U.S. patent application number 11/704773 was filed with the patent office on 2008-01-10 for protein-protein interactions and methods for identifying interacting proteins and the amino acid sequence at the site of interaction.
This patent application is currently assigned to Elias Georges. Invention is credited to Elias Georges.
Application Number | 20080009068 11/704773 |
Document ID | / |
Family ID | 22462515 |
Filed Date | 2008-01-10 |
United States Patent
Application |
20080009068 |
Kind Code |
A1 |
Georges; Elias |
January 10, 2008 |
Protein-protein interactions and methods for identifying
interacting proteins and the amino acid sequence at the site of
interaction
Abstract
The invention relates to protein-protein interactions and
methods for identifying interacting proteins and the amino acid
sequence at the site of interaction. Using overlapping hexapeptides
that encode for the entire amino acid sequences of the linker
domains of human P-glycoprotein gene 1 and 3 (HP-gp1 and HP-gp3), a
direct and specific binding between HP-gp1 and 3 linker domains and
intracellular proteins was demonstrated. Three different stretches
(.sup.617EKGIYFKLVTM.sup.627, (SEQ ID NO: 1)
.sup.658SRSSLIRKRSTRRSVRGSQA.sup.677 (SEQ ID NO: 2) and
.sup.694PVSFWRIMKLNLT.sup.706 (SEQ ID NO: 3) for HP-gp1 and
.sup.618LMKKEGVYFKLVNM.sup.631 (SEQ ID NO: 4),
.sup.648KAATRMAPNGWKSRLFRHSTQKNLKNS.sup.674 (SEQ ID NO: 5), and
.sup.695PVSFLKVLKLNKT.sup.707 (SEQ ID NO: 6) for HP-gp3) in linker
domains bound to proteins with apparent molecular masses of
.about.80 kDa, 57 kDa and 30 kDa. The binding of the 57 kDa protein
was further characterized. Purification and partial N-terminal
amino acid sequencing of the 57 kDa protein showed that it encodes
the N-terminal amino acids of alpha and beta-tubulins. The method
of the present invention was further validated with Annexin. The
present invention thus demonstrates a novel concept whereby the
interactions between two proteins are mediated by strings of few
amino acids with high and repulsive binding energies, enabling the
identification of high affinity binding sites between any
interacting proteins.
Inventors: |
Georges; Elias; (Laval,
CA) |
Correspondence
Address: |
WILMERHALE/BOSTON
60 STATE STREET
BOSTON
MA
02109
US
|
Assignee: |
Georges; Elias
Laval
CA
|
Family ID: |
22462515 |
Appl. No.: |
11/704773 |
Filed: |
February 9, 2007 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10010310 |
Nov 13, 2001 |
7176035 |
|
|
11704773 |
Feb 9, 2007 |
|
|
|
PCT/CA00/00587 |
May 12, 2000 |
|
|
|
10010310 |
Nov 13, 2001 |
|
|
|
60134259 |
May 14, 1999 |
|
|
|
Current U.S.
Class: |
436/86 |
Current CPC
Class: |
C40B 30/04 20130101;
C07K 14/705 20130101; G01N 33/6845 20130101; A61P 35/00
20180101 |
Class at
Publication: |
436/086 |
International
Class: |
G01N 33/00 20060101
G01N033/00 |
Claims
1. A method of identifying a compound that modulates the binding of
a polypeptide to a peptide in a chosen protein, the peptide region
being adjacent to a repulsive peptide region of the chosen protein,
the method comprising: (a) providing a set of short overlapping
peptides spanning a complete sequence of at least a domain of the
chosen protein, the set of short overlapping peptides being
covalently attached to a support; (b) contacting the support to
which the overlapping peptides are covalently attached with a
candidate compound and the polypeptide under conditions enabling
binding between the peptide attached to the support and the
polypeptide; (c) washing the support to remove unbound polypeptides
of the mixture; and (d) detecting binding of the polypeptide to the
peptide attached to the support, wherein a change in the binding of
the polypeptide to the peptide attached to the support in the
presence of the candidate compound compared to the binding of the
polypeptide to the peptide attached to the support in the absence
of the candidate compound identifies the candidate compound as a
compound that modulates binding of the polypeptide to the peptide
in the chosen protein.
2. The method of claim 1, wherein the domain of the chosen protein
is a high affinity domain of the chosen protein.
3. The method of claim 1, wherein the polypeptide is known to bind
to the chosen protein.
4. The method of claim 1, wherein the support is selected from the
group consisting of a chip, a bead, and a plate.
5. The method of claim 1, wherein the overlapping peptides attached
to the support are synthesized synthetically using the amino acid
sequence of the chosen protein.
6. The method of claim 1, wherein each of the overlapping peptides
attached to the support is from about 5 amino acids to about 15
amino acids in length.
7. The method of claim 1, wherein each of overlapping peptides
attached to the support is from about 5 amino acids to about 12
amino acids in length.
8. The method of claim 1, wherein each of the overlapping peptides
attached to the support is from about 5 amino acids to about 10
amino acids in length.
9. The method of claim 1, wherein each of the overlapping peptides
attached to the support is from about 5 amino acids to about 7
amino acids in length.
10. The method of claim 1, wherein the chosen protein is human
P-glycoprotein 1.
11. The method of claim 10, wherein the polypeptide is tubulin.
12. The method of claim 1, wherein the chosen protein is human
P-glycoprotein 3.
13. A method of identifying an agent which modulates an interaction
between high-affinity interacting domains between a set of
overlapping peptides spanning a complete sequence of a chosen
protein, domain thereof or part thereof, covalently bound to a
support, and a mixture of proteins wherein said mixture of proteins
is a mixture of cellular proteins, comprising: (a) incubating the
set of overlapping peptides with the mixture in the presence of at
least one agent, under conditions enabling the binding between the
high-affinity interacting domain in a peptide of the set and one or
more proteins of the mixture to occur; (b) washing off any
protein-protein interaction which is not a high-affinity
interaction in step (a); and (c) identifying which peptide from
step (a) interacts with high-affinity to a protein of the mixture
in the presence of the agent as compared to in the absence of the
agent, thereby identifying the agent as a modulator of
high-affinity interaction when the interaction in the presence of
the agent is measurably different from the interaction in the
absence of the agent.
14. The method of claim 13, wherein said mixture of proteins and/or
the mixture of peptides contains a label.
15. The method of claim 13, wherein the set of overlapping peptides
is synthesized synthetically using the sequence of the chosen
protein.
16. The method of claim 13, wherein the support is chosen from a
chip, a bead, or a plate.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 10/010,310, filed Nov. 13, 2001, entitled, "Protein-Protein
Interactions and Methods for Identifying Interacting Proteins and
the Amino Acid Sequence at the Site of Interaction," now U.S. Pat.
No. 7,176,035, which claims the benefit of PCT Appl. No.
PCT/CA00/0587, filed May 12, 2000, which in turns claims priority
from U.S. Provisional Appl. No. 60/134,259, filed May 14, 1999, the
entire contents of all of which are hereby incorporated by
reference.
FIELD OF THE INVENTION
[0002] The present invention relates to proteonomics. More
specifically, the invention relates to protein-protein interactions
and methods for identifying interacting proteins and the amino acid
sequence at the site of interaction.
BACKGROUND OF THE INVENTION
[0003] Specific protein-protein interactions are critical events in
biological processes. Protein-protein interactions govern
biological processes that handle cellular information flow and
control cellular decisions (e.g., signal transduction, cell cycle
regulation and assembly of cellular structures). The entire network
of interactions between cellular proteins is a biological chart of
functional events that regulate the internal working of living
organisms and their responses to external signals. A necessary step
for the completion of this biological interaction chart is the
knowledge of all the gene sequences in a given living organism. The
entire DNA sequence of the Homo sapiens genome will be completed at
the latest by the year 2003 (112). Unfortunately, the sequence of a
gene does not reveal its biological function nor its position in
the biological chart. Given the expected number of proteins in the
human genome (80,000 to 120,000), the mapping of the biological
chart of protein-protein interactions will be an enormous but a
rewarding task.
[0004] During the past few decades, several techniques have been
developed to determine the interactions between proteins (for
review, see (82)). These techniques include, i) physical methods to
select and detect interacting proteins (e.g., protein affinity
chromatography, co-immunoprecipitation, crosslinking, and affinity
blotting), ii) Library based methods (e.g., Phage display and
two-hybrid systems); and iii) genetic methods (e.g., overproduction
phenotype, synthetic lethal effects and unlinked
noncomplementation). Of the above mentioned methods for detecting
protein-protein interactions, the two-hybrid systems are most
popular and are most extensively used. In the classical two-hybrid
system (30), transcription of reporter genes depends on an
interaction between a DNA-bound "bait" protein and an
activation-domain containing "prey" protein. The two hybrid systems
unfortunately may suffer from a number of disadvantages. For
example, the interaction of proteins is monitored in the nuclear
milieu rather than the cytoplasm where most proteins are found and
it does not allow the simultaneous identification of the precise
amino acid sequences between two interacting proteins and cannot be
easily applied to different cell types or tissues whereby different
interacting proteins may be expressed.
[0005] It has been previously demonstrated that small synthetic
peptides can bind to proteins (1, 18, 55, 102). Nevertheless, the
use of synthetic peptides in a systematic approach to identify
interacting protein domains and sequences has not been proposed or
provided. Certain signature domains have been shown to bind with
high affinity to specific peptide sequences (e.g., the Src
homology-2 or SH2 domain of Src-family kinases bind tightly to a
phosphorylated tyrosine (Y*-EEI) sequence (SEQ ID NO: 9) found in
epidermal growth factor receptor and the focal adhesion kinase)
(61).
[0006] There thus remains a need to provide a method which enables
identification of i) the exact amino acid sequences of at least one
binding partner between interacting proteins; ii) numerous,
possibly all interacting proteins in different cells or tissues;
and iii) the specific domains (or sequences) between two
interacting proteins as targets for isolation of lead drugs. In
addition, there remains a need to provide methods and assays which
enable the identification of the precise amino acid sequence of
interacting domains of proteins which is significantly faster than
conventional methods (e.g., days instead of months).
[0007] The present invention seeks to meet these and other
needs.
[0008] The present description refers to a number of documents, the
content of which is herein incorporated by reference, in their
entirety.
SUMMARY OF THE INVENTION
[0009] The present invention seeks to overcome the drawbacks of the
prior art. More specifically, the invention concerns an approach to
identify protein-protein interaction domains which differ from the
prior art. Moreover, one approach of the present invention is based
on an understanding of the principle that governs protein-protein
interactions. Such understanding therefore, allows the use of
several methods. Such a method is exemplified in detail below to
identify: i) at least one of the exact amino acid sequences between
interacting proteins; ii) a number of, possibly all, interacting
proteins in different cells or tissues; and iii) the specific
domains (or sequences) between two interacting proteins as targets
for isolation of lead drugs. Preferably, the method and assay of
the present invention enable a determination of i), ii) and iii).
Moreover, unlike the approaches of the prior art, the method
described herein allows for the identification of interacting
proteins and the precise amino acid sequences of interactions in
several days as opposed to several months.
[0010] The ability to select proteins (or other molecules) that
block interactions between a gene product and some partners but not
others, should allow sophisticated modulation of cellular signaling
or cell metabolism in human cells and other currently intractable
systems. Indeed, the identification of proteins that interact with
a therapeutically important protein and the identification of the
sites of interaction may be more relevant to drug development than
other genetic approaches such as "knock-outs" (71). The latter
addresses the phenotypic consequences of disrupting all of the
interactions in which a given protein is involved as opposed to
inhibiting the interaction of one protein (at worse of a few
proteins as opposed to all) in a multimeric complex.
[0011] The present invention further relates to a novel approach in
drug discovery. A major obstacle in drug development for the
treatment of diseases has been the identification of target
proteins and their functional sites. In fact, most research and
development (R&D) projects in pharmaceutical companies take
several years to identify a valid target protein. The selection of
drugs that bind to and inhibit the functions of these proteins
takes several years and is generally non-specific and random.
Furthermore, drugs identified by current approaches often target
the active sites in proteins. Such drugs thus often lead to major
side-effects. Therefore, it is not surprising that many R&D
projects never lead to the development of specific drugs even after
three to five years of intensive research efforts. The methods and
assays to identify protein-protein interactions of the present
invention may address three important steps in the development of
drugs:
[0012] 1) the identification of the amino acid sequences of all
interacting domains in target proteins;
[0013] 2) the identification of a set of interacting proteins
(preferably all interacting proteins) for drug development; and
[0014] 3) screening for specific drugs against each of the
interacting domains in a target protein.
[0015] P-glycoprotein (P-gp) has been shown to cause multidrug
resistance in tumor cell lines selected with lipophilic anticancer
drugs. Analysis of P-gp amino acid sequence has lead to a proposed
model of a duplicated molecule with two hydrophobic and hydrophilic
domains linked by a highly charged region of about 90 amino acids,
the linker domain. Although similarly charged domains are found in
other members of the P-gp superfamily, the function(s) of this
domain are not known. Herein, it is demonstrated using the method
of the present invention that this domain binds to other cellular
proteins. Using overlapping hexapeptides that span the entire amino
acid sequences of the linker domains of human P-glycoprotein gene 1
and 3 (HP-gp1 and HP-gp3), a direct and specific binding between
HP-gp1 and 3 linker domains and intracellular proteins is shown
herein. Three different stretches (.sup.617EKGIYFKLVTM.sup.627 (SEQ
ID NO: 1), .sup.658SRSSLIRKRSTRRSVRGSQA.sup.677 (SEQ ID NO: 2) and
.sup.694PVSFWRIMKLNLT.sup.706 (SEQ ID NO: 3) for HP-gp1 and
.sup.618LMKKEGVYFKLVNM.sup.631 (SEQ ID NO: 4),
.sup.648KAATRMAPNGWKSRLFRHSTQKNLKNS.sup.674 (SEQ ID NO: 5) and
.sup.695PVSFLKVLKLNKT.sup.707 (SEQ ID NO: 6) for HP-gp3) in linker
domains specifically bound to proteins with apparent molecular
masses of .about.80 kDa, 57 kDa and 30 kDa. Interestingly, only the
57 kDa protein was bound, to varying degrees, to the three
different sequences in the linker domain. Moreover, the binding
between the overlapping peptides encoding the linker sequence and
the 57 kDa protein were resistant to the zwitterionic detergent,
CHAPS, but were sensitive to SDS. Purification and partial
N-terminal amino acid sequencing of the 57 kDa protein showed that
it encodes the N-terminal amino acids of alpha and beta-tubulins.
Further, Western blot analysis using monoclonal antibodies that
binds to .alpha.- and .beta.-tubulins confirmed the identity of the
57 kDa protein. Taken together, this is the first example showing
protein interactions with the P-gp linker domain. This may of
course be important to the overall function of P-gp. More
importantly, the results in this study demonstrate the novel
concept whereby the interactions between two proteins are mediated
by strings of few amino acids with high and repulsive binding
energies.
[0016] In accordance with one embodiment of the present invention,
there is provided a method of identifying a high affinity
interacting domain in a chosen protein, domain thereof, or part
thereof, and the amino acid sequence thereof comprising: a)
providing a set of overlapping peptides spanning a complete
sequence of the chosen protein, domain thereof, or part thereof,
covalently bound to a support; b) providing a mixture of proteins
and/or a mixture of peptides; c) incubating the set of overlapping
peptides of a), with the mixture of b), under conditions enabling
the binding between a high affinity interacting domain in a peptide
of the set and one or more protein or peptide of b) to occur; d)
washing off any protein-protein interaction which is not a high
affinity interaction of c); and e) identifying which peptide of a)
interacts with high affinity to a protein or peptide of b), thereby
identifying the peptide of e) and the sequence thereof as a high
affinity interacting domain.
[0017] In accordance with another embodiment of the present
invention, there is provided a method of identifying an agent which
modulates an interaction between high affinity interacting domains
between a set of overlapping peptides spanning a complete sequence
of a chosen protein, domain thereof or part thereof, covalently
bound to a support and a mixture of proteins and/or a mixture of
peptides comprising: a) incubating the set of overlapping peptides,
with the mixture in the presence of at least one agent, under
conditions enabling the binding between a high affinity interacting
domain in a peptide of the set and one or more protein or peptide
of the mixture to occur; b) washing off any protein-protein
interaction which is not a high affinity interaction of b); and c)
identifying which peptide of a) interacts with high affinity to a
protein or peptide of the mixture in a presence of the agent as
compared to in an absence thereof; thereby identifying the agent as
a modulator of the high affinity interaction when the interaction
in the presence of the agent is measurably different from in the
absence thereof.
[0018] In accordance with yet another embodiment of the present
invention, there is provided agents identified as modulators of the
high affinity protein interactions of the present invention.
[0019] For the purpose of the present invention, the following
abbreviations and terms are defined below.
DEFINITIONS
[0020] The terminology "overlapping peptides spanning a peptide
sequence" (e.g., a domain, a full length protein sequence or a part
thereof) or the like refers to peptides of a chosen size, based on
the sequence of the protein (or part thereof). Preferably, these
peptides are synthetic peptides.
[0021] As explained hereinbelow, the size of the overlapping
peptides has a significant impact on the workings of the present
invention. For example, peptides of four contiguous amino acids
appear to significantly increase the low affinity binding of
proteins thereto. Moreover, the use of larger peptides, such as 20
amino acids or higher, would be expected to increase the proportion
of repulsive amino acids to high affinity amino acids, thereby
masking or totally inhibiting the binding of specific proteins to
the peptides. Thus, while the person of ordinary skill would
understand that there are trade-offs associated with the choice of
small peptides as opposed to larger ones, the preferred size for
the overlapping peptides of the present invention is between 5 and
15 amino acids, more preferably between 5 and 12, and especially
preferably between 5 and 10 amino acids.
[0022] The term "support" in the context of a support to which the
overlapping peptides of the present invention are covalently bound,
can be chosen from a multitude of supports found in the art. Such
supports include CHIPS, plates (e.g. 96-well plates), glass beads
and the like. The CHIP technology is well-known in the art (10, 19,
24, 26, 85, 97).
[0023] Protein sequences are presented herein using the one letter
or three letter amino acid symbols as commonly used in the art and
in accordance with the recommendations of the IUPAC-IUB Biochemical
Nomenclature Commission.
[0024] Unless defined otherwise, the scientific and technological
terms and nomenclature used herein have the same meaning as
commonly understood by a person of ordinary skill to which this
invention pertains. Generally, the procedures for cell cultures,
infection, molecular biology methods and the like are common
methods used in the art. Such standard techniques can be found in
reference manuals (4, 96).
[0025] The present description refers mainly to proteins, or
recombinant DNA (rDNA) technology terms. Selected examples are
provided for clarity and consistency.
[0026] As used herein, "nucleic acid molecule", refers to a polymer
of nucleotides. Non-limiting examples thereof include DNA (e.g.
genomic DNA, cDNA) and RNA molecules (e.g. mRNA). The nucleic acid
molecule can be obtained by cloning techniques or synthesized. DNA
can be double-stranded or single-stranded (coding strand or
non-coding strand [antisense]).
[0027] The term "recombinant DNA" as known in the art refers to a
DNA molecule resulting from the joining of DNA segments. This is
often referred to as genetic engineering.
[0028] The term "DNA segment," is used herein, to refer to a DNA
molecule comprising a linear stretch or sequence of nucleotides.
This sequence when read in accordance with the genetic code, can
encode a linear stretch or sequence of amino acids which can be
referred to as a polypeptide, protein, protein fragment and the
like.
[0029] The terminology "amplification pair" refers herein to a pair
of oligonucleotides (oligos) of the present invention, which are
selected to be used together in amplifying a selected nucleic acid
sequence by one of a number of types of amplification processes,
preferably a polymerase chain reaction. Other types of
amplification processes include ligase chain reaction, strand
displacement amplification, or nucleic acid sequence-based
amplification, as explained in greater detail below. As commonly
known in the art, the oligos are designed to bind to a
complementary sequence under selected conditions.
[0030] The nucleic acid (e.g. DNA or RNA) for practicing the
present invention may be obtained according to well known
methods.
[0031] As used herein, the term "physiologically relevant" is meant
to describe interactions which can take effect to modulate an
activity or level of one or more proteins in their natural
setting.
[0032] The term "DNA" molecule or sequence (as well as sometimes
the term "oligonucleotide") refers to a molecule comprised of the
deoxyribonucleotides adenine (A), guanine (G), thymine (T) and/or
cytosine (C), in a double-stranded form, and comprises or includes
a "regulatory element" according to the present invention, as the
term is defined herein. The term "oligonucleotide" or "DNA" can be
found in linear DNA molecules or fragments, viruses, plasmids,
vectors, chromosomes or synthetically derived DNA. As used herein,
particular double-stranded DNA sequences may be described according
to the normal convention of giving only the sequence in the 5' to
3' direction.
[0033] "Nucleic acid hybridization" refers generally to the
hybridization of two single-stranded nucleic acid molecules having
complementary base sequences, which under appropriate conditions
will form a thermodynamically favored double-stranded structure.
Examples of hybridization conditions can be found in the two
laboratory manuals referred above (4, 96) and are commonly known in
the art. In the case of hybridization to a nitrocellulose filter,
as for example in the well known Southern blotting procedure, a
nitrocellulose filter can be incubated overnight at 65.degree. C.
with a labeled probe in a solution containing 50% formamide, high
salt (5.times.SSC or 5.times.SSPE), 5.times.Denhardt's solution, 1%
SDS, and 100 .mu.g/ml denatured carrier DNA (e.g., salmon sperm
DNA). The non-specifically binding probe can then be washed off the
filter by several washes in 0.2.times.SSC/0.1% SDS at a temperature
which is selected in view of the desired stringency: room
temperature (low stringency), 42.degree. C. (moderate stringency)
or 65.degree. C. (high stringency). The selected temperature is
based on the melting temperature (Tm) of the DNA hybrid. Of course,
RNA-DNA hybrids can also be formed and detected. In such cases, the
conditions of hybridization and washing can be adapted according to
well-known methods by the person of ordinary skill. Stringent
conditions will be preferably used (96).
[0034] Probes for nucleic acids can be utilized with naturally
occurring sugar-phosphate backbones as well as modified backbones
including phosphorothioates, dithionates, alkyl phosphonates and
.alpha.-nucleotides and the like. Modified sugar-phosphate
backbones are generally taught (73, 75). Probes of the invention
can be constructed of either ribonucleic acid (RNA) or
deoxyribonucleic acid (DNA), and preferably of DNA.
[0035] It is an advantage of the present invention that the
detection of the interaction between proteins and/or peptides be
dependent on a label. Such labels provide sensitivity and often
enable automation. In one embodiment of the present invention,
automation is performed using CHIP technology. For example, the
overlapping peptides spanning a chosen sequence of a protein, are
bound to a CHIP which can then be used to automate a test for
interaction with proteins or peptides. Of course, it should be
understood that the present invention is not strictly dependent on
a design and synthesis of the overlapping set of peptides spanning
a chosen protein sequence. Indeed, banks of peptides are available,
from which this set of overlapping peptides could be
constructed.
[0036] Protein labelling is well known in the art. Non-limiting
examples of labels include .sup.3H, .sup.14C, .sup.32P, and
.sup.35S, Non-limiting examples of detectable markers include
ligands, fluorophores, chemiluminescent agents, enzymes, and
antibodies. It will become evident to the person of ordinary skill
that the choice of a particular label dictates the manner in which
it is bound to the protein.
[0037] The identification of the interaction is not specifically
dependent on labelling of the proteins, since for example, this
interaction could be assessed using proteomic approaches (such as
2-D gels and mass spectrometry) or using a library of
antibodies.
[0038] As commonly known, radioactive amino acids can be
incorporated into peptides or proteins of the invention by several
well-known methods. A non-limiting example thereof includes in
vitro or in vivo labelling of proteins using .sup.35SMet.
[0039] The term "vector" is commonly known in the art and defines a
plasmid DNA, phage DNA, viral DNA and the like, which can serve as
a DNA vehicle into which DNA of the present invention can be
cloned. Numerous types of vectors exist and are well known in the
art.
[0040] The term "expression" defines the process by which a gene is
transcribed into mRNA (transcription), the mRNA then being
translated (translation) into one polypeptide (or protein) or
more.
[0041] The terminology "expression vector" defines a vector or
vehicle as described above, but designed to enable the expression
of an inserted sequence following transformation into a host. The
cloned gene (inserted sequence) is usually placed under the control
of control element sequences such as promoter sequences. The
placing of a cloned gene under such control sequences is often
referred to as being operably linked to control elements or
sequences.
[0042] Operably linked sequences may also include two segments that
are transcribed into the same RNA transcript. Thus, two sequences,
such as a promoter and a "reporter sequence" are operably linked if
transcription commencing in the promoter will produce an RNA
transcript of the reporter sequence. In order to be "operably
linked" it is not necessary that two sequences be immediately
adjacent to one another.
[0043] Expression control sequences will vary depending on whether
the vector is designed to express the operably linked gene in a
prokaryotic or eukaryotic host or both (shuttle vectors) and can
additionally contain transcriptional elements such as enhancer
elements, termination sequences, tissue-specificity elements,
and/or translational initiation and termination sites.
[0044] Prokaryotic expression is useful for the preparation of
large quantities of the protein encoded by the DNA sequence of
interest. This protein can be purified according to standard
protocols that take advantage of the intrinsic properties thereof,
such as size and charge (e.g., SDS gel electrophoresis, gel
filtration, centrifugation, ion exchange chromatography, etc.). In
addition, the protein of interest can be purified via affinity
chromatography using polyclonal or monoclonal antibodies. The
purified protein can be used for therapeutic applications.
[0045] The DNA construct can be a vector comprising a promoter that
is operably linked to an oligonucleotide sequence of the present
invention, which in turn is operably linked to a heterologous gene,
such as the gene for the luciferase reporter molecule. "Promoter"
refers to a DNA regulatory region capable of binding directly or
indirectly to RNA polymerase in a cell and initiating transcription
of a downstream (3' direction) coding sequence. For purposes of the
present invention, the promoter is bound at its 3' terminus by the
transcription initiation site and extends upstream (5' direction)
to include the minimum number of bases or elements necessary to
initiate transcription at levels detectable above background.
Within the promoter will be found a transcription initiation site
(conveniently defined by mapping with S1 nuclease), as well as
protein binding domains (consensus sequences) responsible for the
binding of RNA polymerase. Eukaryotic promoters will often, but not
always, contain "TATA" boxes and "CCAT" boxes. Prokaryotic
promoters contain Shine-Dalgarno sequences in addition to the -10
and -35 consensus sequences.
[0046] As used herein, the designation "functional derivative"
denotes, in the context of a functional derivative of a sequence,
whether a nucleic acid or amino acid sequence, a molecule that
retains a biological activity (either function or structural) that
is substantially similar to that of the original sequence. This
functional derivative or equivalent may be a natural derivative or
may be prepared synthetically. Such derivatives include amino acid
sequences having substitutions, deletions, or additions of one or
more amino acids, provided that the biological activity of the
protein is conserved. The same applies to derivatives of nucleic
acid sequences which can have substitutions, deletions, or
additions of one or more nucleotides, provided that the biological
activity of the sequence is generally maintained. When relating to
a protein sequence, the substituting amino acid has
chemico-physical properties, which are similar to that of the
substituted amino acid. The similar chemico-physical properties
include, similarities in charge, bulkiness, hydrophobicity,
hydrophilicity and the like. The term "functional derivatives" is
intended to include fragments, segments, variants, analogs or
chemical derivatives of the subject matter of the present
invention.
[0047] As well-known in the art, a "conservative mutation or
substitution" of an amino acid refers to mutation or substitution
which maintains: 1) the structure of the backbone of the
polypeptide (e.g. a beta sheet or alpha-helical structure); 2) the
charge or hydrophobicity of the amino acid; or 3) the bulkiness of
the side chain. More specifically, the well-known terminologies
"hydrophilic residues" relate to serine or threonine. "Hydrophobic
residues" refer to leucine, isoleucine, phenylalanine, valine or
alanine. "Positively charged residues" relate to lysine, arginine
or histidine. "Negatively charged residues" refer to aspartic acid
or glutamic acid. Residues having "bulky side chains" refer to
phenylalanine, tryptophan or tyrosine.
[0048] Peptides, protein fragments, and the like in accordance with
the present invention can be modified in accordance with well-known
methods dependently or independently of the sequence thereof. For
example, peptides can be derived from the wild-type sequence
exemplified herein in the figures using conservative amino acid
substitutions at 1, 2, 3 or more positions. The terminology
"conservative amino acid substitutions" is well known in the art,
which relates to substitution of a particular amino acid by one
having a similar characteristic (e.g. aspartic acid for glutamic
acid, or isoleucine for leucine). Of course, non-conservative amino
acid substitutions can also be carried out, as well as other types
of modifications such as deletions or insertions, provided that
these modifications modify the peptide, in a suitable way (e.g.
without affecting the biological activity of the peptide if this is
what is intended by the modification). A list of exemplary
conservative amino acid substitutions is given hereinbelow.
TABLE-US-00001 TABLE 2 CONSERVATIVE AMINO ACID REPLACEMENTS For
Amino Acid Code Replace With Alanine A D-Ala, Gly, Aib, .beta.-Ala,
Acp, L-Cys, D-Cys Arginine R D-Arg, Lys, D-Lys, homo-Arg,
D-homo-Arg, Met, Ile, D-Met, D-Ile, Orn, D-Orn Asparagine N D-Asn,
Asp, D-Asp, Glu, D-Glu, Gln, D-Gln Aspartic Acid D D-Asp, D-Asn,
Asn, Glu, D-Glu, Gln, D-Gln Cysteine C D-Cys, S-Me-Cys, Met, D-Met,
Thr, D-Thr Glutamine Q D-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp
Glutamic Acid E D-Glu, D-Asp, Asp, Asn, D-Asn, Gln, D-Gln Glycine G
Ala, D-Ala, Pro, D-Pro, Aib, .beta.-Ala, Acp Isoleucine I D-Ile,
Val, D-Val, AdaA, AdaG, Leu, D-Leu, Met, D-Met Leucine L D-Leu,
Val, D-Val, AdaA, AdaG, Leu, D-Leu, Met, D-Met Lysine K D-Lys, Arg,
D-Arg, homo-Arg, D-homo-Arg, Met, D-Met, Ile, D-Ile, Orn, D-Orn
Methionine M D-Met, S-Me-Cys, Ile, D-Ile, Leu, D-Leu, Val, D-Val
Phenylalanine F D-Phe, Tyr, D-Thr, L-Dopa, His, D-His, Trp, D-Trp,
Trans-3,4, or 5-phenylproline, AdaA, AdaG, cis-3,4, or
5-phenylproline, Bpa, D-Bpa Proline P D-Pro,
L-I-thioazolidine-4-carboxylic acid, D-or
L-I-oxazolidine-4-carboxylic acid (Kauer, U.S. Pat No. (4,511,390)
Serine S D-Ser, Thr, D-Thr, allo-Thr, Met, D-Met, Met(O), D-Met(O),
L-Cys, D-Cys Threonine T D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met,
Met(O), D-Met(O), Val, D-Val Tyrosine Y D-Tyr, Phe, D-Phe, L-Dopa,
His, D-His Valine V D-Val, Leu, D-Leu, Ile, D-Ile, Met, D-Met,
AdaA, AdaG
[0049] As can be seen in this table, some of these modifications
can be used to render the peptide more resistant to proteolysis. Of
course, modifications of the peptides can also be effected without
affecting the primary sequence thereof using an enzymatic or
chemical treatment well known in the art.
[0050] The term "variant" refers herein to a protein or nucleic
acid molecule, which is substantially similar in structure and
biological activity to the protein or nucleic acid of the present
invention.
[0051] The functional derivatives of the present invention can be
synthesized chemically or produced through recombinant DNA
technology, using methods well known in the art. In one particular
embodiment of the present invention, a variant according to the
present invention can be identified using a method of the present
invention. It can also be designed to formally test for the
conservation of particular amino acids (e.g. by synthesizing a
variant or mutant peptide). These variants can also be tested as
part of the full-length sequence of the protein in order to
validate the interaction. Of course, the skilled artisan will
understand that having identified a region of a chosen protein as a
region which is involved in high affinity protein interaction(s)
enables an in vitro mutagenesis (or a testing of related peptide
sequences) of this region to identify and dissect the
structure/function relation of this region. Such methods are well
known in the art. When the interaction domains of two proteins
having been identified, it is thus possible for the skilled artisan
to identify and/or design variants having a modified affinity for
an interacting protein. Of course, when both interacting sequences
are known, very powerful questions can be asked to dissect the
structure-function relationship, which governs the high affinity
interaction between same.
[0052] As used herein, "chemical derivatives" is meant to cover
additional chemical moieties not normally part of the subject
matter of the invention. Such moieties could affect the
physico-chemical characteristic of the derivative (e.g. solubility,
absorption, half life and the like, decrease of toxicity). Such
moieties are exemplified in Remington's Pharmaceutical Sciences
(88). Methods of coupling these chemical-physical moieties to a
polypeptide are well known in the art.
[0053] The term "allele" defines an alternative form of a gene,
which occupies a given locus on a chromosome.
[0054] As commonly known, a "mutation" is a detectable change in
the genetic material, which can be transmitted to a daughter cell.
As well known, a mutation can be, for example, a detectable change
in one or more deoxyribonucleotide. For example, nucleotides can be
added, deleted, substituted for, inverted, or transposed to a new
position. Spontaneous mutations and experimentally induced
mutations exist. The result of a mutation of a nucleic acid
molecule is a mutant nucleic acid molecule. A mutant polypeptide
can be encoded from this mutant nucleic acid molecule.
[0055] As used herein, the term "purified" refers to a molecule
having been separated from a cellular component. Thus, for example,
a "purified protein" has been purified to a level not found in
nature. A "substantially pure" molecule is a molecule that is
lacking in most other cellular components.
[0056] As used herein, the terms "molecule", "compound" or "ligand"
are used interchangeably and broadly to refer to natural, synthetic
or semi-synthetic molecules or compounds. The term "molecule"
therefore denotes for example chemicals, macromolecules, cell or
tissue extracts (from plants or animals) and the like. Non-limiting
examples of molecules include nucleic acid molecules, peptides,
antibodies, carbohydrates and pharmaceutical agents. The agents can
be selected and screened by a variety of means including random
screening, rational selection and by rational design using for
example protein or ligand modelling methods such as computer
modelling, combinatorial library screening and the like. The terms
"rationally selected" or "rationally designed" are meant to define
compounds, which have been chosen based on the configuration of the
interaction domains of the present invention. As will be understood
by the person of ordinary skill, macromolecules having
non-naturally occurring modifications are also within the scope of
the term "molecule". For example, peptidomimetics, well known in
the pharmaceutical industry and generally referred to as peptide
analogs, can be generated by modelling as mentioned above.
Similarly, in a preferred embodiment, the polypeptides of the
present invention are modified to enhance their stability. It
should be understood that in most cases this modification should
not alter the biological activity of the interaction domain. The
molecules identified in accordance with the teachings of the
present invention have a therapeutic value in diseases or
conditions in which the physiology or homeostasis of the cell
and/or tissue is compromised by a high affinity protein interaction
identified in accordance with the present invention. Alternatively,
the molecules identified in accordance with the teachings of the
present invention find utility in the development of more efficient
agents, which can modulate such interactions.
[0057] Libraries of compounds (publicly available or commercially
available, e.g., a combinatorial library) are well known in the
art. Libraries of peptides are also available. Such libraries can
be used to build an overlapping set of peptide sequences spanning a
chosen domain, protein or part thereof.
[0058] As used herein, the recitation "indicator cells" refers to
cells that express, in one particular embodiment, two interacting
peptide domains of the present invention, and wherein an
interaction between these proteins or interacting domains thereof
is coupled to an identifiable or selectable phenotype or
characteristic such that it provides an assessment or validation of
the interaction between same. Such indicator cells can also be used
in the screening assays of the present invention. In certain
embodiments, the indicator cells have been engineered so as to
express a chosen derivative, fragment, homolog, or mutant of these
interacting domains. The cells can be yeast cells or higher
eukaryotic cells such as mammalian cells (WO 96/41169). In one
particular embodiment, the indicator cell is a yeast cell harboring
vectors enabling the use of the two hybrid system technology, as
well known in the art (4) and can be used to test a compound or a
library thereof. In one embodiment, a reporter gene encoding a
selectable marker or an assayable protein can be operably linked to
a control element such that expression of the selectable marker or
assayable protein is dependent on the interaction of the Protein A
and Protein B interacting domains. Such an indicator cell could be
used to rapidly screen at high-throughput a vast array of test
molecules. In a particular embodiment, the reporter gene is
luciferase or .beta.-Gal.
[0059] In one embodiment, at least one of the two interacting
proteins or domains of the present invention may be provided as a
fusion protein. The design of constructs therefor and the
expression and production of fusion proteins are well known in the
art (4, 96). In a particular embodiment, both interaction domains
are part of fusion proteins. A non-limiting example of such fusion
proteins includes a LexA-Protein A fusion (DNA-binding
domain-Protein A; bait) and a B42-Protein B fusion (transactivator
domain-Protein B; prey). In yet another particular embodiment, the
LexA-Protein A and B42-Protein B fusion proteins are expressed in a
yeast cell also harboring a reporter gene operably linked to a LexA
operator and/or LexA responsive element. Of course, it will be
recognized that other fusion proteins can be used in such two
hybrid systems. Furthermore, it will be recognized that the fusion
proteins need not contain the full-length interacting proteins.
Indeed, fragments of these polypeptides, provided that they
comprise the interacting domains, can be used in accordance with
the present invention, as evidenced with the peptide spanning
method of the present invention.
[0060] Non-limiting examples of such fusion proteins include
hemagglutinin fusions, gluthione-S-transferase (GST) fusions and
maltose binding protein (MBP) fusions. In certain embodiments, it
might be beneficial to introduce a protease cleavage site between
the two polypeptide sequences which have been fused. Such protease
cleavage sites between two heterologously fused polypeptides are
well known in the art.
[0061] In certain embodiments, it might also be beneficial to fuse
the interaction domains of the present invention to signal peptide
sequences enabling a secretion of the fusion protein from the host
cell. Signal peptides from diverse organisms are well known in the
art. Bacterial OmpA and yeast Suc2 are two non limiting examples of
proteins containing signal sequences. In certain embodiments, it
might also be beneficial to introduce a linker (commonly known)
between the interaction domain and the heterologous polypeptide
portion. Such fusion protein find utility in the assays of the
present invention as well as for purification purposes, detection
purposes and the like.
[0062] For certainty, the sequences and polypeptides useful to
practice the invention include, without being limited thereto,
mutants, homologs, subtypes, alleles and the like. It shall be
understood that generally, the sequences of the present invention
should encode a functional (albeit defective) domain. It will be
clear to the person of ordinary skill that whether an interaction
domain, variant, derivative, or fragment thereof, of the present
invention retains its function in binding to its partner can be
readily determined by using the teachings and assays of the present
invention and the general teachings of the art.
[0063] As exemplified herein below, the interaction domains of the
present invention can be modified, for example by in vitro
mutagenesis, to dissect the structure-function relationship thereof
and permit a better design and identification of modulating
compounds. However, some derivative or analogs having lost their
biological function of interacting with their respective
interaction partner may still find utility, for example for raising
antibodies. Such analogs or derivatives could be used for example
to raise antibodies to the interaction domains of the present
invention. These antibodies could be used for detection or
purification purposes. In addition, these antibodies could also act
as competitive or non-competitive inhibitors and be found to be
modulators of an interaction identified in accordance with the
present invention.
[0064] A host cell or indicator cell has been "transfected" by
exogenous or heterologous DNA (e.g. a DNA construct) when such DNA
has been introduced inside the cell. The transfecting DNA may or
may not be integrated (covalently linked) into chromosomal DNA
making up the genome of the cell. In prokaryotes, yeast, and
mammalian cells for example, the transfecting DNA may be maintained
on a episomal element such as a plasmid. With respect to eukaryotic
cells, a stably transfected cell is one in which the transfecting
DNA has become integrated into a chromosome so that it is inherited
by daughter cells through chromosome replication. This stability is
demonstrated by the ability of the eukaryotic cell to establish
cell lines or clones comprised of a population of daughter cells
containing the transfecting DNA. Transfection methods are well
known in the art (4, 96). The use of a mammalian cell as indicator
can provide the advantage of furnishing an intermediate factor,
which permits or modulates the interaction of two polypeptides
which are tested, that might not be present in lower eukaryotes or
prokaryotes. Of course, an advantage might be rendered moot if both
polypeptides tested directly interact. It will be understood that
extracts from mammalian cells for example could be used in certain
embodiments, to compensate for the lack of certain factors in a
chosen indicator cell. It shall be realized that the field of
translation provides ample teachings of methods to prepare and
reconstitute different types of extracts.
[0065] In general, techniques for preparing antibodies (including
monoclonal antibodies and hybridomas) and for detecting antigens
using antibodies are well known in the art (12). The present
invention also provides polyclonal, monoclonal antibodies, or
humanized versions thereof, chimeric antibodies and the like which
inhibit or neutralize their respective interaction domains and/or
are specific thereto.
[0066] From the specification and appended claims, the term
"therapeutic agent" should be taken in a broad sense so as to also
include a combination of at least two such therapeutic agents. The
DNA segments or proteins according to the present invention can be
introduced into individuals in a number of ways. For example,
erythropoietic cells can be isolated from the afflicted individual,
transformed with a DNA construct according to the invention and
reintroduced to the afflicted individual in a number of ways,
including intravenous injection. Alternatively, the DNA construct
can be administered directly to the afflicted individual, for
example, by injection in the bone marrow. The therapeutic agent can
also be delivered through a vehicle such as a liposome, which can
be designed to be targeted to a specific cell type, and engineered
to be administered through different routes.
[0067] For administration to humans, the prescribing medical
professional will ultimately determine the appropriate form and
dosage for a given patient, and this can be expected to vary
according to the chosen therapeutic regimen (e.g. DNA construct,
protein, molecule), the response and condition of the patient as
well as the severity of the disease.
[0068] Compositions within the scope of the present invention
should contain the active agent (e.g. protein, nucleic acid, or
molecule) in an amount effective to achieve the desired therapeutic
effect while avoiding adverse side effects. Typically, the nucleic
acids in accordance with the present invention can be administered
to mammals (e.g. humans) in doses ranging from 0.005 to 1 mg per kg
of body weight per day of the mammal which is treated.
Pharmaceutically acceptable preparations and salts of the active
agent are within the scope of the present invention and are well
known in the art (88). For the administration of polypeptides,
antagonists, agonists and the like, the amount administered should
be chosen so as to avoid adverse side effects. The dosage will be
adapted by the clinician in accordance with conventional factors
such as the extent of the disease and different parameters from the
patient. Typically, 0.001 to 50 mg/kg/day will be administered to
the mammal.
[0069] The methods and assays of the present invention have also
been validated with Annexin. This protein is significantly
different from P-glycoprotein in both structure and function.
Consequently, together with the knowledge of protein chemistry and
molecular biology, these validations support the utility of the
instant assays and methods for all proteins (from viruses, living
cells, animals, plants, etc.)
BRIEF DESCRIPTION OF THE DRAWINGS
[0070] Having thus generally described the invention, reference
will now be made to the accompanying drawings, showing by way of
illustration a preferred embodiment thereof, and in which:
[0071] FIG. 1 shows the principle of protein-protein interaction.
The plus signs (+) indicate the regions of high affinity binding.
The minus signs (-) indicate the regions of high-repulsive forces.
As indicated in the text, interactions between two proteins are
made up of discontinuous regions of high affinity binding and
high-repulsive forces that are almost in equilibrium with high
affinity binding being more favoured while proteins are
together.
[0072] FIG. 2 is a schematic representation of a method of
identification of high affinity binding sequences according to one
embodiment of the present invention. A, the different shapes
represent different proteins in a total cell lysate. The signs are
like for FIG. 1. B, small overlapping peptides that cover the
entire sequence (or a segment) of protein. A will be synthesized
directly on derivatized wells of 96-well polypropylene plates.
Following peptide synthesis, metabolically radiolabeled total cell
lysate is added to each well containing the various peptides and
incubated in an incubator buffer. C, the dark filled circles
represent the radiolabeled proteins from total cell lysate isolated
from metabolically radiolabeled cells added to all the wells of the
96-well plates to identify high affinity binding sequences on
Protein A. D, after an extensive washing, the high affinity binding
sequences (overlapping peptides from Protein A) are in those wells
that bind radiolabeled proteins (in dark). Four high affinity
binding sequences between Protein A and another protein(s) are
identified in rows 1, 3, 6 and 8. The wells that contain the high
affinity binding sequences are identified by radiolabeled counting
and SDS-PAGE.
[0073] FIG. 3 is a schematic representation of a method of
identification of high affinity binding sequences according to
another embodiment of the present invention. A shows a schematic
representation of the interaction between Protein A and Protein B.
B, small overlapping peptides that cover the entire sequence (or a
segment) of Protein A will be synthesized directly on derivatized
wells of 96-well polypropylene plates. Following peptide synthesis,
a radiolabelled Protein B (synthesized from in vitro
transcription-translation reaction mix) are added to each well
containing the various peptides and incubated in an incubation
buffer. C, the dark filled circles represent the radiolabeled
Protein B that has been added to all the wells of the 96-well
plates to identify high affinity binding sequences on Protein A. D,
after a washing procedure, the high affinity binding sequences are
in those wells in which Protein B (radiolabeled protein in dark) is
still bound to the peptides from Protein A. The wells that contain
the high affinity binding sequences are identified by radiolabeled
counting and SDS-PAGE.
[0074] FIG. 4 is a schematic representation of a method of
selection of drugs that specifically inhibit the binding of protein
A to B according to one embodiment of the present invention. A
shows a schematic representation of the interaction between Protein
A and Protein B. B, peptides that encode high affinity binding
sequences are used as LEAD sequences for the selection of specific
drugs that inhibit the association between Protein A and Protein B
and ultimately the function of the complex. To target the high
affinity binding sequences that were identified in FIG. 2 or 3,
peptides encoding one of the high affinity binding sequences are
synthesized in every well of the 96-well plate. Grey circles
represent one of four high affinity binding sequences identified in
FIGS. 2 and 3. C, following the addition of a compound to be tested
to each well of the 96-well plate, a radiolabeled Protein B are
added to each of the wells. Of course, combinatorial libraries can
be screened to identify drugs that bind specifically to the high
affinity binding sequences of Protein A. As previously stated,
radiolabeled Protein B from transcription-translation reaction mix
are represented. Plates are washed and drugs that specifically bind
to high affinity sequences of Protein A are found in those wells
that do not contain radiolabeled Protein B. D, wells containing
drugs/compounds that bind specifically to one of the high affinity
binding sequence in Protein A and therefore prevent the binding of
Protein B are identified by the absence of a dark circle (i.e.,
wells 28, 70 and 75). Selected drugs/compounds represent invaluable
LEAD compounds that can be used in biological assays to confirm
their mechanism of action. Validated drugs can proceed toward in
vivo studies.
[0075] FIG. 5 shows a P-glycoprotein predicted secondary structure
and amino acid of the linker domain. A schematic representation of
P-gp predicted secondary structure. The twelve filled squares
represent the twelve putative transmembrane domains. The two ATP
binding domains are represented by two circles in the N- and
C-terminal halves of P-gp. The inset represents the linker domain.
The amino acid sequence of the linker domains of Human P-gp 1
(HP-gp1) (SEQ ID NO: 15) and HP-gp3 (residues 1-88 of SEQ ID NO:
14) is indicated as a single-letter amino acid code. The numbers in
brackets at the beginning and end of each amino acid sequence of
HP-gp1 and HP-gp3 shows the length of the linker domains (1-90 and
1-88 for HP-gp1 and HP-gp3, respectively). The numbered lines
underneath the amino acid sequence show the sequences of the
overlapping hexapeptides, which differ by one amino acid. For
HP-gp3, the last hexapeptide is number 88.
[0076] FIG. 6 shows the protein binding to overlapping hexapeptides
encoding HP-gp1 linker domain. Overlapping hexapeptides that encode
the linker domain of HP-gp1 were synthesized on polypropylene rods
and used to identify proteins that bind to these peptides. A total
of 90 plus two control hexapeptides for HP-gp1 were incubated with
total cell lysate from [.sup.35S] methionine metabolically labeled
cells (see methods). All bound proteins were eluted from the
peptide-fixed rods and resolved on 10% SDS-PAGE. Lanes 1 to 92 show
the [.sup.35S] methionine bound proteins from HP-gp1. The migration
of the molecular weight markers is shown to the left of gels.
[0077] FIG. 7 shows the effects of different detergents or high
salt on the binding of proteins to HP-gp1 hexapeptides.
Metabolically radiolabeled proteins bound to hexapeptides
(hexapeptides 50 to 53) from HP-gp1 linker domain were eluted in
the presence of increasing concentrations of anionic detergent
(0.12%-0.5% SDS), zwitterionic detergent (20 mM-80 mM CHAPS) or
salt (0.3 M-1.2 M KCl). The y-axis represents the amount of
radioactivity eluted from a pool of three hexapeptides (50 to
53).
[0078] FIG. 8 shows the effects of CHAPS on the binding of proteins
to the overlapping hexapeptides encoding HP-gp1 linker domain.
Overlapping hexapeptides of the linker domain of HP-gp1 were
incubated with total cell lysate from [.sup.35S] methionine
metabolically labeled cells extracted with 10 mM CHAPS. Bound
proteins were eluted from the peptide-fixed rods and resolved by
10% SDS-PAGE. Lanes 1 to 92 show the [.sup.35S] methionine bound
proteins to HP-gp1 linker domain. The migration of the molecular
weight markers is shown to the left of gels.
[0079] FIG. 9 shows the protein binding to overlapping hexapeptides
encoding HP-gp3 linker domain. Overlapping hexapeptides that encode
the linker domain of HP-gp3 were synthesized on polypropylene rods
and used to identify proteins that bind to these peptides. A total
of 88 plus two control hexapeptides for HP-gp3 were incubated with
total cell lysate from [.sup.35S] methionine metabolically labeled
cells. All bound proteins were eluted from the peptide-fixed rods
and resolved on 10% SDS-PAGE. Lanes 1 to 90 show the [.sup.35S]
methionine bound proteins from HP-gp3. The migration of the
molecular weight markers is shown to the left of gels.
[0080] FIG. 10 shows the sequence alignment of three binding
regions of HP-gp1 and HP-gp3 linker domains. Alignment of HP-gp1
(SEQ ID NO: 15) and HP-gp3 (SEQ ID NO: 14) linker domains is shown
using a single-letter code for amino acids. The regions of high
binding affinities for HP-gp3 and HP-gp1 are shown in bold.
Identical amino acids are shown by single letter code between the
two aligned sequences. Conserved amino acids are indicated by plus
(+) sign. The numbers on each side of the amino acid sequence of
the linker domains refer to the amino acid sequence of human P-gp1
and 3 as in (90, 111).
[0081] FIG. 11 shows the two high affinity binding hexapeptides.
Two high affinity binding sequences .sup.658RSSLIR.sup.663 (SEQ ID
NO: 7) and .sup.669SVRGSQ.sup.674 (SEQ ID NO: 8) from HP-gp1 linker
domain were resynthesized and incubated with total cell lysate from
[.sup.35S] methionine metabolically labeled cells following 24 hour
or 48 hour incubation times. Bound proteins were eluted from
peptide-fixed rods and resolved by 10% SDS-PAGE. The migration of
the molecular weight markers is shown to the left of the
figure.
[0082] FIG. 12 shows the effects of different carrier proteins as
blocking agent of unspecific binding. Total cell lysates from
[.sup.35S] methionine metabolically labeled CEM cells were used as
is or made 1% gelatin, 0.3% BSA or 3% BSA. The cell lysates were
incubated with a high affinity binding hexapeptide
.sup.658RSSLIR.sup.663 (SEQ ID NO: 7) from HP-gp1 linker domain.
The bound proteins were eluted with SDS sample buffer and resolved
on 10% SDS-PAGE. The migration of the molecular weight markers is
shown to the left of the figure.
[0083] FIG. 13 shows the purification of a 57 kDa protein. Total
cell lysate was incubated with fifty HP-gp1 hexapeptides
.sup.658RSSLIR.sup.663 (SEQ ID NO: 7) and .sup.669SVRGSQ.sup.674
(SEQ ID NO: 8). Samples containing the 57 kDa protein (P57) from
one hundred hexapeptide incubation mix were pooled and resolved by
10% SDS-PAGE. The resolved proteins were transferred to PVDF
membrane and stained with Ponceau S. The migration of the molecular
weight markers is shown to the right of the figure.
[0084] FIG. 14 shows a Western blot analysis with anti-tubulin
monoclonal antibodies. Total cell lysate from CEM cells and
proteins eluted from the high affinity binding hexapeptides of
HP-gp1 linker domain (P57) were resolved on SDS-PAGE and
transferred to nitrocellulose membrane. One half of the membrane
was probed with anti-.alpha. and anti-.beta. tubulin monoclonal
antibodies. The migration of the molecular weight markers is shown
to the left of the figure.
[0085] FIG. 15 shows the helical wheel presentations of the high
affinity binding region of HP-gp1 and HP-gp3 linker domains. The
single-letter amino acid code for the high affinity binding region
of HP-gp1 (SEQ ID NO: 12) and HP-gp3 (SEQ ID NO: 13) linker domains
are shown. The positively charged amino acids on one side of the
helix have been circled.
[0086] Other objects, advantages and features of the present
invention will become more apparent upon reading of the following
non-restrictive description of preferred embodiments with reference
to the accompanying drawing, which is exemplary and should not be
interpreted as limiting the scope of the present invention.
DESCRIPTION OF THE PREFERRED EMBODIMENT
[0087] The function or functions of proteins is mediated through an
interaction thereof with other cellular or extracellular proteins.
Until now it was thought that interactions between two proteins
involve large segments of polypeptides that have complementary
amino acid sequences. However, it is not known how these
complementary sequences mediate the interactions between proteins.
In this application, a novel concept to explain the principle of
protein-protein interactions is proposed. Briefly, interactions
between any two or more proteins are mediated by strings of
discontinuous sequences with high affinity binding and
high-repulsive forces (see FIG. 1). The sum of these forces over
the entire exposed sequence of proteins determines the nature and
extent of the interactions between proteins. The sizes of these
interacting domains can vary from 5 to 25 amino acids in length.
The attractive forces between two small high affinity binding
sequences are generally larger than the sum of all the high
affinity binding and repulsive-forces between two proteins.
Therefore, using the present approach, it is possible to isolate
interacting proteins from a mixture of proteins using a short
peptide (almost six amino acids) that encodes only the high
affinity binding sequence. Indeed, with this in mind, it is now
easy to see why many methods attempting to isolate interacting
proteins have failed. The use of large fragments or proteins to
isolate interacting proteins is less efficient since the sum of
attractive/repulsive forces are much weaker than any string of
attractive forces. The herein proposed principle is also consistent
with the fact that protein-protein interactions can be modulated by
post-translation modifications (e.g., by phosphorylation (29)) and
the presence of other interacting proteins (60). Hence, the
addition or loss of weak forces following post-translation
modification can disrupt the tenuous balance between high affinity
binding and high-repulsive forces that hold proteins together or
prevent their association. Support for the magnitude of attractive
forces between two high affinity binding sequences is demonstrated
in antibody-antigen binding whereby the antigen can be only of a
few amino acids (36, 37). Furthermore, numerous examples exist in
biology where cellular interactions between proteins occur due to
the presence of small consensus sequence of five to ten amino
acids. Non-limiting examples of such small consensus sequences
include the leucine zipper (63), and SH2 and SH3 binding sequences
(63, 80). In addition to the domains of interactions between two or
more proteins (indicated above), protein-protein interactions can
have many measurable effects, such as: i) changes in the kinetic
properties of one or both proteins (83, 84); ii) formation of new
binding or functional sites (65, 104); and iii) the inactivation of
function(s) (106, 114). In other words, a given protein could
expose different functional domains or sequences in the presence
(as opposed to the absence) of any interacting proteins. Thus, in
the presence of protein B, protein A can expose other sequences not
previously exposed for interactions with other proteins (65, 83,
84, 104, 106, 114). The latter concept is very important as it
argues against the effectiveness of some structural studies (i.e.,
X-ray and NMR) in predicting functional or surface exposed domains
from the resolved crystal structure of proteins. By enabling the
measurement and the identification of potentially all the high
affinity binding sites of a given protein, the present invention
seeks to overcome the drawbacks of the results obtained from such
structural studies.
[0088] Further to the above examples of protein-protein
interactions, a subset of protein-protein interactions is
dimerization. There is an abundance of examples in biology whereby
protein-protein interactions are essential for activation or
inhibition of function (59). Non-limiting examples of homo- or
heterodimers include; growth factor receptors (52); membrane
transport proteins (9, 36, 76); tumor suppressor proteins (72); and
proteins that mediate apoptosis (87). In fact, dynamic dimerization
is a common theme in the regulation of signal transduction. Some of
the functional consequences of dimerization include, increased
proximity for activation of single transmembrane cell surface
receptors (e.g., EGF receptor (52)) and differential regulation by
heterodimerization [e.g., BCL2 family of proteins (87)].
[0089] The protein concentration in living cells is very high and
is in the range of 10-30 mg/ml. At this high protein concentration,
most if not all proteins should interact precisely and specifically
with other cellular proteins. Some of the interacting proteins act
as inhibitors of function, while others may be activators (e.g.,
The BCL2-BAX family of proteins, (87)). Moreover, the cycling of a
given protein between activator and inhibitor association will
require the association-dissociation process to occur rapidly. For
example, when protein X is associated with an inhibitor protein I,
the domains (small sequences) that are required for the association
of protein X with an activator protein A may not be easily
accessible in the X-I complex. Therefore, current methods to
identify associated protein (i.e., the two-hybrid system and
similar approaches) may not be able to identify all associated
proteins. In other words, current methods, when successful, may
only identify some but not all functional domains and their
associated proteins. By contrast, using the peptide scanning
approach, the method of the present invention is capable of
identifying all functional domains or high affinity interacting
domains of protein X and its associated proteins. Once the
associated proteins are identified, their biological functions as
it relates to the target protein X can be tested. Thus, for a given
interacting protein, should its interaction with one or many
possible associated proteins prove to be important for function,
the high affinity binding sequences (between protein X and Protein
I or A) can be easily identified and can be used as a target site
in a high throughput drug screening assays (see below) or other
assays.
[0090] This invention includes the concept (described in FIGS.
1A-1D) that protein-protein interactions are made-up of
discontinuous high affinity binding and high-repulsive forces
scattered throughout the 3-D sequence of proteins and that these
sequences can be isolated using one of many possible approaches
indicated herein (e.g., the overlapping peptide approach).
Although, in this application, the overlapping peptide approach is
exemplified, other approaches can be envisioned that give similar
results. It should be stressed that the approach described herein
is immune to conformational changes resulting from interacting
proteins that could affect other commonly used methods to identify
protein-protein interactions (e.g., two-hybrid system, affinity
blotting, and crosslinking). In the two hybrid system, for example,
Protein A is fused with another protein sequence (the DNA-bound
"bait" protein) and the other interacting protein is fused to the
activation-domain containing "prey" protein. The fusion of
interacting proteins to protein A could expose regions other than
those found in the native conformation which will affect their
interactions. Furthermore, the two-hybrid system has several
disadvantages, some of which are listed below,
[0091] i. The interaction of proteins is monitored in the nuclear
milieu rather than the cytoplasm where most proteins are found.
[0092] ii. Proteins can be toxic when expressed in different cells
or organisms.
[0093] iii. The interactions between two proteins in a complex in
the two-hybrid system can sterically exclude the binding of other
interacting proteins.
[0094] iv. The post-translational modification of one protein can
exclude its interaction with other proteins.
[0095] v. The two-hybrid system does not allow the simultaneous
identification of the precise amino acid sequences between two
interacting proteins.
[0096] vi. The application of the two-hybrid system is associated
with high percentage of false positives.
[0097] vii. The two-hybrid system cannot be easily applied to
different cell types or tissues whereby different interacting
proteins may be expressed (this can be a critical drawback of this
system).
Method to Identify Interacting Proteins and Sites of Interactions
for Protein A
[0098] The present approach and methodology used to identify
discontinuous strings of sequences between two or more interactive
proteins is a scanning overlapping peptide approach. Using this
approach, a large number of short overlapping peptides which cover
the entire amino acid sequence of the given protein, "the bait,"
are synthesized in parallel on an inert solid support (see FIG. 2).
The rationale for synthesizing a large number of overlapping
peptides as opposed to a discontinuous peptide library is based on
the fact that one does not know a priori what exact sequence of a
given protein will contain the high affinity binding sites and the
repulsive sequences. Therefore, a discontinuous peptide approach
will often lead to the presence of both high affinity binding
sequences and repulsive sequences in the same peptide. Such
peptides will not bind to potential interacting proteins with high
affinity. Moreover, the use of overlapping peptides also provides
internal controls for unspecific binding. For example, using
overlapping peptides, the high affinity binding sequences will give
a peak of signal when peptides within the high affinity domain will
have the high affinity amino acid sequences but will lack amino
acids which provide the repulsive forces (see FIG. 6 in Example I).
Of course, it should be understood that the present invention is
not dependent on a spanning of the full peptide sequence. Indeed,
sub-region(s) of a protein can be used. In addition, overlapping
peptides can be derived from a chosen domain of a protein. Also, it
would be envisageable to probe an overlapping peptide side set of a
first protein with an overlapping peptide set of a second
protein.
[0099] To demonstrate how one can use this approach of overlapping
peptides as "a bait" to isolate interacting proteins "the prey" or
"preys" from a mixture of total cell proteins, the following
example can be considered. P-glycoprotein is a membrane protein
(46) that confers resistance to anticancer drugs and therefore is
responsible for the failure of chemotherapy. Although,
P-glycoprotein has been shown to function by preventing the
accumulation of chemotherapeutic drugs in tumor cells; the exact
mechanism of how this protein functions and what are the associated
proteins that modulate its function are not known. Thus, it is of
interest to identify proteins that interact with P-glycoprotein,
such as to enable an inhibition of binding between P-glycoprotein
and its associated proteins, thereby potentially modulating its
function in resistant tumor cells. In this example, it was of
interest to identify those proteins which bind to the linker domain
of P-glycoprotein. Thus, in this particular example, a domain of a
chosen protein was used. The linker domain, encodes a region of
about 90 amino acids. Thus, overlapping hexapeptides covering this
entire linker sequence of P-glycoprotein were synthesized onto a
solid support using standard F-moc chemistry (74). The covalently
fixed peptides (on a solid support) were incubated with a total
cell lysate isolated from cells metabolically with
[.sup.35S]methionine. The peptides and total cell lysate were
incubated in the presence of a carrier substrate (1-3% bovine serum
albumin, or 1-3% gelatin, 1-3% skim milk, etc.) for 18 hours at
4.degree. C. Following this incubation period, the covalently fixed
peptides were washed extensively with isotonic buffer. Any proteins
from the radiolabeled total cell lysate which maintained their
association with the overlapping hexapeptides following the washing
step are eluted in SDS-contain sample buffer and analyzed by SDS
polyacrylamide gel electrophoresis (SDS-PAGE) (62). The presence of
radiolabeled proteins on SDS polyacrylamide gels following gel
drying and signal enhancement, provides the following
information:
[0100] 1) those specific overlapping peptides represent high
affinity binding sequences in the P-glycoprotein linker domain (or
other chosen domains or non-chosen domains); and
[0101] 2) the proteins bound to the specific overlapping peptides
are associated proteins (see FIG. 6).
[0102] The associated proteins which bound to the high affinity
binding sequences, can be isolated in large quantities for the
purpose of determining their identity by N-terminal amino acid
sequencing by Edman degradation (27) or the like. Briefly, the
sequences of the overlapping peptides that bound a given protein
are resynthesized on a solid support and kept fixed thereto. Total
cell lysate from [.sup.35S]methionine metabolically-radiolabeled
cells is added to the solid support containing the fixed high
affinity sequence peptides and incubated as described above.
Following washing steps to remove unbound material, the associated
protein is isolated in large amounts following an elution step with
SDS-containing buffers (see below). The purified associated protein
is now ready for amino acid sequencing. Of course, should further
purification steps be required, they are well known to the skilled
artisan. The purified protein is run on SDS polyacrylamide gels and
the resolved protein is transferred to PVDF membrane as previously
described (108). Other methods for amino acid sequence
determination can also be easily applied (27, 33).
Method to Identify the Amino Acid Sequences Between Two Interacting
Proteins
[0103] The same concept as described above can be applied if one is
only interested in identifying the high affinity binding sequences
between two proteins. A non-limiting example of such two proteins
are the regions of interactions between p53 and MDM (28, 103).
Specifically therefore, the purpose of this exercise is to identify
the high affinity binding sequences between proteins A (p53) and
protein B (MDM) in order to use these sequences as target sites for
the identification of compounds that modulate this interaction and
more particularly for the development of drugs. Thus, in one
embodiment, when a given drug is bound to one of these high
affinity binding sites on protein A, it will prevent the formation
of the active complex (protein A+B) and therefore inhibit the
functions of the complex. To isolate the string of high affinity
binding sequences between Protein A and B (see FIG. 3), small
overlapping peptides (5 to 7 amino acids) that cover the entire
amino acid sequence of protein A, "the bait" will be synthesized in
parallel onto a solid support (as mentioned above and described in
more detail in Example 3). Note that, in this particular
embodiment, only the primary amino acid sequence of protein A, "the
bait," is needed. Once the peptides are synthesized (peptide
synthesis is done parallel on a solid support in 96-well plates),
an enriched and radiolabeled full-length protein B (the
radiolabeled protein B is easily obtained from in vitro
transcription-translation reactions), (118) "the prey," is added to
each well of the 96-well plate that contain the covalently fixed
overlapping peptides. The peptides encoding protein A are incubated
with radiolabeled protein B to allow for binding to occur.
Following an incubation period (5 to 24 hours), unbound
radiolabeled protein B will be removed by extensive washing in
isotonic buffer. Any overlapping peptides which bound to
radiolabeled protein B will be eluted in the presence of denaturing
agents. The eluant from each of the 96-well plates are analyzed for
the presence of radiolabeled protein B by running the samples by
SDS-PAGE (62). High affinity binding peptides will be identified as
those that retain the radiolabeled Protein B.
[0104] The use of metabolically radiolabeled proteins as "the prey"
to interact with the overlapping peptides of "the bait" increases
the sensitivity of this technique and allows the identification of
interacting proteins with binding affinities of
10.sup.-10.sup.-10-10.sup.-12 M for a standard 50 kDa protein which
encodes one to ten radiolabeled methionine residues (82).
Method to Use High Affinity Binding Sequences in High Throughput
Assays to Screen for Lead Compounds
[0105] The approach, described herein, to identify high affinity
binding sequences or target sites for drug development can also be
used in high throughput assays to screen for small molecules from
combinatorial libraries. For example, to select drugs that
specifically inhibit the binding of protein A to B (see FIG. 4),
one or more target sites (the high affinity binding sequences) are
synthesized in each of the 96-well plates as described earlier. In
this example (FIG. 4) the same high affinity binding sequence is
synthesized in all of the wells. To each well containing the high
affinity binding sequence, one or more small molecules from
combinatorial library are added. Following the addition of drug(s),
a radiolabeled protein B from an in vitro transcription-translation
mix, for example, is added and allowed to incubate as indicated
above. Following several washes, bound protein B is eluted with
SDS-sample buffer. Wells containing radiolabeled protein B
indicates that the drug had no effect on the binding between the
high affinity binding sequence and protein B. Alternatively, if one
or more wells do not contain radiolabeled protein B in the presence
of a drug, then that drug has inhibited the interactions between
the high affinity binding sequence on A and protein B. Hence, the
latter drug is a good LEAD compound. These drugs can now enter the
second phase of their analysis to determine if they prevent the
formation of the active complex of full length protein A and B.
Active drugs that are identified will be tested in vivo to further
confirm their mechanism of action. In this manner, more specific
drugs with fewer or no side-effects will be developed.
[0106] The latter point provides an advantage since most proteins
have more than one biological function. For example, if protein A
interacts with itself, it will do one function, while the same
protein interacting with a different protein will do a different
function. Moreover, protein A, when part of a given complex of
associated proteins, will mediate several functions, inhibiting the
interactions between protein A and B, while leaving the
interactions between proteins A and C, D, or F intact will inhibit
one or few cellular pathways. By contrast, inhibiting the function
of protein A will inhibit the functions of the entire complex. In
this respect, the identification, isolation and development of
drugs that will inhibit specifically interactions between two
proteins within a complex of proteins should result in more
specific drugs with fewer side effects. In addition, as different
proteins are differentially expressed in different tissues or
organs, the composition of a given protein complex will be
different between different tissues. Hence, the approach of
developing drugs that inhibit protein-protein interactions will
also lead to drugs that are organ or tissue-specific.
[0107] Of course, it will be understood that the present invention
also provides quantitative assays to measure the protein-protein
interaction and the modulation thereof by compounds.
[0108] In conclusion, the approach described in this application
for the identification interacting proteins, the precise amino acid
sequence between interacting proteins, and targeting of such
specific sequences in proteins with drugs that inhibit
protein-protein interactions has tremendous potential in dictating
future drug discovery in the pharmaceutical industry.
[0109] The present invention is illustrated in further detail by
the following non-limiting examples.
Example 1
P-glycoprotein Binding to Tubulin is Mediated by Sequences in the
Linker Domain
[0110] The successful treatment of cancer patients with
chemotherapeutic drugs is often limited by the development of
drug-resistant tumors. Tumor cell lines selected, in vitro, with a
single anticancer drug become resistant to a broad spectrum of
chemotherapeutic drugs, termed multidrug resistant (or MDR) tumor
cells (for review, (21, 45, 66). Moreover, the expression of MDR in
these tumor cells has been associated with the overexpression of
two membrane proteins; the MDR1 P-glycoprotein (P-gp) and the
multidrug resistance-associated protein (MRP1) (21, 45, 66). Both
P-gp and MRP are members of a large family of membrane transporter
proteins known as ATP binding cassette proteins or ABC membrane
transporters (54). Although, the structure of P-gp1 remains a
matter speculation (91), cumulative topological evidence suggest a
tandemly duplicated structure of six transmembrane domains and a
large cytoplasmic domain encoding an ATP binding sequence (58, 68).
The two halves of P-gp1 are linked by a stretch of 90 residues rich
in polar or charged amino acids, termed the "linker domain."
[0111] The P-gp gene family is made up of three structurally
similar isoforms in rodents (classes I, II, and III) and two
isoforms in humans (classes I and III) (20). Gene transfer studies
suggest functional differences among these structurally similar
isoforms. For example, only the P-gp isoforms of classes I and II
confer the MDR phenotype (25, 111), while the class III isoforms do
not (11, 98). The class III isoforms mediate the transfer of
phosphatidylcholine from the inner to the outer leaflet of the
plasma membrane (i.e., "flipase") (92, 100). In normal tissues,
P-gp distribution is restricted mainly to tissues with secretory
functions (79, 116). Its polarized localization to apical surfaces
facing a lumen in the adrenal gland, liver, kidney intestine
suggests a normal transport or detoxification mechanism. Moreover,
hematopoietic stem cells and specific lymphocyte subclasses also
express high levels of P-gp (49). The normal function or
substrate(s) of the classes I and II remain undefined; however, the
disruption of the class I or/and II genes from the mouse genome
results in the accumulation of cytostatic drugs or lipophilic
compounds in most normal tissues, but more strikingly in the brain
(99, 100). Based on these results it is speculated that the normal
function of P-gp (the class I and II or the MDR causing P-gp) is
detoxification similar to that seen in MDR cells, especially at the
blood brain barrier (57).
[0112] High levels of P-gp have been found in many intrinsically
drug resistant tumors from colon, kidney, breast and adrenals as
well as in other tumors which had acquired the MDR phenotype after
chemotherapy (for example, in acute non-lymphoblastic leukemia)
(22, 32, 35, 47, 53, 78). Several studies have now established an
inverse correlation of P-gp expression and the response to
chemotherapy (5, 89, 113). Further, Chan et al. (16, 17) have shown
that P-gp expression was prognostic of MDR and durable response in
childhood leukemia, soft tissue sarcomas and neuroblastomas of
children. In light of these studies there appears to be convincing
evidence, at least in some cancers, that P-gp levels predict the
response to chemotherapeutic treatment.
[0113] Direct binding between P-gp and various lipophilic compounds
has been demonstrated using photoactive drug analogues (77, 93,
94). Certain compounds which bind to P-gp were shown to reverse the
MDR phenotype presumably by competing for the same drug binding
site in P-gp (34, 38). These compounds, which have been
collectively labeled as MDR-reversing agents, include verapamil,
quinidine, ivermectin, cyclosporins, and dipyrimadol analogues to
name but few (34, 38). Clinical trials using MDR-reversing agents
(e.g., verapamil or quinidine) have shown some response in tumors
that were otherwise non-responsive to chemotherapy (23, 44, 117).
However, high pharmacological toxicity associated with several
MDR-reversing agents has prevented their use at an effective
concentration (67). A better clinical response has been observed
using other MDR-reversing agents (i.e., cyclosporin A and its
non-immunosuppressive analog PSC833); however toxic effects have
also been seen with cyclosporins (101, 115).
[0114] P-gp was shown to be a substrate for protein kinases C and A
(2, 9). Moreover, it has been demonstrated that agents which
modulate protein kinase C activity, modulate P-gp phosphorylation
and its MDR-mediate phenotype (7, 13). In one study (31), PMA
phorbol ester (a protein kinase C activator) was shown to increase
the MDR phenotype and drug efflux in MCF7 breast cancer cells. In
another study (6), sodium butyrate treatment of SW620 human colonic
carcinoma cells was shown to result in a large increase in P-gp
expression without a concomitant increase in drug-resistance or
efflux. Interestingly, P-gp in SW620 cells was also shown to be
poorly phosphorylated following sodium butyrate treatment (6).
Taken together, the lack of transport function of P-gp in SW620
cells was not clear, however mutations of P-gp phosphorylation
sites within the linker domain was shown not to affect its drug
transport function (40). By contrast, protein kinase C modulation
of serine/threonine residues in the linker domain regulated the
activity of an endogenous chloride channel and thus suggests that
P-gp is a channel regulator (41, 110). Thus, although, it remains
unclear what functions the linker domain of P-gp1 mediates, it was
of interest to identify the proteins that interact with linker
domain using an in vitro assay. The latter assay is based on the
novel understanding of protein interactions provided by the present
invention. The results show hereinbelow that three sequences in the
linker domain bind to proteins with apparent molecular masses of
.about.80 kDa, 57 kDa and 30 kDa. Purification and partial
N-terminal amino acid sequencing of the 57 kDa protein showed that
it encodes the N-terminal amino acids of .alpha. and
.beta.-tubulins.
[0115] Thus, using a protein domain as an example of a validation
of the power of the present invention, it was demonstrated that: i)
this domain is bound specifically to proteins; ii) the specifically
binding proteins can be formally identified; and iii) the sequence
responsible for the specific binding of these proteins formally
identified (together with the interacting domain of this binding
protein, if derived).
Example 2
Materials
[0116] [.sup.35S] methionine (1000 Ci/mmol; Amersham Life Sciences,
Inc.) and [.sup.125] goat anti-mouse antibody were purchased from
Amersham Biochemical Inc. Protein-A Sepharose-4B was purchased from
Bio-Rad Life Science. All other chemicals used were of the highest
commercial grade available.
Example 3
Peptide Synthesis
[0117] Pre-derivatized plastic rods, active ester and polypropylene
trays were purchased from Cambridge Research Biochemicals (Valley
Stream, N.Y.). Peptides were synthesized on solid polypropylene
rods as previously described (36, 37). Briefly, the F-moc
protecting group on the prederivatized polypropylene rods as solid
support (arranged in a 96-well formate) was removed by incubation
with 20% (v/v) piperidine in dimethylformamide (DMF) for 30 minutes
with shaking. Following the deprotection of the .beta.-alanine
spacer on the polypropylene rods, Fmoc protected amino acids were
dissolved in HOBt/DMF and added to the appropriate wells containing
deprotected rods. Coupling of amino acids was allowed to take place
for 18 hours at room temperature after which the rods were washed
in DMF (1.times.2 minutes), methanol (4.times.2 minutes), and DMF
(1.times.2 minutes). The coupling of the second amino acid required
the deprotection of the F-moc amino protecting group of the first
amino acid and incubation of the rods with the second preactivated
F-moc-protected amino acids (pentafluorophenyl derivatives). The
reaction was allowed to proceed for 18 hours, and the rods were
removed and washed as indicated above. The same steps were repeated
for each amino acid coupling until the sixth amino acid was
coupled. Following the last coupling step, the F-moc N-terminal
protecting group was removed with 20% piperidine/DMF and the free
amino group acetylated for 90 minutes in an acetylation cocktail
containing acetic anhydride:diisopropylethylamine (DIEA):DMF
(50:1:50 v/v/v). The side chain protecting groups of the N-terminal
acetylated hexapeptides onto the polypropylene rods were removed by
incubation in a cleavage mixture containing trifluoroacetic
acid:phenol:ethandithiol (95:2.5:2.5 v/v/v) for 4 hours at room
temperature. After the cleavage step the rods were washed with
dichloromethane (DCM) and neutralized in 5% (v/v) DIEA/DCM. The
deprotected peptide-coupled rods were washed in DCM, methanol and
vacuum dried for 18 hours.
Example 4
Tissue Culture and Metabolic Labeling of Cells
[0118] Drug sensitive (CEM) and resistant (CEM/VLB.sup.1.0) cells
were cultured in .alpha.-MEM media supplemented with 10% fetal calf
serum (Hyclone, Inc.) as previously described (8). All cells were
examined for Mycoplasma contamination every three months using the
Mycoplasma PCR kit from Stratagene Inc. (San Diego, Calif.). For
metabolic labeling of cells, CEM or CEM/VLB.sup.1.0 cells at 70-80%
confluency were metabolically labeled with [.sup.35S] methionine
(100 .mu.Ci/ml) for 6 hours at 37.degree. C. in methionine-free
A-MEM media.
Example 5
Cell Extraction and Binding Assay
[0119] Following metabolic labeling of proteins with [.sup.35S]
methionine, cells were washed 3 times with phosphate buffered
saline (PBS) and resuspended in hypotonic buffer (10 mM KCl, 1.5 mM
MgCl.sub.2, 10 mM Tris-HCl, pH 7.4) containing protease inhibitors
(2 mM PMSF, 3 .mu.g/ml Leupeptin, 4 .mu.g/ml pepstatin A and 1
.mu.g/ml aprotinin) and kept on ice for 30 minutes. Cells were
lysed by homogenization in a hypotonic buffer and the cell lysate
was sequentially centrifuged at 6000.times. g for 10 minutes.
Following the latter centrifugation, the supernatant was removed
and made 0.5 M NaCl final concentration from a stock solution of 4
M NaCl. The cell lysate was incubated on ice for 30 minutes. The
sample was mixed and brought back to 0.1 M NaCl final
concentration. The cell lysate was centrifuged for 10 minutes at
15,000.times. g at 4.degree. C. The latter supernatant was removed
and recentrifuged at 100,000.times. g for 60 minutes in a Beckman
ultracentrifuge using SW55 rotor. The amount of protein in the
above samples was determined by the method of Lowry (69).
[0120] For a binding assay, [.sup.35S] methionine labeled proteins
from total cell lysate were mixed with equal volume of 3-6% BSA in
phosphate buffered saline (PBS) and incubated with overlapping
hexapeptides covalently fixed to polypropylene rods. The peptides
and total cell lysate were incubated overnight at 4.degree. C. The
rods were then removed and washed four times in PBS. The bound
proteins were eluted by incubating the peptide-fixed rods in
1.times. SDS sample buffer for 60 minutes at room temperature with
shaking. The peptides-fixed rods, were regenerated by incubation in
PBS, containing 2% SDS and 1 mM .beta.-mercaptoethanol at
65.degree. C. in a sonicator for 30 minutes. Following the latter
incubation, the rods were washed for five minutes in 65.degree. C.
ionized water and two minutes in 65.degree. C. methanol. The
peptides-fixed rods are now ready for the next round of screening.
In cases where the effects of various detergents on binding was
tested, [.sup.35S] methionine labeled proteins from total cell
lysate were mixed with equal volume of 3% BSA in phosphate buffered
saline containing KCl (300 mM to 1200 mM), SDS (0.12% to 2%), or
CHAPS (20 mM to 160 mM) and incubated with covalently fixed
peptides as described above.
Example 6
Polyacrylamide Gel Electrophoresis and Western Blotting
[0121] Protein fractions (100-15011) were resolved on SDS-PAGE
using the Laemmli gel system (62). Briefly, proteins were dissolved
in 1.times. solubilization sample buffer I (62.5 mM Tris-HCl, pH
6.8, containing 2% (w/v) SDS, 10% (w/v) glycerol and 5%
.beta.-mercaptoethanol) and samples were electrophoresed at
constant current. Gel slabs containing the resolved proteins were
fixed in 50% methanol and 10% acetic acid. Polyacrylamide gels
containing [.sup.35S] methionine proteins were exposed to Kodak
x-ray film following a thirty-minute incubation in an Amplify.TM.
solution (Amersham Inc.).
[0122] Alternatively, proteins were transferred to nitrocellulose
membrane in Tris-glycine buffer in the presence of 20% methanol for
Western blot analysis according to the procedure of Towbin et al.
(108). Nitrocellulose membrane was incubated in 5% skim milk/PBS
prior to the addition of anti-.alpha. or anti-.beta. tubulin
monoclonal antibodies (0.5 .mu.g/ml in 3% BSA; Amersham, Inc.).
Following several washes with PBS, the nitrocellulose membrane was
incubated with goat anti-mouse peroxidase-conjugated antibody and
immunoreactive proteins were visualized by chemiluminescence using
ECL method (Amersham, Inc.).
Example 7
Protein Purification and N-terminal Sequencing
[0123] The 57 kDa associated protein was purified using a block of
polypropylene rods with two high affinity binding peptides.
Briefly, the peptide-fixed rods were incubated with total cell
lysate as indicated above; however, in this case the carrier
substance was gelatin (1%). The bound proteins were eluted in 100
mM phosphate buffer, pH 7.4 containing 2% SDS and 0.1%
.beta.-mercaptoethanol. The eluted proteins were precipitated by
mixing with 9 volumes of ice cold ethanol and incubated at
-20.degree. C. Following a high speed centrifugation of the latter
sample (15 minute centrifugation at 15,000.times. g, at 4.degree.
C.), the precipitated proteins were resuspended in 1% SDS in PBS
and mixed with equal volume of 2.times. SDS Laemmli sample buffer
(62). Protein samples were resolved by 10% SDS-PAGE and transferred
to PVDF membrane. The migration of the 57 kDa band was visualized
by staining the PVDF membrane with Ponceau S. The PVDF membrane
containing the 57 kDa band was excised and submitted to the protein
sequencing facility at the Biotechnology Service Centre in Toronto,
Ontario. Amino acid sequencing of peptides was performed according
to the method of Edman and Begg (27) using an applied biosystems
gas-phase Model 470A sequenator.TM. according to the procedure
described by Flynn (33).
Example 8
Identification of P-gp Interacting Proteins
[0124] As explained above, P-gp is a tandemly duplicated molecule
made up of two halves with each encoding for six transmembrane
domains and an ATP binding domain. The two halves of P-gp are
linked by a linker domain. Of the 90 amino acids that make up the
linker domain, 32 amino acid are either positively or negatively
charged at physiological pH. While P-gp phosphorylation sites
appear to have relevance to P-gp function, the function of the
linker domain of P-gp remains unknown. To identify and dissect the
role of this domain in MDR, the overlapping peptides method of the
present invention was used. A novel approach was developed to
isolate interacting proteins using overlapping synthetic
hexapeptides. The use of overlapping peptides to isolate
interacting proteins allows the specific identification of
interacting proteins and bypasses many of the problems associated
with the use of random peptides. FIG. 5 shows the amino acid
sequences of the linker domain of HP-gp 1 and HP-gp 3. The two
linker domains of HP-gp1 and HP-gp3 share 41% amino acid sequence
identity and 66% sequence homology. Overlapping hexapeptides were
synthesized in parallel on derivatized polypropylene rods as
previously described (36, 37). 92 and 90 hexapeptides were
synthesized to cover the entire linker sequence of HP-gp1 and
HP-gp3, respectively. The hexapeptides remain covalently attached
to the polypropylene rods.
[0125] To identify the interacting proteins with the various
hexapeptides of the linker domains, the peptide-fixed rods were
incubated with total cell lysate from [.sup.35S] methionine
metabolically labeled CEM or CEM/VLB.sup.1.0 cells. After washing
off non-specifically binding lysate proteins, the specifically
bound proteins were eluted with SDS containing buffers and resolved
by SDS-PAGE. FIG. 6 shows the proteins specifically bound to the 92
overlapping hexa-peptides from HP-gp1 linker sequence. Three
regions in HP-gp1 linker domain (.sup.617EKGIYFKLVTM.sup.627 (SEQ
ID NO: 1), .sup.657SRSSLIRKRSTRRSVRGSQA.sup.676 (SEQ ID NO: 2) and
.sup.693PVSFWRIMKLNLT.sup.705 SEQ ID NO: 3) bound a 57 kDa protein.
The hexapeptides numbers 46-60, 81-89 and 5-9 (see FIG. 5) bound
with decreasing affinities to the 57 kDa protein (FIG. 6).
Moreover, peptides 46-60 showed binding to two other proteins with
apparent molecular masses of 80 kDa and 30 kDa, however much weaker
than that of the 57 kDa protein. It is likely that the latter
proteins (80 kDa and 30 kDa) are associated with the 57 kDa, since
these proteins are detected when the intensity of the 57 kDa
protein signal is high (FIG. 6, peptides 50-56). Comparison of the
amino acid sequences of the three 57 kDa binding proteins did not
reveal significant sequence homology among them to account for
their binding to the same protein. Interestingly, however, the
amino acid sequence of the second region (peptides 46-60) encodes
for protein kinase C consensus sequences (15). In addition, the
third region (peptides 81-89) was also shown to encode for a
protein kinase A site (43).
[0126] To determine the affinity of binding between the sequences
of the hexapeptides and the 57 kDa protein, it was of interest to
determine the effects of high salt (0.3-2.4 M KCl), zwitterionic
detergent (10-160 mM CHAPS) and ionic detergents (0.1%-2% SDS) on
the interactions between the hexapeptides encoded by
.sup.657SRSSLIRKRSTRRSVRGSQA.sup.676 (SEQ ID NO: 2) and the 57 kDa
protein. Our results show the binding to be stable to high salt,
moderately stable to high concentrations of CHAPS, but sensitive to
low concentrations of SDS (FIG. 7). Given the stability of protein
binding to covalently attached peptides, in the presence of 10 mM
CHAPS, it was of interest to determine the binding of the
hexapeptides from HP-gp1 linker domain to CHAPS soluble proteins
that could include integral membrane proteins. The results in FIG.
8 show bound proteins to the same overlapping hexapeptides that
codes for the linker domain of HP-gp1. Although the hexapeptides
numbers 46-60, 81-89 and 5-9 (see FIG. 5) bound to the 57 kDa
protein (FIG. 7); other proteins were found to interact with the
same or different hexapeptides which did not bind proteins in the
absence of 10 mM CHAPS. For example, hexapeptides 3-10 bound to
.about.210 kDa protein that was not detected previously in the
absence of CHAPS. Similarly, hexapeptides 16-20, which did not bind
any proteins in the absence of CHAPS, bound to the same high
molecular weight protein (FIG. 7). Peptides 40-60 bound more
strongly to several low molecule weight proteins (.about.45-25 kDa)
in the presence of CHAPS. The hexapeptides 80-89 bound to two other
proteins in addition to the 57 kDa protein. Taken together, the
results in FIG. 8 demonstrate that the binding between the various
hexapeptides to the 57 kDa protein is resistant to mild
zwitterionic detergents such as CHAPS. Moreover, the solubilization
of membrane proteins in 10 mM CHAPS show binding to other proteins
not seen in the absence or 10 mM CHAPS. One possibility is that 10
mM CHAPS allows integral membrane proteins to interact with the
various hexapeptides of HP-gp 1 linker domain. Alternatively, CHAPS
exposes new domains that in turn allows for binding to hexapeptides
of HP-gp1 linker domain. In addition, some of the lower molecular
weight proteins that bound to hexapeptides 40-60 and 80-89 may be
degradation products of the 57 kDa protein (FIG. 8).
[0127] The P-gp gene family in man is encoded by two isoforms,
HP-gp 1 and HP-gp 3 (or mdr 1 and mdr 3; (20)). However, as
indicated earlier, only HP-gp 1 confers an MDR phenotype. Moreover,
although HP-gp 1 and 3 share about 80% amino acid sequence homology
(111); the linker domain is the most variable domain among the two
isoforms with 66% amino acid sequence homology. To determine if the
HP-gp 3 linker domain binds to the same or different proteins,
overlapping hexapeptides encoding HP-gp 3 linker domain were
synthesized on polypropylene rods and their binding to soluble
proteins was examined as indicated above. FIG. 9 shows the profile
of binding proteins to the hexapeptides of HP-gp 3. Interestingly,
a similar molecular weight protein (57 kDa) also bound to the
hexapeptides from HP-gp 3. However, the binding to some
hexapeptides was different from that seen with HP-gp 1 (FIG. 6
versus FIG. 9). For HP-gp 3, three larger stretches of amino acids
(.sup.618LMKKEGVYFKLVNM.sup.631 (SEQ ID NO: 4),
.sup.648KAATRMAPNGWKSRLFRHSTQKNLKNS.sup.674 (SEQ ID NO: 5) and
.sup.695PVSFLKVLKLNKT.sup.707 (SEQ ID NO: 6) bound to the 57 kDa
protein. The first and third regions of HP-gp 3 linker domain share
considerable sequence identity with the first and third regions of
HP-gp 1 linker domain (FIG. 10). Hence, it is not surprising that
the same hexapeptides bound to the same protein. The second region
of HP-gp 1 and HP-gp 3 linker domains are different (FIG. 10).
Consequently, although both the HP-gp1 and HP-gp3 sequences bound
to a 57 kDa, the region of interaction between HP-gp 3 and the 57
kDa protein is larger than that of HP-gp 1 (FIG. 6 and FIG. 9). A
comparison of the amino acid sequences from HP-gp 1 and HP-gp 3
binding hexapeptides is shown in FIG. 10.
Example 9
Purification and Sequencing of the 57 kDa Protein
[0128] To determine the identity of the 57 kDa proteins, several
copies of two hexapeptides (.sup.658RSSLIR.sup.663 (SEQ ID NO: 7)
and .sup.669SVRGSQ.sup.671 (SEQ ID NO: 8) from the second region of
HP-gp 1 linker domain were synthesized. The latter hexapeptide
sequences were those that bound with the highest affinity to the 57
kDa protein. FIG. 11 shows the binding of these two peptides to
total cell lysate from [.sup.35S] methionine metabolically labeled
cells. Both hexapeptides bound specifically to the 57 kDa protein
and another protein of an apparent molecular mass of .about.41 kDa.
Interestingly, longer incubation times of the total cell lysate led
to an increase in the level of the 41 kDa protein (FIG. 11). Thus,
the 41 kDa band is likely a degradation product of the 57 kDa
protein.
[0129] To purify the 57 kDa protein using the two hexapeptides, it
was of interest to determine if other carrier proteins than BSA can
be used. FIG. 12 shows the effects of no blocking carrier, 1%
gelatin and 0.3% or 3% BSA on the binding of the hexapeptides to
the 57 kDa protein. The results of this experiment were surprising
in that no carrier protein was required to reduce the unspecific
binding (FIG. 12). The latter established binding conditions were
used to isolate large amounts of 57 kDa protein that bound to
several copies of hexapeptides .sup.658RSSLIR.sup.663 (SEQ ID NO:
7) and .sup.669SVRGSQ.sup.614 (SEQ ID NO: 8). FIG. 13 shows
purified 57 kDa protein on SDS-PAGE stained with Coomassie blue.
The latter purified protein was transferred to PVDF membrane and
stained with Ponceau S to localize the position of the 57 kDa
protein. The Ponceau S-stained band that migrated with the expected
molecular mass was cut out and used for direct N-terminal
sequencing (33). The first seven rounds of Edman degradation showed
two sequences of MREVISI (SEQ ID NO: 10) and MREIVHI (SEQ ID NO:
11). These two sequences differed only by three amino acids (VIS
instead of IVH). Comparison of the two sequence with known protein
sequences using FastA protein search engine, showed the latter
sequences to encode the first seven N-terminal amino acids of
.alpha.- and .beta.-tubulins. The identification of tubulins, as
the 57 kDa protein was consistent with the apparent molecular mass
and the potential degradation products that were observed following
long incubation periods. To further confirm the identity of the 57
kDa protein as tubulins, Western blot analysis was preformed on
hexapeptide-bound 57 kDa protein and total cell lysate resolved by
SDS-PAGE and transferred to nitrocellulose membrane. The
nitrocellulose membrane was then probed with anti .alpha.-tubulin
and anti-.beta.-tubulin monoclonal antibodies, respectively. FIG.
14 shows the results of the Western blot analysis. Consistent with
the sequencing results, both tubulin subunits (.alpha. and .beta.)
were recognized in the lanes containing the hexapeptide bound
proteins. Thus, establishing the identity of the 57 kDa protein as
.alpha. and .beta.-tubulin.
Example 10
[0130] The power of the overlapping peptide spanning method
invention was thus validated with P-gp. As shown above, the
overlapping peptide-based method of the present invention provides
the proof of principle to the hypothesis which states that the
region between two interacting proteins consists of high affinity
binding sequences and repulsive sequences as well as the fact that
such a method can be used efficiently and successfully to identify
and characterize domains and sequences of interacting proteins. The
balance of high affinity and repulsive forces determine whether two
proteins will form stable complex. The use of short overlapping
peptides allows the identification of such high affinity binding
sequences between bait and prey proteins. The rationale for using
short overlapping peptides to isolate high affinity binding
sequences is essential to the success and efficiency of the proof
of the principle described herein. For instance, larger peptides
could contain both high affinity and repulsive binding sequences in
one peptide sequence such that the net force of interaction is
negative. Moreover, the use of overlapping peptides that differ by
one amino acid from the previous or next peptide reduces the
possibility of unspecific binding. Thus, overlapping peptides often
demonstrate a peak in the binding affinity of various peptides (see
FIGS. 7 and 4). The skilled artisan will understand that longer
overlapping peptides could also be used. Unfortunately, such larger
peptides increase the risk of missing the identification of
interacting proteins due to a change in the balance between high
affinity and repulsive amino acids.
[0131] The binding of 57 kDa protein to three different regions in
HP-gp1 and HP-gp3 linker domains is consistent with the herein
proposed hypothesis to explain protein interactions (see principle
of protein-protein interactions). The high affinity binding domains
vary in sizes from 10-26 amino acids in length. In the case of
HP-gp1 and HP-gp3 linker domains, two of the three high affinity
binding domains shared considerable sequence identity. The third
high affinity binding region of the linker domains
(.sup.658SRSSLIRKRSTRRSVRGSQA.sup.677 (SEQ ID NO: 2) versus
.sup.648KAATRMAPNGWKSRLFRHSTQKNLKNS.sup.674 (SEQ ID NO: 5)) shared
no homology in their primary amino acid sequence. However, helical
wheel presentation of these two domains show a cluster of
positively charged residues on one face of the helix while a
cluster of serine/threonine residues on the other side (see FIG.
15). Interestingly, the region of highest binding affinity to the
57 kDa protein encodes the three putative phosphorylation sites in
HP-gp 1 (15). The positions of the phosphorylation sites in HP-gp3
have not been determined experimentally, however they encode for
the consensus sequence of protein kinase C. In this respect, it is
possible that HP-gp1 and HP-gp3 interactions at the linker domains
is modulated by phosphorylation of this domain. Thus, although
mutations of P-gp phosphorylation sites within the linker domain
were shown not to affect its drug transport function (40), other
proposed functions of HP-gp1 (e.g., regulator of endogenous
chloride channel) was shown to be affected by its phosphorylation
state (41, 110). Indeed, a member of the ABC transporters, CFTR
(the cystic fibrosis transmembrane conductance regulator), which
encodes a similar linker domain was found to co-localize with the
microtubule network (107). Furthermore, microtubule-dependent acute
recruitment of CFTR to the apical plasma membrane of T84 cells was
responsive to elevations in intracellular cAMP and phosphorylation
of the linker domain (107). Taken together, although it is not
clear if phosphorylation plays a role in modulating P-gp functions
in a tubulin dependent manner, given the co-localization of HP-gp1
phosphorylation and binding to tubulin, such a possibility is
likely. Work is in progress to determine if phosphorylated
hexapeptides bind to tubulin using the assay described herein.
Thus, the present invention opens the door to the validation of a
physiologically relevant interaction between proteinaceous
domains.
[0132] The possibility that the 57 kDa protein binds to the
polypropylene rods or their derivatized moieties is unlikely since
all other rods which are similarly derivatized did not bind the 57
kDa protein. Moreover, hexapeptides synthesized on at least four
different times bound to the same proteins. Finally, hexapeptides
encoding the first and third high affinity binding regions of the
linker domains of HP-gp1 and HP-gp3 bound to the 57 kDa protein. In
addition to the 57 kDa protein, other proteins with apparent
molecular masses of .about.80 kDa and 30 kDa also bound to some of
the hexapeptides in the linker domains. However, the binding of
these proteins was much weaker than the 57 kDa and maybe associated
proteins. Although direct measurements of binding affinities
between the various hexapeptides and the 57 kDa protein have not
been done, it is interesting that this interaction is resistant to
10 mM CHAPS and high salt. Moreover, the presence of 10 mM CHAPS in
the incubation mix lead to the binding of other proteins (most
notably the .about.210 kDa protein) to several stretches of
hexapeptides which did not bind in the absence of 10 mM CHAPS. The
binding of the latter proteins to the hexapeptides 15-28 are likely
due to the extraction of proteins from the membranous material
which were excluded in the absence of CHAPS. In absence of CHAPS,
the cell lysate contained soluble proteins and membrane associated
proteins only.
[0133] The physiological significance of HP-gp1 or HP-gp3 binding
to tubulin is not clear. However, tubulin has been shown to
interact with several membrane proteins (42, 50, 81, 86). HP-gp1 or
HP-gp3 interactions with tubulin and possibly microtubules maybe an
example of the membrane-skeleton fence model (56). In this model, a
small fraction of membrane receptors seem to be fixed to the
underlying cytoskeleton (95). It is interesting in this respect
that increase in the stability and expression of P-gp in rat liver
tumors in vivo are associated with similar increases in the
stability of several cytoskeleton proteins, including
.alpha.-tubulin, .beta.-actin, and cytokeratins 8/18 (64). Work is
in progress to determine the functional significance of P-gp
interactions with tubulin in vivo.
Example 11
The Overlapping Peptides Spanning Method is not Limited to
Pgp-Interacting Proteins
[0134] The overlapping peptide approach of the present invention
has been further validated with Annexin I, a soluble and membrane
associated protein, as opposed to P-glycoprotein, a strictly
transmembrane protein. Annexin is thus structurally and
functionally different from P-glycoprotein.
[0135] Using this approach, several proteins that interact with
Annexin I, and the precise amino acid sequences of Annexin I which
mediate these interactions, were identified. Annexin I is a member
of a large family of intracellular soluble and membrane associated
proteins that bind phospholipids in a reversible and
calcium-dependent manner. Various members of the Annexin family
have been implicated in a number of different intracellular
processes including vesicular trafficking, membrane fusion
exocytosis, signal transduction, and ion channel formation and drug
resistance. Given the many possible physiological functions of
Annexin I, the method of the present invention was set out to
identify its interacting proteins and the precise amino acid
sequences that mediate Annexin I interactions thereto.
[0136] Briefly, as described earlier, overlapping peptides
corresponding to the entire amino acid sequence of Annexin I (total
of .about.340 peptides plus controls) were synthesized on a solid
support as described above. In this case, overlapping
heptapeptides, as opposed to hexapeptides were used. The peptides
were then incubated with total cellular proteins isolated from MCF7
breast tumor cells that were metabolically labeled with [.sup.35S]
methionine. Following several washes, the bound proteins were
eluted and resolved on SDS-PAGE as outlined above. The results are
consistent with previous results with P-glycoprotein, as the method
leads to the identification of several islands of Annexin I amino
acid sequences (data not shown) which interacted with five proteins
ranging in molecular masses from 10 kDa to 200 kDa (specifically,
.about.10 kDa; .about.29 kDa; .about.85 kDa; .about.106 kDa and
.about.200 kDa). Briefly, eight interacting domains having high
affinity for the cellular proteins of the extract were identified.
Two of these high affinity islands were located in the tail domain
of Annexin (residues 1-36) and six in the .alpha. helical bundles
of Annexin I (residues 37-to the end; see for example WO 99/21980).
The identity of the latter interacting proteins is presently under
study. However, the interaction of a 10 kDa protein with Annexin I
is consistent with earlier works which demonstrated a direct
interaction between Annexin I and S100C protein (70).
[0137] Thus, the present invention is shown to enable the simple
and efficient identification of high affinity protein interaction
as well as enabling the simultaneous identification of the precise
amino acid sequence of at least one of the interacting
partners.
CONCLUSIONS
[0138] In conclusion, a simple approach to identify P-gp
interacting proteins from a total cell lysate has been used.
Moreover, this approach allows for the identification of the
precise amino acid sequences in P-gp1 and P-gp3 linker domains that
mediate the protein interactions with tubulins. In addition,
knowledge of the high affinity binding sequences allow for the
subsequent purification of the interacting proteins from a total
mixture of cellular proteins, as further exemplified with Annexin
I. Indeed, given the simplicity of this approach to study
protein-protein interactions, it is easily applied to other
proteins. Finally, our approach is rapid and has several advantages
over other currently used approaches.
[0139] Although the present invention has been described
hereinabove by way of preferred embodiments thereof, it can be
modified, without departing from the spirit and nature of the
subject invention as defined in the appended claims.
REFERENCES
[0140] 1. Adorini, L., 1993, Clin Exp Rheumatol 8:S41-44. [0141] 2.
Ahmad et al., 1994, Biochemistry 33:10313-10318. [0142] 3. Alba et
al., 1998, Electrophoresis 19:2407-2411. [0143] 4. Ausubel, et al.,
1994, Current Protocols in Molecular Biology, Wiley, New York.
[0144] 5. Bates et al., 1995, Cancer Chemotherapy &
Pharmacology. 35:457-463. [0145] 6. Bates et al., 1992,
Biochemistry 31:6366-6372. [0146] 7. Bates et al., 1993,
Biochemistry 37:9156-9164. [0147] 8. Beck, W. T., 1983, Cancer
Treat. Rep. 67:875-882. [0148] 9. Boscoboinik et al., 1990,
Biochimica et Biophysica Acta 1027:225-228. [0149] 10. Brown et
al., Nat. Genet. 1999 January; 21 (1 Suppl):33-7, Review. [0150]
11. Buschman et al., 1994, Cancer Research 54:4892-4898. [0151] 12.
Campbell, et al., 1984, Monoclonal Antibody Technology: Laboratory
Techniques in Biochemistry and Molecular Biology, Elsevier Science
(Publ.), Amsterdam, The Netherlands. [0152] 13. Chambers et al.,
1990, Biochemical and Biophysical Research Communications
169:253-259. [0153] 14. Chambers et al., 1994, Biochemical Journal
299:309-315. [0154] 15. Chambers et al., 1993, The Journal of
Biological Chemistry 268:4592-4595. [0155] 16. Chan et al., 1995,
Hematology--Oncology Clinics of North America 9:275-318. [0156] 17.
Chan et al., 1991, New England Journal of Medicine 325:1608-1614.
[0157] 18. Chen et al., 1997, Curr Opin Chem Biol 1:458-466. [0158]
19. Cheung et al., Nat. Genet. 1999 January; 21 (1 Suppl): 15-9,
Review. [0159] 20. Childs et al., 1994, Important Adv Oncol, 21-36.
[0160] 21. Cole et al., 1996, Cancer Treatment & Research
87:39-62. [0161] 22. Cornelissen et al., 1994, Journal of Clinical
Oncology 12:115-119. [0162] 23. Dalton et al., 1995, Cancer
75:815-820. [0163] 24. Debouck et al., Nat. Genet. 1999 January; 21
(1 Suppl):48-50, Review. [0164] 25. Devault et al., 1990, Molecular
and Cellular Biology 10: 1652-1663. [0165] 26. Duggan et al., Nat.
Genet. 1999 January; 21 (1 Suppl): 10-4, Review 27. Edman et al.,
1967, Eur J Biochem 1:80-91. [0166] 28. Ehrmann et al, 1997,
Neoplasma 44:299-304. [0167] 29. Felder et al., 1993, Mol Cell Biol
13:1449-1455. [0168] 30. Fields et al., 1994, Trends Genet.
10:286-292. [0169] 31. Fine et al., 1988, Proceedings of the
National Academy of Science USA 85:582-586. [0170] 32. Fitscher et
al., 1993, Analytical Biochemistry 213:414-421. [0171] 33. Flynn et
al., 1983, Biochem Biophys Res Commun 117:859-65. [0172] 34. Ford
et al., 1990, Pharmacological Reviews 42:155-199. [0173] 35.
Futscher et al, 1993, Analytical Biochemistry 213:414-421. [0174]
36. Georges et al., 1993, The Journal of Biological Chemistry
268:1792-1798. [0175] 37. Georges et al., 1990, Proceedings of the
National Academy of Science USA 87:152-156. [0176] 38. Georges et
al., 1990, Advances in Pharmacology 21:185-220. [0177] 39. Georges
et al., 1991, Journal of Cellular Physiology 148:479-484. [0178]
40. Germann et al., 1996, J Biol Chem 271:1708-16. [0179] 41. Gill
et al., 1992, Cell 71:23-32. [0180] 42. Giustetto et al., J Comp
Neurol 395:231-244. [0181] 43. Glavy et al., 1997, J Biol Chem
272:5909-5914. [0182] 44. Goldstein, L. J., 1995, Curr Probl Cancer
19:65-124. [0183] 45. Gottesman et al., 1995, Annu Rev Genet.
29:607-649. [0184] 46. Gottesman et al., 1993, Annual Review of
Biochemistry 62:385-427. [0185] 47. Grogan et al., 1990, Laboratory
Investigation 63:815-824. [0186] 48. Gros et al., 1986, Cell
47:371-380. [0187] 49. Gupta, S., 1996, "P-glycoprotein Expression
in Normal Hematopoietic Progenitors and Cells of the Immune System"
in Multidrug Resistance in Cancer Cells: Molecular, Biochemical,
Physiological and Biological Aspects. Editors: Gupta, S, and
Tsuruo, T., John Wiley & Sons, NY., 293-302. [0188] 50. Haga et
al., 1988, Eur J Biochem 255:363-368. [0189] 51. Hardy et al.,
1995, The EMBO Journal 14:68-75. [0190] 52. Heldin, C. H., 1995,
Cell 80:213-223. [0191] 53. Herweijer et al., 1990, Journal of the
National Cancer Institute 82:1133-1140. [0192] 54. Higgins, C. F.,
1992, Annual Review of Cell Biology 8:67-113. [0193] 55. Hoogenboom
et al., 1998, Immunotechnology 4:1-20. [0194] 56. Jacobson et al.,
1995, Sciences 268:1441-1442. [0195] 57. Jolliet-Riant et al.,
1999, Fundam Clin Pharmacol 13:16-26. [0196] 58. Kast et al., 1997,
J. Biological Chemistry 272:26479-26487. [0197] 59. Klemm et al.,
1998, Annu Rev Immunol 16:569-592. [0198] 60. Klotz et al., 1975,
In H. Neurath and R. L. Hill (ed.), The Proteins. Academic Press,
Inc. New York:293-411. [0199] 61. Kuriyan et al., 1997, Annu Rev
Biophys Biomol Struct 26:259-288. [0200] 62. Laemmli, U. K., 1970,
Nature 227:680-685. [0201] 63. Landschulz et al., 1988, Science
240:1759-1764. [0202] 64. Lee et al., 1998, J Cell Physiol
177:1-12. [0203] 65. Li et al., 1993, Nature 363:85-88. [0204] 66.
Ling, V., 1997, Cancer Chemother Pharmacol 40:Suppl:S3-8. [0205]
67. List et al., 1993, Journal of Clinical Oncology 11:1652-1660.
[0206] 68. Loo et al., 1995, The Journal of Biological Chemistry
270:843-848. [0207] 69. Lowry et al., 1951, Journal of Biological
Chemistry 193:265-275. [0208] 70. Mailliard, W. S., et al., 1996,
The Journal of Biological Chemistry, 271:719-725. [0209] 71.
Martini et al., 1998, Curr Opin Neurol 11:545-556. [0210] 72. McCoy
et al., 1997, EMBO J. 16:6230-6236. [0211] 73. Miller, et al.,
1988, Ann. Reports Med. Chem. 23:295. [0212] 74. Molina et al.,
1996, Pept Res 9:151-155. [0213] 75. Morgan, et al., 1987, Nucleic
Acids Research, 14:5019. [0214] 76. Naito et al., 1992, Biochemical
and Biophysical Research Communications 185:284-290. [0215] 77.
Nare et al., 1994, Biochemical Pharmacology 48:2215-2222. [0216]
78. Nooter et al., 1994, Leukemia Research 18:233-243. [0217] 79.
O'Brien et al., 1996, "P-glycoprotein Expression in Normal Human
Tissues," in Multidrug Resistance in Cancer Cells: Molecular,
Biochemical, Physiological and Biological Aspects. Editors: Gupta,
S, and Tsuruo, T., John Wiley & Sons, NY., 285-292. [0218] 80.
Pawson et al., 1992, Cell 71:359-362. [0219] 81. Perrot-Applanat et
al, 1995, J Cell Sci 108:2037-2051. [0220] 82. Phizicky et al.,
1995, Microbiological Reviews 59:94-123. [0221] 83. Porpaczy et
al., 1983, Biochem. Biophysica. Acta. 749:172-179. [0222] 84.
Prelich et al., 1989, Nature 326:517-520. [0223] 85. Ramsay et al.,
Nat. Biotechnol. 1998 January; 16(1):40-4, Review. [0224] 86.
Ravindra, R., 1997, Endocrine 7:127-143. [0225] 87. Reed et al.,
1996, J Cell Biochem 60: 23-32. [0226] 88. Remington,
Pharmaceutical Science, 16.sup.th Edition, Mack, Ed. [0227] 89. Ro
et al., 1990, Human Pathology. 21:787-791. [0228] 90. Roninson et
al., 1986, Proceedings of the National Academy of Sciences USA
83:4538-4542. [0229] 91. Rosenberg et al., 1997, Journal of
Biological Chemistry. 272:10685-10694. [0230] 92. Ruetz et al.,
1994, The Journal of Biological Chemistry 269:12277-12284. [0231]
93. Safa et al., 1986, The Journal of Biological Chemistry
261:6137-6140. [0232] 94. Safa, A. R., 1993, Cancer Investigation
11:46-56. [0233] 95. Sako et al., 1995, J Cell Biol 129:1559-1574.
[0234] 96. Sambrook, et al., 1989, Molecular Cloning--A Laboratory
Manual, Cold Spring Harbor Laboratories. [0235] 97. Schena et al.,
Trends Biotechnol. 1998 July; 16(7):301-6, Review [0236] 98.
Schinkel et al., 1991, Cancer Research 51:2628-2635. [0237] 99.
Schinkel et al., 1994, Cell 77:491-502. [0238] 100. Smit et al.,
1993, Cell 75:451-462. [0239] 101. Sonneveld et al., 1994, Journal
Clinical Oncology 12:1584-91. [0240] 102. Stanfield et al., 1995,
Curr Opin Struct Biol 5:103-113. [0241] 103. Stefanou et al., 1998,
Anticancer Res 18:4673-4681. [0242] 104. Steitz et al., 1977, J
Biol Chem 252:4494-4500. [0243] 105. Stevenson et al., 1998, Anal
Biochem 262:99-109. [0244] 106. Susskind et al., 1983, In R. W.
Hendrix, J. W. Roberts, F. W. Stahl, and R. A. Weisberg (ed.),
Lambda II. Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y.:347-363. [0245] 107. Tousson et al., 1996, J Cell Sci
109:1325-34. [0246] 108. Towbin et al., 1979, Proceedings of the
National Academy of Sciences of the United States of America
76:4350-4354. [0247] 109. Tschesche et al., 1975, Eur J Biochem
58:439-451. [0248] 110. Valverde et al., 1992, Nature 355:830-833.
[0249] 111. Van der Bliek et al., 1987, The EMBO Journal
6:3325-3331. [0250] 112. Venter et al., 1998, Science
280:1540-1542. [0251] 113. Verrelle et al., 1991, Journal of the
National Cancer Institute 83:111-116. [0252] 114. Vincent et al.,
1972, Biochemistry 11:2967-2977. [0253] 115. Watanabe et al., 1995,
Acta Oncologica 34:235-241. [0254] 116. Weinstein et al, 1990,
Human Pathology. 21:34-48. [0255] 117. Wigler, P. W., 1996, J
Bioenerg Biomembr 28:279-84. [0256] 118. Wilson et al., 1995,
Methods Enzymol 250:79-91. [0257] 119. EP-A-0818467 [0258] 120. WO
98 15833A [0259] 121. WO 84 03564A
Sequence CWU 1
1
15 1 11 PRT Homo sapiens 1 Glu Lys Gly Ile Tyr Phe Lys Leu Val Thr
Met 1 5 10 2 20 PRT Homo sapiens 2 Ser Arg Ser Ser Leu Ile Arg Lys
Arg Ser Thr Arg Arg Ser Val Arg 1 5 10 15 Gly Ser Gln Ala 20 3 13
PRT Homo sapiens 3 Pro Val Ser Phe Trp Arg Ile Met Lys Leu Asn Leu
Thr 1 5 10 4 14 PRT Homo sapiens 4 Leu Met Lys Lys Glu Gly Val Tyr
Phe Lys Leu Val Asn Met 1 5 10 5 27 PRT Homo sapiens 5 Lys Ala Ala
Thr Arg Met Ala Pro Asn Gly Trp Lys Ser Arg Leu Phe 1 5 10 15 Arg
His Ser Thr Gln Lys Asn Leu Lys Asn Ser 20 25 6 13 PRT Homo sapiens
6 Pro Val Ser Phe Leu Lys Val Leu Lys Leu Asn Lys Thr 1 5 10 7 6
PRT Homo sapiens 7 Arg Ser Ser Leu Ile Arg 1 5 8 6 PRT Homo sapiens
8 Ser Val Arg Gly Ser Gln 1 5 9 4 PRT Homo sapiens 9 Tyr Glu Glu
Ile 1 10 7 PRT Homo sapiens 10 Met Arg Glu Val Ile Ser Ile 1 5 11 7
PRT Homo sapiens 11 Met Arg Glu Ile Val His Ile 1 5 12 18 PRT Homo
sapiens 12 Ser Arg Ser Ser Leu Ile Arg Lys Arg Ser Thr Arg Arg Ser
Val Arg 1 5 10 15 Gly Ser 13 17 PRT Homo sapiens 13 Asn Gly Trp Lys
Ser Arg Leu Phe Arg His Ser Thr Gln Lys Asn Leu 1 5 10 15 Lys 14 93
PRT Homo sapiens 14 Leu Met Lys Lys Glu Gly Val Tyr Phe Lys Leu Val
Asn Met Gln Thr 1 5 10 15 Ser Gly Ser Gln Ile Gln Ser Glu Glu Phe
Glu Leu Asn Asp Glu Lys 20 25 30 Ala Ala Thr Arg Met Ala Pro Asn
Gly Trp Lys Ser Arg Leu Phe Arg 35 40 45 His Ser Thr Gln Lys Asn
Leu Lys Asn Ser Gln Met Cys Gln Lys Ser 50 55 60 Leu Asp Val Glu
Thr Asp Gly Leu Glu Ala Asn Val Pro Pro Val Ser 65 70 75 80 Phe Leu
Lys Val Leu Lys Leu Asn Lys Thr Glu Trp Pro 85 90 15 95 PRT Homo
sapiens 15 Leu Met Lys Glu Lys Gly Ile Tyr Phe Lys Leu Val Thr Met
Gln Thr 1 5 10 15 Ala Gly Asn Glu Val Glu Leu Glu Asn Ala Ala Asp
Glu Ser Lys Ser 20 25 30 Glu Ile Asp Ala Leu Glu Met Ser Ser Asn
Asp Ser Arg Ser Ser Leu 35 40 45 Ile Arg Lys Arg Ser Thr Arg Arg
Ser Val Arg Gly Ser Gln Ala Gln 50 55 60 Asp Arg Lys Leu Ser Thr
Lys Glu Ala Leu Asp Glu Ser Ile Pro Pro 65 70 75 80 Val Ser Phe Trp
Arg Ile Met Lys Leu Asn Leu Thr Glu Trp Pro 85 90 95
* * * * *