis, In:
Essential Molecular Biology: A Practical Approach, Vol. 2, Brown,
ed., pp. 111-156, IRL Press at Oxford University Press, Oxford,
U.K.).
[0097] In one embodiment, a nucleic acid which is hybridizable to
another nucleic acid under conditions of high stringency is
provided. In another embodiment, a nucleic acid which is
hybridizable to another nucleic acid under conditions of medium
stringency is provided. By way of example and not limitation,
hybridization procedures using conditions of high stringency may be
as follows. Prehybridization of filters containing DNA is carried
out for 8 h to overnight at 65.degree. C. in buffer composed of
6.times.SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02%
Ficoll, 0.02% BSA, and 500 .mu.g/ml denatured salmon sperm DNA.
Filters are hybridized for 48 h at 65.degree. C. in
prehybridization mixture containing 100 .mu.g/ml denatured salmon
sperm DNA and 5-20.times.10.sup.6 cpm of .sup.32P-labeled probe.
Washing of filters is done at 37.degree. C. for 1 h in a solution
containing 2.times.SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA.
This is followed by a wash in 0.1.times.SSC at 50.degree. C. for 45
min before autoradiography.
[0098] In yet another embodiment, a nucleic acid which is
hybridizable to another nucleic acid under conditions of low
stringency is provided. Again by way of example and not limitation,
procedures using conditions of low stringency may be as follows
(see also Shilo and Weinberg, 1981, Proc. Natl. Acad. Sci. U.S.A.
78, 6789-6792). Filters containing DNA are pretreated for 6 h at
40.degree. C. in a solution containing 35% formamide, 5.times.SSC,
50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA,
and 500 .mu.g/ml denatured salmon sperm DNA. Hybridizations are
carried out in the same solution with the following modifications:
0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 .mu.g/ml salmon sperm DNA,
10% (wt/vol) dextran sulfate, and 5-20.times.10.sup.6 cpm
.sup.32P-labeled probe. Filters are incubated in hybridization
mixture for 18-20 h at 40.degree. C., and then washed for 1.5 h at
55.degree. C. in a solution containing 2.times.SSC, 25 mM Tris-HCl
(pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution is replaced
with fresh solution and incubated an additional 1.5 h at 60.degree.
C. Filters are blotted dry and exposed for autoradiography. If
necessary, filters are washed for a third time at 65-68.degree. C.
and re-exposed to film.
[0099] 5.3.3. Nucleic Acid Amplification
[0100] The polymerase chain reaction (PCR) may be used in
connection with the invention to amplify any desired sequence from
any given source (e.g., a cultured cell, a tissue sample, a genomic
library, a cDNA library, a purified plasmid, a purified phagemid,
etc.). Oligonucleotide primers representing known sequences are
used as primers in PCR. PCR may be carried out using a thermal
cycler (e.g., from Perkin-Elmer Cetus) and a thermostable
polymerase (e.g., Gene Amp.TM. brand of Taq polymerase). The
nucleic acid being amplified may include but is not limited to
mRNA, cDNA or genomic DNA from any species. The PCR amplification
method is quite well known in the art (see e.g., U.S. Pat. Nos.
4,683,202, 4,683,195 and 4,889,818; Gyllenstein et al., 1988, Proc.
Natl. Acad. Sci. U.S.A. 85, 7652-7656; Ochman et al., 1988,
Genetics 120, 621-623; Loh et al., 1989, Science 243, 217-220).
[0101] The rolling circle amplification (RCA) method may also be
used for nucleic acid amplification. One such method utilizing
rolling circle replication by DNA polymerase under isothermal
conditions has recently been described by Lizardi et al. (1998,
Nature Genetics 19, 225-232; see also references therein).
[0102] Any prokaryotic cell, eukaryotic cell, or virus, can serve
as the nucleic acid source. For example, nucleic acid sequences may
be obtained from the following sources: human, porcine, bovine,
feline, avian, equine, canine, insect (e.g., D. melanogaster),
invertebrate (e.g., C. elegans), plant, etc. The DNA may be
obtained by standard procedures known in the art (see e.g.,
Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2d
Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.;
Glover (ed.), 1985, DNA Cloning: A Practical Approach, MRL Press,
Ltd., Oxford, U.K. Vol. I, II).
[0103] 5.4. Diseases, Disorders and Conditions
[0104] Various diseases, disorders and conditions to which
peptides, peptide derivatives, other small molecules, and methods
of the invention may be applied include but are not limited to the
following examples. Disease states include acquired
immunodeficiency syndrome (AIDS), angina, arteriosclerosis,
arthritis, asthma, blood pressure dysregulation, bronchitis, cancer
(all forms), cholesterol imbalance, cerebral circulatory,
cirrhosis, clotting disorder, depression, dermatologic disease,
diabetes, diarrhea, dysmenorrhea, dyspepsia, emphysema,
gastrointestinal distress, hemorrhoids, hepatitis, hypertension,
hyperprolactinemia, immunomodulation, resistance to bacterial
infection, resistance to viral infection, inflammation, insomnia,
lactation disorders, lipidemia, migraine, pain prevention or
management, peripheral vascular disease, platelet aggregation,
premenstrual syndrome, prostatic disorders, elevated triglycerides,
respiratory tract infection, retinopathy, sinusitus, rheumatic
disease, impaired wound healing, tinnitus, urinary tract infection
and venous insufficiency.
[0105] Other indications include cardiovascular disorders, nervous
system disorders, hypercholesterolemia, inflammation, antipyretic,
analgesic, slowing the aging process, accelerated convalescence,
anemia, indigestion, impotence and menstrual disorders.
[0106] 5.5. Pharmaceutical Compositions
[0107] The methods of the present invention comprise administering
to a subject in need thereof an effective amount of a peptide or
derivative thereof (e.g. a small molecule mimetic), or a
composition comprising a peptide or peptide derivative, to the
subject to modulate (i.e. stimulate or inhibit) an intracellular
protein-protein interaction, such as a signal transduction event.
In one embodiment, an effective amount of a therapeutic composition
comprising a peptide or derivative thereof and a pharmaceutical
carrier is administered systemically to a subject to modulate a
signal transduction event or to treat a disease, disorder or
condition. In another embodiment, an effective amount of a
therapeutic composition comprising a peptide or derivative thereof
and a pharmaceutical carrier is applied locally to a site to
modulate signal transduction or to treat a disease, disorder or
condition at the site.
[0108] The peptides and derivatives thereof and pharmaceutical
compositions of the present invention are used in the treatment of
or amelioration of symptoms in any disease, condition or disorder
where modulation of a signal transduction event would be
beneficial. Non-limiting examples of diseases, disorders or
conditions in which the peptides, peptide derivatives and
pharmaceutical compositions of the present invention can be used
for treatment are set forth in Section 5.4 herein.
[0109] The methods of the present invention also provide for the
treatment of a subject by administration of a therapeutic
composition comprising a peptide or derivative thereof and a
pharmaceutically acceptable carrier. The subject is preferably an
animal, including but not limited to animals such as dogs, cats,
cows, sheep, pigs, chickens, etc., is preferably a mammal, and most
preferably a human.
[0110] Various delivery systems are known and can be used to
administer a peptide or derivative thereof or a pharmaceutical
composition of the invention. For example, a pharmaceutical
composition of the invention can be administered systemically by,
e.g., intravenous or intramuscular injection. In another example, a
pharmaceutical composition of the invention can be introduced to a
site by any suitable route including sub-cutaneously, orally,
topically, subconjunctivally, etc. In yet another example, a
pharmaceutical composition of the invention can be introduced into
the central nervous system by any suitable route, including
intraventricular or intrathecal injection, etc. Intraventricular
injection may be facilitated by an intraventricular catheter, for
example, attached to a reservoir, such as an Ommaya reservoir. For
veterinary or other purposes the composition may be administered
intraperitoneally.
[0111] Further, delivery systems are well known and can be used to
administer a pharmaceutical composition of the invention, e.g., via
aqueous solution, encapsulation in liposomes, microparticles,
microcapsules, and by way of receptor-mediated endocytosis (see
e.g., Wu and Wu, 1987, J. Biol. Chem. 262, 4429-4432). Other
methods of administration include but are not limited to direct
application to the skin, intradermal, intranasal and epidural
routes. A pharmaceutical composition of the invention may be
administered by any convenient route, for example, by infusion or
bolus injection, by absorption through epithelial or mucocutaneous
linings (e.g., oral mucosa, rectal and intestinal mucosa). In a
preferred embodiment, intravenous administration is used.
[0112] In a specific embodiment, a therapeutic or pharmaceutical
composition of the invention is administered locally to the area in
need of treatment. This may be achieved by, for example and not by
way of limitation, local infusion during surgery, topical
application (e.g. cream or ointment), in conjunction with a wound
dressing after surgery, or directly onto the eye, by injection, by
means of a catheter, or by means of an implant, said implant being
of a porous or gelatinous material, including membranes, such as
silastic membranes, or fibers. In one embodiment, administration
can be by direct injection at the site of treatment. In another
embodiment, a therapeutic or pharmaceutical composition can be
administered to the eye by eye drops.
[0113] In yet another embodiment, a therapeutic or pharmaceutical
composition can be delivered in a vesicle, in particular, a
liposome (see Langer, 1990, Science 249, 1527-1533; Treat et al.,
1989, in Liposomes In The Therapy Of Infectious Disease And Cancer,
Lopez-Berestein and Fidler, eds., Liss, New York, pp. 353-365;
Lopez-Berestein, ibid., pp. 317-327). A vesicle or liposome
delivery system is particularly preferred for delivery of a peptide
or other small molecule which does not easily cross cell membranes
to reach an intracellular site of action.
[0114] In yet another embodiment, a therapeutic or pharmaceutical
composition can be delivered in a controlled release system. In one
embodiment, a pump may be used (see Langer, supra; Sefton, 1987,
CRC Crit. Rev. Biomed. Eng. 14, 201; Buchwald et al., 1980, Surgery
88, 507; Saudek et al., 1989, N. Engl. J. Med. 321, 574). In
another embodiment, polymeric materials can be used (see Medical
Applications of Controlled Release, 1974, Langer and Wise, eds.,
CRC Press, Boca Raton, Fla.; Controlled Drug Bioavailability, Drug
Product Design and Performance, 1984, Smolen and Ball, eds., Wiley,
New York; Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol.
Chem. 23, 61; see also Levy et al., 1985, Science 228, 190; During
et al., 1989, Ann. Neurol. 25, 351; Howard et al., 1989, J.
Neurosurg. 71, 105). In yet another embodiment, a controlled
release system can be placed in proximity of the therapeutic
target, i.e., the brain (see, e.g., Goodson, 1984, in Medical
Applications of Controlled Release, supra, vol. 2, pp.
115-138).
[0115] The present invention also provides for therapeutic or
pharmaceutical compositions comprising a peptide or a peptide
derivative of the invention in combination with a pharmaceutically
acceptable carrier, which compositions can be administered as
described above. The term "carrier" refers to a diluent, adjuvant,
excipient, or vehicle with which the peptide is administered. Such
pharmaceutical carriers can be sterile liquids, such as water and
oils, including those of petroleum oil such as mineral oil,
vegetable oil such as peanut oil, soybean oil, and sesame oil,
animal oil, or oil of synthetic origin. Saline solutions and
aqueous dextrose and glycerol solutions can also be employed as
liquid carriers, particularly for injectable solutions.
Particularly preferred pharmaceutical carriers for treatment of or
amelioration of inflammation in the central nervous system are
carriers that can penetrate the blood/brain barrier.
[0116] Suitable pharmaceutical excipients include starch, glucose,
lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel,
sodium stearate, glycerol monostearate, talc, sodium chloride,
dried skim milk, glycerol, propylene, glycol, water, ethanol, and
the like. The therapeutic composition, if desired, can also contain
minor amounts of wetting or emulsifying agents, or pH buffering
agents. These compositions can take the form of solutions,
suspensions, emulsion, tablets, capsules, powders,
sustained-release formulations, and the like. The composition can
be formulated with traditional binders and carriers such as
triglycerides. Examples of suitable pharmaceutical carriers are
described in "Remington's Pharmaceutical Sciences" by E. W. Martin.
Such compositions contain a therapeutically effective amount of the
therapeutic composition, together with a suitable amount of carrier
so as to provide the form for proper administration to the subject.
The formulation should suit the mode of administration.
[0117] In a preferred embodiment, the composition is formulated in
accordance with routine procedures as a pharmaceutical composition
adapted for local injection administration to human beings.
Typically, compositions for local injection administration are
solutions in sterile isotonic aqueous buffer. Where necessary, the
composition may also include a solubilizing agent and a local
anesthetic such as lidocaine to ease pain at the site of the
injection. Generally, the ingredients are supplied either
separately or mixed together in unit dosage form, for example, as a
dry lyophilized powder or water free concentrate in a hermetically
sealed container such as an ampoule or sachet indicating the
quantity of active agent. Where the composition is administered by
injection, an ampoule of sterile water for injection or saline can
be provided so that the ingredients may be mixed prior to
administration.
[0118] The therapeutic or pharmaceutical compositions of the
invention can be formulated as neutral or salt forms.
Pharmaceutically acceptable salts include those formed with free
amino groups such as those derived from hydrochloric, phosphoric,
acetic, oxalic, tartaric acids, etc., and those formed with free
carboxyl groups such as those derived from sodium, potassium,
ammonium, calcium, ferric hydroxides, isopropylamine,
triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
[0119] The composition, if desired, can also contain minor amounts
of wetting or emulsifying agents, or pH buffering agents. The
composition can be a liquid solution, suspension, emulsion, tablet,
pill, capsule, sustained release formulation, cream, gel or powder.
The composition can be formulated as a suppository, with
traditional binders and carriers such as triglycerides.
[0120] The present invention also provides for the modification of
the peptide or peptide derivative such that it is more stable once
administered to a subject (i.e., once administered, it has a longer
period of effectiveness as compared to the unmodified form). Such
modifications are well know to those of skill in the art (e.g.,
polyethylene glycol derivatization a.k.a. PEGylation,
microencapsulation, etc.).
[0121] The amount of the therapeutic or pharmaceutical composition
of the invention which is effective in the treatment of a
particular disease, condition or disorder will depend on the nature
of the disease, condition or disorder and can be determined by
standard clinical techniques. In general, the dosage ranges from
about 0.001 mg/kg to about 2 mg/kg. In addition, in vitro assays
such as those set forth in the Examples of Section 6 herein may
optionally be employed to help identify optimal dosage ranges. The
precise dose to be employed in the formulation will also depend on
the route of administration, and the seriousness of the disease,
condition or disorder, and should be decided according to the
judgment of the practitioner and each patient's circumstances.
Effective doses may be extrapolated from dose-response curves
derived from in vitro or animal model test systems. For example, in
order to obtain an effective mg/kg dose for humans based on data
generated from rat studies, the effective mg/kg dosage in rats is
divided by six.
[0122] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients (e.g., peptide or small molecule derivative thereof
plus carrier) of the pharmaceutical compositions of the invention.
In another embodiment, the invention comprises kits containing an
effective amount of a pharmaceutical composition of the invention.
Thus, the kit is contemplated to comprise one or more containers
containing at least one pharmaceutical composition of the
invention. Simply by way of example, the kit will contain such a
composition formulated for application to the skin, or for
administration by intradermal, intramuscular, intravenous,
intranasal, epidural and oral routes of administration. The kits
may contain a liquid solution, suspension, emulsion, tablet, pill,
capsule, sustained release formulation, cream, gel or powder.
[0123] 5.6. Peptide Derivatives
[0124] Peptide derivatives (e.g. small molecule mimetics) may
include cyclic peptides, peptides obtained by substitution of a
natural amino acid residue by the corresponding D-stereoisomer or
by a non-natural amino acid residue, chemical derivatives of the
peptides, dual peptides, multimers of the peptides, and peptides
fused to other proteins or carriers (e.g. cell permeable
carriers).
[0125] The term "cyclic peptide" as used herein refers to a cyclic
derivative of a peptide of the invention to which, e.g., two or
more additional amino acid residues suitable for cyclization have
been added, often at the carboxyl terminus and at the amino
terminus. A cyclic peptide may contain either an intramolecular
disulfide bond, i.e., --S--S--, an intramolecular amide bond
between the two added residues, i.e., --CONH-- or --NHCO-- or
intramolecular S-alkyl bonds, i.e., --S--(CH.sub.2).sub.n--CONH--
or --NH--CO(CH.sub.2).sub.n--S--, wherein n is 1, 2, or more.
[0126] A cyclic derivative containing an intramolecular disulfide
bond may be prepared by conventional solid phase synthesis
(Merrifield et al., 1982) while incorporating suitable S-protected
cysteine or homocysteine residues at the positions selected for
cyclization such as the amino and carboxyl termini (Sahm et al.,
1996, J. Pharm. Pharmacol. 48, 197). Following completion of the
chain assembly, cyclization can be performed either by selective
removal of the S-protecting groups with a consequent on-support
oxidation of free corresponding two SH-functions, to form S--S
bonds, followed by conventional removal of the product from the
support and appropriate purification procedure, or by removal of
the peptide from the support along with complete side-chain
deprotection, followed by oxidation of the free SH-functions in
highly dilute aqueous solution.
[0127] The cyclic derivatives containing an intramolecular amide
bond may be prepared by conventional solid phase synthesis while
incorporating suitable amino and carboxyl side-chain protected
amino acid derivatives at the positions selected for cyclization.
The cyclic derivatives containing intramolecular --S-alkyl bonds
can be prepared by conventional solid phase synthesis while
incorporating an amino acid residue with a suitable amino-protected
side chain, and a suitable S-protected cysteine or homocysteine
residue at the positions selected for cyclization.
[0128] According to another embodiment, a peptide derivative of the
invention may have one or more amino acid residues replaced by the
corresponding D-amino acid residue. Thus, a peptide or peptide
derivative of the invention may be all-L, all-D, or a mixed
D,L-peptide. In another embodiment, an amino acid residue may be
replaced by a non-natural amino acid residue. Examples of
non-naturally occurring or derivatized non-naturally occurring
amino acids include N.alpha.-methyl amino acids, C.alpha.-methyl
amino acids, and .beta.-methyl amino acids. Amino acid analogs in
general may include but are not limited to .beta.-alanine
(.beta.-Ala), norvaline (Nva), norleucine (Nle), 4-aminobutyric
acid (.gamma.-Abu), 2-aminoisobutyric acid (Aib), 6-aminohexanoic
acid (.epsilon.-Ahx), ornithine (Orn), hydroxyproline (Hyp),
sarcosine, citrulline, cysteic acid, and cyclohexylalanine.
Further, such amino acids may include but are not limited to,
.alpha.-amino isobutyric acid, t-butylglycine, t-butylalanine and
phenylglycine.
[0129] A chemical derivative of a peptide of the invention
includes, but is not limited to, a derivative containing additional
chemical moieties not normally a part of the peptide, provided that
the derivative retains the desired functional activity of the
peptide. Examples of such derivatives include: (a) N-acyl
derivatives of the amino terminal or of another free amino group,
wherein the acyl group may be either an alkanoyl group, e.g.,
acetyl, hexanoyl, octanoyl, an aroyl group, e.g., benzoyl, or a
blocking group such as Fmoc (fluorenylmethyl-O--CO--), carbobenzoxy
(benzyl-O--CO--), monomethoxysuccinyl, naphthyl-NH--CO--,
acetylamino-caproyl, adamantyl-NH--CO--; (b) esters of the carboxyl
terminal or of another free carboxyl or hydroxy groups; (c) amides
of the carboxyl terminal or of another free carboxyl groups
produced by reaction with ammonia or with a suitable amine; (d)
glycosylated derivatives; (e) phosphorylated derivatives; (f)
derivatives conjugated to lipophilic moieties, e.g., caproyl,
lauryl, stearoyl; and (g) derivatives conjugated to an antibody or
other biological ligand.
[0130] Also included among the chemical derivatives are those
derivatives obtained by modification of the peptide bond
--CO--NH--, for example, by: (a) reduction to --CH.sub.2--NH--; (b)
alkylation to --CO--N (alkyl)-; and (c) inversion to
--NH--CO--.
[0131] A dual peptide according to the invention consists of two of
the same, or two different, peptides of the invention covalently
linked to one another, either directly or through a spacer, such as
by a short stretch of alanine residues, or by a putative site for
proteolysis (e.g. by cathepsin, see U.S. Pat. No. 5,126,249 and
European Patent No. 495,049 with respect to such sites).
[0132] Multimers according to the invention consist of polymer
molecules formed from a number of the same or different peptides or
derivatives thereof. The polymerization is carried out with a
suitable polymerization agent, such as 0.1% glutaraldehyde
(Audibert et al., 1981, Nature 289, 593).
[0133] In one aspect of the invention, the peptide derivative is
more resistant to proteolytic degradation than the corresponding
non-derivatized peptide. For example, a peptide derivative having
D-amino acid substitution(s) in place of one or more L-amino acid
residue(s) resists proteolytic cleavage when administered to a
mammal. In a preferred aspect of the invention, the peptide
derivative has increased permeability across a cell membrane as
compared to the corresponding non-derivatized peptide. For example,
a peptide derivative may have a lipophilic moiety coupled at the
amino terminus and/or carboxyl terminus and/or an internal site.
Such derivatives are highly preferred when targeting intracellular
protein-protein interactions, provided they retain the desired
functional activity. In yet another aspect, a dualized or
multimerized peptide or peptide derivative has enhanced functional
activity.
[0134] The peptides or peptide derivatives of the invention are
obtained by any method of peptide synthesis known to those skilled
in the art, including synthetic and recombinant techniques. For
example, the peptides or peptide derivatives can be obtained by
solid phase peptide synthesis which, in brief, consists of coupling
the carboxyl group of the C-terminal amino acid to a resin and
successively adding N-alpha protected amino acids. The protecting
groups may be any such groups known in the art. Before each new
amino acid is added to the growing chain, the protecting group of
the previous amino acid added to the chain is removed. The coupling
of amino acids to appropriate resins has been described by Rivier
et al. (U.S. Pat. No. 4,244,946). Such solid phase syntheses have
been described, for example, by Merrifield, 1964, J. Am. Chem. Soc.
85, 2149; Vale et al. 1981, Science 213, 1394-1397; Marki et al.,
1981, J. Am. Chem. Soc. 103, 3178, and in U.S. Pat. Nos. 4,305,872
and 4,316,891. In a preferred aspect, an automated peptide
synthesizer is employed.
[0135] Purification of the synthesized peptides or peptide
derivatives is carried out by standard methods, including
chromatography (e.g., ion exchange, affinity, and sizing column
chromatography), centrifugation, differential solubility,
hydrophobicity, or by any other standard technique for the
purification of proteins. In one embodiment, thin layer
chromatography is employed. In another embodiment, reverse phase
HPLC (high performance liquid chromatography) is employed.
[0136] Finally, structure-function relationships determined from
the peptides, peptide derivatives, and other small molecules of the
invention may also be used to prepare analogous molecular
structures having similar properties. Thus, the invention is
contemplated to include molecules in addition to those expressly
disclosed that share the structure, hydrophobicity, charge
characteristics and side chain properties of the specific
embodiments exemplified herein.
[0137] In a specific embodiment, the peptide or other small
molecule, e.g., derivative or analog, is functionally active, i.e.,
capable of exhibiting one or more of the identified functional
activities associated with a peptide of the invention. As one
example, such derivatives or analogs which have the desired
immunogenicity or antigenicity can be used in immunoassays, for
immunization, for activation or inhibition of effector activity,
etc. As another example, such derivatives or analogs which have the
desired binding activity can be used for binding to a molecule or
other target of interest. As yet another example, such derivatives
or analogs which have the desired binding activity can be used for
binding to a binding partner specific for another known protein
(see e.g., Clemmons, 1993, Mol. Reprod. Dev. 35, 368-374; Loddick
et al., 1998, Proc. Natl. Acad. Sci. U.S.A. 95, 1894-1898).
Derivatives or analogs that retain, or alternatively lack or
inhibit, a desired property-of-interest (e.g., binding to a protein
binding partner), can be used as activators, or inhibitors,
respectively, of such property and its physiological correlates. A
specific embodiment relates to a peptide or other small molecule
that can be bound by an anti-peptide antibody. Derivatives or
analogs of a peptide can be tested for the desired activity by any
functional assay known in the art, including but not limited to the
assays described in Section 6 below.
[0138] In particular, peptide derivatives can be made by altering
amino acid sequences by substitutions, additions or deletions that
provide for functionally equivalent molecules, or for functionally
enhanced or diminished molecules, as desired. Due to the degeneracy
of the genetic code, other nucleic acid sequences which encode
substantially the same amino acid sequence may be used for the
production of recombinant peptides. These include but are not
limited to nucleotide sequences comprising all or portions of a
peptide of the invention which is altered by the substitution of
different codons that encode a functionally equivalent amino acid
residue within the sequence, thus producing a silent change.
Likewise, the derivatives of the invention include, but are not
limited to, those containing, as a primary amino acid sequence, all
or part of the amino acid sequence of a protein including altered
sequences in which functionally equivalent amino acid residues are
substituted for residues within the sequence resulting in a silent
change. For example, one or more amino acid residues within the
sequence can be substituted by another amino acid of a similar
polarity which acts as a functional equivalent, resulting in a
silent alteration. Substitutions for an amino acid within the
sequence may be selected from other members of the class to which
the amino acid belongs. For example, the nonpolar (hydrophobic)
amino acids include alanine, leucine, isoleucine, valine, proline,
phenylalanine, tryptophan and methionine. The polar neutral amino
acids include glycine, serine, threonine, cysteine, tyrosine,
asparagine, and glutamine. The positively charged (basic) amino
acids include arginine, lysine and histidine. The negatively
charged (acidic) amino acids include aspartic acid and glutamic
acid. Such substitutions are generally understood to be
conservative substitutions.
[0139] In a specific embodiment of the invention, proteins
comprising a part (i.e. fragment) of a peptide of the invention
having at least 3, at least 6, or at least 9 (continuous) amino
acids of the peptide of the invention is provided. In other
embodiments, the fragment consists of at least 10 or at least 20 or
at least 50 amino acids of the peptide. In specific embodiments,
such fragments are not larger than 35, 100 or 200 amino acids.
Derivatives or analogs of peptides include but are not limited to
those molecules comprising regions that are substantially
homologous to a peptide or fragment thereof (e.g., in various
embodiments, at least 60% or 70% or 80% or 90% or 95% or 98% or 99%
identity over an amino acid sequence of identical size or when
compared to an aligned sequence in which the alignment is done by a
computer homology program known in the art) or whose encoding
nucleic acid is capable of hybridizing to a coding gene sequence,
under high stringency, moderate stringency, or low stringency
conditions.
[0140] The derivatives and analogs of the invention can be produced
by various methods known in the art. The manipulations which result
in their production can occur at the gene or protein level. For
example, a cloned nucleic acid sequence can be modified by any of
numerous strategies known in the art (Sambrook et al., 1989,
Molecular Cloning, A Laboratory Manual, 2d ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.). The sequence can be
cleaved at appropriate sites with restriction endonuclease(s),
followed by further enzymatic modification if desired, isolated,
and ligated in vitro.
[0141] Additionally, a nucleic acid sequence can be mutated in
vitro or in vivo, to create and/or destroy translation, initiation,
and/or termination sequences, or to create variations in coding
regions and/or to form new restriction endonuclease sites or
destroy preexisting ones, to facilitate further in vitro
modification. Any technique for mutagenesis known in the art can be
used, including but not limited to, chemical mutagenesis, in vitro
site-directed mutagenesis (Hutchinson et al., 1978, J. Biol. Chem.
253:6551), use of TAB.RTM. linkers (Pharmacia), etc. See also
Section 5. herein which sets forth general cloning techniques.
[0142] Manipulations of a protein sequence may also be made at the
protein level. Included within the scope of the invention are
peptide fragments or other derivatives or analogs which are
differentially modified during or after translation, e.g., by
glycosylation, acetylation, phosphorylation, amidation,
derivatization by known protecting/blocking groups, proteolytic
cleavage, linkage to an antibody molecule or other cellular ligand,
etc. Any of numerous chemical modifications may be carried out by
known techniques, including but not limited to specific chemical
cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8
protease, NaBH.sub.4, acetylation, formylation, oxidation,
reduction, metabolic synthesis in the presence of tunicamycin,
etc.
[0143] In a preferred embodiment, a peptide derivative is a
chimeric or fusion protein comprising a peptide of the invention or
fragment thereof joined at its amino- or carboxy-terminus, or both,
via a peptide bond to an amino acid sequence of a different
protein. Such a chimeric or fusion protein may be produced by
recombinant expression of a nucleic acid encoding the protein. In
another preferred embodiment, such a chimeric or fusion protein
comprises a fragment of at least six (6) amino acids of a peptide
of the invention. In a most preferred embodiment, such a chimeric
or fusion protein not only comprises a fragment of at least six (6)
amino acids of a peptide of the invention but also has a functional
activity equivalent to or greater than the peptide of the
invention.
[0144] 5.7. Antibodies
[0145] According to the invention, a peptide, peptide fragment,
peptide derivative, peptide analog, or a small molecule derivative
thereof (e.g., a peptide mimetic), may be used as an immunogen to
generate antibodies which immunospecifically bind such an
immunogen. Such antibodies may in turn be used as diagnostic or
therapeutic agents and include but are not limited to polyclonal,
monoclonal, humanized or chimeric antibodies, single chain
antibodies, Fab fragments and 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.
[0146] Various procedures well known in the art may be used for the
production of polyclonal antibodies to a peptide or derivative or
analog. In a particular embodiment, rabbit polyclonal antibodies to
an epitope of a protein encoded by a peptide of the invention, or a
subsequence thereof of at least three amino acids, can be obtained.
For the production of antibody, various host animals can be
immunized by injection with the native protein, or a synthetic
version, or derivative or fragment thereof, including but not
limited to rabbits, mice, rats, etc. Various adjuvants may be used
to increase the immunological response, depending on the host
species, and including but not limited to Freund's (complete and
incomplete), mineral gels such as aluminum hydroxide, surface
active substances such as lysolecithin, polyanions, peptides, oil
emulsions, keyhole limpet hemocyanins, dinitrophenol, and
potentially useful human adjuvants such as BCG (bacille
Calmette-Guerin) and corynebacterium parvum.
[0147] For preparation of monoclonal antibodies directed to a
protein sequence or analog thereof, any technique which provides
for the production of antibody molecules by continuous cell lines
in culture may be used. For example, the hybridoma technique
originally developed by Kohler and Milstein (1975, Nature 256,
495-497), as well as the trioma technique, the human B-cell
hybridoma technique (Kozbor et al., 1983, Immunology Today 4, 72),
and the EBV-hybridoma technique to produce human monoclonal
antibodies (Cole et al., 1985, in Monoclonal Antibodies and Cancer
Therapy, Alan R. Liss, Inc., pp. 77-96). In an additional
embodiment of the invention, monoclonal antibodies can be produced
in germ-free animals utilizing recent technology (see e.g.,
PCT/US90/02545). According to the invention, human antibodies may
be used and can be obtained by using human hybridomas (Cole et al.,
1983, Proc. Natl. Acad. Sci. U.S.A. 80, 2026-2030) or by
transforming human B cells with EBV virus in vitro (Cole et al.,
1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,
pp. 77-96).
[0148] The monoclonal antibodies which may be. used in the methods
of the invention include but are not limited to human monoclonal
antibodies or chimeric human-mouse (or other species) monoclonal
antibodies. Human monoclonal antibodies may be made by any of
numerous techniques known in the art (e.g., Teng et al., 1983,
Proc. Natl. Acad. Sci. U.S.A. 80, 7308-7312; Kozbor et al., 1983,
Immunology Today 4, 72-79; Olsson et al., 1982, Meth. Enzymol. 92,
3-16).
[0149] 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. Techniques have been developed for
the production of "chimeric antibodies" (Morrison et al., 1984,
Proc. Natl. Acad. Sci. U.S.A. 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.
[0150] Briefly, humanized antibodies are antibody molecules from
non-human species having one or more complementarity determining
regions (CDRs) from the non-human species and a framework region
from a human immunoglobulin molecule. Various techniques have been
developed for the production of humanized antibodies (see e.g.,
Queen, U.S. Pat. No. 5,585,089, which is incorporated herein by
reference in its entirety). An immunoglobulin light or heavy chain
variable region consists of a "framework" region interrupted by
three hypervariable regions, referred to as complementarily
determining regions (CDRs). The extent of the framework region and
CDRs have been precisely defined (see, Kabat et al., 1983)
"Sequences of Proteins of Immunological Interest", U.S. Department
of Health and Human Services.
[0151] According to the invention, techniques described for the
production of single chain antibodies (U.S. Pat. No. 4,946,778) can
be adapted to produce peptide-specific single chain antibodies. An
additional embodiment of the invention utilizes the techniques
described for the construction of Fab expression libraries (Huse et
al., 1989, Science 246, 1275-1281) to allow rapid and easy
identification of monoclonal Fab fragments with the desired
specificity for proteins, derivatives, or analogs of the
invention.
[0152] Antibody fragments which contain the idiotype of the
molecule can be generated by known techniques. For example, such
fragments include but are not limited to: the F(ab').sub.2 fragment
which can be produced by pepsin digestion of the antibody molecule;
the Fab' fragments which can be generated by reducing the disulfide
bridges of the F(ab').sub.2 fragment, the Fab fragments which can
be generated by treating the antibody molecule with papain and a
reducing agent, and Fv fragments.
[0153] Antibodies raised against a peptide can, in turn, be
utilized to generate anti-idiotype antibodies that "mimic" the
peptide, using techniques well known in the art (see, e.g.,
Greenspan and Bona, 1993, FASEB J. 7, 437-444; and Nissinoff, 1991,
J. Immunol. 147, 2429-2438). For example, antibodies which bind to
the peptide and competitively inhibit the binding of peptide to its
receptor can be used to generate anti-idiotypes that "mimic" the
peptide receptor and, therefore, bind the peptide.
[0154] In the production of antibodies, screening for the desired
antibody can be accomplished by techniques known in the art (e.g.,
enzyme-linked immunosorbent assay or ELISA). For example, to select
antibodies which recognize a specific domain of a protein, one may
assay generated hybridomas for a product which binds to a fragment
containing such domain. For selection of an antibody that
specifically binds a first homolog but which does not specifically
bind a different homolog, one can select on the basis of positive
binding to the first homolog and a lack of binding to the second
homolog.
[0155] Antibody molecules may be purified by many well known
techniques, e.g., immunoabsorption or immunoaffinity
chromatography, chromatographic methods such as HPLC (high
performance liquid chromatography), or a combination thereof,
etc.
[0156] The functional activity of peptides and other small
molecules of the invention, and derivatives and analogs thereof,
can be assayed by various antibody methods known to one skilled in
the art. For example, where one is assaying for the ability to bind
to or compete with another molecule for binding to an anti-peptide
antibody, assays known in the art which can be used include but are
not limited to competitive and non-competitive assays using
techniques such as: radioimmunoassays, ELISA (enzyme linked
immunosorbent assay), "sandwich" immunoassays, immunoradiometric
assays, gel diffusion precipitin reactions, immunodiffusion assays,
in situ immunoassays (e.g., using colloidal gold, enzyme or
radioisotope labels), western blots, precipitation reactions,
agglutination assays (e.g., gel agglutination assays,
hemagglutination assays), complement fixation assays,
immunofluorescence assays, protein A assays, and
immunoelectrophoresis assays, etc. In one embodiment, antibody
binding is detected by detecting a label on the primary antibody.
In another embodiment, the primary antibody is detected by
detecting binding of a secondary antibody or reagent to the primary
antibody. In a further embodiment, the secondary antibody is
labeled.
[0157] The methods of antibody production and use employed herein
can, for example, be such as those described in Harlow and Lane
(Harlow and Lane, 1988, "Antibodies: A Laboratory Manual", Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), which is
incorporated herein by reference in its entirety.
[0158] 5.8. Structure of Peptides and Nucleic Acids
[0159] The structure of peptides and other small molecules of the
invention, of fragments, derivatives and analogs thereof, and,
where applicable, of the nucleic acids encoding them, can be
analyzed by any of various methods well known in the art. Examples
of such methods include but are not limited to those described
below.
[0160] 5.8.1. Peptide Structural Analysis
[0161] Well known structural analysis methods (e.g., Chou and
Fasman, 1974, Biochemistry 13, 222) may be performed to identify
candidate regions of a peptide that assume specific secondary
structures. Further secondary structure prediction may be
accomplished using computer software programs available in the
art.
[0162] Additional well known methods of structural analysis can
also be employed. These include but are not limited to X-ray
crystallography (Engstom, 1974, Biochem. Exp. Biol. 11, 7-13),
nuclear magnetic resonance spectroscopy (Clore and Gonenborn, 1989,
CRC Crit. Rev. Biochem. 24, 479-564) and computer modeling
(Fletterick and Zoller, eds., 1986, Computer Graphics and Molecular
Modeling, in Current Communications in Molecular Biology, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
[0163] The single-letter amino acid code as used herein corresponds
to the three-letter amino acid code of the Sequence Listing herein,
as follows: A, Ala, alanine; R, Arg, arginine; N, Asn, asparagine;
D, Asp, aspartic acid; B, Asx, asparagine or aspartic acid; C, Cys,
cysteine; Q, Gln, glutamine; E, Glu, glutamic acid; Z, Glx,
glutamine or glutamic acid; G, Gly, glycine; H, His, histidine; I,
Ile, isoleucine; L, Leu, leucine; K, Lys, lysine; M, Met,
methionine; F, Phe, phenylalanine; P, Pro, proline; S, Ser, serine;
T, Thr, threonine; W, Trp, tryptophan; Y, Tyr, tyrosine; V, Val,
valine; and X, Xaa, unknown or other or any amino acid.
[0164] 5.8.2. Nucleic Acid Structural Analysis
[0165] A nucleic acid encoding a recombinant peptide of the
invention can be analyzed, as needed, by any number of methods well
known in the art including but not limited to Southern
hybridization (Southern, 1975, J. Mol. Biol. 98, 503-517), Northern
hybridization (see e.g., Freeman et al., 1983, Proc. Natl. Acad.
Sci. U.S.A. 80, 4094-4098), restriction endonuclease mapping
(Maniatis, 1982, Molecular Cloning, A Laboratory Manual, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), and DNA
sequence analysis. Accordingly, this invention provides nucleic
acid probes recognizing a nucleic acid encoding a peptide of the
invention. For example, polymerase chain reaction (PCR; U.S. Pat.
Nos. 4,683,202, 4,683,195 and 4,889,818; Gyllenstein et al., 1988,
Proc. Natl. Acad. Sci. U.S.A. 85, 7652-7656; Ochman et al., 1988,
Genetics 120, 621-623; Loh et al., 1989, Science 243, 217-220)
followed by Southern hybridization with an nucleic acid-specific
probe can allow the detection of an nucleic acid in DNA from
various cell types. Methods of amplification other than PCR are
commonly known and can also be employed.
[0166] In one embodiment, Southern hybridization can be used to
determine the genetic linkage of a given nucleic acid. Northern
hybridization analysis can be used to determine the expression of
an nucleic acid. Various cell types, at various states of
development or activity can be tested for nucleic acid expression.
The stringency of the hybridization conditions for both Southern
and Northern hybridization can be manipulated to ensure detection
of nucleic acids with the desired degree of relatedness to the
specific nucleic acid probe used. Modifications of these methods
and other methods commonly known in the art can be used.
[0167] 5.9. Expression of Recombinant Peptides
[0168] For expression of recombinant peptides of the invention, any
nucleotide sequence encoding such peptides predicted from the
genetic code can be inserted into an appropriate expression vector
(i.e., a vector which contains the necessary elements for the
transcription and translation of the inserted protein-coding
sequence). A variety of host-vector systems may be utilized to
express the protein-coding sequence. These include but are not
limited to mammalian cell systems infected with virus (e.g.,
vaccinia virus, adenovirus, etc.); insect cell systems infected
with virus (e.g., baculovirus); microorganisms such as yeast
containing yeast vectors, or bacteria transformed with
bacteriophage, DNA, plasmid DNA, or cosmid DNA. The expression
elements of vectors vary in their strengths and specificities.
Depending on the host-vector system utilized, any one of a number
of suitable transcription and translation elements may be used. In
yet another embodiment, a fragment of a protein comprising one or
more domains of the protein is expressed.
[0169] Any of the methods well known in the art for the insertion
of DNA fragments into a vector may be used to construct expression
vectors containing a chimeric nucleic acid consisting of
appropriate transcriptional/translational control signals and the
protein coding sequences. These methods may include in vitro
recombinant DNA and synthetic techniques and in vivo recombinants
(genetic recombination). Expression of a nucleic acid sequence
encoding a peptide of the invention may be regulated by a second
nucleic acid sequence so that the peptide is expressed in a host
transformed with the recombinant DNA molecule. For example,
expression of a peptide may be controlled by any promoter/enhancer
element known in the art. Promoters which may be used to control
nucleic acid expression include, but are not limited to, the SV40
early promoter region (Benoist and Chambon, 1981, Nature
290:304-310), the promoter contained in the 3' long terminal repeat
of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797), the
herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl.
Acad. Sci. U.S.A. 78:1441-1445), the regulatory sequences of the
metallothionein nucleic acid (Brinster et al., 1982, Nature
296:39-42); prokaryotic expression vectors such as the
.beta.-lactamase promoter (Villa-Kamaroff et al., 1978, Proc. Natl.
Acad. Sci. U.S.A. 75:3727-3731), or the lac promoter (DeBoer et
al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:21-25); see also
"Useful proteins from recombinant bacteria" in Scientific American,
1980, 242:74-94; plant expression vectors comprising the nopaline
synthetase promoter region (Herrera-Estrella et al., Nature
303:209-213) or the cauliflower mosaic virus 35S RNA promoter
(Gardner et al., 1981, Nucl. Acids Res. 9:2871), and the promoter
of the photosynthetic enzyme ribulose biphosphate carboxylase
(Herrera-Estrella et al., 1984, Nature 310:115-120); promoter
elements from yeast or other fungi such as the Gal 4 promoter, the
ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase)
promoter, alkaline phosphatase promoter, and the following animal
transcriptional control regions, which exhibit tissue specificity
and have been utilized in transgenic animals: elastase I gene
control region which is active in pancreatic acinar cells (Swift et
al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor
Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology
7:425-515); a gene control region which is active in pancreatic
beta cells (Hanahan, 1985, Nature 315:115-122), an immunoglobulin
gene control region which is active in lymphoid cells (Grosschedl
et al., 1984, Cell 38:647-658; Adames et al., 1985, Nature
318:533-538; Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444),
mouse mammary tumor virus control region which is active in
testicular, breast, lymphoid and mast cells (Leder et al., 1986,
Cell 45:485-495), albumin gene control region which is active in
liver (Pinkert et al., 1987, Genes and Devel. 1:268-276),
alpha-fetoprotein gene control region which is active in liver
(Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et
al., 1987, Science 235:53-58; alpha 1-antitrypsin gene control
region which is active in the liver (Kelsey et al., 1987, Genes and
Devel. 1:161-171), beta-globin gene control region which is active
in myeloid cells (Mogram et al., 1985, Nature 315:338-340; Kollias
et al., 1986, Cell 46:89-94; myelin basic protein gene control
region which is active in oligodendrocyte cells in the brain
(Readhead et al., 1987, Cell 48:703-712); myosin light chain-2 gene
control region which is active in skeletal muscle (Sani, 1985,
Nature 314:283-286), and gonadotropic releasing hormone gene
control region which is active in the hypothalamus (Mason et al.,
1986, Science 234:1372-1378).
[0170] In a specific embodiment, a vector is used that comprises a
promoter operably linked to a nucleic acid encoding a peptide of
the invention, one or more origins of replication, and, optionally,
one or more selectable markers (e.g., an antibiotic resistance
gene).
[0171] Expression vectors containing nucleic acid inserts can be
identified by any of a number of well known methods. Three general
approaches are: (a) nucleic acid hybridization; (b) presence or
absence of "marker" gene functions; and (c) expression of inserted
sequences. In the first approach, the presence of a gene inserted
in an expression vector can be detected by nucleic acid
hybridization using probes comprising sequences that are homologous
to an inserted gene. In the second approach, the recombinant
vector/host system can be identified and selected based upon the
presence or absence of certain "marker" gene functions (e.g.,
thymidine kinase activity, resistance to antibiotics,
transformation phenotype, occlusion body formation in baculovirus,
etc.) caused by the insertion of a nucleic acid of interest in the
vector. For example, if the nucleic acid is inserted within the
marker gene sequence of the vector, recombinants containing the
insert can be identified by the absence of the marker gene
function. In the third approach, recombinant expression vectors can
be identified by assaying the product expressed by the recombinant.
Such assays can be based, for example, on the physical or
functional properties of an expressed peptide in in vi tro assay
systems (e.g., binding with an anti-peptide antibody of the
invention).
[0172] A host cell strain for expressing a nucleic acid encoding a
peptide of the invention may be chosen which modulates the
expression of the inserted sequences, or modifies and processes the
nucleic acid product in the specific fashion desired. Expression
from certain promoters can be elevated in the presence of certain
inducers; thus, expression of a genetically-engineered peptide of
the invention may be controlled. Furthermore, different host cells
have characteristic and specific mechanisms for the translational
and post-translational processing and modification (e.g.,
glycosylation, phosphorylation). Appropriate cell lines or host
systems can be chosen to ensure the desired modification and
processing of the foreign peptide or protein expressed. For
example, expression in a bacterial system can be used to produce a
non-glycosylated core protein product. Expression in yeast will
produce a glycosylated product. Expression in mammalian cells can
be used to ensure "native" glycosylation of a heterologous protein.
Furthermore, different vector/host expression systems may effect
processing reactions to different extents.
[0173] 5.10. Identification of Molecules Having Binding
Capacity
[0174] This invention provides screening methods useful in the
identification of molecules (e.g. proteins or other compounds)
which bind to, or otherwise directly interact with, the identified
peptides and other small molecules of the invention. Such screening
methods are well known in the art (see e.g., PCT International
Publication No. WO 96/34099, published Oct. 31, 1996, which is
incorporated by reference herein in its entirety). Such proteins
and compounds may include endogenous cellular components which
interact with the identified peptides in vivo and which, therefore,
may provide new targets for pharmaceutical and therapeutic
interventions, as well as recombinant, synthetic, and otherwise
exogenous compounds which may have binding capacity and, therefore,
may be candidates for pharmaceutical agents. Thus, in one series of
embodiments, cell lysates or tissue homogenates may be screened for
proteins or other compounds which bind to a peptide of the
invention. Alternatively, any of a variety of exogenous compounds,
both naturally occurring or synthetic (e.g., libraries of small
molecules), may be screened for binding capacity.
[0175] As will be apparent to one of ordinary skill in the art,
there are numerous other methods of screening individual proteins
or other compounds, as well as large libraries of proteins or other
compounds (e.g., phage display libraries) to identify molecules
which bind to peptides or other small molecules of the invention,
or fragments, derivatives or analogs thereof. All of these methods
comprise the step of mixing such a molecule with test compounds,
allowing time for any binding to occur, and assaying for any bound
complexes. All such methods are contemplated by the present
disclosure of substantially pure peptides and other small
molecules, substantially pure functional domain fragments, fusion
proteins, antibodies, and methods of making and using the same.
[0176] In a preferred embodiment, a peptide of the invention having
a desired functional activity in a functional assay can be
evaluated for cross reactivity with other cellular components by
using the peptide, or a chimeric or fusion protein thereof, as
"bait" in a yeast two-hybrid assay system (Fields and Song, 1989,
Nature 340:245-246; U.S. Pat. No. 5,283,173) or a variation
thereof. In this way, other potential interactions or functional
effects of a peptide of the invention can be identified prior to
pharmaceutical development and clinical use.
[0177] The yeast two-hybrid method has been used to analyze
IGF-1-receptor interactions (see Zhu and Kahn, 1997, Proc. Natl.
Acad. Sci. U.S.A. 94, 13063-13068). Because interactions are
screened for in yeast, the protein-protein interactions detected
occur under physiological conditions that mimic conditions in
eukaryotic cells, including vertebrates or invertebrates (Chien et
al., 1991, Proc. Natl. Acad. Sci. U.S.A. 88, 9578-9581). This
feature facilitates identification of proteins capable of
interaction with peptides, derivatives, or fusion proteins of the
invention having a desired functional activity.
[0178] Identification of interacting proteins by the improved yeast
two-hybrid system is based upon the detection of expression of a
reporter gene, the. transcription of which is dependent upon the
reconstitution of a transcriptional regulator by the interaction of
two proteins, each fused to one half of the transcriptional
regulator. The "bait" (i.e., peptide or fusion protein or
derivative or analog thereof) and "prey" (proteins to be tested for
ability to interact with the bait) are expressed as fusion proteins
to a DNA binding domain and to a transcriptional regulatory domain,
respectively, or vice versa. In various specific embodiments, the
prey has a complexity of at least about 50, about 100, about 500,
about 1,000, about 5,000, about 10,000, or about 50,000; or has a
complexity in the range of about 25 to about 100,000, about 100 to
about 50,000, about 50,000 to about 100,000, or about 100,000 to
about 500,000. For example, the prey population can be one or more
nucleic acids encoding mutants of a protein (e.g., as generated by
site-directed mutagenesis or another method of making mutations in
a nucleotide sequence). Preferably, the prey populations are
proteins encoded by DNA, e.g., cDNA or genomic DNA or
synthetically-generated DNA. For example, the populations can be
expressed from chimeric genes comprising cDNA sequences from an
un-characterized sample of a population of cDNA from mRNA.
[0179] One characteristic of the yeast two-hybrid system is that
proteins examined in this system are expressed as cytoplasmic
proteins, and therefore facilitating identification of interactors
with the peptides and derivatives thereof of the invention.
[0180] In one embodiment, recombinant biological libraries
expressing random peptides can be used as the source of prey
nucleic acids.
[0181] In another embodiment, the invention provides methods of
screening for inhibitors or enhancers of the protein interactants
identified herein. Briefly, the protein-protein interaction assay
can be carried out as described herein, except that it is done in
the presence of one or more candidate molecules. An increase or
decrease in reporter gene activity relative to that present when
the one or more candidate molecules are absent indicates that the
candidate molecule has an effect on the interacting pair. In a
preferred method, inhibition of the interaction is selected for
(i.e., inhibition of the interaction is necessary for the cells to
survive), for example, where the interaction activates the URA3
gene, causing yeast to die in medium containing the chemical
5-fluoroorotic acid (Rothstein, 1983, Meth. Enzymol. 101, 167-180).
The identification of inhibitors of such interactions can also be
accomplished, for example, but not by way of limitation, using
competitive inhibitor assays, as described above.
[0182] In general, proteins of the bait and prey populations are
provided as fusion (chimeric) proteins (preferably by recombinant
expression of a chimeric coding sequence) comprising each protein
contiguous to a pre-selected sequence. For one population, the
pre-selected sequence is a DNA binding domain. The DNA binding
domain can be any DNA binding domain, as long as it specifically
recognizes a DNA sequence within a promoter. For example, the DNA
binding domain is of a transcriptional activator or inhibitor. For
the other population, the pre-selected sequence is an activator or
inhibitor domain of a transcriptional activator or inhibitor,
respectively. The regulatory domain alone (not as a fusion to a
protein sequence) and the DNA-binding domain alone (not as a fusion
to a protein sequence) preferably do not detectably interact (so as
to avoid false positives in the assay). The assay system further
includes a reporter gene operably linked to a promoter that
contains a binding site for the DNA binding domain of the
transcriptional activator (or inhibitor).
[0183] Accordingly, in the present method of the invention, binding
of a peptide of the invention to a fusion protein leads to
reconstitution of a transcriptional activator (or inhibitor) which
activates (or inhibits) expression of the reporter gene. The
activation (or inhibition) of transcription of the reporter gene
occurs intracellularly, e.g., in prokaryotic or eukaryotic cells,
preferably in cell culture.
[0184] The promoter that is operably linked to the reporter gene
nucleotide sequence can be a native or non-native promoter of the
nucleotide sequence, and the DNA binding site(s) that are
recognized by the DNA binding domain portion of the fusion protein
can be native to the promoter (if the promoter normally contains
such binding site(s)) or non-native to the promoter. Thus, for
example, one or more tandem copies (e.g., four or five copies) of
the appropriate DNA binding site can be introduced upstream of the
TATA box in the desired promoter (e.g., in the area of about
position -100 to about -400). In a preferred aspect, 4 or 5 tandem
copies of the 17 bp UAS (GAL4 DNA binding site) are introduced
upstream of the TATA box in the desired promoter, which is upstream
of the desired coding sequence for a selectable or detectable
marker. In a preferred embodiment, the GALl-10 promoter is operably
fused to the desired nucleotide sequence; the GALl-10 promoter
already contains 4 binding sites for GAL4.
[0185] Alternatively, the transcriptional activation binding site
of the desired gene(s) can be deleted and replaced with GAL4
binding sites (Bartel et al., 1993, BioTechniques 14, 920-924;
Chasman et al., 1989, Mol. Cell. Biol. 9, 4746-4749). The reporter
gene preferably contains the sequence encoding a detectable or
selectable marker, the expression of which is regulated by the
transcriptional activator, such that the marker is either turned on
or off in the cell in response to the presence of a specific
interaction. Preferably, the assay is carried out in the absence of
background levels of the transcriptional activator (e.g., in a cell
that is mutant or otherwise lacking in the transcriptional
activator).
[0186] In one embodiment, more than one reporter gene is used to
detect transcriptional activation, e.g., one reporter gene encoding
a detectable marker and one or more reporter genes encoding
different selectable markers. The detectable marker can be any
molecule that can give rise to a detectable signal, e.g., a
fluorescent protein or a protein that can be readily visualized or
that is recognizable by a specific antibody. The selectable marker
can be any protein molecule that confers the ability to grow under
conditions that do not support the growth of cells not expressing
the selectable marker, e.g., the selectable marker is an enzyme
that provides an essential nutrient and the cell in which the
interaction assay occurs is deficient in the enzyme and the
selection medium lacks such nutrient. The reporter gene can either
be under the control of the native promoter that naturally contains
a binding site for the DNA binding protein, or under the control of
a heterologous or synthetic promoter.
[0187] The activation domain and DNA binding domain used in the
assay can be from a wide variety of transcriptional activator
proteins, as long as these transcriptional activators have
separable binding and transcriptional activation domains. For
example, the GAL4 protein of S. cerevisiae (Ma et al., 1987, Cell
48, 847-853), the GCN4 protein of S. cerevisiae (Hope and Struhl,
1986, Cell 46, 885-894), the ARD1 protein of S. cerevisiae (Thukral
et al., 1989, Mol. Cell. Biol. 9, 2360-2369), and the human
estrogen receptor (Kumar et al., 1987, Cell 51, 941-951), have
separable DNA binding and activation domains. The DNA binding
domain and activation domain that are employed in the fusion
proteins need not be from the same transcriptional activator. In a
specific embodiment, a GAL4 or LEXA DNA binding domain is employed.
In another specific embodiment, a GAL4 or herpes simplex virus VP16
(Triezenberg et al., 1988, Genes Dev. 2, 730-742) activation domain
is employed. In a specific embodiment, amino acids 1-147 of GAL4
(Ma et al., 1987, Cell 48, 847-853; Ptashne et al., 1990, Nature
346, 329-331) is the DNA binding domain, and amino acids 411-455 of
VP16 (Triezenberg et al., 1988, Genes Dev. 2, 730-742; Cress et
al., 1991, Science 251, 87-90) comprise the activation domain.
[0188] In a preferred embodiment, the yeast transcription factor
GAL4 is reconstituted by protein-protein interaction and the host
strain is mutant for GAL4. In another embodiment, the DNA-binding
domain is Ace1N and/or the activation domain is Ace1, the DNA
binding and activation domains of the Ace1 protein, respectively.
Ace1 is a yeast protein that activates transcription from the CUP1
operon in the presence of divalent copper. CUP1 encodes
metallothionein, which chelates copper, and the expression of CUP1
protein allows growth in the presence of copper, which is otherwise
toxic to the host cells. The reporter gene can also be a CUP1-lacZ
fusion that expresses the enzyme beta-galactosidase (detectable by
routine chromogenic assay) upon binding of a reconstituted Ace1N
transcriptional activator (see Chaudhuri et al., 1995, FEBS Letters
357, 221-226). In another specific embodiment, the DNA binding
domain of the human estrogen receptor is used, with a reporter gene
driven by one or three estrogen receptor response elements (Le
Douarin et al., 1995, Nucl. Acids. Res. 23, 876-878).
[0189] The DNA binding domain and the transcriptional
activator/inhibitor domain each preferably has a nuclear
localization signal (see Ylikomi et al., 1992, EMBO J. 11,
3681-3694; Dingwall and Laskey, 1991, TIBS 16, 479-481) functional
in the cell in which the fusion proteins are to be expressed.
[0190] To facilitate isolation of the encoded proteins, the fusion
constructs can further contain sequences encoding affinity tags
such as glutathione-S-transferase or maltose-binding protein or an
epitope of an available antibody, for affinity purification (e.g.,
binding to glutathione, maltose, or a particular antibody specific
for the epitope, respectively) (Allen et al., 1995, TIBS 20,
511-516). In another embodiment, the fusion constructs further
comprise acterial promoter sequences for recombinant production of
the fusion protein in bacterial cells.
[0191] The host cell in which the interaction assay occurs can be
any cell, prokaryotic or eukaryotic, in which transcription of the
reporter gene can occur and be detected, including, but not limited
to, mammalian (e.g., monkey, mouse, rat, human, bovine), chicken,
bacterial, or insect cells, and is preferably a yeast cell.
Expression constructs encoding and capable of expressing the
binding domain fusion proteins, the transcriptional activation
domain fusion proteins, and the reporter gene product(s) are
provided within the host cell, by mating of cells containing the
expression constructs, or by cell fusion, transformation,
electroporation, microinjection, etc. In a specific embodiment in
which the assay is carried out in mammalian cells (e.g., hamster
cells, HeLa cells), the DNA binding domain is the GAL4 DNA binding
domain, the activation domain is the herpes simplex virus VP16
transcriptional activation domain, and the reporter gene contains
the desired coding sequence operably linked to a minimal promoter
element from the adenovirus E1B gene driven by several GAL4 DNA
binding sites (see Fearon et al., 1992, Proc. Natl. Acad. Sci.
U.S.A. 89, 7958-7962). The host cell used should not express an
endogenous transcription factor that binds to the same DNA site as
that recognized by the DNA binding domain fusion population. Also,
preferably, the host cell is mutant or otherwise lacking in an
endogenous, functional form of the reporter gene(s) used in the
assay. Various vectors and host strains for expression of the two
fusion protein populations in yeast are known and can be used (see
e.g., U.S. Pat. No. 5,1468,614; Bartel et al., 1993, "Using the
two-hybrid system to detect protein-protein interactions" In
Cellular Interactions in Development, Hartley, ed., Practical
Approach Series xviii, IRL Press at Oxford University Press, New
York, N.Y., pp. 153-179; Fields and Sternglanz, 1994, Trends In
Genetics 10, 286-292). By way of example but not limitation, yeast
strains or derivative strains made therefrom, which can be used are
N105, N106, N1051, N1061, and YULH. Other exemplary strains that
can be used in the assay of the invention also include, but are not
limited to, the following:
[0192] Y190: MATa, ura3-52, his3-200, lys2-801, ade2-101, trp1-901,
leu2-3,112, gal4.alpha., gal80.alpha., cyh.sup.r2,
LYS2::GAL1.sub.UAS-HIS3.sub.TATAHIS3,
URA3::GAL1.sub.UAS-GAL1.sub.TATA-la- cZ; Harper et al., 1993, Cell
75, 805-816, available from Clontech, Palo Alto, Calif. Y190
contains HIS3 and lacZ reporter genes driven by GAL4 binding
sites.
[0193] CG-1945: MATa, ura3-52, his3-200, lys2-801, ade2-101,
trpl-901, leu2-3,112, gal4-542, gal80-538, cyh.sup.r2,
LYS2::GALl.sub.UAS-HIS3.sub.- TATAHIS3,
URA3::GALl.sub.UAS17mers(x3)-CYC1.sub.TATA-lacZ, available from
Clontech, Palo Alto, Calif. CG-1945 contains HIS3 and lacZ reporter
genes driven by GAL4 binding sites.
[0194] Y187: MAT-.alpha., ura3-52, his3-200, ade2-101, trp1-901,
leu2-3,112, gal4.alpha., gal80.alpha.,
URA3::GAL1.sub.UAS-GAL1.sub.TATA-l- acZ, available from Clontech,
Palo Alto, Calif. Y187 contains a lacZ reporter gene driven by GAL4
binding sites.
[0195] SFY526: MATa, ura3-52, his3-200, lys2-801, ade2-101,
trp1-901, leu2-3,112, gal4-542, gal80-538, can.sup.r,
URA3::GAL1-lacZ, available from Clontech, Palo Alto, Calif. SFY526
contains HIS3 and lacZ reporter genes driven by GAL4 binding
sites.
[0196] HF7c: MATa, ura3-52, his3-200, lys2-801, ade2-101, trp1-901,
leu2-3,112, gal4-542, gal80-538, LYS2::GAL1-HIS3,
URA3::GAL1.sub.UAS 17 MERS(x3)-CYC1-lacZ, available from Clontech,
Palo Alto, Calif. HF7c contains HIS3 and lacZ reporter genes driven
by GAL4 binding sites.
[0197] YRG-2: MATa, ura3-52, his3-200, lys2-801, ade2-101,
trp1-901, leu2-3,112, gal4-542, gal80-538,
LYS2::GAL1.sub.UAS-GAL1.sub.TATA-HIS3,
URA3::GAL1.sub.UAS17mers(x3)-CYC1-lacZ, available from Stratagene,
La Jolla, Calif. YRG-2 contains HIS3 and lacZ reporter genes driven
by GAL4 binding sites. Many other strains commonly known and
available in the art can be used.
[0198] If not already lacking in endogenous reporter gene activity,
cells mutant in the reporter gene may be selected by known methods,
or the cells can be made mutant in the target reporter gene by
known gene-disruption methods prior to introducing the reporter
gene (Rothstein, 1983, Meth. Enzymol. 101, 202-211).
[0199] In a specific embodiment, plasmids encoding the different
fusion protein populations can be introduced simultaneously into a
single host cell (e.g., a haploid yeast cell) containing one or
more reporter genes, by co-transformation, to conduct the assay for
protein-protein interactions. Or, preferably, the two fusion
protein populations are introduced into a single cell either by
mating (e.g., for yeast cells) or cell fusions (e.g., of mammalian
cells). In a mating type assay, conjugation of haploid yeast cells
of opposite mating type that have been transformed with a binding
domain fusion expression construct (preferably a plasmid) and an
activation (or inhibitor) domain fusion expression construct
(preferably a plasmid), respectively, will deliver both constructs
into the same diploid cell. The mating type of a yeast strain may
be manipulated by transformation with the HO gene (Herskowitz and
Jensen, 1991, Meth. Enzymol. 194, 132-146).
[0200] In a preferred embodiment, a yeast interaction mating assay
is employed using two different types of host cells, strain-type a
and alpha of the yeast Saccharomyces cerevisiae. The host cell
preferably contains at least two reporter genes, each with one or
more binding sites for the DNA-binding domain (e.g., of a
transcriptional activator). The activator domain and DNA binding
domain are each parts of chimeric proteins formed from the two
respective populations of proteins. One strain of host cells, for
example the a strain, contains fusions of the library of nucleotide
sequences with the DNA-binding domain of a transcriptional
activator, such as GAL4. The hybrid proteins expressed in this set
of host cells are capable of recognizing the DNA-binding site in
the promoter or enhancer region in the reporter gene construct. The
second set of yeast host cells, for example, the alpha strain,
contains nucleotide sequences encoding fusions of a library of DNA
sequences fused to the activation domain of a transcriptional
activator.
[0201] In a preferred embodiment, the fusion protein constructs are
introduced into the host cell as a set of plasmids. These plasmids
are preferably capable of autonomous replication in a host yeast
cell and preferably can also be propagated in E. coli. The plasmid
contains a promoter directing the transcription of the DNA binding
or activation domain fusion genes, and a transcriptional
termination signal. The plasmid also preferably contains a
selectable marker gene, permitting selection of cells containing
the plasmid. The plasmid can be single-copy or multi-copy.
Single-copy yeast plasmids that have the yeast centromere may also
be used to express the activation and DNA binding domain fusions
(Elledge et al., 1988, Gene 70, 303-312).
[0202] In another embodiment, the fusion constructs are introduced
directly into the yeast chromosome via homologous recombination.
The homologous recombination for these purposes is mediated through
yeast sequences that are not essential for vegetative growth of
yeast, e.g., the MER2, MER1, ZIPI, REC102, or ME14 gene.
[0203] Bacteriophage vectors can also be used to express the DNA
binding domain and/or activation domain fusion proteins. Libraries
can generally be prepared faster and more easily from bacteriophage
vectors than from plasmid vectors.
[0204] In a specific embodiment, the present invention provides a
method of detecting one or more protein-protein interactions
comprising (a) recombinantly expressing a peptide of the invention
having a desired functional activity, or a derivative or analog
thereof, in a first population of yeast cells being of a first
mating type and comprising a first fusion protein containing the
peptide amino acid sequence and a DNA binding domain, wherein said
first population of yeast cells contains a first nucleotide
sequence operably linked to a promoter driven by one or more DNA
binding sites recognized by said DNA binding domain such that an
interaction of said first fusion protein with a second fusion
protein, said second fusion protein comprising a transcriptional
activation domain, results in increased transcription of said first
nucleotide sequence; (b) recombinantly expressing in a second
population of yeast cells of a second mating type different from
said first mating type, a plurality of said second fusion proteins,
each second fusion protein comprising a sequence of a fragment,
derivative or analog of a protein and an activation domain of a
transcriptional activator, in which the activation domain is the
same in each said second fusion protein; (c) mating said first
population of yeast cells with said second population of yeast
cells to form a third population of diploid yeast cells, wherein
said third population of diploid yeast cells contains a second
nucleotide sequence operably linked to a promoter driven by a DNA
binding site recognized by said DNA binding domain such that an
interaction of a first fusion protein with a second fusion protein
results in increased transcription of said second nucleotide
sequence, in which the first and second nucleotide sequences can be
the same or different; and (d) detecting said increased
transcription of said first and/or second nucleotide sequence,
thereby detecting an interaction between a first fusion protein and
a second fusion protein. In a preferred aspect, between step (a)
and (b), a step is carried out of negatively selecting to eliminate
those yeast cells in said first population which said increased
transcription of said first nucleotide sequence occurs in the
absence of said second fusion protein (see e.g. PCT International
Publication No. WO 97/47763, published Dec. 18, 1997, which is
incorporated by reference-herein in its entirety).
[0205] In a preferred embodiment, the bait peptide sequence and the
prey library of chimeric genes are combined by mating the two yeast
strains on solid media, such that the resulting diploids contain
both kinds of chimeric genes, i.e., the DNA-binding domain fusion
and the activation domain fusion.
[0206] Preferred reporter genes include the URA3, HIS3 and/or the
lacZ genes (see e.g., Rose and Botstein, 1983, Meth. Enzymol. 101,
167-180) operably linked to GAL4 DNA-binding domain recognition
elements. Other reporter genes include but are not limited to,
Green Fluorescent Protein (GFP) (Cubitt et al., 1995, Trends
Biochem. Sci. 20, 448-455), luciferase, LEU2, LYS2, ADE2, TRP1,
CAN1, CYH2, GUS, CUP1 or chloramphenicol acetyl transferase (CAT).
Expression of the reporter genes can be detected by techniques
known in the art (see e.g. PCT International Publication No. WO
97/47763, published Dec. 18, 1997, which is incorporated by
reference herein in its entirety).
[0207] In a specific embodiment, transcription of the reporter gene
is detected by a linked replication assay. For example, as
described by Vasavada et al., 1991, Proc. Natl. Acad. Sci. U.S.A.
88, 10686-10690, expression of SV40 large T antigen is under the
control of the ElB promoter responsive to GAL4 binding sites. The
replication of a plasmid containing the SV40 origin of replication,
indicates a protein-protein interaction. Alternatively, a polyoma
virus replicon can be used (Id.).
[0208] In another embodiment, the expression of reporter genes that
encode proteins can be detected by immunoassay, i.e., by detecting
the immunospecific binding of an antibody to such protein, which
antibody can be labeled, or incubated with a labeled binding
partner to the antibody, to yield a detectable signal. Alam and
Cook disclose non-limiting examples of detectable marker genes that
can be operably linked to a transcriptional regulatory region
responsive to a reconstituted transcriptional activator, and thus
used as reporter genes (Alam and Cook, 1990, Anal. Biochem. 188,
245-254).
[0209] The activation of reporter genes like URA3 or HIS3 enables
the cells to grow in the absence of uracil or histidine,
respectively, and hence serves as a selectable marker. Thus, after
mating, the cells exhibiting protein-protein interactions are
selected by the ability to grow in media lacking a nutritional
component, such as uracil or histidine (see Le Douarin et al.,
1995, Nucl. Acids Res. 23, 876-878; Durfee et al., 1993, Genes Dev.
7, 555-569; Pierrat et al., 1992, Gene 119, 237-245; Wolcott et
al., 1966, Biochem. Biophys. Acta 122, 532-534). In other
embodiments of the present invention, the activities of the
reporter genes like GFP or lacZ are monitored by measuring a
detectable signal (e.g., fluorescent or chromogenic, respectively)
that results from the activation of these reporter genes. LacZ
transcription, for example, can be monitored by incubation in the
presence of a substrate, such as X-gal
(5-bromo-4-chloro-3-indolyl-.beta.-D-galactoside), of its encoded
enzyme, .beta.-galactosidase.
[0210] In a preferred embodiment of the present invention, false
positives arising from transcriptional activation by the DNA
binding domain fusion proteins in the absence of a transcriptional
activator domain fusion protein are prevented or reduced by
negative selection prior to exposure to the activation domain
fusion population (see e.g. PCT International Publication No. WO
97/47763, published Dec. 18, 1997, which is incorporated by
reference herein in its entirety). By way of example, if such cell
contains URA3 as a reporter gene, negative selection is carried out
by incubating the cell in the presence of 5-fluoroorotic acid
(5-FOA, which kills URA+cells (Rothstein, 1983, Meth. Enzymol. 101,
167-180). Hence, the metabolism of 5-FOA will lead to cell death of
self-activating DNA-binding domain hybrids.
[0211] In a preferred aspect, negative selection involving a
selectable marker as a reporter gene can be combined with the use
of a toxic or growth inhibitory agent to allow a higher rate of
processing than other methods. Negative selection can also be
carried out on the activation domain fusion population prior to
interaction with the DNA binding domain fusion population, by
similar methods, either alone or in addition to negative selection
of the DNA binding fusion population. Negative selection can be
carried out on the recovered protein-protein complex by known
methods (see e.g., Bartel et al., 1993, BioTechniques 14, 920-924;
PCT International Publication No. WO 97/47763, published Dec. 18,
1997).
[0212] In a preferred embodiment of the invention the DNA sequences
encoding the pairs of interactive proteins are isolated by a method
wherein either the DNA-binding domain hybrids or the activation
domain hybrids are amplified, in separate respective reactions.
Preferably, the amplification is carried out by polymerase chain
reaction (PCR) (see U.S. Pat. Nos. 4,683,202; 4,683,195; and
4,889,818; Gyllenstein et al., 1988, Proc. Natl. Acad. Sci. U.S.A.
85:7652-7656; Ochman et al., 1988, Genetics 120:621-623; Loh et
al., 1989, Science 243:217-220; Innis et al., 1990, PCR Protocols,
Academic Press, Inc., San Diego, Calif.) using pairs of
oligonucleotide primers specific for either the DNA-binding domain
hybrids or the activation domain hybrids. Other amplification
methods known in the art can be used, including but not limited to
ligase chain reaction (see EP 320,308), use of Q.beta. replicase,
or methods listed in Kricka et al., 1995, Molecular Probing,
Blotting, and Sequencing, Academic Press, New York, Chapter 1 and
Table IX.
[0213] The plasmids encoding the DNA-binding domain hybrid and the
activation domain hybrid proteins can also be isolated and cloned
by any of the methods well known in the art. For example, but not
by way of limitation, if a shuttle (yeast to E. coli) vector is
used to express the fusion proteins, the genes can be recovered by
transforming the yeast DNA into E. coli and recovering the plasmids
from E. coli (see e.g., Hoffman et al., 1987, Gene 57, 267-272).
Alternatively, the yeast vector can be isolated, and the insert
encoding the fusion protein subcloned into a bacterial expression
vector, for growth of the plasmid in E. coli.
6. EXAMPLES
[0214] The invention described and claimed herein can be further
appreciated by one skilled in the art through reference to the
examples which follow. These examples are provided merely to
illustrate several aspects of the invention and shall not be
construed to limit the invention in any way.
[0215] 6.1. A Surface on the G Protein .beta. Subunit Involved in
Interactions with Adenylyl Cyclases
[0216] Receptor activation of heterotrimeric G proteins dissociates
G.alpha. from the G.beta..gamma. complex, allowing both to regulate
effectors. Little is known about the effector-interaction regions
or domains of G.beta..gamma.. We had used molecular modeling to
dock a peptide encoding residues 956-982 of adenylyl cyclase (AC) 2
(SEQ ID NO:3) onto G.beta. to identify residues of G.beta. that may
interact with effectors. Based on predictions from the model, we
synthesized peptides encoding residues 86-105 (G.beta.86-105) (SEQ
ID NO:5) and 115-135 (G.beta.115-135) (SEQ ID NO:6) of G.beta. (SEQ
ID NO:1). The G.beta.86-105 peptide inhibited G.beta..gamma.
stimulation of AC2 (SEQ ID NO:3) and blocked G.beta..gamma.
inhibition of AC1 (SEQ ID NO:2) and by itself inhibited
calmodulin-stimulated AC1, thus displaying partial agonist
activity. Substitution of Met-101 with Asn in G.beta.86-105
resulted in the loss of both the inhibitory and partial agonist
activities. Most activities of the G.beta.115-135 peptide were
similar to those of G.beta.86-105, but G.beta.115-135 was less
effective in blocking G.beta..gamma. inhibition of AC1.
Substitution of Tyr-124 with Val in the G.beta.115-135 peptide
diminished all of its activities. These results identify the region
encoded by amino acids 84-143 of G.beta. (SEQ ID NO:1) as a surface
that is involved in transmitting signals to effectors.
[0217] 6.1.1. Introduction
[0218] Heterotrimeric G proteins serve as signal transducers for a
wide variety of receptors. Both G.alpha. and G.beta..gamma.
subunits can communicate receptor signals (Fung et al., 1981, Proc.
Natl. Acad. Sci. U.S.A. 78, 152-156; Northup et al., 1983, J. Biol.
Chem. 258, 11369-11376; Logothetis et al., 1987, Nature 325,
321-326; Tang and Gilman, 1991, Science 254, 1500-1503; Dietzel and
Kurjan, 1987, Cell 50, 1000-1010). Regions of G.beta..gamma.
complex involved in communicating the signal to effectors have not
been well characterized. We had identified the region of residues
956-982 of adenylyl cyclase (AC) 2 (SEQ ID NO:3) as being involved
in receiving signals from G.beta..gamma. (Chen et al., 1995,
Science 268, 1166-1169). By using the yeast two-hybrid system, the
AC2 region of residues 956-982 has been subsequently shown to
interact with G.beta. but not G.gamma. subunits (Yan and Gautam,
1996, J. Biol. Chem. 271, 17597-17601). In recent studies we found
that the peptide encoding residues 956-982 of AC2 (SEQ ID NO:3) can
be crosslinked to G.beta. when it is part of the free
G.beta..gamma. complex but not when it is part of the heterotrimer,
indicating that the putative binding surface on G.beta. for the AC2
peptide is occluded by interactions with G.alpha.. On the basis of
constraints deduced from the crosslinking studies and other
biophysical criteria, we docked the AC2 of G.beta. by using
molecular modeling techniques (Weng et al., 1996, J. Biol. Chem.
271, 26445-26448). From this docking model, we have identified the
regions of G.beta. that are predicted to interact with the AC2
peptide. Herein we have tested whether peptides encoding the
effector-interaction surface of G.beta. predicted from the modeling
(Weng et al., 1996, J. Biol. Chem. 271, 26445-26448) can modulate
G.beta..gamma. regulation of AC1 and AC2.
[0219] 6.1.2. Materials and Methods
[0220] Materials. Reagents for peptide synthesis were from Bachem.
[.alpha.-.sup.32P]ATP was from New England Nuclear. Tissue culture
reagents and fetal calf serum was from GIBCO. All other chemicals
used were the highest grade available.
[0221] Peptide Synthesis. Peptides were synthesized on an Applied
Biosystems peptide synthesizer (model 431A) and purified by HPLC on
acetonitrile gradients. Purified peptides were lyophilized and
stored at -20.degree. C. When required peptides were dissolved in
water to final concentration of 1-3 mM. Identity of the peptides
was verified by mass spectrometry.
[0222] Expression Of G-Protein Subunits And Adenylyl Cyclases.
G.beta..gamma. was purified from bovine brain (Dingus et al., 1994,
Meth. Enzymol. 237, 457-471). Q227L-G.alpha..sub.s was expressed in
rabbit reticulocyte lysates. AC2 was expressed in Sf9 cells by
infection with recombinant baculovirus (Jacobowitz and Iyengar,
1994, Proc. Natl. Acad. Sci. U.S.A. 91, 10630-10634). AC2 assays
have been described (Chen et al., 1995, Science 268, 1166-1169).
Bovine AC1 (Jacobowitz et al., 1993, J. Biol. Chem. 268, 3829-3832)
was epitope tagged at the N terminus with the FLAG epitope
(Jacobowitz and Iyengar, 1994, Proc. Natl. Acad. Sci. U.S.A. 91,
10630-10634) and expressed in Sf9 cells by baculovirus
infection.
[0223] Adenylyl Cyclase Assays. AC2 assays have been described
(Chen et al., 1995, Science 268, 1166-1169). When required the
peptides were mixed with adenylyl cyclase containing membranes and
held on ice for 10 min prior to assays. Approximately 1-4 .mu.g of
AC2 Sf9 cell membranes per assay tube was used. All assays
contained a mixture of protease inhibitors. The final concentration
of each inhibitor was leupeptin at 3.2 .mu.g/ml, aprotinin at 2
.mu.g/ml, phenanthroline at 1.0 mM, and phenylmethylsulfonyl
fluoride at 1.0 mM. To study G.beta..gamma. inhibition,
AC1-containing Sf9 cell membranes (1-4 .mu.g per assay tube) was
used. In these assays, in addition to the other standard reagents,
the assay mixture contained either 1 mM EGTA or 50 .mu.M CaCl.sub.2
plus 100 nM calmodulin (CaM). All experiments were repeated two or
more times with qualitatively similar results. Typical experiments
are shown. Values are mean.+-.SD of triplicate determinations.
[0224] Molecular Modeling. Procedures for molecular modeling have
been described (Weng et al., 1996, J. Biol. Chem. 271,
26445-26448). Briefly, a secondary structure prediction of the AC2
peptide containing residues 956-982 (AC2 956-982) was obtained and
used to construct an energy minimized three-dimensional model of
the peptide. To identify likely interaction surfaces, the
electrostatic potentials of the AC2 956-982 peptide and the G.beta.
protein (Sondek et al., 1996, Nature 379, 369-374) were visualized
with the GRASP program. Long-range electrostatic interactions were
then used as guides in the initial docking of the peptide to
G.beta.. The structure of the AC2 956-982 peptide docked to G.beta.
was subjected to energy minimization followed by conformational
explorations with a novel Monte Carlo-based method (Guarnieri and
Weinstein, 1996, J. Am. Chem. Soc. 118, 5580-5589). The most
favorable structure of the docked AC2 peptide interacting with
G.beta. was thus obtained within the imposed constraints. Contact
residues on G.beta. were identified with the LOOK software (MAC,
Palo Alto, Calif.) as residues within 4 .ANG. of the AC2
peptide.
[0225] 6.1.3. Results
[0226] We used the docking model (Weng et al., 1996, J. Biol. Chem.
271, 26445-26448) to obtain predicted contact points between the
G.beta. and the AC2 956-982 peptide. FIG. 1A shows the backbone of
G.beta.. The regions of G.beta. predicted to interact with the AC2
peptide are shown in pink. Predicted contacts between residues of
the AC2 peptide and G.beta. (see Molecular Modeling in Section
6.1.2) are shown in FIG. 1B. Since the peptide encodes a region of
AC2, we reasoned that the predicted contact residues on G.beta.
could be involved in communicating signals to effectors. To test
this idea, we synthesized peptides encoding sequences from G.beta.
and determined whether these peptides modulated G.beta..gamma.
regulation of AC2 and AC1. Two peptides were designed based on the
predicted contact interactions between G.beta. and AC2 peptide. The
first peptide (TTN) encodes the region of residues 86-105 of
G.beta., which includes the stretch of residues 91-99 predicted by
the model to be important for effector interactions (FIG. 2A). The
effects of TTN peptide on the activity of recombinant AC2 expressed
in Sf9 cells are shown in FIG. 2 at 100 .mu.M, the peptide did not
inhibit basal or activated .alpha..sub.s. (.alpha..sub.s*)
stimulated activities; however, it significantly inhibited
G.beta..gamma.-stimulated activity, which is seen only in the
presence of .alpha..sub.s* (Fung et al., 1981, Proc. Natl. Acad.
Sci. U.S.A. 78, 152-156). To ascertain the specificity of the
peptide effect, we substituted the residue corresponding to Met-101
in G.beta. with Asn. This Met is conserved in most G.beta. proteins
from different species (Sondek et al., 1996, Nature 379, 369-374)
and mutation of the residue at this position in yeast abolishes
G.alpha. interactions (Whiteway et al., 1994, Mol. Cell. Biol. 14,
3223-3229). The "mutated" peptide (m-TTN) containing Asn at the
position corresponding to G.beta.-101 was much less efficacious
than the TTN peptide in inhibiting G.beta..gamma. stimulation (FIG.
2B). The half-maximal concentration at which the TTN peptide
inhibited G.beta..gamma. stimulation of AC2 was in the range of
30-60 .mu.M (FIG. 2C). Since G.beta..gamma. also inhibits AC1, we
tested whether the TTN peptide's ability to block G.beta..gamma.
interactions with effectors could be extended to modulation of
G.beta..gamma. inhibition of AC1. The recombinant AC1 expressed in
Sf9 cells was used in the assays. The TTN peptide did not affect
basal activity of AC1. G.beta..gamma. inhibited the
Ca.sup.2+/CaM-stimulated AC1 activity whereas m-TTN did not affect
G.beta..gamma. inhibition (FIG. 2D). Increasing concentrations of
TTN peptide further did not result in greater blockade of
G.beta..gamma. inhibition. The reason for this became apparent when
the effect of TTN peptide by itself was evaluated on the
Ca.sup.2+/CaM-stimulated activation of AC1 (FIG. 2E). At 100 .mu.M,
TTN peptide inhibited AC1 activity by 50-70%. The M101N "mutant"
peptide had greatly reduced capacity to inhibit AC1 (FIG. 2E).
[0227] Two other regions of G.beta. predicted by the model to be in
contact with the crosslinked AC2 peptide are between residues
117-119 and 129-135 (FIG. 1B). Hence, we designed a second eptide
(GGL) encoding the region of residues 115-135 of G.beta. (SEQ ID
NO:6) (FIG. 3A). The GGL peptide did not affect basal AC2 activity
and did not significantly inhibit .alpha..sub.s*-stimulated
activity, but it did inhibit G.beta..gamma. stimulated activity
(FIG. 3B). To assess the specificity of this peptide, we converted
the residue corresponding to Tyr-124 in G.beta. to a Val. This Tyr
is conserved in all currently known G.beta. from different species
(Sondek et al., 1996, Nature 379, 369-374). This "mutated" peptide
(m-GGL) was less effective in inhibiting G.beta..gamma. stimulation
of AC2 (FIG. 3B). In contrast to its effect on AC2, the GGL peptide
was not efficacious in blocking G.beta..gamma.-induced inhibition
of AC1 (FIG. 3C). The m-GGL peptide also showed no effect on
G.beta..gamma. inhibition of AC2 (FIG. 3C). Like the TTN peptide,
the GGL peptide alone was also capable of inhibiting
Ca.sup.2+/CaM-stimulated AC1 activity, but the m-GGL peptide did
not inhibit the AC1 activity as extensively as the GGL peptide
(FIG. 3C).
[0228] 6.1.4. Discussion
[0229] The results indicate that we have identified a surface on
G.beta. that is involved in effector interactions. The location of
this region at the interface of G.alpha. and G.beta..gamma. (Weng
et al., 1996, J. Biol. Chem. 271, 26445-26448; Sondek et al., 1996,
Nature 379, 369-374) is consistent with the ability of G.alpha. to
block effector regulation by G.beta..gamma., as many residues of
G.beta. that are involved in interactions with G.alpha., such as
Trp-99, Met-101, Leu-117, and Asn-119 (Sondek et al., 1996, Nature
379, 369-374; Lambright et al., 1996, Nature 379, 311-319), are
also predicted by our model to interact with effectors. We have
explicitly tested the importance of Met-101 that, as shown by the
experiments in FIG. 2, is critical for regulation of effector
function. We have also shown that the conserved Tyr-124 of G.beta.1
is important for effector regulation. FIG. 4 shows how the G.alpha.
binding region on G.beta. identified from the crystal structure
overlaps with an adenylyl cyclase (effector) interaction domain we
have identified by molecular modeling.
[0230] One issue that arises from these studies is whether the
surface on G.beta. where the AC2 peptide docks is sufficient for
full effector contact. Our experiments indicate that the affinity
provided by the interaction of the peptide from this surface is not
sufficient to achieve full blockade of G.beta..gamma. stimulation
of AC2 or to elicit full agonist activity of the G.beta. peptides
in regulating AC1. Interactions with additional regions of G.beta.
might be necessary. Alternatively, the remainder of the
interactions required to achieve full contact with effectors could
involve G.gamma.. Mutational analyses in yeast have identified
three-amino acid residues in the N-terminal part of G.gamma. that
are required for effector function (Grishin et al., 1994, Mol.
Cell. Biol. 14, 4571-4578). The importance of the protein portion
of G.gamma. in effector regulation remains to be investigated in
biochemical experiments. It has also been shown that the
posttranslational modification of G.gamma. that results in
farnesylation (.gamma.1 and possibly .gamma.11) or
geranylgeranylation (other .gamma.s) is required for effector
interactions as assessed by biochemical assays with resolved
components (Iniguez-Lluhi et al., 1992, J. Biol. Chem. 267,
23409-23417). These results suggest that the specific hydrophobic
properties of the acyl group may be required for complete
G.beta..gamma. action on effectors. Thus, a more complete model for
the mode of interaction of G.beta..gamma. with effectors may
involve both the select protein regions in G.beta. and the lipid
moiety in G.gamma..
[0231] 6.2. Resolution of a Signal Transfer Region from a General
Binding Domain in G.beta. for Stimulation of Phospholipase
C-.beta.2
[0232] Transmembrane signal transfer in heterotrimeric G protein
coupled pathways involves sequential protein-protein interactions.
We have studied interactions between G.beta..gamma. subunits and
one of their effectors, phospholipase C-.beta.2 (PLC-.beta.2)(SEQ
ID NO:4), to determine if all of the contact points on G.beta. (SEQ
ID NO:1) are required for signal transfer. A peptide encoding
residues 86-105 of G.beta. (SEQ ID NO:5) was able to specifically
stimulate phospholipase C-.beta.2, and a six amino acid stretch
within this sequence (G.beta. residues 96-101) (SEQ ID NO:8) was
sufficient for signal transfer and thus could be considered as a
core signal transfer region. Another peptide encoding G.beta.
115-135 (SEQ ID NO:6) did not substantially stimulate PLC-.beta.2
by itself but inhibited G.beta..gamma. stimulation of PLC-.beta.2,
indicating that the 115-135 amino acid stretch of G.beta. may be
part of a general binding domain. This resolution of signal
transfer regions from general binding domains indicates that not
all of the interactions in protein-protein contact may be required
for signal transfer, and it may be feasible to synthesize agonists
and antagonists that regulate signal flow at intracellular
sites.
[0233] 6.2.1. Introduction
[0234] Transmembrane signaling in heterotrimeric G protein coupled
systems occurs through protein-protein interactions. Agonist
occupied receptors interact with G proteins to promote nucleotide
exchange and subunit dissociation. The G.alpha. subunits as well as
the G.beta..gamma. complex interact with and regulate effectors
(Gilman, 1987, Ann. Rev. Biochem. 57, 615; Hamm, J. Biol. Chem.
273, 669). The G.beta..gamma. complex regulates numerous effectors
including K+ channels, adenylyl cyclase 2 (AC2), phospholipase
C-.beta.2 and Ca.sup.2+ channels. A general issue that arises in
this mode of signal transduction involving protein-protein
interactions is whether all of the contacts between the protein
partners are required information flow. In this study, we have
addressed this issue in G.beta..gamma. regulation of phospholipase
C-.beta.2.
[0235] We had identified a region within AC2 that was involved in
receiving signals from G.beta. (Chen et al., 1995, Science 268,
1166). With a peptide encoding this region we had used crosslinking
studies and molecular modeling to identify the region 85-145 of
G.beta. as being involved in effector interactions (Weng et al.,
1996, J. Biol. Chem. 271, 26445; and Example 6.1 herein).
Independent studies by Yan and Gautam had also identified the first
one hundred amino acids of G.beta. as being involved in effector
action (Yan and Gautam, 1996, J. Biol. Chem. 271, 17597; Yan and
Gautam, 1997, J. Biol. Chem. 272, 2056). A detailed site-directed
mutagenesis study of G.beta. has also confirmed that the region
60-150 is involved in interactions with multiple effectors (Ford et
al., 1998, Science 280, 1271). Since a relatively large area of
G.beta. is involved in effector interactions, we chose one
effector, phospholipase C-.beta.2 (PLC-.beta.2), and determined a
minimal region of G.beta. required for stimulation. We also
determined if there were regions of G.beta. that are involved in
effector interactions but are not required for signal transfer.
[0236] 6.2.2. Methods
[0237] Peptides were synthesized on an Applied Biosystems peptide
synthesizer (model 431A) and purified by HPLC on acetonitrile
gradients. Purified peptides were lyophilized and stored at -20
degrees C. When needed, peptides were dissolved in HED buffer (10
mM Hepes pH 7.0, 1 mM EDTA pH 8.0, 1 mM DTT). Identity of peptides
was verified by mass spectrometry.
[0238] Recombinant PLC-.beta.2 was expressed in High 5 cells by
infection with recombinant baculovirus. Three to four days after
infection, the cells were lysed by par bombing to 600 psi. The
lysate was then centrifuged, and the cytosolic fraction was
collected. Approximately 10-15 .mu.g of cytosolic fraction was used
per 100 .mu.l reaction volume. Phospholipid substrate is a mixture
of [3H]PIP2 and unlabeled phospholipids. Unlabeled phospholipids,
from Sigma (P-6023), are crude phospholipids from bovine brain. The
total diphosphoinositide and triphosphoinositide content is 20-40%.
The remainder is a mixture of phosphotidylinositol and
phosphotidylserine. Phospholipids are sonicated in 10 mM Hepes pH
7.0 to form micelles. A total of 0.01 .mu.Ci of [3H]PIP2,
corresponding to approximately 7000 cpm, and 5 .mu.g of unlabeled
mixed phospholipids are used per reaction.
[0239] The PLC assay was performed as previously described (De
Vivo, 1994, Meth. Enzymol. 238, 131). Briefly, substrate,
PLC-.beta.2, peptide, and G.beta..gamma. subunits are mixed on ice
in a 100 .mu.l volume buffer containing 10 mM Hepes pH 7.0, 1 mM
DTT, 100 mM KCl, 10 mM NaCl, 2 mM EGTA, 1 mM EDTA, and 1 mM
MgCl.sub.2. Reactions are started by the addition of 25 .mu.l 5 mM
CaCl.sub.2 and incubated at 32 degrees C. for 15 minutes. Reactions
are stopped by the addition of 1 ml CMH
(chloroform:methanol:H.sub.2O=100:100:1 by volume) and 250 .mu.l 10
mM EDTA. After extraction, 400 .mu.l aqueous phase is counted using
a Beckman scintillation counter. All experiments were repeated at
least thrice with very similar results. For concentration-effect
curves, typical experiments are shown.
[0240] For fluorescent resonance energy transfer experiments,
recombinant PLC-.beta.2 was expressed in Sf9 cells and purified as
described (Runnel et al., 1996, Biochem. 35, 16824) and labeled
with the amine-reactive probe, Cascade Blue acetyl azide (Molecular
Probes, Eugene Oreg.) by raising the pH to 8.0 and adding a 4-fold
excess of probe from a freshly prepared concentrated DMF solution.
The reaction was kept on ice for 30 minutes before extensive
dialysis in a solution comprising 20 mM Hepes, 0.16 M NaCl, 1 mM
DTT, pH 7.2 to remove excess probe. Peptides were labeled with
DABMI (4-dimethyl-aminophenylazophenyl-4'-maleimeide) using an
equimolar amount of dye in the absence of reducing agents. The
reaction was allowed to proceed for 30 minutes at room temperature
before quenching with 5 mM DTT. The final labeling ratios, as
determined by absorption, were 1:1 for CB-PLC-.beta.2 and 0.8 for
the two DABMI-peptides. Fluorescence spectra were taken on an
ISS-PC7 (ISS Champaign, IL) photon counting spectrofluorometer
using a 3.times.3 mm cuvette and exciting at 380 nm and scanning
from 400-560 nm.
[0241] 6.2.3. Results and Discussion
[0242] We had previously synthesized two peptides encoding regions
86-105 and 115-135 of G.beta. that were capable of modulating
G.beta..gamma. stimulation of AC1 and AC2 (Weng et al., 1997, J.
Biol. Chem. 271, 26445; and Example 6.1). We tested the G.beta.
86-105 peptide (SEQ ID NO:5) on G.beta..gamma. stimulation of
PLC-.beta.2 (SEQ ID NO:4). In initial experiments we used a
sub-saturating concentration of G.beta. and looked for inhibition
of G.beta. stimulation by the G.beta. 86-105 peptide. Much to our
surprise we found that the peptide robustly stimulated PLC-.beta.2
both in the absence and presence of sub-saturating concentrations
of G.beta..gamma.. The stimulation by maximal concentration of
peptide was non-additive with G.beta..gamma. stimulation (FIG. 5A,
upper panel). Substitution of Methionine at position 101 renders
this peptide inactive for interactions with AC2 and AC1 (Weng et
al., 1997, J. Biol. Chem. 271, 26445; and Example 6.1). The G.beta.
86-105 M101N substituted peptide was not capable of activating
PLC-.beta.2 (FIG. 5B, lower panel), indicating that the 101
position could be important for interactions with PLC-.beta.2. To
determine if the stimulation resulted from direct interactions
between the peptide and PLC-.beta.2 we tested the binding of the
G.beta. 86-105 peptide to PLC-.beta.2 and compared it to the
binding of the G.beta. 86-105 M101N substituted peptide by
fluorescent resonance energy transfer (see Methods in Section
6.2.2). The G.beta. 86-105 peptide binds to PLC-.beta.2 with a
K.sub.d of approximately 1 .mu.M (FIG. 5B), while the G.beta.
86-105 M101N substituted peptide did not display measurable
binding. This binding experiment was conducted both in the presence
and absence of phospholipids with identical results, indicating
that the binding of the peptide to PLC-.beta.2 is independent of
substrate. This is consistent with the ability of the G.beta.
86-105 peptide to stimulate PLC-.beta.2, while the G.beta. 86-105
M101N substituted peptide does not stimulate on its own, nor does
it inhibit G.beta..gamma. stimulation.
[0243] Complementary charge interactions are often key determinants
for protein-protein interactions. The G.beta. 86-105 peptide
contains two charged residues K89 and R96, and one histidine, H91.
We evaluated the importance of each of these residues for the
G.beta. 86-105 peptide stimulation of PLC-.beta.2. Substitution of
each of these residues individually decreased the affinity of the
peptide but did not affect maximal stimulation (FIG. 6A).
Particularly noteworthy was the large shift in affinity when R96
was substituted (FIG. 6A, lower panel). Also noteworthy is the
agreement in effect when the K89A substitution is made in the
peptide or the G.beta. subunit through site-directed mutagenesis
(Li et al., 1998, J. Biol. Chem. 273, 16265; Panchenko et al.,
1998, J. Biol. Chem. 273, 28298). When all three residues were
substituted simultaneously, the peptide did not stimulate
PLC-.beta.2 (FIG. 6B, upper panel) and did not affect
G.beta..gamma. stimulation of PLC-.beta.2 (FIG. 6B, lower panel).
These results indicate that charge interactions may be crucial for
both interactions and signal transfer from G.beta..gamma. to
PLC-.beta.2.
[0244] The experiments in FIG. 6B also raise the possibility that
charged peptides non-specifically activate PLC-.beta.2. Hence we
tested the effects of an unrelated peptide, FLLT, on PLC-.beta.2
activity. FLLT encodes region 660-688 of adenylyl cyclase 6 and has
the same overall change (+2 at pH 6.8-6.9) as the G.beta. 86-105
peptide. While the G.beta. 86-105 peptide stimulates, the FLLT
peptide has no measurable effects (FIG. 6C). These results
demonstrate that the stimulatory effects of the G.beta. 86-105
peptide. on PLC-.beta.2 are not solely due to the charge of the
peptide. To ascertain whether the G.beta. 86-105 peptide
stimulation of PLC-.beta.2 was selective, we tested the ability of
this peptide to stimulate PLC-X.beta., an isoform of PLC.beta. from
Xenopus that is stimulated poorly by G.beta..gamma. subunits under
our assay conditions. While the G.beta. 86-105 peptide stimulates
PLC-.beta.2 robustly, it has relatively little ability to stimulate
PLC-X.beta. (FIG. 6D). This experiment shows that the G.beta.
86-105 peptide selectively stimulates an isoform of PLC.beta. that
is regulated by G.beta..gamma. subunits.
[0245] We next analyzed the importance of the serines at positions
97 and 98 in signal transfer. Site directed mutagenesis studies
have shown that S98A mutants of G.beta. stimulate PLC-.beta.2 more
extensively (Ford et al., 1998, Science 280, 1271). We studied the
effects of four types of substitutions at this position. When S98
was substituted with alanine (FIG. 7, upper panel) there is
approximately a 2-fold increase in the affinity with which the
peptide stimulates. This is consistent with the site-directed
mutagenesis experiment (Ford et al., 1998, Science 280, 1271). When
both serines were substituted with arginine there was a five-fold
increase in affinity of the peptide (FIG. 7, middle panel). In
contrast, substitution with asparagine resulted in an inactive
peptide while substitution with cysteine resulted in greatly
reduced affinity (FIG. 7, lower panel). These experiments suggested
that the region around 96-101 was crucial for signal transfer, and
peptides encoding shorter regions of G.beta. might be capable of
stimulating PLC-.beta.2. Hence, we tested several short peptides
including a three amino acid peptide encoding residues 96-98, a six
amino acid peptide encoding residues 96-101 (SEQ ID NO:8) and a
thirteen amino acid peptide encoding residues 89-101 (SEQ ID NO:7).
The three amino acid peptide did not stimulate PLC-.beta.2, but the
six amino acid peptide as well as the thirteen amino acid peptide
did stimulate (FIG. 8A). Since the G.beta. 96-101 six amino acid
peptide was the smallest peptide we had found that stimulated
PLC-.beta.2, we analyzed it further. Concentration-effect curves
showed that it had considerably lower affinity than the G.beta.
86-105 peptide (FIG. 8B, upper panel). However, when the serines
corresponding to position 97 and 98 were substituted by arginines
(SEQ ID NO:9), the six amino acid peptide stimulated with an
apparent K.sub.act of 30 .mu.M (FIG. 8B, upper panel) as compared
to 5-10 .mu.M K.sub.act for the G.beta. 86-105 peptide (FIG. 5
through FIG. 7). In contrast, when the serines were substituted
with Asp the six amino acid peptide did not stimulate PLC-.beta.2
(FIG. 8B, lower panel). The relative role of the two serines was
further investigated by individually substituting them with
arginine. While both substitutions increase affinity of
stimulation, substitution at position 97 (SEQ ID NO:10) results in
stimulation of PLC-.beta.2 with both a higher affinity and a higher
efficacy than the substitution at position 98 (FIG. 8C, upper
panel). In fact, the efficacy of the S97R substituted peptide (SEQ
ID NO:10) is the same as that for the full length G.beta. 86-105
peptide (SEQ ID NO:5) (FIG. 8C, lower panel), indicating that this
stretch of six amino acids retains the full capacity to transmit
signals, albeit with lower affinity. These results also indicate
that it is the relative positions of the amino acids which
contributes to their effect, not simply the amino acid composition.
The data in FIG. 8 show that amino acids 96 to 101 of G.beta.
constitute a core signal transfer region for activation of
PLC-.beta.2. If this region is sufficient for signal transfer then
what is the role of the other regions of G.beta. that interact with
PLC-.beta.2? One role may be to contribute to the overall affinity
of the interactions but not be involved in signal transfer. If this
were the case then a peptide encoding such a region should inhibit
G.beta..gamma. stimulation of PLC-.beta.2, but by itself would not
stimulate PLC-.beta.2. We tested a peptide encoding residues
115-135 of G.beta. for such effects. We had previously shown that
the G.beta. 115-135 peptide modulated G.beta..gamma. stimulation of
both AC2 and AC1 (Weng et al., 1996, J. Biol. Chem. 271, 26445; and
Example 6.1). The G.beta. 115-135 peptide marginally (.about.20%)
stimulated PLC-.beta.2 by itself, but when added with
G.beta..gamma., substantially inhibited G.beta..gamma. stimulation
of PLC-.beta.2. When the conserved tyrosine at position 124 was
substituted, the peptide was inactive (FIG. 9A). The G.beta.
115-135 peptide inhibits with an apparent K.sub.act of 5 .mu.M
(FIG. 9B). Thus we conclude that the 115-135 region of G.beta.
constitutes a general binding domain involved in G.beta..gamma.
interactions with PLC-.beta.2, but not required for signal
transfer.
[0246] The position of the residues identified in these studies
relative to the remainder of the protein is summarized in a ribbon
diagram of G.beta..gamma. (FIG. 10). In FIG. 10, G.beta. is shown
in khaki, G.gamma. is shown in grey, residues 96-101 of G.beta. are
shown in pink, and residues 115-135 of G.beta. are shown in
aqua.
[0247] These studies demonstrate that all of the contacts between
two proteins are not required for signal transfer. In the case of
G.beta..gamma. and PLC-.beta.2, our data show that a relatively
short stretch of six amino acids (i.e. residues 96-101 of G.beta.)
(SEQ ID NO:8) is sufficient to transfer the signal (in this case,
enzyme activation). Substitution of residues within the six amino
acid peptide produces a more potent peptide than the naturally
occurring sequence. Thus the naturally occurring residues in signal
transfer regions may not be optimized for this particular set of
interactions. Such sub-optimal interactions may be one mechanism to
achieve regulated reversibility. It should also be noted that the
G.beta. 86-105 peptide does not stimulate AC2 in the presence of
G.alpha.s (Weng et al., 1996, J. Biol. Chem. 271, 26445; and
Example 6.1), suggesting that there may be different signal
transfer regions on G.beta. for different effectors. The G.beta.
115-135 peptide minimally stimulates PLC-.beta.2, but is very
(.about.80%) effective in inhibiting G.beta..gamma. stimulation of
PLC-.beta.2 presumably by interacting with PLC-.beta.2. This
indicates that 115-135 region of G.beta. is not crucial for signal
transfer but is part of a general binding domain that participates
in interactions with PLC-.beta.2. The core signal transfer region
and general binding domain we have identified is shown with a
ribbon diagram of G.beta..gamma. derived from the crystal structure
(Sondek et al., 1996, Nature 279, 369).
[0248] What is the relevance of such a functional resolution
between signal transfer regions and general binding domains within
the overall interaction area? From the perspective of protein
engineering, it offers a built-in capability to regulate the
affinity of interaction between the protein partners and thus make
reversibility feasible. Peptide hormones have long been recognized
to have distinct address and message regions (Schwyzer, 1980, Proc.
R. Soc. Lond. 210, 5; Portoghese, 1989, TIPS, 10, 230) that are
involved in binding interactions with receptors and activation of
intracellular signaling pathways, respectively. This functional
resolution of peptide hormones has been used for the design of
peptidomimetic antagonists (Portoghese et al., 1990, J. Med. Chem.
33, 1714). Similarly, our resolution of a signal transfer region
from general binding domain for interactions between intracellular
proteins provides an approach to identifying molecular interactions
relevant for development of agonists and antagonists at
intracellular protein interaction sites. The interactions between
the signal transfer region peptide and PLC-.beta.2 could form the
basis for synthesis of agonists that mimic receptor-dependent
activation of PLC-.beta.2. In contrast, the interactions between
the G.beta. 115-135 peptide and PLC-.beta.2 would form the basis
for synthesis of antagonists that block receptor-dependent
activation of PLC-.beta.2. Signaling pathways are major targets for
therapeutic agents. Until now, agonists and antagonists have
largely focused on extracellular receptor sites. These studies, for
the first time, indicate that it may be feasible to design agonists
and antagonists directed at the interface between signaling
components inside the cell.
[0249] The invention described and claimed herein is not to be
limited in scope by the specific embodiments herein disclosed since
these embodiments are intended as illustrations of the several
aspects of the invention. Any equivalent embodiments are intended
to be within the scope of this 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. Such modifications are also
intended to fall within the scope of the appended claims.
Throughout this application various references are cited, the
contents of each of which is hereby incorporated by reference into
the present application in its entirety. stimulating factor,
Science 281, 257-259). Further, methods have been developed for
determining whether a candidate compound is an agonist of a peptide
hormone receptor (see Kopin et al., U.S. Pat. No. 5,750,353, issued
May 12, 1998, Assay for non-peptide agonists to peptide hormone
receptors). Peptides and mimetics have also been developed based on
the transmembrane domains of G-protein-coupled receptors (Bouvier
et al., Jan. 8, 1998, Peptides and peptidomimetic compounds
affecting the activity of G-protein-coupled receptors by altering
receptor oligomerization, International Publication No. WO
98/00538). Examples of other extracellular ligands for which
peptide mimetics have been developed include erythropoietin and
TNF.alpha. (Wrighton et al., 1997, Increased potency of an
erythropoietin peptide mimetic through covalent dimerization,
Nature Biotechnology 15, 1261-1265; Takasaki et al., 1997,
Structure-based design and characterization of exocyclic
peptidomimetics that inhibit TNF.alpha. binding to its receptor,
Nature Biotechnology 15, 1266-1270). Finally, distinct regions of
peptide hormones have even been considered for design of receptor
antagonists (Portoghese et al., 1990, Design of peptidomimetic
.delta. opioid receptor antagonists using the message-address
concept, J. Med. Chem. 33, 1714-1720).
[0250] Heterotrimeric GTP-binding proteins (G proteins) consisting
of G.alpha..beta..gamma. subunits are ubiquitous signal
transduction proteins that play essential roles in intracellular
communication (see e.g. DeVivo and Iyengar, 1994, G protein
pathways: signal processing by effectors, Molec. Cell. Endocrinol.
100, 65-70). For example, the enzymatic production of cyclic AMP
(cAMP) via adenylyl cyclases is regulated by G proteins (Smit and
Iyengar, 1998, Mammalian adenylyl cyclases, Adv. Sec. Mess.
Phosphoprot. Res. 32, 1-21; Iyengar, 1993, Multiple families of
Gs-regulated adenylyl cyclases, Adv. Sec. Mess. Phosphoprot. Res.
28, 27-36; Pieroni et al., 1993, Signal recognition and integration
by Gs-stimulated adenylyl cyclases, Curr. Opin. Neurobiol. 3,
345-351; Weng et al., 1996, G beta subunit interacts with a peptide
encoding region 956-982 of adenylyl cyclase 2, cross-linking of the
peptide to free G beta gamma but not the heterotrimer, J. Biol.
Chem. 271, 26445-264488; Harry et al., 1997, Differential
regulation of adenylyl cyclases by G alphas, J. Biol. Chem. 272,
19017-19021). G proteins provide a versatile system for
investigation of intracellular protein-protein interactions by
virtue of their interactions with multiple downstream effectors.
For example, G protein .beta..gamma. subunits regulate the activity
of not on adenylyl cyclase but also phospholipase C-.beta.2,
calcium channels, potassium channels, and .beta.-adrenergic
receptor kinase (see e.g. Ford et al., 1998, Molecular basis for
interactions of G protein .beta..gamma. subunits with effectors,
Science 280, 1271-1274).
[0251] Drug intervention beyond the cell surface, i.e. at
intracellular protein-protein interaction sites, would broaden the
array of potential targets for achieving a desired therapeutic
effect. Intracellular targets may also provide intervention points
having enhanced specificity compared to drugs targeted strictly at
cell surface receptors. The ability to use intracellular
interacting proteins as therapeutic targets for drug design has
been less clearly established, however. One reason may be that an
intracellular protein-protein interaction, unlike a typical cell
surface hormone-receptor interaction, will often involve a
multiplicity of proteins. Thus, resolution of specific interactions
among three or more proteins will often be necessary to carry out
design of safe and effective drugs. Accordingly, a need exists for
a generally-applicable approach for identification of peptides and
mimetics thereof having selective activity at a chosen
intracellular site of action.
3. SUMMARY OF THE INVENTION
[0252] This invention provides peptides and other small molecules
derived from regions of intracellular interacting proteins and
methods for identification of such molecules. More specifically,
the present invention provides peptides and other small molecules
derived from regions of G.beta. proteins which function as agonists
or antagonists of adenylyl cyclase or phospholipase C-.beta.2. The
invention is based, at least in part, on the discovery of the
inventors that it is possible to resolve, within a given
intracellular signal transduction protein, a signal transfer region
from a general binding domain. Such resolution provides a rational
basis for design of agonists and antagonists of virtually any
desired intracellular protein-protein interaction. The drug design
methods of the invention utilize three-dimensional structural
information for prediction of protein-protein interactions followed
by evaluation of predictions in functional assays.
[0253] The present invention relates generally to the field of
peptides and peptide mimetics as pharmaceutical and/or therapeutic
agents. More particularly, the present invention relates to
peptides and other small molecules (e.g. peptide mimetics) derived
from regions of G.beta. proteins and their use as pharmaceutical
and/or therapeutic agents. For example, peptides and derivatives
thereof for modulating adenylyl cyclase and phospholipase C-.beta.2
activities are provided. Still further, methods for identification
of peptides and derivatives thereof useful for modulating a chosen
effector-of-interest among various effectors are provided. One
advantage of the methods of the invention is the use of structural
modeling information to predict and validate pharmacologic and
therapeutic agents.
[0254] Predictions about effector interactions of G.beta. proteins
have been made using a combination of molecular modeling and
experimental validation in which the predictions of the model are
tested. Through an iterative process involving cycles of structural
modeling followed by experimental testing, precise definition of
individual effector domains within a G.beta. signaling protein has
been achieved. This validated procedure has general applicability
for drug design targeted at other intracellular protein-protein
interactions in virtually any intracellular signal transduction
pathway.
[0255] This invention provides an isolated G.beta. peptide or
derivative thereof. This invention provides a peptide having an
amino acid sequence selected from the group consisting of SEQ ID
NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID
NO:10. In one embodiment, a derivative of a peptide is capable of
immunospecific binding to an anti-peptide antibody. In a preferred
embodiment, a peptide or a derivative thereof displays only one
functional activity of an intracellular signaling protein from
which it is derived. This invention provides a purified fragment of
a peptide, which fragment displays one or more functional
activities of an intracellular signaling protein. This invention
provides a purified fragment of a peptide comprising a region of
the peptide selected from the group consisting of an adenylyl
cyclase interaction region and a phospholipase C interaction
region. This invention provides a purified molecule comprising the
fragment. This invention provides a chimeric peptide comprising the
fragment, which fragment consists of at least 6 amino acids fused
by a covalent bond to an amino acid sequence of a second
peptide.
[0256] This invention provides a purified antibody or an
antigen-binding derivative thereof capable of immunospecific
binding to a peptide selected from the group consisting of SEQ ID
NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID
NO:10 and not to a protein from which the peptide was derived. In
one embodiment, the antibody is polyclonal. In another embodiment,
the antibody is monoclonal.
[0257] This invention provides a method of making a recombinant
protein comprising: (a) growing a recombinant cell containing a
nucleic acid comprising a nucleotide sequence encoding an amino
acid sequence selected from the group consisting of SEQ ID NO:5,
SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10
such that the recombinant protein is expressed by the cell; and (b)
recovering the expressed recombinant protein. Further, this
invention provides a purified recombinant protein produced by said
method. Any method known in the art may be used for growing the
recombinant cell (see e.g. Freshney, 1994, Culture of animal cells,
A manual of basic technique, 3d ed., Wiley-Liss, Inc., New York).
Any method known in the art may be used for recovering the
recombinant protein, such as routine size exclusion chromatography,
molecular tagging with histidine and purification on a nickel
column, etc.
[0258] This invention provides a pharmaceutical composition
comprising: (a) a peptide or derivative thereof selected from the
group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID
NO:8, SEQ ID NO:9 and SEQ ID NO:10; and (b) a pharmaceutically
acceptable carrier. The pharmaceutically acceptable carrier can be
any carrier known to one skilled in the art.
[0259] This invention provides a method of identifying a peptide or
derivative thereof having a biological activity of interest
comprising: (a) providing a molecular model of an intracellular
protein-protein interaction, which model predicts one or more
interaction surfaces among a plurality of interacting proteins from
three-dimensional structure information; and (b) testing a
candidate interaction surface predicted by the molecular model by
determining whether a peptide encoding at least a portion of the
surface has a functional activity in a functional assay. In one
embodiment, the functional activity is an agonist activity. In
another embodiment, the functional activity is an antagonist
activity.
[0260] This invention provides a method of identifying a functional
activity of a G.beta. peptide comprising: (a) expressing a protein
comprising a peptide selected from the group consisting of SEQ ID
NO:9 and SEQ ID NO:10 in a biological system; and (b) measuring an
effect of expression in a biological assay. In one embodiment, the
biological system is selected from the group consisting of an
animal cell culture and an experimental animal. In another
embodiment, the experimental animal is selected from the group
consisting of a fly (e.g. D. melanogaster), a worm (e.g. C.
elegans), a fish (e.g. zebrafish), a rat, a mouse and a guinea pig.
In yet another embodiment, the biological assay is selected from
the group consisting of an adenylyl cyclase assay, a phospholipase
C assay, a potassium channel assay, a calcium channel assay and a
.beta.-adrenergic receptor kinase assay.
[0261] This invention provides a method of detecting an effect of
expression of a recombinant protein comprising a peptide selected
from the group consisting of SEQ ID NO:9 and SEQ ID NO:10 on a
signal transduction pathway, the method comprising: (a) expressing
the recombinant protein in a cell culture or experimental animal
already having a mutation in the signal transduction pathway; and
(b) detecting the effect of expression in a biological assay. In
one embodiment, the biological assay is selected from the group
consisting of an adenylyl cyclase assay, a phospholipase C.beta.
assay, a potassium channel assay and a calcium channel assay. In
another embodiment, the mutation in the signal transduction pathway
is in a gene selected from the group consisting of an adenylyl
cyclase gene, a phospholipase C gene, a potassium channel gene and
a calcium channel gene.
[0262] This invention provides a method of identifying a molecule
that specifically binds to a peptide or derivative thereof selected
from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,
SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, the method comprising:
(a) contacting the peptide or derivative thereof with a plurality
of molecules under conditions conducive to binding; and (b)
identifying a molecule from the plurality of molecules that
specifically binds to the peptide or derivative thereof.
4. BRIEF DESCRIPTION OF THE DRAWINGS
[0263] The file of this patent contains at least one drawing
executed in color. Copies of this patent with color drawings will
be provided by the Patent and Trademark Office upon request and
payment of the necessary fee.
[0264] FIG. 1. Regions of G.beta. involved in contacts with the AC2
956-982 peptide. (FIG. 1A) Ribbon diagram of the G.beta. backbone
from the crystal structure of G.beta..gamma. (Sondek et al., 1996,
Nature 379, 369-374; Lambright et al., 1996, Nature 379, 311-319);
the residues in contact with the AC2 peptide are shown in pink
(Weng et al., 1996, J. Biol. Chem. 271, 26445-26448). (FIG. 1B)
Predicted core contacts between the AC2 956-982 peptide and
G.beta.. The AC2 peptide residues are in the blue boxes. The AC2
peptide residues are numbered 1-27 from the N terminus. G.beta.1
residues are in green boxes. The G.beta.1 residues are shown in the
spatial sequence in which they are predicted to interact with the
AC2 peptide.
[0265] FIG. 2. Effects of the G.beta.86-105 peptides on AC2 and AC1
activities. (FIG. 2A) Ribbon diagram of the G.beta. backbone with
residues 86-105 in yellow. Other residues in contact with the AC2
peptide are shown in pink. (FIG. 2B) Effect of the G.beta.86-105
peptide (TTN) and the M101N G.beta.86-105 mutant peptide (m-TTN) on
basal, .alpha..sub.s* (2 nM), and various concentrations of TTN
peptide on G.beta..gamma.-stimulated AC2 activity in the presence
of .alpha..sub.s* (2 nM) plus G.beta..gamma. (50 nM) stimulated AC2
activities. (FIG. 2C) Effect of various concentrations of TTN
peptide on G.beta..gamma.-stimulated AC2 activity in the presence
of .alpha..sub.s* (2 nM). (FIG. 2D) Effect of TTN and m-TTN
peptides on basal and CaM (100 nM) plus G.beta..gamma. (30 nM)
regulated AC1 activities. (FIG. 2E) Effect of TTN and m-TTN
peptides on basal and CaM (100 nM) stimulated AC1 activities.
[0266] FIG. 3. Effects of the G.beta. 115-135 peptide on AC2 and
AC1 activities. (FIG. 3A) Ribbon diagram of the G.beta.1 backbone
with residues 115-135 in yellow. Other residues in contact with the
AC2 peptide are shown in pink. (FIG. 3B) Effect of the
G.beta.115-135 peptide (GGL) and the Y124V G.beta.115-135 mutant
peptide (m-GGL) on basal, .alpha..sub.s* (2 nM), and .alpha..sub.s*
(2 NM) plus G.beta..gamma. (50 nM) stimulated AC2 activities. (FIG.
3C) Effect of GGL and m-GGL peptides on basal, CaM (100 nM), or CaM
(100 nM) plus G.beta..gamma. (30 nM) regulated AC1 activities.
[0267] FIG. 4. Schematic representation of the regions of G.beta.
involved in interactions with G.alpha. (outlined in green) and some
regions that may interact with adenylyl cyclases 1 and 2 (outlined
in red). The space-filling model of G.beta. was obtained from the
crystallographic coordinates; G.alpha. contact regions are those
identified by Sigler and coworkers (Sondek et al., 1996, Nature
379, 369-374; Lambright et al., 1996, Nature 379, 311-319) from the
crystal structure of the heterotrimer. The AC2 peptide interaction
region was deduced from molecular modeling studies (Weng et al.,
1996, J. Biol. Chem. 271, 26445-26448) and the functional data in
FIG. 2 and FIG. 3 indicate that these regions may be involved in
interactions with AC1 and AC2.
[0268] FIG. 5. Effects of varying concentrations of G.beta. 86-105
peptide on PLC-.beta.2 activity. FIGS. 5A and 5B: Effects of
G.beta. 86-105 peptide on basal and G.beta..gamma. (100 nM)
stimulated PLC-.beta.2 activity. FIG. 5B: Effects of G.beta. 86-105
peptide and M101N G.beta. 86-105 peptide on PLC-.beta.2 basal
activity.
[0269] FIG. 6. Effects of varying concentrations of G.beta. 86-105
peptide and (FIGS. 6A-6C) K89A, H91A, and R96A substituted peptides
on PLC-.beta.2 activity (FIGS. 6D and E) K89A, H91A, and R96A
triple substituted peptide on basal FIG. 6D and G.beta..gamma. (100
nM) FIG. 6E stimulated PLC-.beta.2 activity. (FIG. 6F) Effects of
varying concentrations of G.beta. 86-105 peptide and FLLT peptide
on PLC-.beta.2 activity. (FIG. 6G) Effects of 100 nM G.beta..gamma.
and varying concentrations of G.beta. 86-105 peptide on PLC-.beta.2
and PLCX.beta. activity.
[0270] FIG. 7. Effects of varying concentrations of G.beta. 86-105
peptide and (FIG. 7A) S97A G.beta. 86-105 peptide, (FIG. 7B)
S97,98R G.beta. 86-105 peptide, and (FIG. 7C) S97,98D and S97,98C
peptides on PLC-.beta.2 activity.
[0271] FIG. 8. Effects of shorter peptides from G.beta. 86-105
region on PLC-.beta.2 activity. (FIG. 8A) Effects of 100 .mu.M
G.beta. 96-98, G.beta. 96-101, and G.beta. 89-101 peptides on
PLC-.beta.2 activity. (FIGS. 8B and 8C) Effects of varying
concentrations of G.beta. 96-101 peptide and S97, 98R (FIG. 8B) and
S97, 98D (FIG. 8C) G.beta. 96-101 peptides on PLC-.beta.2 basal
activity. Values for (FIG. 8A) are given as mean.+-.SEM of three
experiments.
[0272] FIG. 9. Effects of G.beta. 115-135 peptide on PLC-.beta.2
activity. (FIG. 9A) Effects of 30 nM G.beta. 115-135 peptide and
Y124V G.beta. 115-135 peptide on basal and G.beta..gamma. (100 nM)
stimulated PLC-.beta.2 activity. (FIG. 9B) Effect of varying
concentrations of G.beta. 115-135 peptide on G.beta..gamma. (100
nM) stimulated PLC-.beta.2 activity. Values for (FIG. 9A) are given
as mean.+-.SEM of three experiments.
[0273] FIG. 10. Ribbon diagram of G.beta..gamma.. G.beta. is shown
in khaki. G.gamma. is shown in grey. Residues 96-101 of G.beta. are
shown in pink, and residues 115-135 of G.beta. are shown in
aqua.
5. DETAILED DESCRIPTION OF THE INVENTION
[0274] The present invention relates to peptides and other small
molecules (e.g. peptide mimetics) derived from regions of
intracellular interacting proteins (e.g. signal transduction
proteins) and to their use as pharmaceutics. The present invention
also relates to methods for identifying peptides and derivatives
thereof as candidate pharmaceutics. Such methods combine molecular
modeling of surface interactions between two or more intracellular
proteins with experimental validation of model predictions. More
specifically, modeling of surface interactions is based on
three-dimensional structure information and validation of model
predictions is based on measuring activities of peptides or
derivatives thereof encoding at least a portion of a predicted
interaction surface in a functional assay.
[0275] The invention further relates to fragments and analogs of
identified peptides. Nucleic acids encoding such peptides are also
within the scope of the invention. Production of peptides and
derivatives thereof, e.g., by recombinant or chemical synthetic
methods, is provided. Antibodies specifically immunoreactive with
identified peptides and derivatives are additionally provided.
[0276] The invention is illustrated by way of Examples set forth in
Section 6 below which disclose, inter alia, the identification and
characterization of peptides derived from a G.beta. protein, human
G.beta.1, which have specific interactions with adenylyl cyclase
and phospholipase C-.beta.2. The complete G.beta.1 protein amino
acid sequence, which is identical in humans, dogs, cows and mice,
is set forth in SEQ ID NO:1 (Codina et al., 1986, Beta-subunits of
the human liver Gs/Gi signal-transducing proteins and those of
bovine retinal rod cell transducin are identical, FEBS Lett. 207,
187-192).
[0277] Any functional assay known to one skilled in the art may be
used to measure a functional activity of a peptide of the
invention. For example, an adenylyl cyclase activity or a
phospholipase C-.beta.2 activity may be measured. Such enzyme
activities may be measured in in vivo or in vitro experimental
systems. Functional assays used to determine an activity of a
peptide may employ any cloned, recombinant enzyme available. Many
such enzymes are known in the art. Examples include but are not
limited to: the bovine adenylyl cyclase 1 (AC1) amino acid sequence
set forth in SEQ ID NO:2 (Krupinski et al., 1989, Science 244,
1558-1564; the rat adenylyl cyclase 2 (AC2) amino acid sequence is
set forth in SEQ ID NO:3 (Feinstein et al., 1991, Proc. Natl. Acad.
Sci. U.S.A. 88, 10173-10177); and the human phospholipase C-.beta.2
(PLC-.beta.2) amino acid sequence is set forth in SEQ ID NO:4 (Park
et al., 1992, J. Biol. Chem. 267, 16048-16055).
[0278] In particular aspects, the invention provides amino acid
sequences of peptides, fragments and derivatives thereof, and other
small molecules, and fragments and derivatives thereof, which
comprise an antigenic determinant (i.e., can be recognized by an
antibody) or which are otherwise functionally active. In the case
of peptides, nucleic acid sequences encoding them are also
provided. "Functionally active" material as used herein refers to
material displaying one or more functional activities associated
with an identified peptide or other small molecule of the
invention, e.g., activation or inhibition of a downstream effector
(e.g., adenylyl cyclase 1 or 2, phospholipase C-.beta.2, etc.) or
binding to another protein binding partner, antigenicity (binding
to an antibody of the invention), immunogenicity, etc.
[0279] In specific embodiments, the invention provides fragments of
a peptide or derivative thereof consisting of at least 3 amino
acids, 6 amino acids, 10 amino acids, 15 amino acids, 20 amino
acids, 30 amino acids, or 50 amino acids. Nucleic acids encoding
the foregoing are also provided.
[0280] Once a peptide of the invention is identified, it may be
isolated and purified by any number of standard methods including
but not limited to chromatography (e.g., ion exchange, affinity,
and sizing column chromatography), centrifugation, differential
solubility, etc. The functional properties of an identified peptide
of interest may be evaluated using any functional assay known in
the art. In preferred embodiments, assays for evaluating downstream
effector functions in intracellular signal transduction pathways
are used (see Examples in Section 6).
[0281] In other specific embodiments, a peptide, fragment, analog,
or derivative may be expressed as a fusion, or chimeric protein
product (comprising the peptide, fragment, analog, or derivative
joined via a peptide bond to a heterologous protein sequence of a
different protein). Such a chimeric product can be made by ligating
the appropriate nucleic acid sequences encoding the desired amino
acid sequences to each other by methods known in the art, in the
proper reading frame, and expressing the chimeric product by
methods known in the art. Such exemplary but not limiting methods
are described below. Alternatively, a chimeric product may be made
by protein synthetic techniques, e.g., by use of a peptide
synthesizer. Standard chemical methods for peptide synthesis are
also well known in the art (see e.g. Hunkapiller et al., 1984,
Nature 310, 105-111). The terms "peptide", "polypeptide" and
"protein" are used synonymously herein.
[0282] This invention provides methods for identification of
peptides and peptide mimetics. In a preferred embodiment, the
methods of the invention provide for identification of peptides
(and/or fragments, analogs, derivatives, and mimetics thereof, i.e.
other small molecules) by first modeling an interaction surface
from three-dimensional structural information of one or more
interacting proteins. In a preferred embodiment, interactions of a
heterotrimeric G protein .beta. subunit with one or more downstream
effectors is modeled to predict one or more interaction regions.
Predicted interaction regions are next evaluated using synthetic or
recombinant peptides (or other small molecules) in functional
assays. Through an iterative process which may involve, for
example, changing one or more residues of a given peptide, the
method can be used to identify peptides having very specific
functional effects. For example, peptide agonists or antagonists of
a specific pathway are identified by activation or inhibition,
respectively, of the functional pathway with a given peptide or
derivative thereof. For an intracellular protein having more than
one interaction partner, interaction regions specific for each
interaction partner may be identified. In a preferred embodiment,
the methods of the invention are used to resolve a specific signal
transfer region from a general binding domain within an
intracellular signaling protein. Such resolution permits design of
selective agonists and antagonists of the identified
interactions.
[0283] 5.1 Peptides Derived from Regions of G.beta. Proteins
[0284] The peptides of the invention described herein which have
been derived from regions of G.beta. proteins include but are not
limited to peptides having amino acid sequences as set forth in SEQ
ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ
ID NO:10.
[0285] 5.1.1 Adenylyl Cyclase Effector Pathway
[0286] Peptides which modulate the adenylyl cyclase effector
pathway include but are not limited to peptides having amino acid
sequences as set forth in SEQ ID NO:5 and SEQ ID NO:6.
[0287] 5.1.2 Phospholipase C Effector Pathway
[0288] Peptides which modulate the phospholipase C effector pathway
include but are not limited to peptides having amino acid sequences
as set forth in SEQ ID NO:9 and SEQ ID NO:10.
[0289] 5.1.3 Other Effector Pathways
[0290] The methods of the invention may be applied to virtually any
intracellular signal transduction pathway. For example, many
cancers have been linked to perturbations in regulation of the cell
cycle. Cell cycle gene products are amenable to the methods of the
invention for identification of peptides and other small molecules
which may act as agonists or antagonists. Such molecules are of
potentially great benefit for cancer treatment.
[0291] Briefly, the cell cycle consists of four stages: G1 (for
Gap1) phase, the resting stage prior to DNA synthesis; S (for
synthesis) phase, in which DNA synthesis occurs; G2 (for Gap2)
phase, the resting stage after DNA synthesis and prior to mitosis;
and M phase, mitosis, in which cell division occurs. For a review
of the cell cycle, including a list of genes encoding intracellular
interacting proteins of the cell cycle, see Murray and Hunt ("The
Cell Cycle, An Introduction", 1993, Oxford University Press, New
York, pp. 1-251, incorporated by reference herein in its
entirety).
[0292] Progression of a cell through the cell cycle is driven by a
group of cyclin-dependent kinases (CDKs) (see e.g. Elledge, 1996,
Science 274, 1664-1672; Nasmyth, 1996, Science 274, 1643-1645). The
kinase activities of CDKs require their positive subunits, the
cyclins. Further, the activities of specific CDK/cyclin complexes
are in turn positively and negatively regulated by phosphorylation
events and CDK inhibitors (CKIs) (see Hunter and Pines, 1995, Cell
80, 225-236; Morgan, 1995, Nature 374, 131-134). While specific
CDKs (CDK2, CDK4 and CDK6) and cyclins D and E regulate the
progression from G1 into S phase, cdc2 and cyclins A and B regulate
the cell cycle progression from G1 into mitosis (see Hunter and
Pines, 1995, Cell 80, 225-236).
[0293] Human tumor suppressor genes often act as negative
regulators of the cell cycle, and several tumor suppressors are
known to influence activities of CDK/cyclin complexes. For example,
p53 activates transcription of the p21 CDK inhibitor
(p21.sup.WAF1/CIP1) in response to DNA damage signals, and p21 in
turn binds and inactivates the CDK4 and CDK6 cyclin D complexes
(Gartel et al., 1996, Proc. Soc. Exp. Biol. Med. 213, 138-149).
Another CDK inhibitor, p16, is itself a potent tumor suppressor
(Biggs and Kraft, 1995, J. Mol. Med. 73, 509-514).
[0294] By systematically applying the methods of the invention to
intracellular protein-protein interactors such as the cyclins and
CDKs, it is possible to identify peptides and derivatives thereof
having functional activity in disease states such as cancer. In
this way, application of the methods of the invention may identify
important pharmacologic and therapeutic cancer drugs.
[0295] 5.2 Troubleshooting
[0296] If any given signal transduction protein or pathway is
initially resistant to the above-described approaches for
identifying peptides and other small molecules therefrom for use as
pharmaceutics, the following troubleshooting discussion may be
helpful. A resistant intracellular signal transduction protein may
be indicated by the identification of no peptide or other small
molecule capable of modulating a downstream effector in a specific
fashion. Consider a case where an initial molecular model of a
given effector interaction does not identify a peptide or other
small molecule when tested experimentally using functional assays
for cyclins, CDKs, or such as those described in the Examples set
forth in Section 6. In this instance, careful attention should be
paid to refining the molecular model.
[0297] For example, a synergistic effect between two or more
domains of a given signal transduction protein, or two or more
domains of more than one signal transduction protein, may be
required to elicit an experimental manifestation of an effector
interaction using a peptide or derivative thereof of the invention.
In this instance, it is desirable to identify and enumerate in a
systematic fashion any and all protein-protein interaction domains
which may have an influence in the downstream effector pathway. In
this way, an accounting is made for the possibility of multiple
molecular determinants in any given effector pathway.
[0298] In this regard, a current review of the literature is often
warranted in an effort to determine whether all possible signal
transduction proteins (and other biologic signaling agents) have
been considered in the design of prospective peptides and peptide
mimetics to be experimentally evaluated. This is particularly so in
the present post-genomic era where vast catalogs of genes encoding
predicted proteins having known or predicted functions are publicly
available in computer databases.
[0299] An effective literature review generally involves reviewing
the relevant chemical, biological, and medical literature
(including clinical data) in connection with a signal transduction
pathway or other biological event of interest. In this regard,
reference to a variety of frequently-updated computer databases is
often the best course to follow (e.g. Medline.RTM., GenBank.RTM.,
etc.).
[0300] 5.3 Methods of Use with the Invention
[0301] Any method known to one of ordinary skill in the art may be
used together with the peptides, derivatives, and methods of the
invention. Set forth below are well known methods for nucleic acid
cloning, hybridization, and amplification which are of general use
together with the invention. These methods enable the production
of, e.g., synthetic and recombinant peptides and derivatives
thereof, including fusion proteins.
[0302] 5.3.1 Nucleic Acid Cloning Methods
[0303] Methods for cloning nucleic acids are very well known in the
art. Several examples of use with the invention are set forth
below. These methods shall not be construed to limit the invention
in any way. The following description sets forth methods by which
clones of any desired nucleic acid may be obtained.
[0304] Any prokaryotic or eukaryotic cell may serve as the nucleic
acid source for molecular cloning. For example, the nucleic acid
sequences encoding proteins and fragments thereof may be isolated
from vertebrate, mammalian, human, porcine, bovine, feline, avian,
equine, canine, as well as additional primate sources, insects
(e.g., Drosophila), invertebrates (e.g., C. elegans), plants, etc.
The DNA may be obtained by standard procedures known in the art
from cloned DNA (e.g., a DNA "library"), by chemical synthesis, by
cDNA cloning, or by the cloning of genomic DNA, or fragments
thereof, purified from the desired cell (see e.g., Sambrook et al.,
1989, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y.; see also Glover,
ed., 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd.,
Oxford, U.K. Vol. I, II.). Clones derived from genomic DNA may
contain regulatory and intron DNA regions in addition to coding
regions; clones derived from cDNA will contain only exon
sequences.
[0305] Once nucleic acid fragments are generated, identification of
the specific nucleic acid fragment of interest may be accomplished
in a number of ways. For example, if a portion of a nucleic acid is
available and can be purified and labeled, the generated nucleic
acid fragments may be screened by hybridization to the labeled
probe (Benton and Davis, 1977, Science 196, 180; Grunstein and
Hogness, 1975, Proc. Natl. Acad. Sci. U.S.A. 72, 3961). Those
fragments with substantial homology to the probe will hybridize. It
is also possible to identify the appropriate fragment by
restriction enzyme digestion(s) and comparison of fragment sizes
with those expected according to a known restriction map if such is
available.
[0306] Alternatively, the presence of the desired nucleic acid may
be detected by assays based on the physical, chemical, or
immunological properties of any expressed product. For example,
cDNA clones, or DNA clones which hybrid-select the cognate mRNAs,
can be selected and expressed to produce a protein that has, e.g.,
similar or identical electrophoretic migration, isoelectric
focusing behavior, proteolytic digestion maps, hormonal activity,
binding activity, or antigenic properties as known for a protein of
interest. Using an antibody to a known protein, other proteins may
be identified by binding of the labeled antibody to expressed
putative proteins, e.g., in an ELISA (enzyme-linked immunosorbent
assay)-type procedure. Further, using a binding protein specific to
a known protein, other proteins may be identified by binding to
such a protein (see e.g., Clemmons, 1993, "IGF binding proteins and
their functions," Mol. Reprod. Dev. 35, 368-374; Loddick et al.,
1998, "Displacement of growth factors from their binding proteins
as a potential treatment for stroke," Proc. Natl. Acad. Sci. U.S.A.
95, 1894-1898).
[0307] An identified and isolated nucleic acid may be inserted into
an appropriate cloning vector. Any of a large number of vector-host
systems known in the art may be used. Possible vectors include, but
are not limited to, plasmids or modified viruses, but the vector
system must be compatible with the host cell used. Such vectors
include, but are not limited to, bacteriophages such as lambda
derivatives, or plasmids such as PBR322 or pUC plasmid derivatives
or the Bluescript vector (Stratagene). The insertion into a cloning
vector can, for example, be accomplished by ligating the DNA
fragment into a cloning vector which has complementary cohesive
termini. However, if the complementary restriction sites used to
fragment the DNA are not present in the cloning vector, the ends of
the DNA molecules may be enzymatically modified. Alternatively, any
site desired may be produced by ligating nucleotide sequences
(linkers) onto the DNA termini; these ligated linkers may comprise
specific chemically synthesized oligonucleotides encoding
restriction endonuclease recognition sequences. In an alternative
method, the cleaved vector and an gene may be modified by
homopolymeric tailing. Recombinant molecules can be introduced into
host cells via transformation, transfection, infection,
electroporation, etc., so that many copies of the desired sequence
are generated.
[0308] In specific embodiments, transformation of host cells with
recombinant DNA molecules that incorporate an isolated nucleic acid
sequence enables generation of multiple copies of the nucleic acid.
Thus, the nucleic acid may be obtained in large quantities by
growing transformants, isolating the recombinant DNA molecules from
the transformants and, when necessary, retrieving the inserted
nucleic acid from the isolated recombinant DNA (e.g. by restriction
digestion or PCR).
[0309] 5.3.2 Nucleic Acid Hybridization
[0310] Nucleic acid hybridization under various stringency
conditions (e.g. low, moderate, or high stringency conditions) is
quite well known to one skilled in the art. Guidelines for nucleic
acid hybridization are widely available, including detailed
protocols for determination and use of an appropriate stringency
(see e.g., Sambrook et al., 1989, Molecular Cloning, A Laboratory
Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.; see also, Ausubel et al., eds., in the Current
Protocols in Molecular Biology series of laboratory technique
manuals, .COPYRGT. 1987-1994 Current Protocols, (.COPYRGT.
1994-1997 John Wiley and Sons, Inc.; see especially, Dyson, 1991,
Immobilization of nucleic acids and hybridization analysis, In:
Essential Molecular Biology: A Practical Approach, Vol. 2, Brown,
ed., pp. 111-156, IRL Press at Oxford University Press, Oxford,
U.K.).
[0311] In one embodiment, a nucleic acid which is hybridizable to
another nucleic acid under conditions of high stringency is
provided. In another embodiment, a nucleic acid which is
hybridizable to another nucleic acid under conditions of medium
stringency is provided. By way of example and not limitation,
hybridization procedures using conditions of high stringency may be
as follows. Prehybridization of filters containing DNA is carried
out for 8 h to overnight at 65.degree. C. in buffer composed of
6.times.SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02%
Ficoll, 0.02% BSA, and 500 .mu.g/ml denatured salmon sperm DNA.
Filters are hybridized for 48 h at 65.degree. C. in
prehybridization mixture containing 100 .mu.g/ml denatured salmon
sperm DNA and 5-20.times.10.sup.6 cpm of .sup.32P-labeled probe.
Washing of filters is done at 37.degree. C. for 1 h in a solution
containing 2.times.SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA.
This is followed by a wash in 0.1.times.SSC at 5.degree. C. for 45
min before autoradiography.
[0312] In yet another embodiment, a nucleic acid which is
hybridizable to another nucleic acid under conditions of low
stringency is provided. Again by way of example and not limitation,
procedures using conditions of low stringency may be as follows
(see also Shilo and Weinberg, 1981, Proc. Natl. Acad. Sci. U.S.A.
78, 6789-6792). Filters containing DNA are pretreated for 6 h at
40.degree. C. in a solution containing 35% formamide, 5.times.SSC,
50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA,
and 500 .mu.g/ml denatured salmon sperm DNA. Hybridizations are
carried out in the same solution with the following modifications:
0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 .mu.g/ml salmon sperm DNA,
10% (wt/vol) dextran sulfate, and 5-20.times.10.sup.6 cpm
.sup.32P-labeled probe. Filters are incubated in hybridization
mixture for 18-20 h at 40.degree. C., and then washed for 1.5 h at
55.degree. C. in a solution containing 2.times.SSC, 25 mM Tris-HCl
(pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution is replaced
with fresh solution and incubated an additional 1.5 h at 60.degree.
C. Filters are blotted dry and exposed for autoradiography. If
necessary, filters are washed for a third time at 65-68.degree. C.
and re-exposed to film.
[0313] 5.3.3 Nucleic Acid Amplification
[0314] The polymerase chain reaction (PCR) may be used in
connection with the invention to amplify any desired sequence from
any given source (e.g., a cultured cell, a tissue sample, a genomic
library, a cDNA library, a purified plasmid, a purified phagemid,
etc.). Oligonucleotide primers representing known sequences are
used as primers in PCR. PCR may be carried out using a thermal
cycler (e.g., from Perkin-Elmer Cetus) and a thermostable
polymerase (e.g., Gene Amp.TM. brand of Taq polymerase). The
nucleic acid being amplified may include but is not limited to
mRNA, cDNA or genomic DNA from any species. The PCR amplification
method is quite well known in the art (see e.g., U.S. Pat. Nos.
4,683,202, 4,683,195 and 4,889,818; Gyllenstein et al., 1988, Proc.
Natl. Acad. Sci. U.S.A. 85, 7652-7656; Ochman et al., 1988,
Genetics 120, 621-623; Loh et al., 1989, Science 243, 217-220).
Sequence CWU 1
1
10 1 340 PRT Homo sapiens 1 Met Ser Glu Leu Asp Gln Leu Arg Gln Glu
Ala Glu Gln Leu Lys Asn 1 5 10 15 Gln Ile Arg Asp Ala Arg Lys Ala
Cys Ala Asp Ala Thr Leu Ser Gln 20 25 30 Ile Thr Asn Asn Ile Asp
Pro Val Gly Arg Ile Gln Met Arg Thr Arg 35 40 45 Arg Thr Leu Arg
Gly His Leu Ala Lys Ile Tyr Ala Met His Trp Gly 50 55 60 Thr Asp
Ser Arg Leu Leu Val Ser Ala Ser Gln Asp Gly Lys Leu Ile 65 70 75 80
Ile Trp Asp Ser Tyr Thr Thr Asn Lys Val His Ala Ile Pro Leu Arg 85
90 95 Ser Ser Trp Val Met Thr Cys Ala Tyr Ala Pro Ser Gly Asn Tyr
Val 100 105 110 Ala Cys Gly Gly Leu Asp Asn Ile Cys Ser Ile Tyr Asn
Leu Lys Thr 115 120 125 Arg Glu Gly Asn Val Arg Val Ser Arg Glu Leu
Ala Gly His Thr Gly 130 135 140 Tyr Leu Ser Cys Cys Arg Phe Leu Asp
Asp Asn Gln Ile Val Thr Ser 145 150 155 160 Ser Gly Asp Thr Thr Cys
Ala Leu Trp Asp Ile Glu Thr Gly Gln Gln 165 170 175 Thr Thr Thr Phe
Thr Gly His Thr Gly Asp Val Met Ser Leu Ser Leu 180 185 190 Ala Pro
Asp Thr Arg Leu Phe Val Ser Gly Ala Cys Asp Ala Ser Ala 195 200 205
Lys Leu Trp Asp Val Arg Glu Gly Met Cys Arg Gln Thr Phe Thr Gly 210
215 220 His Glu Ser Asp Ile Asn Ala Ile Cys Phe Phe Pro Asn Gly Asn
Ala 225 230 235 240 Phe Ala Thr Gly Ser Asp Asp Ala Thr Cys Arg Leu
Phe Asp Leu Arg 245 250 255 Ala Asp Gln Glu Leu Met Thr Tyr Ser His
Asp Asn Ile Ile Cys Gly 260 265 270 Ile Thr Ser Val Ser Phe Ser Lys
Ser Gly Arg Leu Leu Leu Ala Gly 275 280 285 Tyr Asp Asp Phe Asn Cys
Asn Val Trp Asp Ala Leu Lys Ala Asp Arg 290 295 300 Ala Gly Val Leu
Ala Gly His Asp Asn Arg Val Ser Cys Leu Gly Val 305 310 315 320 Thr
Asp Asp Gly Met Ala Val Ala Thr Gly Ser Trp Asp Ser Phe Leu 325 330
335 Lys Ile Trp Asn 340 2 1134 PRT Bos taurus 2 Met Ala Gly Ala Pro
Arg Gly Arg Gly Gly Gly Gly Gly Gly Gly Gly 1 5 10 15 Ala Gly Glu
Ser Gly Gly Ala Glu Arg Ala Ala Gly Pro Gly Gly Arg 20 25 30 Arg
Gly Leu Arg Ala Cys Asp Glu Glu Phe Ala Cys Pro Glu Leu Glu 35 40
45 Ala Leu Phe Arg Gly Tyr Thr Leu Arg Leu Glu Gln Ala Ala Thr Leu
50 55 60 Lys Ala Leu Ala Val Leu Ser Leu Leu Ala Gly Ala Leu Ala
Leu Ala 65 70 75 80 Glu Leu Leu Gly Ala Pro Gly Pro Ala Pro Gly Leu
Ala Lys Gly Ser 85 90 95 His Pro Val His Cys Val Leu Phe Leu Ala
Leu Leu Val Val Thr Asn 100 105 110 Val Arg Ser Leu Gln Val Pro Gln
Leu Gln Gln Val Gly Gln Leu Ala 115 120 125 Leu Leu Phe Ser Leu Thr
Phe Ala Leu Leu Cys Cys Pro Phe Ala Leu 130 135 140 Gly Gly Pro Ala
Gly Ala His Ala Gly Ala Ala Ala Val Pro Ala Thr 145 150 155 160 Ala
Asp Gln Gly Val Trp Gln Leu Leu Leu Val Thr Phe Val Ser Tyr 165 170
175 Ala Leu Leu Pro Val Arg Ser Leu Leu Ala Ile Gly Phe Gly Leu Val
180 185 190 Val Ala Ala Ser His Leu Leu Val Thr Ala Thr Leu Val Pro
Ala Lys 195 200 205 Arg Pro Arg Leu Trp Arg Thr Leu Gly Ala Asn Ala
Leu Leu Phe Leu 210 215 220 Gly Val Asn Val Tyr Gly Ile Phe Val Arg
Ile Leu Ala Glu Arg Ala 225 230 235 240 Gln Arg Lys Ala Phe Leu Gln
Ala Arg Asn Cys Ile Glu Asp Arg Leu 245 250 255 Arg Leu Glu Asp Glu
Asn Glu Lys Gln Glu Arg Leu Leu Met Ser Leu 260 265 270 Leu Pro Arg
Asn Val Ala Met Glu Met Lys Glu Asp Phe Leu Lys Pro 275 280 285 Pro
Glu Arg Ile Phe His Lys Ile Tyr Ile Gln Arg His Asp Asn Val 290 295
300 Ser Ile Leu Phe Ala Asp Ile Val Gly Phe Thr Gly Leu Ala Ser Gln
305 310 315 320 Cys Thr Ala Gln Glu Leu Val Lys Leu Leu Asn Glu Leu
Phe Gly Lys 325 330 335 Phe Asp Glu Leu Ala Thr Glu Asn His Cys Arg
Arg Ile Lys Ile Leu 340 345 350 Gly Asp Cys Tyr Tyr Cys Val Ser Gly
Leu Thr Gln Pro Lys Thr Asp 355 360 365 His Ala His Cys Cys Val Glu
Met Gly Leu Asp Met Ile Asp Thr Ile 370 375 380 Thr Ser Val Ala Glu
Ala Thr Glu Val Asp Leu Asn Met Arg Val Gly 385 390 395 400 Leu His
Thr Gly Arg Val Leu Cys Gly Val Leu Gly Leu Arg Lys Trp 405 410 415
Gln Tyr Asp Val Trp Ser Asn Asp Val Thr Leu Ala Asn Val Met Glu 420
425 430 Ala Ala Gly Leu Pro Gly Lys Val His Ile Thr Lys Thr Thr Leu
Ala 435 440 445 Cys Leu Asn Gly Asp Tyr Glu Val Glu Pro Gly His Gly
His Glu Arg 450 455 460 Asn Ser Phe Leu Lys Thr His Asn Ile Glu Thr
Phe Phe Ile Val Pro 465 470 475 480 Ser His Arg Arg Lys Ile Phe Pro
Gly Leu Ile Leu Ser Asp Ile Lys 485 490 495 Pro Ala Lys Arg Met Lys
Phe Lys Thr Val Cys Tyr Leu Leu Val Gln 500 505 510 Leu Met His Cys
Arg Lys Met Phe Lys Ala Glu Ile Pro Phe Ser Asn 515 520 525 Val Met
Thr Cys Glu Asp Asp Asp Lys Arg Arg Ala Leu Arg Thr Ala 530 535 540
Ser Glu Lys Leu Arg Asn Arg Ser Ser Phe Ser Thr Asn Val Val Gln 545
550 555 560 Thr Thr Pro Gly Thr Arg Val Asn Arg Tyr Ile Gly Arg Leu
Leu Glu 565 570 575 Ala Arg Gln Met Glu Leu Glu Met Ala Asp Leu Asn
Phe Phe Thr Leu 580 585 590 Lys Tyr Lys Gln Ala Glu Arg Glu Arg Lys
Tyr His Gln Leu Gln Asp 595 600 605 Glu Tyr Phe Thr Ser Ala Val Val
Leu Ala Leu Ile Leu Ala Ala Leu 610 615 620 Phe Gly Leu Val Tyr Leu
Leu Ile Ile Pro Gln Ser Val Ala Val Leu 625 630 635 640 Leu Leu Leu
Val Phe Cys Ile Cys Phe Leu Val Ala Cys Val Leu Tyr 645 650 655 Leu
His Ile Thr Arg Val Gln Cys Phe Pro Gly Cys Leu Thr Ile Gln 660 665
670 Ile Arg Thr Val Leu Cys Ile Phe Ile Val Val Leu Ile Tyr Ser Val
675 680 685 Ala Gln Gly Cys Val Val Gly Cys Leu Pro Trp Ser Trp Ser
Ser Ser 690 695 700 Pro Asn Gly Ser Leu Val Val Leu Ser Ser Gly Gly
Arg Asp Pro Val 705 710 715 720 Leu Pro Val Pro Pro Cys Glu Ser Ala
Pro His Ala Leu Leu Cys Gly 725 730 735 Leu Val Gly Thr Leu Pro Leu
Ala Ile Phe Leu Arg Val Ser Ser Leu 740 745 750 Pro Lys Met Ile Leu
Leu Ala Val Leu Thr Thr Ser Tyr Ile Leu Val 755 760 765 Leu Glu Leu
Ser Gly Tyr Thr Lys Ala Met Gly Ala Gly Ala Ile Ser 770 775 780 Gly
Arg Ser Phe Glu Pro Ile Met Ala Ile Leu Leu Phe Ser Cys Thr 785 790
795 800 Leu Ala Leu His Ala Arg Gln Val Asp Val Lys Leu Arg Leu Asp
Tyr 805 810 815 Leu Trp Ala Ala Gln Ala Glu Glu Glu Arg Asp Asp Met
Glu Lys Val 820 825 830 Lys Leu Asp Asn Lys Arg Ile Leu Phe Asn Leu
Leu Pro Ala His Val 835 840 845 Ala Gln His Phe Leu Met Ser Asn Pro
Arg Asn Met Asp Leu Tyr Tyr 850 855 860 Gln Ser Tyr Ser Gln Val Gly
Val Met Phe Ala Ser Ile Pro Asn Phe 865 870 875 880 Asn Asp Phe Tyr
Ile Glu Leu Asp Gly Asn Asn Met Gly Val Glu Cys 885 890 895 Leu Arg
Leu Leu Asn Glu Ile Ile Ala Asp Phe Asp Glu Leu Met Asp 900 905 910
Lys Asp Phe Tyr Lys Asp Leu Glu Lys Ile Lys Thr Ile Gly Ser Thr 915
920 925 Tyr Met Ala Ala Val Gly Leu Ala Pro Thr Ala Gly Thr Lys Ala
Lys 930 935 940 Lys Cys Ile Ser Ser His Leu Ser Thr Leu Ala Asp Phe
Ala Ile Glu 945 950 955 960 Met Phe Asp Val Leu Asp Glu Ile Asn Tyr
Gln Ser Tyr Asn Asp Phe 965 970 975 Val Leu Arg Val Gly Ile Asn Val
Gly Pro Val Val Ala Gly Val Ile 980 985 990 Gly Ala Arg Arg Pro Gln
Tyr Asp Ile Trp Gly Asn Thr Val Asn Val 995 1000 1005 Ala Ser Arg
Met Asp Ser Thr Gly Val Gln Gly Arg Ile Gln Val Thr 1010 1015 1020
Glu Glu Val His Arg Leu Leu Arg Arg Gly Ser Tyr Arg Phe Val Cys
1025 1030 1035 1040 Arg Gly Lys Val Ser Val Lys Gly Lys Gly Glu Met
Leu Thr Tyr Phe 1045 1050 1055 Leu Glu Gly Arg Thr Asp Gly Asn Gly
Ser Gln Thr Arg Ser Leu Asn 1060 1065 1070 Ser Glu Arg Lys Met Tyr
Pro Phe Gly Arg Ala Gly Leu Gln Thr Arg 1075 1080 1085 Leu Ala Ala
Gly His Pro Pro Val Pro Pro Ala Ala Gly Leu Pro Val 1090 1095 1100
Gly Ala Gly Pro Gly Ala Leu Gln Gly Ser Gly Leu Ala Pro Gly Pro
1105 1110 1115 1120 Pro Gly Gln His Leu Pro Pro Gly Ala Ser Gly Lys
Glu Ala 1125 1130 3 1090 PRT Rattus norvegicus 3 Met Arg Arg Arg
Arg Tyr Leu Arg Asp Arg Ala Glu Ala Ala Ala Ala 1 5 10 15 Ala Ala
Ala Gly Gly Gly Glu Gly Leu Gln Arg Ser Arg Asp Trp Leu 20 25 30
Tyr Glu Ser Tyr Tyr Cys Met Ser Gln Gln His Pro Leu Ile Val Phe 35
40 45 Leu Leu Leu Ile Val Met Gly Ala Cys Leu Ala Leu Leu Ala Val
Phe 50 55 60 Phe Ala Leu Gly Leu Glu Val Glu Asp His Val Ala Phe
Leu Ile Thr 65 70 75 80 Val Pro Thr Ala Leu Ala Ile Phe Phe Ala Ile
Phe Ile Leu Val Cys 85 90 95 Ile Glu Ser Val Phe Lys Lys Leu Leu
Arg Val Phe Ser Leu Val Ile 100 105 110 Trp Ile Cys Leu Val Ala Met
Gly Tyr Leu Phe Met Cys Phe Gly Gly 115 120 125 Thr Val Ser Ala Trp
Asp Gln Val Ser Phe Phe Leu Phe Ile Ile Phe 130 135 140 Val Val Tyr
Thr Met Leu Pro Phe Asn Met Arg Asp Ala Ile Ile Ala 145 150 155 160
Ser Ile Leu Thr Ser Ser Ser His Thr Ile Val Leu Ser Val Tyr Leu 165
170 175 Ser Ala Thr Pro Gly Ala Lys Glu His Leu Phe Trp Gln Ile Leu
Ala 180 185 190 Asn Val Ile Ile Phe Ile Cys Gly Asn Leu Ala Gly Ala
Tyr His Lys 195 200 205 His Leu Met Glu Leu Ala Leu Gln Gln Thr Tyr
Arg Asp Thr Cys Asn 210 215 220 Cys Ile Lys Ser Arg Ile Lys Leu Glu
Phe Glu Lys Arg Gln Gln Glu 225 230 235 240 Arg Leu Leu Leu Ser Leu
Leu Pro Ala His Ile Ala Met Glu Met Lys 245 250 255 Ala Glu Ile Ile
Gln Arg Leu Gln Gly Pro Lys Ala Gly Gln Met Glu 260 265 270 Asn Thr
Asn Asn Phe His Asn Leu Tyr Val Lys Arg His Thr Asn Val 275 280 285
Ser Ile Leu Tyr Ala Asp Ile Val Gly Phe Thr Arg Leu Ala Ser Asp 290
295 300 Cys Ser Pro Gly Glu Leu Val His Met Leu Asn Glu Leu Phe Gly
Lys 305 310 315 320 Phe Asp Gln Ile Ala Lys Glu Asn Glu Cys Met Arg
Ile Lys Ile Leu 325 330 335 Gly Asp Cys Tyr Tyr Cys Val Ser Gly Leu
Pro Ile Ser Leu Pro Asn 340 345 350 His Ala Lys Asn Cys Val Lys Met
Gly Leu Asp Met Cys Glu Ala Ile 355 360 365 Lys Lys Val Arg Asp Ala
Thr Gly Val Asp Ile Asn Met Arg Val Gly 370 375 380 Val His Ser Gly
Asn Val Leu Cys Gly Val Ile Gly Leu Gln Lys Trp 385 390 395 400 Gln
Tyr Asp Val Trp Ser His Asp Val Thr Leu Ala Asn His Met Glu 405 410
415 Ala Gly Gly Val Pro Gly Arg Val His Ile Ser Ser Val Thr Leu Glu
420 425 430 His Leu Asn Gly Ala Tyr Lys Val Glu Glu Gly Asp Gly Glu
Ile Arg 435 440 445 Asp Pro Tyr Leu Lys Gln His Leu Val Lys Thr Tyr
Phe Val Ile Asn 450 455 460 Pro Lys Gly Glu Arg Arg Ser Pro Gln His
Leu Phe Arg Pro Arg His 465 470 475 480 Thr Leu Asp Gly Ala Lys Met
Arg Ala Ser Val Arg Met Thr Arg Tyr 485 490 495 Leu Glu Ser Trp Gly
Ala Ala Lys Pro Phe Ala His Leu His His Arg 500 505 510 Asp Ser Met
Thr Thr Glu Asn Gly Lys Ile Ser Thr Thr Asp Val Pro 515 520 525 Met
Gly Gln His Asn Phe Gln Asn Arg Thr Leu Arg Thr Lys Ser Gln 530 535
540 Lys Lys Arg Phe Glu Glu Glu Leu Asn Glu Arg Met Ile Gln Ala Ile
545 550 555 560 Asp Gly Ile Asn Ala Gln Lys Gln Trp Leu Lys Ser Glu
Asp Ile Gln 565 570 575 Arg Ile Ser Leu Leu Phe Tyr Asn Lys Asn Ile
Glu Lys Glu Tyr Arg 580 585 590 Ala Thr Ala Leu Pro Ala Phe Lys Tyr
Tyr Val Thr Cys Ala Cys Leu 595 600 605 Ile Phe Leu Cys Ile Phe Ile
Val Gln Ile Leu Val Leu Pro Lys Thr 610 615 620 Ser Ile Leu Gly Phe
Ser Phe Gly Ala Ala Phe Leu Ser Leu Ile Phe 625 630 635 640 Ile Leu
Phe Val Cys Phe Ala Gly Gln Leu Leu Gln Cys Ser Lys Lys 645 650 655
Ala Ser Thr Ser Leu Met Trp Leu Leu Lys Ser Ser Gly Ile Ile Ala 660
665 670 Asn Arg Pro Trp Pro Arg Ile Ser Leu Thr Ile Val Thr Thr Ala
Ile 675 680 685 Ile Leu Thr Met Ala Val Phe Asn Met Phe Phe Leu Ser
Asn Ser Glu 690 695 700 Glu Thr Thr Leu Pro Thr Ala Asn Thr Ser Asn
Ala Asn Val Ser Val 705 710 715 720 Pro Asp Asn Gln Ala Ser Ile Leu
His Ala Arg Asn Leu Phe Phe Leu 725 730 735 Pro Tyr Phe Ile Tyr Ser
Cys Ile Leu Gly Leu Ile Ser Cys Ser Val 740 745 750 Phe Leu Arg Val
Asn Tyr Glu Leu Lys Met Leu Ile Met Met Val Ala 755 760 765 Leu Val
Gly Tyr Asn Thr Ile Leu Leu His Thr His Ala His Val Leu 770 775 780
Asp Ala Tyr Ser Gln Val Leu Phe Gln Arg Pro Gly Ile Trp Lys Asp 785
790 795 800 Leu Lys Thr Met Gly Ser Val Ser Leu Ser Ile Phe Phe Ile
Thr Leu 805 810 815 Leu Val Leu Gly Arg Gln Ser Glu Tyr Tyr Cys Arg
Leu Asp Phe Leu 820 825 830 Trp Lys Asn Lys Phe Lys Lys Glu Arg Glu
Glu Ile Glu Thr Met Glu 835 840 845 Asn Leu Asn Arg Val Leu Leu Glu
Asn Val Leu Pro Ala His Val Ala 850 855 860 Glu His Phe Leu Ala Arg
Ser Leu Lys Asn Glu Glu Leu Tyr His Gln 865 870 875 880 Ser Tyr Asp
Cys Val Cys Val Met Phe Ala Ser Ile Pro Asp Phe Lys 885 890 895 Glu
Phe Tyr Thr Glu Ser Asp Val Asn Lys Glu Gly Leu Glu Cys Leu 900 905
910 Arg Leu Leu Asn Glu Ile Ile Ala Asp Phe Asp Asp Leu Leu Ser Lys
915 920 925 Pro Lys Phe Ser Gly Val Glu Lys Ile Lys Thr Ile Gly Ser
Thr Tyr 930 935 940 Met Ala Ala Thr Gly Leu Ser Ala Ile Pro Ser Gln
Glu His Ala Gln 945 950 955 960 Glu Pro Glu Arg Gln Tyr Met His Ile
Gly
Thr Met Val Glu Phe Ala 965 970 975 Tyr Ala Leu Val Gly Lys Leu Asp
Ala Ile Asn Lys His Ser Phe Asn 980 985 990 Asp Phe Lys Leu Arg Val
Gly Ile Asn His Gly Pro Val Ile Ala Gly 995 1000 1005 Val Ile Gly
Ala Gln Lys Pro Gln Tyr Asp Ile Trp Gly Asn Thr Val 1010 1015 1020
Asn Val Ala Ser Arg Met Asp Ser Thr Gly Val Leu Asp Lys Ile Gln
1025 1030 1035 1040 Val Thr Glu Glu Thr Ser Leu Ile Leu Gln Thr Leu
Gly Tyr Thr Cys 1045 1050 1055 Thr Cys Arg Gly Ile Ile Asn Val Lys
Gly Lys Gly Asp Leu Lys Thr 1060 1065 1070 Tyr Phe Val Asn Thr Glu
Met Ser Arg Ser Leu Ser Gln Ser Asn Leu 1075 1080 1085 Ala Ser 1090
4 1181 PRT Homo sapiens 4 Met Ser Leu Leu Asn Pro Val Leu Leu Pro
Pro Lys Val Lys Ala Tyr 1 5 10 15 Leu Ser Gln Gly Glu Arg Phe Ile
Lys Trp Asp Asp Glu Thr Thr Val 20 25 30 Ala Ser Pro Val Ile Leu
Arg Val Asp Pro Lys Gly Tyr Tyr Leu Tyr 35 40 45 Trp Thr Tyr Gln
Ser Lys Glu Met Glu Phe Leu Asp Ile Thr Ser Ile 50 55 60 Arg Asp
Thr Arg Phe Gly Lys Phe Ala Lys Met Pro Lys Ser Gln Lys 65 70 75 80
Leu Arg Asp Val Phe Asn Met Asp Phe Pro Asp Asn Ser Phe Leu Leu 85
90 95 Lys Thr Leu Thr Val Val Ser Gly Pro Asp Met Val Asp Leu Thr
Phe 100 105 110 His Asn Phe Val Ser Tyr Lys Glu Asn Val Gly Lys Ala
Trp Ala Glu 115 120 125 Asp Val Leu Ala Leu Val Lys His Pro Leu Thr
Ala Asn Ala Ser Arg 130 135 140 Ser Thr Phe Leu Asp Lys Ile Leu Val
Lys Leu Lys Met Gln Leu Asn 145 150 155 160 Ser Glu Gly Lys Ile Pro
Val Lys Asn Phe Phe Gln Met Phe Pro Ala 165 170 175 Asp Arg Lys Arg
Val Glu Ala Ala Leu Ser Ala Cys His Leu Pro Lys 180 185 190 Gly Lys
Asn Asp Ala Ile Asn Pro Glu Asp Phe Pro Glu Pro Val Tyr 195 200 205
Lys Ser Phe Leu Met Ser Leu Cys Pro Arg Pro Glu Ile Asp Glu Ile 210
215 220 Phe Thr Ser Tyr His Ala Lys Ala Lys Pro Tyr Met Thr Lys Glu
His 225 230 235 240 Leu Thr Lys Phe Ile Asn Gln Lys Gln Arg Asp Ser
Arg Leu Asn Ser 245 250 255 Leu Leu Phe Pro Pro Ala Arg Pro Asp Gln
Val Gln Gly Leu Ile Asp 260 265 270 Lys Tyr Glu Pro Ser Gly Ile Asn
Ala Gln Arg Gly Gln Leu Ser Pro 275 280 285 Glu Gly Met Val Trp Phe
Leu Cys Gly Pro Glu Asn Ser Val Leu Ala 290 295 300 Gln Asp Lys Leu
Leu Leu His His Asp Met Thr Gln Pro Leu Asn His 305 310 315 320 Tyr
Phe Ile Asn Ser Ser His Asn Thr Tyr Leu Thr Ala Gly Gln Phe 325 330
335 Ser Gly Leu Ser Ser Ala Glu Met Tyr Arg Gln Val Leu Leu Ser Gly
340 345 350 Cys Arg Cys Val Glu Leu Asp Cys Trp Lys Gly Lys Pro Pro
Asp Glu 355 360 365 Glu Pro Ile Ile Thr His Gly Phe Thr Met Thr Thr
Asp Ile Phe Phe 370 375 380 Lys Glu Ala Ile Glu Ala Ile Ala Glu Ser
Ala Phe Lys Thr Ser Pro 385 390 395 400 Tyr Pro Ile Ile Leu Ser Phe
Glu Asn His Val Asp Ser Pro Arg Gln 405 410 415 Gln Ala Lys Met Ala
Glu Tyr Cys Arg Thr Ile Phe Gly Asp Met Leu 420 425 430 Leu Thr Glu
Pro Leu Glu Lys Phe Pro Leu Lys Pro Gly Val Pro Leu 435 440 445 Pro
Ser Pro Glu Asp Leu Arg Gly Lys Ile Leu Ile Lys Asn Lys Lys 450 455
460 Asn Gln Phe Ser Gly Pro Thr Ser Ser Ser Lys Asp Thr Gly Gly Glu
465 470 475 480 Ala Glu Gly Ser Ser Pro Pro Ser Ala Pro Ala Val Trp
Ala Gly Glu 485 490 495 Glu Gly Thr Glu Leu Glu Glu Glu Glu Val Glu
Glu Glu Glu Glu Glu 500 505 510 Glu Ser Gly Asn Leu Asp Glu Glu Glu
Ile Lys Lys Met Gln Ser Asp 515 520 525 Glu Gly Thr Ala Gly Leu Glu
Val Thr Ala Tyr Glu Glu Met Ser Ser 530 535 540 Leu Val Asn Tyr Ile
Gln Pro Thr Lys Phe Val Ser Phe Glu Phe Ser 545 550 555 560 Ala Gln
Lys Asn Arg Ser Tyr Val Ile Ser Ser Phe Thr Glu Leu Lys 565 570 575
Ala Tyr Asp Leu Leu Ser Lys Ala Ser Val Gln Phe Val Asp Tyr Asn 580
585 590 Lys Arg Gln Met Ser Arg Ile Tyr Pro Lys Gly Thr Arg Met Asp
Ser 595 600 605 Ser Asn Tyr Met Pro Gln Met Phe Trp Asn Ala Gly Cys
Gln Met Val 610 615 620 Ala Leu Asn Phe Gln Thr Met Asp Leu Pro Met
Gln Gln Asn Met Ala 625 630 635 640 Val Phe Glu Phe Asn Gly Gln Ser
Gly Tyr Leu Leu Lys His Glu Phe 645 650 655 Met Arg Arg Pro Asp Lys
Gln Phe Asn Pro Phe Ser Val Asp Arg Ile 660 665 670 Asp Val Val Val
Ala Thr Thr Leu Ser Ile Thr Val Ile Ser Gly Gln 675 680 685 Phe Leu
Ser Glu Arg Ser Val Arg Thr Tyr Val Glu Val Glu Leu Phe 690 695 700
Gly Leu Pro Gly Asp Pro Lys Arg Arg Tyr Arg Thr Lys Leu Ser Pro 705
710 715 720 Ser Thr Asn Ser Ile Asn Pro Val Trp Lys Glu Glu Pro Phe
Val Phe 725 730 735 Glu Lys Ile Leu Met Pro Glu Leu Ala Ser Leu Arg
Val Ala Val Met 740 745 750 Glu Glu Gly Asn Lys Phe Leu Gly His Arg
Ile Ile Pro Ile Asn Ala 755 760 765 Leu Asn Ser Gly Tyr His His Leu
Cys Leu His Ser Glu Ser Asn Met 770 775 780 Pro Leu Thr Met Pro Ala
Leu Phe Ile Phe Leu Glu Met Lys Asp Tyr 785 790 795 800 Ile Pro Gly
Ala Trp Ala Asp Leu Thr Val Ala Leu Ala Asn Pro Ile 805 810 815 Lys
Phe Phe Ser Ala His Asp Thr Lys Ser Val Lys Leu Lys Glu Ala 820 825
830 Met Gly Gly Leu Pro Glu Lys Pro Phe Pro Leu Ala Ser Pro Val Ala
835 840 845 Ser Gln Val Asn Gly Ala Leu Ala Pro Thr Ser Asn Gly Ser
Pro Ala 850 855 860 Ala Arg Ala Gly Ala Arg Glu Glu Ala Met Lys Glu
Ala Ala Glu Pro 865 870 875 880 Arg Thr Ala Ser Leu Glu Glu Leu Arg
Glu Leu Lys Gly Val Val Lys 885 890 895 Leu Gln Arg Arg His Glu Lys
Glu Leu Arg Glu Leu Glu Arg Arg Gly 900 905 910 Ala Arg Arg Trp Glu
Glu Leu Leu Gln Arg Gly Ala Ala Gln Leu Ala 915 920 925 Glu Leu Gly
Pro Pro Gly Val Gly Gly Val Gly Ala Cys Lys Leu Gly 930 935 940 Pro
Gly Lys Gly Ser Arg Lys Lys Arg Ser Leu Pro Arg Glu Glu Ser 945 950
955 960 Ala Gly Ala Ala Pro Gly Glu Gly Pro Glu Gly Val Asp Gly Arg
Val 965 970 975 Arg Glu Leu Lys Asp Arg Leu Glu Leu Glu Leu Leu Arg
Gln Gly Glu 980 985 990 Glu Gln Tyr Glu Cys Val Leu Lys Arg Lys Glu
Gln His Val Ala Glu 995 1000 1005 Gln Ile Ser Lys Met Met Glu Leu
Ala Arg Glu Lys Gln Ala Ala Glu 1010 1015 1020 Leu Lys Ala Leu Lys
Glu Thr Ser Glu Asn Asp Thr Lys Glu Met Lys 1025 1030 1035 1040 Lys
Lys Leu Glu Thr Lys Arg Leu Glu Arg Ile Gln Gly Met Thr Lys 1045
1050 1055 Val Thr Thr Asp Lys Met Ala Gln Glu Arg Leu Lys Arg Glu
Ile Asn 1060 1065 1070 Asn Ser His Ile Gln Glu Val Val Gln Val Ile
Lys Gln Met Thr Glu 1075 1080 1085 Asn Leu Glu Arg His Gln Glu Lys
Leu Glu Glu Lys Gln Ala Ala Cys 1090 1095 1100 Leu Glu Gln Ile Arg
Glu Met Glu Lys Gln Phe Gln Lys Glu Ala Leu 1105 1110 1115 1120 Ala
Glu Tyr Glu Ala Arg Met Lys Gly Leu Glu Ala Glu Val Lys Glu 1125
1130 1135 Ser Val Arg Ala Cys Leu Arg Thr Cys Phe Pro Ser Glu Ala
Lys Asp 1140 1145 1150 Lys Pro Glu Arg Ala Cys Glu Cys Pro Pro Glu
Leu Cys Glu Gln Asp 1155 1160 1165 Pro Leu Ile Ala Lys Ala Asp Ala
Gln Glu Ser Arg Leu 1170 1175 1180 5 20 PRT Homo sapiens 5 Thr Thr
Asn Lys Val His Ala Ile Pro Leu Arg Ser Ser Trp Val Met 1 5 10 15
Thr Cys Ala Tyr 20 6 21 PRT Homo sapiens 6 Gly Gly Leu Asp Asn Ile
Cys Ser Ile Tyr Asn Leu Lys Thr Arg Glu 1 5 10 15 Gly Asn Val Arg
Val 20 7 13 PRT Homo sapiens 7 Lys Val His Ala Ile Pro Leu Arg Ser
Ser Trp Val Met 1 5 10 8 6 PRT Homo sapiens 8 Arg Ser Ser Trp Val
Met 1 5 9 6 PRT Homo sapiens 9 Arg Arg Arg Trp Val Met 1 5 10 6 PRT
Homo sapiens 10 Arg Arg Ser Trp Val Met 1 5
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