U.S. patent application number 13/009228 was filed with the patent office on 2012-06-07 for novel bak binding protein, dna encoding the protein, and methods of use thereof.
This patent application is currently assigned to GENENTECH, INC.. Invention is credited to Philip J. Barr, Paul A. Fitzpatrick, Helen L. Gibson, Michael C. Kiefer.
Application Number | 20120142099 13/009228 |
Document ID | / |
Family ID | 26718025 |
Filed Date | 2012-06-07 |
United States Patent
Application |
20120142099 |
Kind Code |
A1 |
Kiefer; Michael C. ; et
al. |
June 7, 2012 |
NOVEL BAK BINDING PROTEIN, DNA ENCODING THE PROTEIN, AND METHODS OF
USE THEREOF
Abstract
The present invention provides polynucleotide sequences (bbp)
encoding a Bak Binding Protein (BBP) and fragments thereof that
bind to Bak. The invention also provides a BBP which binds to Bak.
The invention also provides recombinant host cells containing
polynucleotides encoding BBP. The invention further provides
antibodies that specifically bind to BBP. The invention further
provides methods for detecting agents such as drugs that alter the
binding of a BBP with a Bak protein. The invention further provides
methods for detecting the presence of bbp or BBP in a biological
sample, and further provides methods for modulating the levels of
BBP in a cell. This invention additionally encompasses novel
peptides, designated the "BBP Binding Domains" and the respective
nucleotides, designated "bbpbd-1" and "bbpbd-2" which are involved
in the interaction between Bak and BBP.
Inventors: |
Kiefer; Michael C.;
(Clayton, CA) ; Fitzpatrick; Paul A.; (Albany,
CA) ; Gibson; Helen L.; (Oakland, CA) ; Barr;
Philip J.; (Oakland, CA) |
Assignee: |
GENENTECH, INC.
South San Francisco
CA
|
Family ID: |
26718025 |
Appl. No.: |
13/009228 |
Filed: |
January 19, 2011 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11539859 |
Oct 9, 2006 |
7888038 |
|
|
13009228 |
|
|
|
|
10189294 |
Jul 1, 2002 |
7316915 |
|
|
11539859 |
|
|
|
|
09381488 |
Feb 11, 2000 |
6441135 |
|
|
PCT/US98/04079 |
Mar 3, 1998 |
|
|
|
10189294 |
|
|
|
|
60041328 |
Mar 20, 1997 |
|
|
|
60071097 |
Jan 9, 1998 |
|
|
|
Current U.S.
Class: |
435/375 |
Current CPC
Class: |
G01N 2510/00 20130101;
A61K 48/00 20130101; C07K 2319/00 20130101; G01N 2500/02 20130101;
C07K 14/47 20130101; G01N 2333/47 20130101; A61K 38/00
20130101 |
Class at
Publication: |
435/375 |
International
Class: |
C12N 5/02 20060101
C12N005/02 |
Claims
1-62. (canceled)
63. A method of modulating apoptosis-induced cell death comprising
modulating the level of BBP in a cell, wherein the method comprises
transfecting the cell with a recombinant nucleic acid molecule
comprising an isolated polynucleotide selected from the group
consisting of: a) a polynucleotide comprising a nucleotide sequence
encoding the polypeptide of SEQ ID NO:6; b) a polynucleotide
consisting of a nucleotide sequence encoding a fragment of SEQ ID
NO:6, wherein the fragment consists of at least six linear amino
acid residues of SEQ ID NO:6, and wherein the fragment binds to a
Bak protein; and c) a polynucleotide consisting of the full
complement of the nucleotide sequence of (a) or (b).
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent is a
continuation of U.S. patent application Ser. No. 11/539,859, filed
Oct. 9, 2006 which, is a continuation of U.S. patent application
Ser. No. 10/189,294, filed Jul. 1, 2002, which is a continuation of
U.S. patent application Ser. No. 09/381,488, filed Feb. 11, 2000,
now U.S. Pat. No. 6,441,135, which is a .sctn.371 of PCT
Application No. PCT US98/04079, filed Mar. 3, 1998, which claims
priority to U.S. Provisional Applications 60/041,328, filed Mar.
20, 1997 and 60/071,097, filed Jan. 9, 1998, each of which are
incorporated herein by this reference.
STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH
[0002] (Not Applicable)
FIELD OF THE INVENTION
[0003] The present invention relates to DNA encoding a protein that
binds to a protein involved in apoptosis. The invention further
relates to methods for identifying agents that modulate activity
levels of a protein binding to a protein involved in apoptosis. The
inventions additionally encompasses novel peptides, designated the
"BBP Binding Domains" which are involved in the interaction between
a protein involved in apoptosis and a protein that binds to it.
SUMMARY OF THE INVENTION
[0004] Substantially purified DNA encoding a novel Bak binding
protein, termed Bak Binding Protein, or BBP, is provided. The
substantially purified BBP protein and compositions thereof are
also provided. Diagnostic and therapeutic methods utilizing the DNA
and proteins are also provided. Methods of screening for
pharmaceutical agents that modify Bak and BBP activity levels are
also provided. The invention additionally encompasses novel
peptides, designated the "BBP Binding Domains" and novel
nucleotides, designated "bbpbd-1" and "bbpbd-2" encoding the
peptides, which are involved in the interaction between a protein
involved in apoptosis and a protein that binds to it.
[0005] The present invention encompasses an isolated polypeptide
comprising SEQ ID NO:7, isolated polypeptides comprising a linear
sequence of six or more amino acids of SEQ ID NO:7, isolated
polypeptides having at least one of the biological functions of the
polypeptide of SEQ ID NO:7, isolated polypeptides comprising a
fragment of SEQ ID NO:7, wherein said fragment binds to Bak protein
under appropriate conditions and fusion polypeptides comprising the
polypeptide of SEQ ID NO:7 or fragments thereof.
[0006] The present invention also encompasses isolated
polynucleotides comprising SEQ ID NO:6 and polynucleotide sequences
complementary thereto, isolated polynucleotides comprising a
fragment of at least 18 consecutive nucleotides of SEQ ID NO:6,
isolated polynucleotides encoding the polypeptide of SEQ ID NO:7,
isolated polynucleotides comprising a sequence that encodes a
polypeptide having at least one of the biological functions of the
polypeptide of SEQ ID NO:7 and a polynucleotide complementary
thereto, isolated polynucleotides comprising a fragment of SEQ ID
NO:6, wherein said fragment encodes a polypeptide that binds to Bak
protein under appropriate conditions, and any of the aforementioned
isolated polynucleotide, which are operably linked to control
sequences for expression.
[0007] Also encompassed by the present invention are recombinant
vectors comprising any of the aforementioned polynucleotides, as
well as recombinant host cells modified to contain the
polynucleotides, wherein he recombinant host cells specifically can
be bacterial or eukaryotic.
[0008] Also encompassed by the present invention are methods for
screening potential therapeutic agents that modulate the
interaction between Bak and BBP comprising the steps of: (a)
combining a Bak and a BBP under conditions in which they interact,
to form a test sample; (b) exposing the test sample to a potential
therapeutic agent and; (c) monitoring the interaction of the Bak
and the BBP; wherein a potential therapeutic agent is selected for
further study when it modifies the interaction compared to a
control test sample to which no potential therapeutic agent has
been added. In one embodiment, the potential therapeutic agent is
selected from the group consisting of a pharmaceutical agent, a
cytokine, a small molecule drug, a cell-permeable small molecule
drug, a hormone, a combination of interleukins, a lectin, a
stimulating agent, a bispecific antibody, a peptide mimetic, and an
antisense oligonucleotide. In another embodiment, the Bak is
selected from the group consisting of Bak, a fragment of Bak
sufficient to effect binding to a BBP, and a fusion protein
comprising a portion of Bak sufficient to effect binding to a BBP.
The fusion protein can comprise epitope-tagged Bak. In one
embodiment, the BBP is selected from the group consisting of
epitope-tagged BBP and proteins homologous to SEQ ID NO:7. In one
embodiment of the present invention, the monitoring step is
selected from the group consisting of co-precipitation, protein
interactive trapping and ELISA.
[0009] The present invention also encompasses compositions
comprising a monoclonal or polyclonal antibody or an
antigen-binding fragment thereof which forms a complex with a BBP
but is substantially unreactive with dissimilar proteins.
[0010] The present invention further encompasses a method of
detecting the presence of a BBP protein in a biological sample
comprising the steps of: a) obtaining a cell sample; b) exposing
the contents of the cells to antibodies; c) adding
anti-BBP-specific antibodies to the cell sample; d) maintaining the
cell sample under conditions that allow the antibodies to complex
with the BBP; and e) detecting the antibody-BBP complexes
formed.
[0011] In one embodiment, a method is provided for detecting the
expression of a bbp gene in a biological sample comprising the
steps of identifying the presence of RNA encoding the bbp. In one
embodiment, identification comprises Northern blotting.
[0012] Also encompassed by the present invention are methods
identifying bbp mRNA comprising the steps of: (a) obtaining a cell
sample; (b) obtaining RNA from the cell sample; (c) performing a
polymerase chain reaction on the RNA using primers corresponding to
unique regions of bbp; and (d) detecting the presence of products
of the polymerase chain reaction.
[0013] The present invention also provides methods of modulating
apoptosis-induced cell death comprising modulating the endogenous
levels of BBP. In a specific embodiment, the BBP levels are
increased or decreased by modulating expression of an endogenous
bbp gene. In one embodiment, the BBP is encoded by an endogenous
gene. Alternatively, the BBP is encoded by a recombinant gene,
wherein in a specific embodiment, expression of the recombinant
gene is under the control of an inducible promoter. In one
embodiment, the recombinant gene is transfected into cells ex vivo
and further comprising the steps of reintroducing the transfected
cells into an animal. Alternatively, the recombinant gene is
transfected into cells in vivo.
[0014] The present invention also encompasses methods of inducing
apoptosis in a patient in need thereof comprising administering a
therapeutically effective amount of the BBP.
[0015] The present invention additionally encompasses isolated
polypeptides comprising amino acids 138-156 of SEQ ID NO:2, or
derivatives thereof.
[0016] Also encompassed are isolated and purified peptides
comprising a BBP Binding Domain, isolated polypeptides comprising a
linear sequence of six or more amino acids of a BBP Binding Domain,
isolated polypeptides having at least one of the biological
functions of a BBP Binding Domain, or isolated polypeptides
comprising a fragment of a BBP Binding Domain wherein said fragment
binds to BBP protein under appropriate conditions. Also encompassed
are fusion polypeptides comprising a BBP Binding Domain or
fragments thereof.
[0017] The present invention further encompasses isolated
polynucleotides comprising nucleotides 507-578 of SEQ. ID NO:1, and
polynucleotide sequences complementary thereto, isolated
polynucleotides comprising a fragment of at least 18 consecutive
nucleotides of bbpbd-1, isolated polynucleotides comprising
nucleotides 611-668 of SEQ. ID NO:1, and polynucleotide sequences
complementary thereto, isolated polynucleotides comprising a
fragment of at least 18 consecutive nucleotides of bbpbd-2,
isolated polynucleotides encoding a BBP Binding Domain, isolated
polynucleotide comprising a sequence that encodes a polypeptide
having at least one of the biological functions of a BBP Binding
Domain and a polynucleotide complementary thereto and any of these
isolated polynucleotides which is operably linked to control
sequences for expression.
[0018] In one embodiment, a recombinant vector comprises these
polynucleotides. In a specific embodiment, recombinant host cells
are modified to contain the polynucleotides. In a specific
embodiment, these host cells are bacterial or eukaryotic.
[0019] Also encompassed by the present invention are methods of
modulating apoptosis-induced cell death comprising modulating the
endogenous levels of a BBP Binding Domain, wherein the BBP Binding
Domain levels can be increased or decreased by modulating
expression of an endogenous bak gene. In alternative embodiments,
the BBP Binding Domain is encoded by an endogenous gene or a
recombinant gene. In one embodiment, the expression of the
recombinant gene is under the control of an inducible promoter. In
alternative embodiments, the recombinant gene is transfected into
cells ex vivo and further comprising the steps of reintroducing the
transfected cells into an animal, or the recombinant gene is
transfected into cells in vivo.
[0020] Also encompassed by the present invention are methods of
modulating apoptosis in a patient in need thereof comprising
administering a therapeutically effective amount of a BBP Binding
Domain.
[0021] The present invention additionally encompasses isolated
polypeptides comprising amino acids 138-156 of SEQ ID No:2, or
derivatives thereof.
BACKGROUND
[0022] Apoptosis, or programmed cell death, is a normal physiologic
process that leads to individual cell death. This process of
programmed cell death is involved in a variety of normal and
pathogenic biological events and can be induced by a number of
unrelated stimuli. Changes in the biological regulation of
apoptosis also occur during aging and are responsible for many of
the conditions and diseases related to aging. Recent studies of
apoptosis have implied that a common metabolic pathway leading to
cell death can be initiated by a wide variety of signals, including
hormones, serum growth factor deprivation, chemotherapeutic agents,
ionizing radiation and infection by human immunodeficiency virus
(HIV). Wyllie (1980) Nature 284:555-556; Kanter et al. (1984)
Biochem. Biophys. Res. Commun. 118:392-399; Duke and Cohen (1986)
Lymphokine Res. 5:289-299; Tomei et al. (1988) Biochem. Biophys.
Res. Commun. 155:324-331; Kruman et al. (1991) J. Cell. Physiol.
148:267-273; Ameisen and Capron (1991) Immunology Today 12:102; and
Sheppard and Ascher (1992) J. AIDS 5:143. Agents that modulate the
biological control of apoptosis thus have therapeutic utility in a
wide variety of conditions.
[0023] Apoptotic cell death is characterized by cellular shrinkage,
chromatin condensation, cytoplasmic blebbing, increased membrane
permeability and interchromosomal DNA cleavage. Kerr et al. (1992)
FASEB J. 6:2450; and Cohen and Duke (1992) Ann. Rev. Immunol.
10:267.
[0024] While apoptosis is a normal cellular event, it can also be
induced by pathological conditions and a variety of injuries.
Apoptosis is involved in a wide variety of conditions including but
not limited to, cardiovascular disease; cancer regression; immune
disorders, including but not limited to systemic lupus
erythematosus; viral diseases; anemia; neurological disorders;
diabetes; hair loss; rejection of organ transplants; prostate
hypertrophy; obesity; ocular disorders; stress; aging; and
gastrointestinal disorders, including but not limited to, diarrhea
and dysentery. In the myocardium, apoptotic cell death follows
ischemia and reperfusion.
[0025] In Alzheimer's disease, Parkinson's disease, Huntington's
chorea, epilepsy, amyotrophic lateral sclerosis, stroke, ischemic
heart disease, spinal cord injury and many viral infections, for
example, abnormally high levels of cell death occur. In at least
some of these diseases, there is evidence that the excessive cell
death occurs through mechanisms consistent with apoptosis. Among
these are 1) spinal cord injury, where the severing of axons
deprives neurons of neurotrophic factors necessary to sustain
cellular viability; 2) stroke, where after an initial phase of
necrotic cell death due to ischemia, the rupture of dead cells
releases excitatory neurotransmitters such as glutamate and oxygen
free radicals that stimulate apoptosis in neighboring healthy
neurons; and 3) Human Immunodeficiency Virus (HIV) infection, which
induces apoptosis of T-lymphocytes.
[0026] In contrast, the level of apoptosis is decreased in cancer
cells, which allows the cancer cells to survive longer than their
normal cell counterparts. As a result of the increased number of
surviving cancer cells, the mass of a tumor can increase even if
the doubling time of the cancer cells does not increase.
Furthermore, the high level of expression in a cancer cell of the
bcl-2 gene, which is involved in regulating apoptosis and, in some
cases, necrotic cell death, renders the cancer cell relatively
resistant to chemotherapeutic agents and to radiation therapy.
[0027] In recent years, a family of proteins has been discovered
that controls apoptosis. The prototype of this family is Bcl-2, a
protein that inhibits most types of apoptotic cell death and is
thought to function by regulating an antioxidant pathway at sites
of free radical generation. Hockenbery et al. (1993) Cell
75:241-251. More recent data suggests that Bcl-2 can also function
as a channel protein and as an adaptor/docking protein. Reed, et
al. (1997) Nature 387:773-776. Together, the Bcl-2 family of
proteins are important intracellular modulators of apoptosis and
can be divided into two groups based on their effect on apoptosis.
Thus, in a general sense, Bcl-2, Bcl-X.sub.L, Mc1-1, BHRF-1 and
E1B19K are cell death inhibitors (anti-apoptotic), while Bak, Bax
and Bcl-x.sub.S accelerate cell death (pro-apoptotic).
[0028] Bcl-2 family members are generally localized to the outer
mitochondrial membrane, the nuclear membrane and the endoplasmic
reticulum, where they associate with membranes by virtue of their
C-terminal hydrophobic tail. All members of the family have two
highly conserved regions, called BH1 and BH2, that permit specific
interactions between two members to form stable dimers. Their
mechanism of action is presently unclear; however, it is known that
the ratio of anti-apoptotic to pro-apoptotic Bcl-2 family members
in a cell is critical to the cell's survival following initiation
of an apoptotic signal.
[0029] Proteins that interact with and alter the activity of Bcl-2
have been described. For example, BAG-1 binds Bcl-2, enhances the
anti-apoptotic effect of Bcl-2 and furthermore activates Raf-1.
Wang et al. (1996) Proc. Natl. Acad. Sci. USA 93:7063-7068;
Takayama et al. (1995) Cell 80:279-284. Other proteins with bcl-2
binding activity include the ras-related R-ras p23, BAP and Bad.
Fernandez-Sarabia and Bischoff (1993) Nature 366:274-275; U.S. Pat.
No. 5,539,085; U.S. Pat. No. 5,539,094; PCT Application WO
96/13614. Identification of such proteins is of great importance,
since an understanding of the protein-protein interactions in which
apoptosis-related proteins are involved can not only provide
insights into the mechanisms of action of these proteins, but can
also provide a focal point toward which apoptosis-modulating
therapies can be designed. For example, disruption of a
protein-protein interaction between an apoptosis-related protein
and a protein which enhances its function can decrease the level of
apoptosis in a cell. This may be a desired effect in a tissue which
displays an inappropriately high level of apoptosis.
[0030] Bak is a member of the Bcl-2 family and is expressed in
heart and other tissues. Bak protein is capable of either killing
cells, or actively protecting cells from cell death, depending on
how this protein interacts with other cellular proteins. Bcl-2
family members are extremely important in determining the fate of a
cell following an apoptotic signal, and Bak may be the most
important in the major organs such as heart. In the treatment of
heart disease, viral infection and cancer, modulation of the
interactions between proteins that control apoptosis is a major
focal point.
[0031] Accordingly, there is a need to identify the proteins that
operate to control cell death, and to develop therapeutic reagents
that modify the actions of those proteins. The present invention
relates to a novel Bak binding protein (BBP), the gene encoding the
novel protein, methods for detecting substances that alter the
specific binding between Bak and BBP, as well as diagnostic and
therapeutic methods utilizing BBP. The invention additionally
encompasses novel peptides, designated the "BBP Binding Domains"
and novel nucleotides, designated "bbpbd-1" and "bbpbd-2"
(collectively "bbpbd") encoding the peptides, which are involved in
the interaction between a protein involved in apoptosis and a
protein that binds to it.
[0032] All references cited herein are hereby incorporated by
reference in their entirety.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] FIG. 1 shows the strategy used to isolate BBP clones.
[0034] FIGS. 2A-2C depict the cDNA sequence (SEQ ID NO:1 and 3) of
bak and the translated amino acid sequence of Bak (SEQ ID
NO:2).
[0035] FIG. 3 depicts the Bak (SEQ ID NO:2) derivative proteins
Bak.DELTA.2-TM (SEQ ID NO:4) and Bak.DELTA.3 (SEQ ID NO:5). The
N-terminal residues are indicated by rightward arrows. The
C-terminus of Bak.DELTA.2-TM is indicated by the leftward
arrow.
[0036] FIG. 4 depicts the cDNA sequence (SEQ ID NO:6) of bbp and
the translated amino acid sequence (SEQ ID NO:7) of BBP.
[0037] FIG. 5 depicts the results of a Northern blot analysis of
multiple tissues with probes specific for BBP.
[0038] FIG. 6 summarizes the interactions of BBP with other
proteins.
[0039] FIG. 7 shows the results of Western blot analysis of the
interactions between BBP, BBP derivatives, BHRF-1. E1B19K and
Bak.
[0040] FIG. 8 shows the results of Western blot analysis of the
interactions between BBP-Loop1, BBP-Loop2 and the Bak derivatives
Bak 1-50 (FIG. 8A) and Bak.DELTA.3 (FIG. 8B).
[0041] FIG. 9 depicts the amino acid sequence of Bak and the
regions of Bak that interact with BBP loop 1. Each horizontal line
above the amino acid sequence shown indicate locations on the Bak
protein where BBP loop1 interacted with a 15 amino acid peptide
having the sequence shown directly below the respective line.
MODES OF CARRYING OUT THE INVENTION
[0042] The present invention encompasses substantially purified
nucleotide sequences encoding the novel Bak Binding Protein (BBP);
the protein encoded thereby; methods of screening potential
therapeutic agents that modify the interaction between BBP and
proteins that bind thereto; and methods for detecting substances
that alter the specific binding between Bak and BBP. The invention
additionally encompasses novel peptides, designated the "BBP
Binding Domains" which are involved in the interaction between a
protein involved in apoptosis and a protein that binds to it.
[0043] The following definitions are for the purpose of clarifying
the terms used herein, and are not meant to be limiting. Where
definitions or techniques are specified with reference to BBP or
bbp, the general teachings provided apply also to BBP Binding
Domains or bbpbd, respectively.
[0044] The present invention encompasses the polypeptide BBP and
modifications of BBP that retain at least one of the biological
functions of BBP. The present invention also encompasses the
polypeptides BBP Binding Domains and modifications of BBP Binding
Domains that retain at least one of the biological functions of BBP
Binding Domains. The terms "polypeptide", "peptide" or "protein"
are used interchangeably herein. By "polypeptide" is meant a linear
sequence of ten or more amino acids.
[0045] By "biologically effective amount" and "therapeutically
effective amount" is meant a concentration of the component able to
improve or ameliorate a condition related to apoptosis.
[0046] The term "BBP" encompasses polypeptide and polypeptide
fragments of BBP (SEQ ID NO:7) containing at least a portion of BBP
that binds to Bak protein or a fragment thereof, or has any other
biological or other useful function characteristic of BBP. The term
"BBP Binding Domains" encompasses polypeptides and polypeptide
fragments of Bak containing at least a portion of Bak protein that
binds to BBP or a fragment thereof, or has any other biological or
other useful function characteristic of BBP Binding Domains. A BBP
is defined primarily by its ability to associate in vitro and in
vivo with a Bak protein. A BBP Binding Domain is defined primarily
by its ability to associate in vitro and in vivo with BBP. As used
herein, "associate" or "interact" or "bind" means that a BBP and a
Bak protein have a binding affinity for each other such that the
BBP and a Bak protein or BBP Binding Domain form a bound complex.
The affinity of binding of a BBP and a Bak protein is sufficiently
specific such that the bound complex can form in vivo in a cell and
can form in vitro under appropriate conditions, as described
herein. The formation or dissociation of a bound complex can be
identified as described in the Examples or using other well known
methods.
[0047] The size of the BBP or BBP Binding Domain polypeptide
fragments can be only the minimum size required to provide a
desired function. It can optionally comprise additional amino acid
sequence, either native to BBP or BBP Binding Domain or from a
heterologous source, as desired. BBP or BBP Binding Domain
fragments may contain only ten consecutive amino acids from a BBP
or BBP Binding Domain amino acid sequence. Polypeptides comprising
ten amino acids, more preferably about 15 amino acids, more
preferably about 25 amino acids, more preferably about 50 amino
acids from the sequence are also included. Even more preferred are
polypeptides comprising the entire BBP or BBP Binding Domain amino
acid sequence.
[0048] The term "BBP" further encompasses a substantially purified
preparation of BBP (SEQ ID NO:7), or a fragment of BBP that is an
antigenic polypeptide containing from six to 151 amino acid
residues of BBP (preferably at least six, more preferably at least
12, and most preferably at least 18), which polypeptide fragment
contains an epitope of BBP such that an antibody raised against the
fragment (or against a conjugate of the polypeptide and a carrier,
including, but not limited to, keyhole limpet hemocyanin) forms an
immune complex with BBP itself.
[0049] The term "BBP Binding Domain" further encompasses a
substantially purified preparation of BBP Binding Domain, or a
fragment of BBP that is an antigenic polypeptide containing from
six to 23 amino acid residues of a BBP Binding Domain (preferably
at least six, more preferably at least 12, and most preferably at
least 18), which polypeptide fragment contains an epitope of a BBP
Binding Domain such that an antibody raised against the fragment
(or against a conjugate of the polypeptide and a carrier,
including, but not limited to, keyhole limpet hemocyanin) forms an
immune complex with a BBP Binding Domain itself.
[0050] Such antibodies can be either monoclonal or polyclonal, and
are generated by standard methods including the step of immunizing
an animal with an antigen containing an antigenic portion of BBP or
BBP Binding Domain.
[0051] The terms "BBP" and "BBP Binding Domains" also include
modified polypeptides that are functionally equivalent to BBP or
BBP Binding Domain, respectively, or fragments thereof.
Modifications include addition, insertion or deletion of amino
acids. Other examples of modified polypeptides include polypeptides
with conservative substitutions in comparison with the prototype
BBP or BBP Binding Domain sequence. Substitutions can include
changing or modifying one or more amino acids, and insertion of
heterologous amino acid sequences. Amino acid substitutions, if
present, include conservative substitutions that do not
deleteriously affect folding or functional properties of the
peptide. Groups of functionally related amino acids within which
conservative substitutions can be made are glycine/alanine;
valine/isoleucine/leucine; asparagine/glutamine; aspartic
acid/glutamic acid; serine/threonine; lysine/arginine; and
phenylalanine/tryptophan/tyr-osine. Polypeptides of this invention
can be modified post-translationally (e.g., acetylation or
phosphorylation) or can be modified synthetically (e.g., the
attachment of a labeling group).
[0052] Some fragments of BBP or BBP Binding Domain or the modified
amino acids encompassed by the present invention may lack some
biological activities characteristic of native proteins (e.g.,
binding to Bak or BBP, respectively) but may have activities, and
therefor be useful for other purposes, such as for raising
antibodies to BBP or BBP Binding Domain epitopes, as an
immunological reagent to detect and/or purify BBP or BBP Binding
Domain-binding antibodies by affinity chromatography, or as a
competitive or noncompetitive agonist, antagonist, or partial
agonist of native BBP or BBP Binding Domain protein function.
[0053] The invention also encompasses BBP or BBP Binding Domain
conjugated to a label capable of producing a detectable signal or
other functional moieties. Suitable labels include, but are not
limited to, radionuclides, enzymes, substrates, cofactors,
inhibitors, fluorescent dyes, chemiluminescent dyes, bioluminescent
compounds and magnetic particles. See, for examples of patents
teaching the use of such labels, U.S. Pat. Nos. 3,817,837;
3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and
4,366,241.
[0054] The invention further comprises fusion proteins comprising
(1) BBP or BBP Binding Domain or fragments thereof, covalently
attached to (2) a second polypeptide. Such fusion polypeptides can
be made by any of a number of standard techniques well known to
those of ordinary skill, including recombinant methods, in which
case the covalent attachment is a peptide bond, or chemical
conjugation, in which case the covalent attachment is another type
of bond, such as a disulfide bond. A BBP or BBP Binding Domain
fusion polypeptide can be prepared, for example, by chemical
synthesis, or by creating and translating a polynucleotide in which
the peptide regions are encoded in the desired relationship. Useful
heterologous sequences for inclusion in a fusion polypeptide
include sequences that provide for secretion from a host cell,
sequences that enhance immunological reactivity relative to BBP or
the BBP fragment, or BBP Binding Domain or the respective fragment,
sequences that facilitate the coupling of the polypeptide to an
immunoassay support or a vaccine carrier, portions of an
immunoglobulin molecule, immunological tags, or sequences that
mediate specific binding to a cell surface or intracellular
receptor.
[0055] The polypeptides described above can be produced in
prokaryotic or eukaryotic host cells by expression of
polynucleotides encoding the desired peptide sequence.
Alternatively, polypeptides can be synthesized by chemical
methods.
[0056] Purification or isolation of BBP or BBP Binding Domain
expressed either by recombinant DNA or from biological sources such
as tissues can be accomplished by any method known in the art.
Protein purification methods are known in the art. Generally, the
terms "substantially purified" or "substantially isolated" or
"isolated" are used interchangeably to indicate proteins which are
free of other, contaminating cellular substances, particularly
proteins. Preferably, the purified BBP or BBP Binding Domain is
more than eighty percent pure and most preferably more than
ninety-five percent pure.
[0057] Suitable methods of protein purification are known in the
art and include, but are not limited to, affinity chromatography,
immunoaffinity chromatography, size exclusion chromatography, HPLC
and FPLC. Any purification scheme that does not result in
substantial degradation of the protein is suitable for use in the
present invention.
[0058] The invention further encompasses isolated polynucleotides
(or nucleic acids) encoding polypeptides substantially identical to
BBP or BBP Binding Domain or portions thereof. The term
polynucleotide as used herein, can be DNA or RNA, either coding or
noncoding strands. Similarly "bbp" includes bbp cDNA, genomic DNA
and synthetic or semi-synthetic DNAs and RNAs and are additional
embodiments of the present invention.
[0059] Also included are isolated polynucleotides encoding
polypeptides substantially identical to allelic variants of SEQ ID
NO:7 or to homologs of BBP or BBP Binding Domain from species other
than man. The isolated polynucleotide preferably contains a DNA
sequence that hybridizes under stringent conditions (as defined
below) with the DNA sequence of SEQ ID NO:6, or the complement
thereof, and can contain the sequence of SEQ ID NO:6. It is
preferably incorporated into a vector (a virus, phage, or plasmid)
which can be introduced by transfection or infection into a cell.
The vector preferably includes one or more expression control
sequences, in which case the cell transfected by the vector is
capable of expressing the polypeptide.
[0060] By "isolated nucleotide" is meant a single- or
double-stranded nucleotide that is free of the genes which, in the
naturally-occurring genome of the animal from which the isolated
nucleotide is derived, flank the bbp gene or the bbpbd sequence.
The term therefore includes, for example, either or both strands of
a cDNA encoding the nucleic acid of interest, or an allelic variant
thereof; a recombinant DNA which is incorporated into a vector,
into an autonomously replicating plasmid or virus, or into the
genomic DNA of a prokaryotic or eukaryotic cell; or a genomic DNA
fragment (e.g., produced by PCR (polymerase chain reaction) or
restriction endonuclease treatment of human or other genomic DNA).
It also includes a recombinant DNA which is part of a hybrid gene
encoding additional polypeptide sequence.
[0061] Stringent conditions for both DNA/DNA and DNA/RNA
hybridization assays are as described by Sambrook et al. Molecular
Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1989, herein
incorporated by reference. For example, see page 7.52 of Sambrook
et al.
[0062] Also within the invention is an isolated DNA at least 15
nucleotides in length (preferably at least 30, more preferably at
least 100, and most preferably at least 450), including (a) a
strand which hybridizes under stringent conditions to a DNA having
the sequence of SEQ ID NO:6, (b) the complement thereof, or (c) a
double-stranded DNA including both (a) and (b). Multiple copies of
this isolated DNA (useful, for example, as a hybridization probe or
PCR primer) can be produced by recombinant means, by transfecting a
cell with a vector containing this DNA.
[0063] The invention includes modifications to bbp or bbpbd DNA
sequences such as deletions, substitutions and additions
particularly in the non-coding regions of genomic DNA. Such changes
are useful to facilitate cloning and modify gene expression.
[0064] Various substitutions can be made within the coding region
that either do not alter the amino acid residues encoded or result
in conservatively substituted amino acid residues. Nucleotide
substitutions that do not alter the amino acid residues encoded are
useful for optimizing gene expression in different systems.
Suitable substitutions are known to those of skill in the art and
are made, for instance, to reflect preferred codon usage in the
particular expression systems.
[0065] The invention encompasses functionally equivalent variants
and derivatives of bbp or bbpbd, which may enhance, decrease or not
significantly affect the properties of BBP or BBP Binding Domain,
respectively. "Derivatives" are defined herein as changes in the
DNA sequence that do not change the encoded amino acid sequence, as
well as those that result in conservative substitutions of amino
acid residues, one or a few amino acid deletions or additions, and
substitution of amino acid residues by amino acid analogs are those
which will not significantly affect its properties.
[0066] The above-mentioned polynucleotides can include control
sequences that facilitate transcription (expression sequences) and
translation of the coding sequences, such that the encoded
polypeptide product is produced. Such control sequences include,
but are not limited to, promoters, transcription termination sites,
polyadenylation sites, ribosome binding sites, enhancers or
sequences necessary for replication of a vector.
[0067] As described herein, hybridization analysis of Chinese
Hamster somatic cell hybrids, each containing one human chromosome,
indicated that bbp or genes closely related thereto, are located on
chromosomes 2, 5 and X.
[0068] The complete Bak nucleotide and protein sequences are
described in PCT Application No. PCT/US94/13930; Kiefer et al.
(1995) Nature 374:736-739. As used herein, "bak" refers to the
polynucleotide molecule, and derivatives thereof, described in the
above-referenced patent application; "Bak" refers to the proteins
encoded thereby and derivatives thereof.
[0069] Techniques for polynucleotide manipulation useful for the
practice of the present invention are described in a variety of
references, including but not limited to, Molecular Cloning: A
Laboratory Manual, 2nd Ed., Vol. 1-3, eds. Sambrook et al. Cold
Spring Harbor Laboratory Press (1989); and Current Protocols in
Molecular Biology, eds. Ausubel et al., Greene Publishing and
Wiley-Interscience: New York (1987) and periodic updates.
[0070] The bbp or bbpbd cDNA or genomic DNA can be incorporated
into vectors for further manipulation. Furthermore, the invention
concerns a recombinant DNA which is a recombinant vector comprising
at least one of the above mentioned genes.
[0071] The recombinant vectors of the invention comprise an origin
of replication or an autonomously replicating sequence, one or more
dominant marker sequences and, optionally, expression control
sequences, signal sequences and additional restriction sites.
[0072] Preferably, the recombinant vector of the invention
comprises an above described nucleic acid insert operably linked to
an expression control sequence, in particular those described
hereinafter.
[0073] Vectors typically perform two functions in collaboration
with compatible host cells. One function is to facilitate the
cloning of the bbp gene or bbpbd, i.e., to produce useable
quantities of the nucleic acid (cloning vectors). The other
function is to provide for replication and expression of the gene
constructs in a suitable host, either by maintenance as an
extrachromosomal element or by integration into the host chromosome
(expression vectors). A cloning vector comprises the DNAs as
described above, an origin of replication or an autonomously
replicating sequence, selectable marker sequences, and optionally,
signal sequences and additional restriction sites. An expression
vector additionally comprises expression control sequences
essential for the transcription and translation of the bbp gene or
bbpbd. Thus, an expression vector refers to a recombinant DNA
construct, such as a plasmid, a phage, recombinant virus or other
vector that, upon introduction into a suitable host cell, results
in expression of the cloned DNA. Suitable expression vectors are
well known in the art and include those that are replicable in
eukaryotic and/or prokaryotic cells.
[0074] Most expression vectors are capable of replication in at
least one class of organisms but can be transfected into another
organism for expression. For example, a vector is cloned in
Escherichia coli and then the same vector is transfected into yeast
or mammalian cells even though it is not capable of replicating
independently of the host cell chromosome. DNA can also be
amplified by insertion into the host genome. However, the recovery,
for example, of the genomic bbp gene is more complex than that of
exogenously replicated vector because restriction enzyme digestion
is required to excise the gene. DNA can be amplified by PCR and be
directly transfected into the host cells without any replication
component.
[0075] Advantageously, expression and cloning vectors contain a
selection gene also referred to as selectable marker. This gene
encodes a protein necessary for the survival or growth of
transformed host cells grown in a selective culture medium. Host
cells not transformed with the vector containing the selection gene
will not survive in the culture medium. Typical selection genes
encode proteins that confer resistance to antibiotics and other
toxins, e.g., ampicillin, neomycin, methotrexate or tetracycline,
complement auxotrophic deficiencies, or supply critical nutrients
not available from complex media.
[0076] Since the amplification of the vectors is conveniently done
in E. coli, an E. coli genetic marker and an E. coli origin of
replication are advantageously included. These can be obtained from
E. coli plasmids, such as pBR322, BLUESCRIPT.RTM. vectors or a pUC
plasmid or any other similar plasmid vector.
[0077] Suitable selectable markers for mammalian cells are those
that enable the identification of cells competent to take up the
bbp gene or bbpbd, such as dihydrofolate reductase (DHFR,
methotrexate resistance), thymidine kinase, or genes conferring
resistance to G418 or hygromycin. The mammalian cell transfectants
are placed under selection pressure which only those transfectants
are uniquely adapted to survive which have taken up and are
expressing the marker.
[0078] Expression and cloning vectors usually contain a promoter
that is recognized by the host organism and is operably linked to
the bbp gene or bbpbd. Suitable promoters can be inducible or
constitutive. The promoters are operably linked to the gene of
interest by removing the promoter from the source DNA by
restriction enzyme digestion and inserting the isolated promoter
sequence into the vector. Both the native promoter sequence and
many heterologous promoters can be used to direct amplification
and/or expression of a gene or fragment of interest. However,
heterologous promoters are preferred, because they generally allow
for greater transcription and higher yields of BBP or BBP Binding
Domain as compared to native promoter.
[0079] Promoters suitable for use with prokaryotic hosts include,
for example, the .beta.-lactamase and lactose promoter systems,
alkaline phosphatase, a tryptophan (trp) promoter system and hybrid
promoters such as the tac promoter. Their nucleotide sequences have
been published thereby enabling the skilled worker to ligate them
to the gene of interest using linkers or adaptors to supply any
required restriction sites. Promoters for use in bacterial systems
will also generally contain a Shine-Dalgarno sequence operably
linked to the gene.
[0080] Gene transcription from vectors in mammalian host cells can
be controlled by promoters compatible with the host cell systems,
e.g., promoters derived from the genomes of viruses. Suitable
plasmids for expression of bbp or fragments of bak, such as bbpbd,
in eukaryotic host cells, particularly mammalian cells, include,
but are not limited to, cytomegalovirus (CMV) promoter-containing
vectors, Rous Sarcoma Virus (RSV) promoter-containing vectors and
SV40 promoter-containing vectors and MMTV LTR promoter-containing
vectors. Depending on the nature of their regulation, promoters can
be constitutive or regulatable by -experimental conditions.
Transcription of bbp or bak fragments according to the invention by
higher eukaryotes can be increased by inserting an enhancer
sequence into the vector. These promoters can also be cell
type-specific, that is, inducible only in a particular cell type
and often only during a specific period of time. The promoter can
further be cell cycle specific, that is, induced or inducible only
during a particular stage in the cell cycle.
[0081] The various DNA segments of the vector DNA are operably
linked, i.e., they are contiguous and placed into a functional
relationship to each other. Construction of vectors according to
the invention employs conventional ligation techniques. Isolated
plasmids or DNA fragments are cleaved, tailored and religated in
the form desired to generate the plasmids required. If desired,
analysis to confirm correct sequences in the constructed plasmids
is performed in a manner known in the art. Suitable methods for
constructing expression vectors, preparing in vitro transcripts,
introducing DNA into host cells, and performing analyses for
assessing bbp expression and function are known to those skilled in
the art. Gene presence, amplification and/or expression can be
measured in a sample directly, for example, by conventional
Southern blotting, northern blotting to quantitate the
transcription of mRNA, dot blotting (DNA or RNA analysis), in situ
hybridization, using an appropriately labeled probe based on a
sequence provided herein, binding assays, immunodetection and
functional assays.
[0082] Nucleotides can also be expressed in non-human transgenic
animals, particularly transgenic warm-blooded animals. Methods for
producing transgenic animals, including mice, rats, rabbits, sheep
and pigs, are known in the art and are disclosed, for example by
Hammer et al. (1985) Nature 315:680-683. An expression unit
including a gene of interest together with appropriately positioned
expression control sequences, is introduced into pronuclei of
fertilized eggs. Introduction can be achieved, e.g., by
microinjection. Integration of the injected DNA is detected, e.g.,
by blot analysis of DNA from suitable tissue samples. It is
preferred that the introduced DNA be incorporated into the germ
line of the animal so that it is passed to the animal's
progeny.
[0083] Furthermore, a knock-out animal can be developed by
introducing a mutation in the bbp or bbpbd sequence, thereby
generating an animal which no longer expresses the functional bbp
gene or bbpbd fragment. Such knock-out animals are useful, e.g.,
for studying the role of the BBP or BBP Binding Domain in apoptosis
or other physiological functions.
[0084] More specifically, a knock-out animal can be developed
(i.e., an animal that does not express the endogenous gene of
interest), in which one introduces a mutated or wild-type gene.
Methods for producing knock-out mice are known in the art. The
knock-out animals are useful not only for studying the role of a
given protein, as exemplified by published studies (see, e.g.,
Conquet et al. (1994) Nature 372:237-243; Aiba et al. (1994) Cell
79:365-375; and Masu et al. (1995) Cell 80:757-765), but also and,
in particular, for providing a mammalian animal model with a
suitable genetic background for introducing and expressing
transgenes encoding the protein.
[0085] The nucleic acid sequences provided herein can be employed
to identify DNAs encoding related polypeptides. A method for
identifying such DNA comprises contacting DNA with a nucleic acid
probe described above and identifying DNA(s) which hybridize to
that probe. Bbp polynucleotides can also be used as probes or
primers to detect a bbp gene or bbp RNA species to diagnose a
phenotype characteristic of cells having elevated or reduced
expression of BBP.
[0086] These methods are well known to those skilled in the art,
and protocols can be found in a variety of sources, such as Ausubel
(1993) Current Protocols in Molecular Biology, Green and Wiley,
U.S.A., and periodic updates.
[0087] For an individual polypeptide related to BBP, the expression
pattern in different tissues may vary. Thus, in order to isolate
cDNA encoding a particular BBP-related polypeptide, it is
advantageous to screen libraries prepared from different suitable
tissues or cells. As a screening probe, there may be employed a DNA
or RNA comprising substantially the entire coding region of bbp or
a suitable oligonucleotide probe based on said DNA. A suitable
oligonucleotide probe (for screening involving hybridization) is a
single stranded DNA or RNA that has a sequence of nucleotides that
includes at least 14 contiguous bases that are the same as (or
complementary to) any 14 or more contiguous bases set forth in SEQ
ID NO:6. The probe can be labeled with a suitable chemical moiety
for ready detection. The nucleic acid sequences selected as probes
should be of sufficient length and be sufficiently unambiguous so
that false positive results are minimized.
[0088] Preferred regions from which to construct probes include 5'
and/or 3' coding sequences, sequences predicted to encode ligand
binding sites, and the like. For example, either the full-length
cDNA clone disclosed herein or fragments thereof can be used as
probes. Preferably, nucleic acid probes of the invention are
labeled with suitable label means for ready detection upon
hybridization. For example, a suitable label means is a radiolabel.
The preferred method of labeling a DNA fragment is by incorporating
.sup.32P-labeled .alpha.-dATP with the Klenow fragment of DNA
polymerase in a random priming reaction, as is well known in the
art. Oligonucleotides are usually end-labeled with .sup.32P-labeled
.gamma.-ATP and polynucleotide kinase. However, other methods
(e.g., non-radioactive) can also be used to label the fragment or
oligonucleotide, including, e.g., enzyme labeling and
biotinylation.
[0089] After screening the library, e.g., with a portion of DNA
including substantially the entire bbp gene or a suitable
oligonucleotide based on a portion of said DNA, positive clones are
identified by detecting a hybridization signal; the identified
clones are characterized by restriction enzyme mapping and/or DNA
sequence analysis, and then examined, e.g., by comparison with the
sequences set forth herein, to ascertain whether they include a
full length bbp gene (i.e., if they include translation initiation
and termination codons). If the selected clones are incomplete,
they can be used to rescreen the same or a different library to
obtain overlapping clones. If the library is genomic, then the
overlapping clones can include exons and introns. If the library is
a cDNA library, then the overlapping clones will include an open
reading frame. In both instances, complete clones can be identified
by comparison with the DNAs and deduced amino acid sequences
provided herein.
[0090] Furthermore, in order to detect any abnormality of an
endogenous bbp or bak, genetic screening can be carried out using a
nucleotide sequence of the invention as hybridization probes.
Genetic defects due to a mutation in a bbp gene or bbpbd sequence
can lead to aberrant expression of BBP or Bak, or to expression of
an aberrant BBP or Bak, which may not associate properly with a
other proteins in the cell. As a result, a genetic defect in a bbp
or bak gene could result in a pathology characterized by increased
or decreased apoptosis. Oligonucleotide probes comprising a
nucleotide sequence of the bbp or bbpbd polynucleotides disclosed
herein can be used to identify cells having a mutation in a bbp or
bak gene using well known hybridization methods. In order to
provide the specificity necessary to identify, for example, a point
mutation in a bbp gene, one skilled in the art would know that an
oligonucleotide probe should be at least about fourteen to sixteen
nucleotides in length. In addition, the probe should incorporate a
detectable moiety such as a radiolabel, a fluorochrome or a
detectable binding agent such as biotin. Various detectable
moieties and methods of incorporating the moiety into an
oligonucleotide probe are well known in the art and are
commercially available.
[0091] Also, based on the nucleic acid sequences provided herein
antisense-type or ribozyme therapeutic agents can be designed and
are included in the scope of the invention. For example,
oligoribonucleotides, including antisense RNA and DNA molecules and
ribozymes can function to inhibit translation of one or more
components of a protein complex. S. T. Crooke and B. Lebleu, eds.
Antisense Research and Applications (1993) CRC Press. Anti-sense
RNA and DNA molecules act to directly block the translation of mRNA
by binding to targeted mRNA and preventing protein translation.
With respect to antisense DNA, oligodeoxyribonucleotides derived
from the translation initiation site, e.g., between -10 and +10
regions of the relevant nucleotide sequence, are preferred.
[0092] Ribozymes are enzymatic RNA molecules capable of catalyzing
the specific cleavage of RNA. The mechanism of ribozyme action
involves sequence specific interaction of the ribozyme molecule to
complementary target RNA, followed by an endonucleolytic cleavage.
Within the scope of the invention are engineered hammerhead or
other motif ribozyme molecules that specifically and efficiently
catalyze endonucleolytic cleavage of RNA sequences encoding protein
complex components.
[0093] Specific ribozyme cleavage sites within any potential RNA
target are initially identified by scanning the target molecule for
ribozyme cleavage sites which include the following sequences, GUA,
GUU and GUC. Once identified, short RNA sequences of between 15 and
20 ribonucleotides corresponding to the region of the target gene
containing the cleavage site can be evaluated for predicted
structural features, such as secondary structure, that may render
the oligonucleotide sequence unsuitable. The suitability of
candidate targets can also be evaluated by testing their
accessibility to hybridization with complementary oligonucleotides,
using ribonuclease protection assays. See, Draper PCT WO
93/23569.
[0094] Both anti-sense RNA and DNA molecules and ribozymes of the
invention can be prepared by any method known in the art for the
synthesis of RNA molecules. See, Draper, id. These include
techniques for chemically synthesizing oligodeoxyribonucleotides
well known in the art including, but not limited to, solid phase
phosphoramidite chemical synthesis. Alternatively, RNA molecules
can be generated by in vitro and in vivo transcription of DNA
sequences encoding the antisense RNA molecule. Such DNA sequences
can be incorporated into a wide variety of vectors which
incorporate suitable RNA polymerase promoters such as the T7 or SP6
polymerase promoters. Alternatively, antisense cDNA constructs that
synthesize antisense RNA constitutively or inducibly, depending on
the promoter used, can be introduced stably into cell lines.
[0095] Various modifications to the DNA molecules can be introduced
as a means of increasing intracellular stability and half-life.
Possible modifications include, but are not limited to, the
addition of flanking sequences or ribo- or deoxyribo-nucleotides to
the 5' and/or 3' O-methyl rather than phosphodiesterase linkages
within the oligodeoxyribonucleotid-e backbone.
[0096] It is envisaged that the nucleic acid of the invention can
be readily modified by nucleotide substitution, nucleotide
deletion, nucleotide insertion or inversion of a nucleotide
stretch, and any combination thereof. Such modified sequences can
be used to produce a mutant BBP which differs from those found in
nature. Mutagenesis can be predetermined (site-specific) or random.
A mutation which is not a silent mutation should not place
sequences out of reading frames and preferably will not create
complementary regions that could hybridize to produce secondary
mRNA structures such as loops or hairpins.
[0097] Given the guidance of the present invention, the nucleic
acids of the invention are obtainable according to the methods well
known in the art. The present invention further relates to a
process for the preparation of such nucleic acids.
[0098] For example, a DNA of the invention is obtainable by
chemical synthesis, by recombinant DNA technology or by PCR.
Preparation by recombinant DNA technology can involve screening a
suitable cDNA or genomic library. A suitable method for preparing a
DNA or of the invention can, e.g., comprise the synthesis of a
number of oligonucleotides, their amplification by PCR methods, and
their splicing to give the desired DNA sequence. Suitable libraries
are commercially available or can be prepared from individual
tissues or cell lines.
[0099] Suitable antibodies for use herein are generated by using
BBP or fragments of BBP as an antigen. Antibodies to a BBP Binding
Domain or Bak are also suitable for use herein. Methods of
detecting proteins using antibodies and of generating antibodies
using proteins or synthetic peptides are known in the art and are
not described in detail herein.
[0100] The present invention further encompasses antibodies or
antigen binding fragments that recognize BBP or BBP Binding Domain,
or fragments thereof. Such antibodies can be useful e.g. for
immunoassays including immunohistochemistry, in the detection of a
substances that bind to BBP or BBP Binding Domain, or,
alternatively, for use in purification or identification of BBP or
BBP Binding Domain or antigenically related polypeptides, or for
diagnostic or therapeutic purposes. For example, an antibody can be
used to detect the presence of a BBP in a biological sample. The
cells can either be lysed or permeabilized to the antibody, both
are acceptable methods of exposing the contents of the cells to
antibodies, using methods known in the art.
[0101] Antibodies which can be used as therapeutic or diagnostic
agents include polyclonal antibodies, monoclonal antibodies (mAbs),
humanized or chimeric antibodies, single chain antibodies, Fab
fragments, F(ab').sub.2 fragments, fragments produced by a Fab
expression library, anti-idiotypic (anti-Id) antibodies, and
epitope-binding fragments of any of the above. Antibodies can be of
any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any
subclass thereof.
[0102] Polyclonal antibodies are heterogeneous populations of
antibody molecules derived from the sera of animals immunized with
an antigen, such as a protein-protein complex, or individual
components. For the production of polyclonal antibodies, various
host animals can be immunized by injection with the antigen such as
rabbits, mice, and rats. Various adjuvants can be used to increase
the immunological response, depending on the host species, such as
Freund's (complete and incomplete), mineral gels (e.g.,
lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, keyhole limpet hemocyanin, dinitrophenol), and
potentially useful human adjuvants such as BCG (bacille
Calmette-Guerin) and Corynebacterium parvum.
[0103] A monoclonal antibody, which is a substantially homogeneous
population of antibodies to a particular antigen, can be obtained
by techniques providing for the production of antibody molecules by
continuous cell lines in culture. Examples of such techniques
include the hybridoma technique described by Kohler and Milstein
(1975) Nature 256:495-497, the human B-cell hybridoma technique
(Olsson et al. J. Immunol. Methods 61:17-32; and Cole et al. (1984)
Mol. Cell. Biochem. 62:109-120), and the EBV-hybridoma technique
(Roder et al. (1986) Methods Enzymol. 121:140-167). The hybridoma
producing the mAb can be cultivated in vitro or in vivo.
[0104] In addition, techniques developed for the production of
"chimeric antibodies" (Morrison et al. (1984) Proc. Natl. Acad.
Sci. USA 81:6851-6855; Neuberger et al. (1984) Nature 312:604-608;
and Takeda et al. (1985) Nature 314:452-454) by splicing the genes
from a mouse antibody molecule of appropriate antigen specificity
together with genes from a human antibody molecule of appropriate
biological activity can be used. A chimeric antibody is a molecule
in which different portions are derived from different animal
species, such as those having a variable region derived from a
murine mAb and a human immunoglobulin constant region.
[0105] Alternatively, techniques described for the production of
single chain antibodies (U.S. Pat. No. 4,946,778; Bird (1988)
Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci.
USA 85:5879-5883; and Ward et al. (1989) Nature 334:544-546) can be
adapted to produce antigen-specific single chain antibodies. Single
chain antibodies are formed by linking the heavy and light chain
fragment of the Fv region via an amino acid bridge, resulting in a
single chain polypeptide.
[0106] Antibody fragments containing specific binding sites of a
complex can be generated by known techniques. For example, such
fragments include the F(ab').sub.2 fragments which can be produced
by pepsin digestion of the antibody molecule and the Fab fragments
which can be generated by reducing the disulfide bridges of the
F(ab').sub.2 fragments. Alternatively, Fab expression libraries can
be constructed (Huse et al. (1989) Science 246:1275-1281) to allow
rapid and easy identification of monoclonal Fab fragments with the
desired specificity to BBP or BBP Binding Domain.
[0107] The invention further provides methods for detecting agents
that can modulate the interaction between a BBP and a Bak protein
or BBP Binding Domain of the Bak protein. "Modulate" in this sense
means to alter the ability of a BBP to associate with a Bak
protein. Thus, modulation includes enhancement of the association
between a BBP and a Bak protein, as well as dissociation of a bound
complex formed by the association of a BBP and a Bak protein.
Methods for detecting an agent that can modulate the interaction
between a Bak and a BBP are known in the art and include, but are
not limited to, yeast two-hybrid screening, phage display and
assays such as the one described in Example 6.
[0108] As used herein, the term "agent" means a chemical or
biological molecule such as a simple or complex organic molecule, a
peptide, a protein or an oligonucleotide. An "effective agent" is
an agent that, in fact, alters an interaction of proteins involved
in apoptosis. An effective agent is therefore also a potential
therapeutic agent.
[0109] The screening assay described herein is particularly useful
in that it can be automated, which allows for high through-put
screening of randomly designed agents to identify useful drugs,
which can alter the ability of a BBP and a Bak protein to
associate. For example, a drug can alter the ability of a BBP and a
Bak protein to associate by decreasing or inhibiting the binding
affinity of a BBP and a Bak protein. Such a drug can be useful
where it is desirable to increase the concentration of unbound Bak
in a cell, for example, so that free Bak is available to induce
apoptosis. Alternatively, a drug can be useful for increasing the
affinity of binding of a BBP and a Bak protein.
[0110] The drug screening assay can utilize BBP or a BBP fusion
protein such as a BBP-glutathione-S-transferase (gst). The BBP or
BBP fusion protein is characterized, in part, by having an affinity
for a solid substrate as well as having an affinity for a Bak
protein. For example, when BBP is used in the assay, the solid
substrate can contain a covalently attached anti-BBP antibody.
Alternatively, a BBP-gst fusion protein can be used in the assay.
Where such a fusion protein is used in the assay, the solid
substrate can contain covalently attached glutathione, which is
bound by the gst component of the BBP-gst fusion protein.
[0111] The drug screening assay can be performed by allowing the
BBP or BBP-fusion protein to bind to the solid support, then adding
a Bak protein and a drug to be tested. Control reactions will not
contain the drug. Following incubation of the reaction mixture
under conditions known to be favorable for the association, for
example, of BBP and Bak in the absence of a drug, the amount of Bak
specifically bound to BBP in the presence of a drug can be
determined. For ease of detecting binding, the Bak protein can be
labeled with a detectable moiety, such as a radionuclide or a
fluorescent label, using methods well known in the art. By
comparing the amount of specific binding of BBP and Bak in the
presence of a drug as compared to the control level of binding, a
drug that increases or decreases the binding of a BBP and a Bak
protein can be identified. Thus, the drug screening assay provides
a rapid and simple method for selecting drugs having a desirable
effect on the association of a BBP and a Bak protein.
[0112] A transcription activation assay such as the yeast
two-hybrid system allows for the identification and manipulation of
protein-protein interactions. Fields and Song (1989) Nature
340:245-246. The conceptual basis for a transcription activation
assay is predicated on the modular nature of transcription factors,
which consist of functionally separable DNA-binding and
trans-activation domains. When expressed as separate proteins,
these two domains fail to mediate gene transcription. However, the
ability to activate transcription can be restored if the
DNA-binding domain and the trans-activation domain are bridged
together through a protein-protein interaction. These domains can
be bridged, for example, by expressing the DNA-binding domain and
trans-activation domain as fusion proteins (hybrids), where the
proteins that are appended to these domains can interact with each
other. The protein-protein interaction of the hybrids can bring the
DNA-binding and trans-activation domains together to create a
transcriptionally competent complex.
[0113] A transcription activation assay such as the yeast two
hybrid system can also be useful as a screening assay to identify
effective agents that alter interactions between a Bak and a BBP. A
transcription activation assay can be used to screen a panel of
agents to identify an effective agent, which can be useful for
increasing or decreasing apoptosis in a cell.
[0114] An effective agent can be identified by detecting an altered
level of transcription of a reporter gene. For example, the level
of transcription of a reporter gene due to the bridging of a
DNA-binding domain and trans-activation domain by a BBP and a Bak
protein can be determined in the absence and in the presence of an
agent. An effective agent that increases the interaction between a
Bak protein and a BBP can be identified by an increased level of
transcription of the reporter gene as compared to the control level
of transcription in the absence of the agent.
[0115] An agent that effectively increases the interaction of Bak
and BBP, as detected by increased transcription of the reporter
gene in a two-hybrid assay, can be used to alter the level of
apoptosis in a cell. An effective agent that decreases the
interaction of Bak and BBP also can be identified, in this case by
detecting a decreased level of transcription of a reporter gene as
compared to the level of transcription in the absence of the agent.
Effective agents that result in decreased levels of apoptosis can
be particularly useful as a medicament for treating a patient
suffering from a disease characterized by a high level of apoptosis
such as a neurodegenerative disease. Effective agents that result
in increased levels of apoptosis can be useful, for example, to
increase the level of apoptosis of a cancer cell, which is
characterized by having a decreased level of apoptosis as compared
to its normal cell counterpart. Thus, effective agents identified
using the methods described herein are particularly useful as
medicaments to increase or decrease the level of apoptosis in a
cell in a subject.
[0116] In some cases, an agent may not be able to cross the yeast
cell wall and, therefore, cannot enter the yeast cell to alter an
interaction among members of the Bak protein family. The use of
yeast spheroplasts, which are yeast cells that lack a cell wall,
can circumvent this problem (Smith and Corcoran, In Current
Protocols in Molecular Biology (ed. Ausubel et al.; Green Publ., NY
1989)). In addition, a potentially effective agent, upon entering a
cell, may require "activation" by a cellular mechanism, which may
not be present in yeast. Activation of an agent can include, for
example, metabolic processing of the agent or a modification such
as phosphorylation of the agent, which can be necessary to convert
the agent into an effective agent. In this case, a mammalian cell
line can be used to screen a panel of agents. A transcription assay
such as the yeast two-hybrid system described in Example 1 can be
adapted for use in mammalian cells using well known methods (Fearon
et al. (1992) Proc. Natl. Acad. Sci. USA 89:7958-7962; see, also,
Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold
Spring Harbor Laboratory Press 1989), and Ausubel et al., Current
Protocols in Molecular Biology (Green Publ., NY 1989).
[0117] It may be advantageous to employ a peptide analog of BBP or
Bak, or a portion thereof, as a pharmaceutical agent or as a
commercial research reagent. For example, a peptide analog of BBP
having high affinity for binding Bak can be used as a competitive
inhibitor of BBP:Bak complex formation by competing with native BBP
for binding to Bak.
[0118] In addition to polypeptides consisting only of
naturally-occurring amino acids, peptidomimetics are also provided.
Peptide analogs are commonly used in the pharmaceutical industry as
non-peptide drugs with properties analogous to those of the
template peptide. These types of non-peptide compound are termed
"peptide mimetics" or "peptidomimetics" and are usually developed
with the aid of computerized molecular modeling. Eichler et al.
(1995) Med. Res. Rev. 15:481-496; Moore et al. (1995) Adv.
Pharmacol. 33:1-41; Moore (1994) Trends Pharmacol. Sci. 15:124-129;
Saragovi et al. (1992) Biotechnol. 10:773-778. Peptide mimetics
that are structurally similar to therapeutically useful peptides
can be used to produce an equivalent therapeutic or prophylactic
effect. Generally, peptidomimetics are structurally similar to a
paradigm polypeptide (i.e., a polypeptide that has a biochemical
property or pharmacological activity), such as Bak, but have one or
more peptide linkages optionally replaced by a linkage such
as:--CH.sub.2NH--, --CH.sub.2S--, --CH.sub.2-CH.sub.2-,
--CH.dbd.CH--(cis and trans), --COCH.sub.2-, --CH(OH)CH.sub.2-, and
--CH.sub.2SO--, by methods known in the art. See, for example,
Spatola (1983) Chemistry and Biochemistry of Amino Acids, Peptides,
and Proteins B. Weinstein eds. Marcel Dekker, New York.
[0119] Such peptide mimetics can have significant advantages over
polypeptide embodiments, including, for example, more economical
production, greater chemical stability, enhanced pharmacological
properties (half-life, absorption, potency, efficacy, etc.),
altered specificity (e.g., a broad-spectrum of biological
activities), reduced antigenicity, and others. Labeling of
peptidomimetics usually involves covalent attachment of one or more
labels, directly or through a spacer (e.g., an amide group), to
non-interfering position(s) on the peptidomimetic that are
predicted by quantitative structure-activity data and/or molecular
modeling. Such non-interfering positions generally are positions
that do not form direct contacts with the macromolecule(s) (e.g.,
are not contact points in Bak:BBP complexes) to which the
peptidomimetic binds to produce the therapeutic effect.
Derivitization (e.g., labeling) of peptidomimetics should not
substantially interfere with the desired biological or
pharmacological activity of the peptidomimetic.
[0120] The invention also provides methods for identifying
polypeptide sequences which bind to a BBP polypeptide. For example,
a yeast two-hybrid screening system can be used for identifying
polypeptide sequences that bind to BBP. Yeast two-hybrid systems
wherein one GAL4 fusion protein comprises a BBP polypeptide
sequence, typically a full-length or near full-length BBP sequence,
and the other GALA fusion protein comprises a cDNA library member
can be used to identify cDNAs encoding proteins which interact with
the BBP polypeptide, can be screened according to the general
method of Chien et al. (1991) Proc. Natl. Acad. Sci. USA
88:9578-9582. Other methods for detecting BBP-binding proteins are
known in the art and include, but are not limited to screening a
phage display library with a BBP. U.S. Pat. No. 5,223,409.
[0121] As used herein, the terms "suitable conditions" or
"appropriate conditions" refer to temperature, pH, ionic strength,
viscosity and biochemical parameters which are compatible with
physiological protein-protein interactions. Suitable in vitro or in
vivo reaction conditions for protein-protein interactions are
generally physiological conditions. In general, in vitro
physiological conditions comprise 50-200 mM NaCl or KCl, pH
6.5-8.5, 20.degree. C.-45.degree. C. and 0.001-10 mM divalent
cation (e.g., Mg.sup.++, Ca.sup.++); preferably about 150 mM NaCl
or KCl, pH 7.2-7.6, 5 mM divalent cation, and often include
0.01-1.0% nonspecific protein (e.g., bovine serum albumin).
Particular aqueous conditions can be selected by the practitioner
according to conventional methods. Addition of other components is
optional and the additional components and reaction conditions can
vary according to a particular need.
[0122] In another embodiment, diagnostic methods are provided to
detect the expression of BBP either at the protein level or the
mRNA level. Any antibody that specifically recognizes BBP is
suitable for use in BBP diagnostics. Abnormal levels of BBP may be
found in the tissues of patients with diseases associated with
inappropriate apoptosis; diagnostic methods are therefore useful
for detecting and monitoring biological conditions associated with
such apoptosis defects. Detection methods are also useful for
monitoring the success of BBP-related therapies.
[0123] BBP protein expression can also be monitored by measuring
the level of bbp mRNA. Any method for detecting specific mRNA
species is suitable for use in this method. This is easily
accomplished using the polymerase chain reaction (PCR).
Alternatively, Northern blots can be utilized to detect bbp mRNA or
bak mRNA by using probes specific to bbp or bbpbd. Methods of
utilizing PCR and Northern blots are known in the art and are not
described in detail herein. The invention further provides methods
for modulating the activity of Bak or a Bak-related protein in a
cell by introducing into the cell a nucleotide sequence encoding
BBP or an antisense nucleotide sequence, which is complementary to
a region of a gene encoding BBP and can hybridize to the gene or to
an mRNA transcribed from the gene. As used herein, the term
"modulate" means the level of a BBP expressed in a cell can be
increased or decreased. Thus, the compounds described herein can be
used as medicaments for the treatment of a pathology caused by an
altered level of apoptosis.
[0124] The level of a gene product such as BBP or BBP Binding
Domain can be increased in a cell using recombinant expression
vectors and gene transfer technology to express a nucleic acid
encoding BBP or BBP Binding Domain or an active fragment of BBP or
BBP Binding Domain. Various expression vectors and methods for
introducing such vectors into a cell are well known in the art and
are described, for example, in Sambrook et al. (1989). Viral
vectors that are compatible with a targeted cell are particularly
useful for introducing a nucleic acid encoding BBP or BBP Binding
Domain into a cell. For example, recombinant adenoviruses having
general or tissue-specific promoters can be used to deliver
expression constructs into a variety of types of tissues and cells,
including non-mitotic cells, and to drive the desired cDNA
expression in the target cells. Recombinant adeno-associated
viruses also are useful and have the added advantage that the
recombinant virus can stably integrate into the chromatin of even
quiescent non-proliferating cells such as neurons of the central
and peripheral nervous systems. Lebkowski et al. (1988) Mol. Cell.
Biol. 8:3988-3996.
[0125] Suitable indications for modulating endogenous levels of BBP
or BBP Binding Domain are any in which BBP or BBP Binding
Domain-mediated apoptosis is involved. These include, but are not
limited to, various types of malignancies and other disorders
resulting in uncontrolled cell growth such as eczema, or
deficiencies in normal programmed cell death such as malignancies,
including, but not limited to, B cell lymphomas.
[0126] Methods of treatment with BBP also include modulating
cellular expression of BBP by increasing or decreasing levels of
bbp mRNA or protein. Suitable methods of increasing cellular
expression of BBP or BBP Binding Domain include, but are not
limited to, increasing endogenous expression and transfecting the
cells with vectors encoding bbp or bbpbd, respectively.
[0127] For purposes of gene therapy, the level of BBP or a BBP
Binding Domain can be increased in a cell using recombinant
expression vectors and gene transfer technology to express bbp or
bbpbd or a portion thereof. Viral vectors that are compatible with
a targeted cell are particularly useful for introducing a bbp or
bbpbd gene into a cell. For example, recombinant adenoviruses
having general or tissue-specific promoters can be used to deliver
bbp constructs into a variety of types of tissues and cells.
Retroviral vectors are often used for in vivo targeting and therapy
procedures. Retroviral vectors can be constructed either to
function as infectious particles or to undergo only a single
initial round of infection. Methods for constructing and using
viral vectors are known in the art and are reviewed, for example,
in Miller and Rosman (1989) Biotechniques 7:980-982.
[0128] The invention encompasses ex vivo transfection with bbp or
bbpbd, in which cells removed from animals including man are
transfected with vectors encoding BBP or BBP Binding Domain and
reintroduced into animals. Suitable transfected cells include
individual cells or cells contained within whole tissues. In
addition, ex vivo transfection can include the transfection of
cells derived from an animal other than the animal or human subject
into which the cells are ultimately introduced. Such grafts
include, but are not limited to, allografts, xenografts, and fetal
tissue transplantation. Essentially any cell or tissue type can be
treated in this manner. Suitable cells include, but are not limited
to, cardiomyocytes and lymphocytes. Transfection methods include,
but are not limited to calcium phosphate precipitation and
DEAE-dextran or lipofectin facilitated transfection; these methods
are well known in the art and are described in, for example,
Sambrook et al. (1989).
[0129] Further, the invention encompasses cells transfected in vivo
by vectors containing bbp or bbpbd cDNA. Suitable methods of in
vivo transfection are known in the art and include, but are not
limited to, that described by Zhu et al. (1993) Science
261:209-211. Receptor-mediated DNA delivery approaches also can be
used to deliver BBP or BBP Binding Domain expression plasmids into
cells in a tissue-specific fashion using a tissue-specific ligand
or antibody non-covalently complexed with DNA via bridging
molecules. Curiel et al. (1992) Hum. Gene Ther. 3:147-154; and Wu
and Wu (1987) J. Biol. Chem. 262:4429-4432. Direct injection of DNA
or DNA encapsulated in cationic liposomes also can be used for
stable gene transfer to non-dividing and dividing cells in vivo
Ulmer et al. (1993) Science 259:1745-1749. In addition, DNA can be
transferred into a variety of cell types using the particle
bombardment method. Heiser (1994) Anal. Biochem. 217:185-196; Yang
et al. (1990) Proc. Natl. Acad. Sci. USA 87:9568-9572. In vivo
transfection by bbp may be particularly useful as a prophylactic
treatment for patients suffering from atherosclerosis. Modulation
of the levels of BBP or BBP Binding Domain could serve as a
prophylaxis for the apoptosis-associated reperfusion damage that
results from cerebral and myocardial infarctions. In these patients
with a high risk of stroke and heart attack, the apoptosis and
reperfusion damage associated with arterial obstruction could be
prevented or at least mitigated.
[0130] Infarctions are caused by a sudden insufficiency of arterial
or venous blood supply due to emboli, thrombi, or pressure that
produces a macroscopic area of necrosis; the heart, brain, spleen,
kidney, intestine, lung and testes are likely to be affected.
Apoptosis occurs to tissues surrounding the infarct upon
reperfusion of blood to the area; thus, modulation of BBP or BBP
Binding Domain levels, achieved by a biological modifier-induced
change in endogenous production or by in vivo transfection, could
be effective at reducing the severity of damage caused by heart
attacks and stroke.
[0131] In addition, increasing endogenous expression of BBP can be
accomplished by exposing the cells to biological modifiers that
directly or indirectly increase levels of BBP either by increasing
expression or by decreasing degradation of bbp mRNA. Suitable
biological modifiers include, but are not limited to, molecules and
other cells. Suitable molecules include, but are not limited to,
drugs, cytokines, small molecules, hormones, combinations of
interleukins, lectins and other stimulating agents e.g. PMA, LPS,
bispecific antibodies and other agents which modify cellular
functions or protein expression. Cells are exposed to such
biological modifiers at physiologically effective concentrations,
and the expression of BBP is measured relative to a control not
exposed to the biological modifiers. Those biological modifiers
which increase expression of BBP relative to the control are
selected for further study.
[0132] The invention further encompasses a method of decreasing
endogenous levels of BBP or BBP Binding Domain. The methods of
decreasing endogenous levels of BBP or BBP Binding Domain include,
but are not limited to, antisense nucleotide therapy and
down-regulation of expression by biological modifiers. Antisense
therapy is known in the art and its application will be apparent to
one of skill in the art. Homologous recombinant gene knock-out
methods can also be used to decrease the level of BBP or BBP
Binding Domain in a cell. Capecchi (1990) Nature 344:105.
[0133] The following examples are provided to illustrate but not
limit the present invention. Unless otherwise specified, all
cloning techniques were essentially as described by Sambrook et al.
(1989) and all reagents were used according to the manufacturer's
instructions.
Example 1
Identification and Cloning of BBP cDNA
[0134] BBP cDNA was discovered in human heart using the yeast two
hybrid system. The requirement of the DNA binding and activation
domains of the yeast transcriptional activator GAL4 for the
induction of the GAL1 promoter and the ability of those functions
to operate even when on separate fusion proteins provides the basis
for the assay and allows detection of associating proteins. The
strategy used to detect proteins with binding affinity for Bak is
shown in FIG. 1.
[0135] The GAL4-Bak fusion protein consisted of amino acids 1-147
of GAL4, a region that directs binding of GAL4 to the upstream
activator sequence of the GAL1 promoter. The Bak protein, or
Bak.DELTA.2-TM, whose amino acid sequences are shown in FIG. 2 and
FIG. 3, respectively, served as the "bait". The lacZ gene under the
control of the GAL1 promoter constituted the first reporter system.
The second reporter system consisted of the histidine reporter gene
similarly under transcriptional control of the GAL1 promoter. Yeast
possessing a dual reporter system responsive to transcriptional
activation through the GAL1 promoter were transformed with a
plasmid expressing a GAL4-Bak fusion protein. A cDNA library from
human heart mRNA was purchased from Clontech and cloned into the
vector pGAD10 such that members of the library would be
constitutively expressed as a fusion protein with amino acids
768-881 of GAL4, a region containing the transcriptional activation
domain. Yeast cells containing library proteins that interact with
the bait protein, Bak or Bak.DELTA.2-TM, turn blue when grown on
medium containing XGa1, which yields a chromogenic product when
acted on by .beta.-galactosidase. Yeast cells were transformed by
the lithium acetate method. Ito et al. (1983) J. Bacteriol.
153:163-168.
[0136] Full length bak or truncated bak.DELTA.2-TM (aa71-187)
constructs were amplified by PCR from cDNA as EcoRI fragments and
cloned as in-frame fusions to the GAL4 DNA binding domain into the
vectors pGBT9 and pAS2-1, respectively (Clontech, Palo Alto,
Calif.). A human heart cDNA library of 3.times.10.sup.6 independent
clones in pGAD 10 (Clontech) was amplified and cotransformed into
yeast strain CG145 (Clontech) with Bak bait plasmids. Histidine
positive transformants were assayed for .beta.-galactosidase
activity by the filter method. Plasmid DNA was isolated from all
His.sup.+/X-Gal.sup.+ clones by the rapid technique recommended by
Clontech or by spheroplasting and subsequent alkaline lysis. This
DNA was amplified by PCR to determine insert size. pGAD10 clones
were isolated by transforming E. coli strain HB101 and selecting on
M9 Leucine-Ampicillin plates. Plasmids were sorted by restriction
enzyme digest and Southern blot and then sequenced.
[0137] Twenty-six clones activated both the .beta.-galactosidase
and histidine reporter genes. Sixteen clones encoded the same
protein, which was termed BBP. DNA sequence analysis of BBP cDNA,
shown in FIG. 4, revealed an open reading frame encoding a 151
amino acid protein, with a predicted relative mass of 16 kD.
TopPredII, a program for predicting membrane protein structure,
indicated that BBP was a three transmembrane protein with
hydrophilic N-terminal 42 amino acid region and a 58 amino acid
loop region, both located on the cytoplasmic side of the
membrane.
Example 2
Northern Blot Analysis of cDNA Clones
[0138] Northern blot analysis was performed according to the method
described by Lehrach et al. (1977) Biochem. 16:4743-4751 and Thomas
(1980) Proc. Natl. Acad. Sci. USA 77:5201-5205. In addition, a
human multiple tissue Northern blot was purchased from Clontech.
The coding regions of BBP cDNA was labeled by the random priming
method described by Feinberg and Vogelstein (1984) Anal. Biochem.
137:266-267. Hybridization and washing conditions were performed
according to standard methods.
[0139] The results, presented in FIG. 5, indicate that BBP shows a
wide tissue distribution among the organs tested (heart, brain,
placenta, lung, liver, skeletal muscle, kidney and pancreas), with
high levels of a 1.2 kb mRNA found in heart, skeletal muscle and
pancreas.
Example 3
BBP Interacts with Itself and with BAK
[0140] Using the yeast two hybrid system described above, the
interactions between BBP and members of the bcl2 family of
apoptosis modulators, as well as with BBP itself, were analyzed.
BBP was expressed as a fusion protein with the activation domain
(AD) of GAL1. Genes encoding BBP, Bak, Bcl2, Bclx, Bax and E1B19k
proteins were inserted into expression vectors containing the
coding sequence for the binding domain of GAL4 such that these
proteins would be expressed as fusion proteins with the binding
domain (BD) of GAL4. Results, summarized in FIG. 6, indicated that
BBP associates with itself and with Bak, but not with the other
members of the bcl2 family that were tested.
Example 4
Copy Number and Chromosomal Location of bbp
[0141] Southern blot analysis was used to determine gene copy
number and size of the bbp gene(s). Restriction analysis of human
genomic DNA with several enzymes, including BamHI, EcoRI and
HindIII, revealed multiple bands hybridizing to the bbp cDNA. EcoRI
generated eight bands ranging in size from 1.5 to 15 kilobase pairs
(kb). Similar patterns were seen with BamHI and HindIII. To
investigate the number and distribution of bbp genes, a Somatic
Cell Hybrid Panel of human chromosomes was obtained from Coriell
Cell Repositories (Camden, N.J.). Each mouse or Chinese Hamster
somatic cell hybrid contains one human chromosome. Genomic DNA from
these hybrids was digested with EcoRI along with human, mouse and
Chinese Hamster genomic DNA controls. Under stringent hybridization
conditions (0.1.times.SSC, 0.1% SDS, 65.degree. C.) with the bbp
cDNA, genes were found to reside on human chromosomes 2, 5 and X
and align with eight bands seen with total human genomic DNA. Thus,
there are at least three gene members in the BBP family.
Example 5
Examination of Bak Interactions with BBP
[0142] To delineate the regions of Bak and BBP that mediate the
association of these two proteins, two series of experiments were
conducted in which portions of BBP fused to
glutathione-S-transferase (gst) were allowed to interact with Bak
or derivatives thereof.
[0143] In the first series of experiments, gst fusion proteins were
made that contained the two hydrophilic regions of BBP, full-length
E1B 19K or full-length BHRF-1. The hydrophilic regions of BBP are
the N-terminal 42 amino acid portion and an internal loop of BBP
consisting of amino acids 86 through 133, and are referred to as
BBP-Loop1 (BBP-L1) and BBP-Loop2 (BBP-L2), respectively. DNA
sequences encoding the hydrophilic regions were amplified by
polymerase chain reaction (PCR) and subcloned into pGEX-5.times.
(Pharmacia) in-frame with gst.
[0144] Gst fusion proteins containing BBP-L1, BBP-L2, E1B19K or
BHRF-1 were immobilized on a glutathione-Sepharose column. Yeast
lysates expressing Bak or control vector were passed over the
column, washed and Bak binding was assessed by Western blot
analysis. As had been previously shown, Bak interacted with BHRF-1
and E1B19K. Bak was also shown to interact with the N-terminal 42
amino acid region of BBP and with the internal loop (FIG. 7).
[0145] To define further the interaction between Bak and BBP, a
peptide consisting of amino acids 1-50 of Bak was used in place of
full-length Bak and binding to immobilized BBP-L1 or BBP-L2 was
assessed. The synthetic peptide of the first 50 residues of Bak
(Bak 1-50) was supplied by Pharmingen. This peptide is soluble in
1.times.phosphate-buffered saline (PBS).
[0146] For each of the three glutathione SEPHAROSE.RTM./gst fusion
protein complexes (representing BBP-L1, BBP-L2 and a control), 100
.mu.g of Bak 1-50 (in 1 ml of PBS/0.1% TRITON.RTM. X-100, a
nonionic surfactant) was added to 50 .mu.L of resin in an Eppendorf
tube and left overnight at 4.degree. C. Duplicate samples were
prepared lacking the peptide in the PBS/0.1% TRITON.RTM. X-100
(nonionic surfactant) buffer. The following day, the resins were
washed 5 times with the PBS/0.1% TRITON.RTM. X-100 (nonionic
surfactant) buffer and then the resins boiled in 50 .mu.L 1.times.
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS
PAGE) sample loading buffer. A western was performed to assess the
protein-protein interactions. The results, shown in FIG. 8A,
demonstrate that a region in the first 50 amino acids of Bak is
capable of interacting with both BBP-L1 and BBP-L2.
[0147] In a second series of experiments, an N-terminal deletion of
Bak was made and analyzed in a similar manner as above for binding
to the hydrophilic regions of BBP. The N-terminal deletion mutant
of Bak is termed Bak.DELTA.3, represents amino acids 96-210 of
full-length Bak and is shown in FIG. 3. Bak.DELTA.3 was cloned into
a baculovirus vector. The PCR amplified Bak.DELTA.3 was subcloned
into the pBlueBac III vector (Invitrogen) and recombinant plasmids
were isolated. Recombinants were sequenced by the dideoxy chain
termination method (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA
74:5463-5467) using sequencing kits purchased from U.S.
Biochemicals (SEQUENASE.RTM. version 2.0). Recombinant viruses were
generated by cotransfecting Spodoptera frugiperda clone 9 (Sf9)
cells with the recombinant plasmid and wild-type AcMNPV viral DNA
using cationic liposomes as described by supplier (Invitrogen).
Recombinant viruses were identified by visual screening and PCR and
propagation of recombinant baculovirus was performed as described
by the supplier (Invitrogen).
[0148] For the production of recombinant Bak.DELTA.3,
1.times.10.sup.7 Sf9 cells were cultured in each of twenty 75
cm.sup.2 tissue culture flasks (Corning) containing 15 ml of
Grace's complete insect cell media (Invitrogen) supplemented with
10% fetal bovine serum. After allowing the insect cells to attach
to the plate for 1 hour, the cells were infected at a multiplicity
of infection of 5 with baculovirus expressing bak.DELTA.3. The
plates were placed on a rocker for 1 hour and then the media was
removed and replaced with 15 ml of fresh media. The plates were
placed at 27.degree. C. and left for 72 hours. After allowing the
Sf9 cells to express the Bak.DELTA.3, the cells were harvested,
centrifuged, and washed with 1.times.PBS. The pellet was collected
into a single 15 ml tube and washed again with 1.times.PBS. The
pellet was then extracted with 0.5% NP-40 extraction buffer (10 mM
HEPES, 142.5 mM KCl, 1 mM EGTA, 5 mM MgCl.sub.2, 0.5% NP-40, pH
7.2). This was left overnight at 4.degree. C. with gentle
shaking.
[0149] The solubilized cell extract was then sonicated,
centrifuged, and one ml of each supernatant was mixed with 50 ml of
a suspension of glutathione SEPHAROSE.RTM. to which either gst,
gst-BBP-L1 or gst-BBP-L2 was bound. The extract containing
Bak.DELTA.3 was kept at 4.degree. C. for 24 hours with the
SEPHAROSE.RTM.-bound gst, gst-BBP-L1 or gst-BBP-L2 suspensions. The
SEPHAROSE.RTM.-protein complexes were collected by centrifugation,
washed three times with the buffer described above, then suspended
in 100 ml each of 1.times.SDS-PAGE buffer. Eight .mu.L of each
sample were run on a 15% SDS PAGE gel and, after the proteins were
satisfactorily separated, transferred to nitrocellulose for
immunoblotting. Complexes containing the Bak derivative were
detected using rabbit IgG anti-Bak antibodies and the blot was
developed using enhanced chemiluminescence (ECL) and anti-rabbit
IgG/peroxidase conjugate. The results, shown in FIG. 8B, indicate
that N-terminal deletion of Bak represented by Bak.DELTA.3 is
capable of interaction with BBP-L1, but is not capable of
associating with the hydrophilic region represented by BBP-L2.
Example 6
Drug Screening Assay
[0150] This example describes an assay useful for screening for
agents such as drugs that alter the affinity of binding of Bak with
BBP.
[0151] Specifically, this example presents a scheme for using a BBP
such as the BBP-Loop1 or BBP-Loop2 in a drug screening assay that
is suitable for automated high through-put random drug screening. A
DNA sequence encoding BBP-Loop1 or BBP-Loop2 is amplified by PCR
and subcloned into the pGEX-5.times.plasmid in frame with gst,
allowing the production of Gst-BBP-L1 or Gst-BBP-L2 fusion proteins
in E. coli. Gst-BBP-L1 or Gst-BBP-L2 are purified by standard
methods using glutathione-Sepharose chromatography, then the fusion
proteins are immobilized onto 96-well microtiter plates. Bak
coupled to a second moiety that can serve as a detectable tag, e.g.
green fluorescent protein (GFP; Prasher (1995) Trends Genet.
11:320-323), is added along with the agent whose ability to affect
the binding between Bak and BBP is to be tested, including, but not
limited to a small molecule or peptide. Control samples include no
test agent. After a suitable incubation period, the test wells are
washed, and fluorescence is monitored by measuring excitation at
488 nm and emission at 511 nm. Test wells showing a significantly
higher or significantly lower fluorescence compared with the
control are then examined further to confirm an effect on Bak-BBP
binding.
Example 7
Analysis of Bak-BBP Interaction
[0152] To further confirm Bak-BBP interactions and to determine
regions of Bak that are important in binding to BBP, SPOTS peptide
binding assays were performed.
[0153] Sixty-three Bak peptides, 15 amino acids in length, were
synthesized on a membrane using a SPOTs kit according to suppliers
instructions (Genosys Biotechnologies, The Woodlands, Tex.). The
peptides were overlapping, off register by 4 amino acids, and
spanned amino acids 1-194 (see SEQ ID NO:2). All regions of Bak
were represented except for the transmembrane region. The order of
synthesis was determined using the SPOTscan and SPOTslot programs
provided by the suppliers.
[0154] SPOTs kit included proprietary membrane for peptide
synthesis, derivatized amino acids, computer program for
determining the order of synthesis, synthesis trough,
10.times.blocking buffer concentrate, and complete instructions.
Some reagents had to be obtained from outside sources.
Dimethylformamide (DMF) was from EM Science (Gibbstown, N.J.) while
N-methylpyrrolidone (NMP) was from Applied Biosystems (Foster City,
Calif.). Both were peptide synthesis grade. Molecular Sieves
(4.ANG.) were obtained from Aldrich (Milwaukee, Wis.).
[0155] Prior to the start of peptide synthesis, the solvents used
(DMF and NMP) had to be further purified to remove any reactive
amines or water. This was done using the molecular sieves which
were activated by heating at 250.degree. C. for 24 hours, followed
by drying in a vacuum dessicator containing DRIERITE.RTM. dessicant
(Aldrich) for another 48 hours. The activated sieves were mixed
with the solvents (at 25% of the total solvent volume) and left for
72 hours.
[0156] Upon completion of peptide synthesis the membrane was washed
in TBS (50 mM Tris, 150 mM NaCl, 5 mM KCl, pH 8) three times and
then incubated in blocking buffer overnight (suppliers blocking
buffer in TBS/0.05% TWEEN-20.RTM. (polysorbate 20), 5% sucrose) at
room temperature. The membrane was washed three times with
TBS/0.05% TWEEN-20.RTM. (polysorbate 20) and incubated overnight at
room temperature in 25 mL of blocking buffer containing 2 mg
purified GST-BBP loop 1, GST-BBP loop 2 or GST (negative control).
The membrane was again washed three times with TBS/0.05%
TWEEN-20.RTM. (polysorbate 20), incubated with rabbit polyclonal
anti-GST (1:1000; Pharmacia) for 1 hour at room temperature, washed
again with TBS/0.05%-20.RTM. (polysorbate 20) and incubated with
goat anti-rabbit IgG/peroxidase conjugate (Sigma, St. Louis, Mo.)
for 1 hour at room temperature. The membrane was then developed
using ECL method (Amersham, Chicago, Ill.).
[0157] The results were as follows. BBP loop 1 interacted most
strongly with two contiguous clusters of Bak peptides (FIG. 9). The
first cluster overlapped the Bak BH3 domain (six peptides spanning
amino acids 70 to 102) and the second cluster began upstream and
extended into the N-terminal region of the BH1 domain (amino acids
103-126). Several weaker interactions occurred between BBP loop 1
and Bak peptides in two regions upstream of the Bak BH3 domain and
a region between the Bak BH1 and BH2 domains. BBP loop 2 did not
interact with any of the Bak peptides in the SPOTS assay (data not
shown).
[0158] The BH3 domain of Bak has been shown to be important for the
cell death promoting activity of Bak (Chittenden et al., 1995 EMBO
J. 14, 5589-5596). Our SPOTS data suggest that BBP can cooperate
with Bak in modulating apoptosis by binding to the BH3 domain of
Bak. Interestingly, it was recently shown that in some cases Bak
does not require its BH3 domain to promote apoptosis (Simonian et
al., 1997, Oncogene 15, 1871-1875). Bak.DELTA.3, a construct which
lacks the N-terminal 95 amino acids, including the BH3 domain, was
shown to be extremely active in promoting cell death in insect
cells (data not shown). Bak.DELTA.3 was also shown to bind to BBP
loop 1 (FIG. 8B).
[0159] Our SPOTS data support that the BBP interaction with
Bak.DELTA.3 occurs in the region of Bak upstream and extending into
the N-terminal region of the BH1 domain (amino acids 103-126 of SEQ
ID NO:2, defined as "BBP Binding Domain 1", nucleic acids 507-578
of SEQ ID NO:1, defined as "bbpbd-1") and also in the region
immediately downstream from the BH1 domain (amino acids 138-156 of
SEQ ID NO:2, defined as "BBP Binding Domain 2", nucleic acids
611-668 of SEQ ID NO:1, defined as "bbpbd-2"). Collectively, BBP
Binding Domains 1 and 2 are defined as "BBP Binding Domains". The
results support that these regions are additional important death
domains of Bak.
[0160] Although the foregoing invention has been described in some
detail for purposes of clarity and understanding, it will be
apparent to those skilled in the art that certain changes and
modifications can be practiced. Therefore, the description and
examples should not be construed as limiting the scope of the
invention, which is delineated by the appended claims.
Sequence CWU 1
1
* * * * *