WO2000026228A1 - Gene et proteine regulant la mort cellulaire - Google Patents

Gene et proteine regulant la mort cellulaire Download PDF

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Publication number
WO2000026228A1
WO2000026228A1 PCT/US1999/025285 US9925285W WO0026228A1 WO 2000026228 A1 WO2000026228 A1 WO 2000026228A1 US 9925285 W US9925285 W US 9925285W WO 0026228 A1 WO0026228 A1 WO 0026228A1
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bbc3
protein
cells
bcl
biologically active
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PCT/US1999/025285
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English (en)
Inventor
Li Zhu
Xudong Yin
Thomas Chittenden
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Clontech Laboratories, Inc.
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Priority to EP99956735A priority Critical patent/EP1129102A1/fr
Publication of WO2000026228A1 publication Critical patent/WO2000026228A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4747—Apoptosis related proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • apoptosis biochemically regulated cell death
  • Regulators of apoptosis both positive and negative, have been identified.
  • the protein known as Bcl-2 counters a variety of apoptotic stimuli (Vaux e t al., 1988; S trasser et al., 1991 ; Garcia et al., 1992).
  • CED-9 th e homolog of Bcl-2 in the nematode Caenorhabditis elegans, is found t o repress apoptosis in cells that are normally expected to die during th e nematode's development.
  • Bcl-2 can substitute for CED-9 functionally in preventing at least some cell death in these nematodes .
  • Vaux et al., 1992; Hengartner and Horvitz, 1994 Many Bcl-2 related proteins share homology within two conserved regions: Bcl-2 homology domains 1 and 2 (referred to a s BH1 and BH2, respectively) (Williams and Smith, 1993; Yin et al., 1994).
  • These proteins include Bax, Bcl-x L , Mcl-1, and Al (Oltvai et al., 1993 ; Boise et al, 1993; Kozopas et al, 1993; Lin et al., 1993).
  • Several of these proteins are cell death regulators; for example, Bcl-x L represses apoptosis, while its short form, Bcl-x s , favors cell death .
  • Bax in excess, interferes with the ability of Bcl-2 t o repress apoptosis. Bax homodimerizes and also heterodimerizes with Bcl-2 (Oltvai et al. , 1993).
  • Bcl-x L /Bcl-2-associated death promoter homolog is conserved within the BH1 and BH2 domains (Yang, E., J. Zha, J . Jockel, L.H. Boise, C.B. Thompson, S.J. Korsmeyer [1995] Cell 80 : 285 - 291).
  • Bad has been shown to heterodimerize with Bcl-xL and Bcl-2, b u t not with other related proteins.
  • One way in which Bad promote s mammalian cell death is by displacing Bax from Bcl-x L as it heterodimerizes with Bcl-x L .
  • the subject invention concerns polynucleotides which encode proteins that regulate mammalian cell death. Specifically exemplified herein is a gene designated bbc3.
  • the subject invention further concerns novel polypeptides encoded by the polynucleotides of the present invention.
  • a further aspect of the subject invention concerns antibodies which can b e raised to the novel proteins of the subject invention.
  • the subject invention further concerns polynucleotides sequences which can be used as probes and primers for the bbc3 gene and homologous polynucleotides.
  • the polynucleotide sequences, proteins, and antibodies of the subject invention are useful for diagnostic and therapeutic procedures.
  • a method of promoting apoptosis in cells comprising th e step of contacting said cells with a BBC3 protein, or biologically active fragment or variant thereof, wherein said contact promotes apoptosis in said cells.
  • a method of inhibiting apoptosis in cells comprising the s tep of contacting said cells with an antibody directed towards a BBC3 protein, or biologically active fragment or variant thereof, wherein said contacting inhibits apoptosis in said cells.
  • SEQ ID NO. 1 is a polynucleotide sequence of the bbc3 gene.
  • SEQ ID NO. 2 is a deduced amino acid sequence (ORF1) of a polypeptide encoded by the polynucleotide sequence of SEQ ID NO. 1.
  • SEQ ID NO. 3 is a deduced amino acid sequence (ORF2) of a polypeptide encoded by the polynucleotide sequence of SEQ ID NO. 1.
  • Figure 1 shows the cytotoxicity of HA-BBC3-ORF1 in Rat- l cells.
  • Rat-1 cells were co-transfected with a ⁇ -galactosidase m arker plasmid in combination with the indicated plasmids (pRcCMV is th e vector control).
  • Cells were fixed and stained with X-gal at 24 hours post-transfection, and the number of blue ( ⁇ -galactosidase positive) cells counted by microscopic examination.
  • Figure 2 is a schematic depiction of the BBC3-ORF1 and
  • Figure 3 shows the specific binding of BBC3-ORF2 to Bcl-x L in vitro.
  • Hemaglutinin (HA) and Flag (FT) epitope-tagged forms o f BBC3-ORF2 were generated by PCR cloning methods.
  • 35 S labeled proteins were produced by translation in vitro and incubated with either GST or GST-Bcl-x fusion proteins. The complexes were captured on glutathione agarose beads and bound proteins detected b y electrophoresis on SDS polyacrylamide gels followed b y autoradiography.
  • Figure 4 shows the BH3 domain within BBC3-ORF2 and its sequence homology to the BH3 domains in other pro-apoptotic proteins . The alanine substitution and BH3 deletion mutations introduced into BBC3-ORF2 are shown at the bottom.
  • Figure 5 shows that the BH3 domain of BBC3-ORF2 is sufficient for binding to Bcl-x L .
  • A) Truncated derivatives of BBC3- ORF2 encompassing BH3 were translated in vitro and incubated with either GST or GST-Bcl-x L (as described in Figure 3).
  • the truncated BBC3-ORF2 proteins encode amino acids 136 to 185, and 136 to 1 65 , respectively. Both proteins bind specifically to GST-Bcl-x L .
  • B Binding of BBC3-ORF2 to Bcl-x L is inhibited by BH3 peptides from either Bak or Bad.
  • the subject invention concerns polynucleotide sequences encoding proteins which regulate mammalian cell death. Specifically exemplified herein is a gene designated bbc3. In a preferred embodiment of the subject invention, the proteins of the subj ect invention regulate cell death through interactions with Bcl-2.
  • the unique polynucleotide sequences of the subj ect invention include sequences which encode the BBC3 polypeptides, a s well as sequences which drive the expression of these proteins.
  • the subject invention also includes those polynucleotide sequences which are antisense to bbc3 gene sequences.
  • the polypeptides encoded by the polynucleotide sequences described herein can be used to generate antibodies to the subject polypeptides . These antibodies can be used in diagnostic or therapeutic applications.
  • the biological activity of the BBC3 polypeptides of the subject invention can b e reduced or eliminated by administering an effective amount of a n antibody to a BBC3 polypeptide.
  • the activity of the BBC3 polypeptide can be controlled by modulation of expression of the bb c 3 mRNA and the polypeptide encoded thereby. This can be accomplished by, for example, the administration of antisense DNA.
  • the BBC3 protein shown in SEQ ID NO. 2 has a molecular weight of about 24.4 kDa, while the BBC3 protein shown in SEQ ID NO. 3 has a molecular weight of about 29 kDa.
  • any of a number of different nucleotide sequences can be used, based on the degeneracy of the genetic code, to produce the cell death regulatory proteins described herein. Accordingly, any nucleotide sequence which encodes the cell death regulatory proteins described herein comes within the scope of this invention and the claims appended hereto.
  • fragments of the cell death regulatory proteins are an aspect of the subject invention so long a s such fragments retain the biological activity so that such fragments are useful in therapeutic and/or diagnostic procedures as described herein. Such fragments can easily and routinely be produced b y techniques well known in the art. For example, time-controlled BaVb 1 exonuclease digestion of the full-length DNA followed by expression o f the resulting fragments and routine screening can be used to readily identify expression products having the desired activity.
  • nucleic acid an d “polynucleotide sequence” refer to a deoxyribonucleotide o r ribonucleotide polymer in either single- or double-stranded form, an d unless otherwise limited, would encompass known analogs of natural nucleotides that can function in a similar manner as naturally- occurring nucleotides.
  • the polynucleotide sequences include both the DNA strand sequence that is transcribed into RNA and the RNA sequence that is translated into protein.
  • the polynucleotide sequences include both full-length sequences as well as shorter sequences derived from the full-length sequences.
  • polynucleotide sequence includes the degenerate codons of the native sequence or sequences which may be introduced to provide co don preference in a specific host cell. Allelic variations of the exemplified sequences also come within the scope of the subject invention.
  • the polynucleotide sequences falling within the scope of the subj ect invention further include sequences which specifically hybridize with the exemplified sequences under stringent conditions.
  • the nucleic acid includes both the sense and antisense strands as either individual strands or in the duplex.
  • hybridize or “hybridizing” refer to the binding of two single-stranded nucleic acids via complementary base pairing.
  • hybridizing specifically to refers to binding, duplexing, or hybridizing of a molecule to a nucleotide sequence under stringent conditions when that sequence is present in a preparation o f total cellular DNA or RNA.
  • stringent conditions refers to conditions under which a polynucleotide probe will hybridize to its target sub-sequence, but not to sequences having little or no homology to the target sequence.
  • stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence a t a defined ionic strength and pH.
  • Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a complementary probe.
  • stringent conditions will be those in which the salt concentration is at least about 0.1 to 1 .0 N Na ion concentration at a pH of about 7.0 to 7.5 and the temperature is at least about 60°C for long sequences (e. g. , greater than about 5 0 nucleotides) and at least about 42°C for shorter sequences (e. g. , about 10 to 50 nucleotides).
  • isolated or “substantially pure” when referring to polynucleotide sequences encoding the cell death regulatory proteins or fragments thereof refers to nucleic acids which encode cell death regulatory proteins or polypeptides and which are no longer in the presence of sequences with which they are associated in nature.
  • isolated or “substantially purified” when referring to the proteins of the subject invention means a chemical composition which is essentially free of other cellular components. It is preferably in a homogenous state and can be in either a dry o r aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein which is the predominant species present in a preparation is substantially purified. Generally, a substantially purified or isolated protein will comprise more than 80% of all macromolecular species present in the preparation. Preferably, the protein is purified t o represent greater than 90% of all macromolecular species present.
  • the protein is purified to greater than 95%, and mos t preferably the protein is purified to essential homogeneity, wherein other macromolecular species are not detected by conventional techniques .
  • the specified antibodies bound to a particular protein do not bind in a significant amount to other proteins present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein.
  • a variety o f immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein.
  • solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein. Harlow and Lan ( 1988), incorporated herein by reference, describe immunoassay formats and conditions that could be used to determine specific immunoreactivity.
  • the subject invention further concerns antibodies raised against the purified BBC3 molecules or their fragments.
  • biological sample refers to any sample obtained from a living organism or from an organism that has died.
  • biological samples include body fluids, tissue specimens, and tissue cultures lines taken from patients.
  • recombinant DNA or “recombinantly-produced DNA” refers to DNA which has been isolated from its native o r endogenous source and modified either chemically or enzymatically t o delete naturally-occurring flanking nucleotides or provide flanking nucleotides that do not naturally occur. Flanking nucleotides are those nucleotides which are either upstream or downstream from the described sequence or sub-sequence of nucleotides.
  • recombinant protein or “recombinantly- produced protein” refers to a polypeptide or protein produced using cells that do not have an endogenous copy of DNA able to express th e protein encoded thereby.
  • the cells produce the protein because they have been genetically altered by the introduction of an appropriate polynucleotide sequence that can be expressed in the host cell.
  • the recombinant protein will not be found in association with proteins an d other subcellular components associated with the cells that normally produce the protein.
  • DNA possesses a fundamental property called base complementarity. In nature, DNA ordinarily exists in the form of pairs of anti-parallel strands, the bases on each strand projecting from that strand toward the opposite strand.
  • the base adenine (A) on one strand will always be opposed to the base thy mine (T) on the other strand, and the base guanine (G) will be opposed t o the base cytosine (C).
  • the bases are held in apposition by their ability to hydrogen bond to their complementary base in this specific manner. Though each individual bond is relatively weak, the net effect of many adjacent hydrogen bonded bases, together with base stacking effects, is a stable joining of the two complementary strands. These bonds c an be broken by treatments such as high pH or high temperature, an d these conditions result in the dissociation, or "denaturation," of the two strands. If the single-stranded DNA is then placed in conditions which make hydrogen bonding of the bases thermodynamically favorable, the DNA strands will anneal, or "hybridize,” and reform the original double stranded DNA. If carried out under appropriate conditions, this hybridization can be highly specific.
  • hybridization may be used to test whether two pieces of DNA are complementary in their base sequences. It is this hybridization mechanism which facilitates the use of probes of the subject invention to readily detect and characterize DNA sequences of interest.
  • PCR Polymerase Chain Reaction
  • Oligonucleotides based on the polynucleotide sequences of the bbc3 gene, that can be used as primers for PCR amplification are also encompassed within the subject invention.
  • a certain degree of mismatch can be tolerated between primer and template. Therefore, mutations, deletions, and insertions (especially additions of nucleotides to the 5' end) of the subject oligonucleotide primers fall within the scope of the subject invention. Mutations, insertions and deletions can be produced in a given primer by methods known to an ordinarily skilled artisan. It is important t o note that the mutational, insertional, and deletional variants generated from a given primer sequence may be more or less efficient than th e original sequences. Notwithstanding such differences in efficiency, these variants are within the scope of the present invention.
  • the DNA can first be obtained in its native, double-stranded form. A number of procedures are currently used to isolate DNA and are well known to those skilled in this art.
  • probes of the subject invention entails first identifying by Southern blot analysis in a DNA library all DNA segments homologous with polynucleotide sequences of the present invention. Thus, it is possible, without the aid of biological analysis, to determine in advance the presence of genes homologous with the polynucleotide sequences described herein. Such an analysis using the subject probes provides a rapid diagnostic method.
  • One hybridization procedure useful according to th e subject invention typically includes the initial steps of isolating the DNA sample of interest and purifying it chemically.
  • total fractionated nucleic acid isolated from a biological sample can b e used.
  • Cells can be treated using known techniques to liberate their DNA (and/or RNA).
  • the DNA sample can be fragmented with a n appropriate restriction enzyme or by other means known in the art.
  • the fragments can be separated by size through electrophoresis in a gel, usually agarose or acrylamide.
  • the nucleic acid fragments c an then be transferred to and immobilized on a membrane in a manner that retains the size relationship of the fragments from the gel.
  • the membrane can then be dried and prehybridized to equilibrate it for later immersion in a hybridization solution.
  • the manner in which the nucleic acid is affixed to a solid support may vary. Fixing the DNA o n the membrane for later processing has great value for the use of this technique in field studies, remote from laboratory facilities.
  • probes can be contacted with the membrane in a hybridization buffer and allowed to hybridize to the DNA fragments under selected conditions of hybridization stringency. After a sufficient period of time has elapsed for annealing of probe to th e immobilized DNA, the membrane is washed free of extraneous, non- hybridized materials.
  • Oligonucleotide probes for use with the methods of the subject invention can be selected and prepared based on th e polynucleotide sequence of the bbc3 gene using standard techniques known in the art. For example, probes can be readily prepared using an automated DNA synthesizer. In addition, PCR-amplified DNA c an serve as a hybridization probe of the present invention.
  • probe molecule and nucleic acid sample hybridize by forming a strong non-covalent bond between the two molecules, it can be reasonably assumed that the probe an d polynucleotide fragment share significant sequence homology.
  • the particular hybridization technique used is not essential to the subj ect invention. As improvements are made in hybridization techniques, they can be readily applied in the subject invention.
  • th e particular probe can be labeled with any suitable detectable label known to those skilled in the art, including radioactive and non- radioactive labels.
  • the probe's detectable label provides a means for determining in a known manner whether hybridization has occurred.
  • Typical radioactive labels include 3 H, 14 C, 32 P, 35 S, or the like.
  • a prob e labeled with a radioactive isotope can be constructed from a nucleotide sequence complementary to the DNA sample by a conventional nick translation reaction, using a DNase and DNA polymerase. Radioactive labeled probes can be detected and quantified by autoradiography and/or liquid scintillation counting. For synthetic probes, it may be most desirable to use enzymes such a s polynucleotide kinase or terminal transferase to end-label the DNA for use as probes.
  • Non-radioactive labels include, for example, ligands such a s biotin or thyroxine, as well as enzymes such as hydrolases o r perixodases, or the various chemiluminescers such as luciferin, o r fluorescent compounds like fluorescein and its derivatives.
  • the probes may be made inherently fluorescent as described in International Application No. WO93/16094.
  • the probe may also be labeled at bo th ends with different types of labels for ease of separation, as, for example, by using an isotopic label at the end mentioned above and a biotin label at the other end.
  • the polynucleotide may be conjugated with ligands, haptens, or antigenic determinants.
  • the conjugated polynucleotide is then contacted with the ligand receptor, an anti-ligand molecule th at binds to the ligands, or with an antibody that binds to the hapten/antigenic determinant, respectively.
  • the polynucleotide can be labelled with digoxygenin and detected with labelled anti-digoxygenin antibodies.
  • the ligand receptor, anti-ligand molecule, or antibody may be directly labeled with a detectable signal system, such as a fluorophore, chemiluminescent molecule, radioisotope, or enzyme. Methods for preparing and detecting labeled moieties are known in the art.
  • the amount of labeled probe which is present in th e hybridization solution will vary widely, depending upon the nature o f the label, the amount of the labeled probe which can reasonably bind to the filter, and the stringency of the hybridization. Generally, substantial excesses of the probe will be employed to enhance the rate of binding of the probe to the fixed DNA.
  • Various degrees of stringency of hybridization can b e employed. The more stringent the conditions, the greater th e complementarity between the strands that is required for duplex formation. Stringency can be controlled by temperature, probe concentration, probe length, ionic strength, time, and the like.
  • hybridization is conducted under stringent conditions using techniques well known in the art, as described, for example, in Keller and Manak, 1987, incorporated herein by reference.
  • th e nucleotide sequences of the subject invention include mutations (b oth single and multiple), deletions, insertions of the described sequences, and combinations thereof, wherein said mutations, insertions and deletions permit formation of stable hybrids with the target polynucleotide of interest. Mutations, insertions, and deletions can b e produced in a given polynucleotide sequence in many ways, and these methods are known to an ordinarily skilled artisan. Other methods may become known in the future.
  • the known methods include, but are not limited to: ( 1 ) synthesizing chemically or otherwise an artificial sequence which is a mutation, insertion or deletion of the known sequence; (2) using a nucleotide sequence of the present invention as a probe to obtain via hybridization a new sequence or a mutation, insertion or deletion o f the probe sequence; and (3) mutating, inserting or deleting a te st sequence in vitro or in vivo.
  • mutational, insertional, and deletional variants generated from a given probe may be more or less efficient than the original probe. Notwithstanding such differences in efficiency, these variants are within the scope of the present invention.
  • nucleic acid sequences can be readily prepared by methods which are well known to those skilled in the art. These variants can be used in the same manner as the instant polynucleotide sequences so long as the variants have substantial sequence homology with the polynucleotides.
  • substantially sequence homology refers to homology which is sufficient to enable the variant polynucleotide to function in the same capacity as the original polynucleotide. Preferably, this homology is greater than 50%; m ore preferably, this homology is greater than 75%; and most preferably, this homology is greater than 90%.
  • the degree of homology needed for the variant to function in its intended capacity will depend up o n the intended use of the polynucleotide sequence. It is well within the skill of a person trained in this art to make mutational, insertional, and deletional mutations which are designed to improve the function of the sequence or otherwise provide a methodological advantage. It is well known in the art that the amino acid sequence of a protein is determined by the nucleotide sequence of the DNA that encodes the protein. Because of the degeneracy of the genetic c ode (i. e. , for most amino acids, more than one nucleotide triplet (codon) codes for a single amino acid), different nucleotide sequences c an code for a particular amino acid, or polypeptide. Thus, the polynucleotide sequences of the subject invention also encompass those degenerate sequences that encode the polypeptides of the subject invention, or a fragment or variant thereof.
  • the subject invention includes probes which hybridize with various polynucleotide sequences encoding a given protein or variations of a given protein. It has been shown that proteins of identified structure and function may be constructed by changing the amino acid sequence if such changes do not alter the protein secondary structure (Kaiser and Kezdy, 1984).
  • a further aspect of the claimed invention are antibodies that are raised by immunization of an animal with a purified protein o f the subject invention. Both polyclonal and monoclonal antibodies can be produced using standard procedures well known to those skilled in the art using the proteins of the subject invention as an immunogen (see, for example, Monoclonal Antibodies: Principles and Practice, 1983; Monoclonal Hybridoma Antibodies: Techniques and
  • kits contemplated by the subj ect invention may include in one or more containers: polynucleotide probes, positive and negative control reagents, and reagents for detecting the probes.
  • the kit may also include polynucleotide primers for performing PCR amplification.
  • a kit of the present invention c an include antibodies to BBC3 polypeptides.
  • the kit may optionally include other reagents or solutions, such as buffering and stabilization reagents, along with any other reagents that may be required for detecting probes, primers, or other reagents.
  • polypeptides of the subject invention include those which are specifically exemplified herein as well as related polypeptides which, for example, are immunoreactive with antibodies which are produced by, or are immunologically reactive with, the polypeptides specifically exemplified herein.
  • proteins and polypeptides described herein can be u sed in therapeutic or diagnostic procedures.
  • the proteins and polypeptides can be used to detect the presence of antibodies immunoreactive with bbc3 gene products.
  • proteins an d polypeptides can also be used as molecular weight standards in protein analysis procedures.
  • the polynucleotide sequences of the subject invention may be composed of either RNA or DNA. More preferably, the polynucleotide sequences are composed of DNA.
  • the polynucleotides of the subject invention can also be used as DNA size markers i n standard electrophoresis methods. Following are examples which illustrate procedures for practicing the invention. These examples should not be construed a s limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted. EXAMPLE 1
  • the MATCHMAKER yeast two-hybrid system (Clontech Laboratories, Palo Alto, CA) was used to evaluate the interaction between bbc3 and Bcl-2. This experiment was done in yeast two- hybrid reporter strain Y190. Y190 was transformed with 12 different combinations of DNA clones, listed below. After the colonies of transformants had grown up in the selection medium plate, individual colonies were patched onto a single selection plate (SD-leu/-Try) an d allowed to grow for 2 additional days at 30°C. All 12 slots of the plate had about an equal amount of yeast cells grown to saturation within 2 days. Then a lift assay was performed according to the standard protocol. A blue color in patched yeast colony represents a positive signal which indicates an interaction between the two DNA clone inserts. A white color represents a negative signal which indicates n o interaction. The results are shown in Table 1.
  • a clone e. g. , p53 + clone-1
  • its bait pVA3 in this case
  • yeast co-transformed with the bait and itself #5 in Table 1
  • Yeasts co-transformed with the empty vector (pGBT9) or unrelated bait (pLAM5.) should not show any positive signals (#4 and #6).
  • yeasts co-transformed with the clone (p53 + clone-2) and either the bait (pVA3), or the empty vector (pGBT9), o r unrelated bait (pLAM5.) will show positive signals.
  • pTDl which encodes SV40 T antigen
  • yeast reporter strain by co- transformation with the empty vector (#7, pGBT9), or its specific bait p53 (#8, pVA3), or an unrelated bait (#9, pLAM5.). Only in the case o f its specific bait p53 (#8, pVA3) was a positive signal observed.
  • bbc3 gene is preferentially expressed in peripheral blood leukocytes and muscle.
  • bbc3 expression is low in spleen, thymus, and ovary, but high in all of the cancer cell lines tested.
  • leukocytes can b e assayed for evidence of bbc3 expression.
  • Such expression can b e determined by detecting the protein itself or the presence of mRNA encoding BBC3.
  • the protein can readily be detected, for example, using antibodies to BBC3 produced as described herein.
  • the bbc3 cDNA insert was subcloned into the pcDNA3 mammalian expression vector (Invitrogen, Inc.), with an amino-terminal HA epitope tag introduced i n frame with BBC3-ORF1 (e.g. in the same frame as the Gal4 activation domain fusion).
  • This HA-BBC3-ORF1 construct was found to b e cytotoxic in transient transfection assays of Rat-1 cells ( Figure 1 ) .
  • Induction of apoptosis by BBC3 in this assay is manifested by a large reduction in the number of transfected cells expressing a co- transfected ⁇ -galactosidase marker plasmid (as described in Chittenden et al., 1995). Cytotoxicity of HA-BBC3-ORF1 was similar t o that of other pro-apoptotic proteins, including Bak and ICE, and could be suppressed by co-transfection of Bcl-x , indicating that BBC3 functions as a pro-apoptotic, Bcl-2/Bcl-x L binding protein.
  • the BBC3-ORF2 coding region (nucleotides 57-628) was cloned by PCR, with an epitope tag (either HA or Flag) introduced in frame at its amino terminus.
  • the HA and Flag tagged BBC3-ORF2 proteins were translated in vitro and produced 35 S labeled proteins o f approximately 32 kD.
  • Both of the epitope tagged BBC3-ORF2 proteins bound to a GST-Bcl-x L fusion protein, but failed to bind GST alone ( Figure 3).
  • the specific interaction of BBC3-ORF2 with Bcl-x L was similar to the binding of Bak with Bcl-x in this assay.
  • HA and Flag epitope tagged BBC3-ORF2 were both found t o be potently cytotoxic in transfection assays of Ratl cells, eliminating virtually all transfected cells. Additionally, the "native" 1.6 kb bb c 3 cDNA, cloned into a mammalian expression vector that provides n o methionine or epitope tag, was also cytotoxic in transfection assays . Therefore, BBC3-ORF2 comprises the pro-apoptotic protein encoded b y the bbc3 cDNA. It is likely that the cytotoxic activity of the HA-tagged BBC3-ORF1 ( Figure 1) construct is due to internal translation initiation and expression of BBC3-ORF2.
  • the protein encoded by the amino acid sequence in frame with the Gal4 activation domain was also cloned as an epitope tagged species and translated in vitro. However, this protein product failed to interact detectably with GST- Bcl-xL in equivalent in vitro binding assays.
  • BH3 for Bcl-2 homology domain 3
  • the BH3 domains of Bak, Bax, Bik and Bad mediate their interactions with Bcl-2 and Bcl-x L (reviewed by Adams et al., 1998).
  • Close examination of the amino acid sequence encoded by BBC3-ORF2 revealed th e presence of a candidate BH3 domain in the C-terminal half of th e protein (amino acids 141 to 150).
  • residues that have been shown to be critical for the function of BH3 in other proteins are largely conserved in the putative BBC3-ORF2 BH3 domain ( Figure 4).
  • BBC3-ORF2 BH3 domain To test the functional importance of the BBC3-ORF2 BH3 domain, three conserved residues (leu 141 , met 144 and asp 147) were each mutated to alanine (BBC3-ORF2ala; Figure 4). Additionally, a second mutant was generated by deleting 9 amino acids (residues 142- 150) within this region (BBC3-ORF2 ⁇ BH3). The BBC3-ala and BBC3- ⁇ BH3 mutants were translated in vitro and incubated with GST-Bcl-x L .
  • BBC3-ORF2ala showed reduced binding to GST-Bcl-x L relative to wild type BBC3-ORF2, whereas BBC3-ORF2 ⁇ BH3 failed to detectably bind t o GST-Bcl-x L .
  • Epitope-tagged BBC3-ORF2 forms were also expressed i n transfected COS cells, and interacted with co-transfected Bcl-x L .
  • Both BBC3-ORF2ala and BBC3-ORF2 ⁇ BH3 failed to interact with Bcl-x L in transfected COS cells.
  • Truncated derivatives of BBC3-ORF2 that encode either 3 0 or 50 amino acids encompassing its BH3 domain retained the ability to bind specifically t o GST-Bcl-x L ( Figure 5A).
  • the interaction of wild type BBC3-ORF2 with GST-Bcl-xL in vitro could be inhibited by the addition of synthetic peptides corresponding to the BH3 domains of either Bak or Bad ( Figure 5B). This result indicates that BBC3-ORF2 requires the s ame binding site in Bcl-x utilized by other BH3-containing, pro-apoptotic proteins.
  • BBC3-ORF2 antibody A portion of the BBC3-ORF2 (amino acids 1-140) was expressed as a GST-fusion protein in bacteria, purified, and used as a n antigen to immunize mice for the production of monoclonal antibodies (Harlow and Lane 1988). A hybridoma clone was isolated th at produces an antibody (designated KM4A5) that reacts with the GST- BBC3-ORF2 fusion protein antigen and detects Flag epitope-tagged BBC3-ORF2 produced in transfected COS cells. This antibody also recognizes a protein of approximately 29 kD in extracts from multiple human tumor cell lines, corresponding to the untagged, endogenous BBC3-ORF2 protein.
  • the present invention is directed to an isolated polynucleotide which comprises a nucleotide sequence encoding a BBC3 protein, or a biologically active fragment or variant thereof.
  • this isolated polynucleotide comprises a nucleotide sequence which hybridizes with the nucleotide sequence shown in SEQ ID NO. 1 or a fragment or variant thereof which encodes an amino acid sequence having BBC3 biological activity.
  • the present invention is also directed to a purified BBC3 protein, or a biologically active fragment or variant thereof.
  • this protein comprises a biologically active portion of the amino acid sequence shown in SEQ ID NO. 3.
  • this protein has at least about 80% homology with the amino acid sequence shown in SEQ ID NO. 3 or a biologically active portion thereof.
  • the present invention is also directed to an antibody which immunoreacts with a BBC3 protein.
  • this antibody is raised to a protein having at least about 80% homology with SEQ ID NO. 3 or a biologically active fragment thereof.
  • the present invention is also directed to a method o f promoting apoptosis in cells, comprising the step of contacting said cells with a BBC3 protein, or biologically active fragment or variant thereof, wherein said contact promotes apoptosis in said cells.
  • this method may be used to induce apoptosis in any cell type, a preferable use would be to induce apoptosis in either diseased cells such as malignant cells or cells causing autoimmune diseases.
  • the present invention is also directed to a method o f inhibiting apoptosis in cells, comprising the step of contacting said cells with an antibody directed towards a BBC3 protein, or biologically active fragment or variant thereof, wherein said contacting inhibits apoptosis in said cells.
  • this method may be used to inhibit apoptosis in any cell type, a preferably use would be to inhibit apoptosis in either diseased cells such as malignant cells or cells causing autoimmune diseases.
  • the present invention is also directed to a vector comprising the isolated polynucleotide disclosed herein. Furthermore, a person having ordinary skill in this art could readily transform a ho st cell with this vector.
  • C. elegans cell survival gene ced-9 encodes a functional homolog of the mammalian proto- oncogene bcl-2," Cell 76:665-676.
  • MCL-1 a gene expressed in programmed myeloid cell differentiation, has sequence similarity to BCL-2," Proc. Natl. Acad. Sci. USA 90 : 35 16- 3520.

Abstract

L'invention concerne de nouveaux polypeptides intervenant dans la régulation de la mort cellulaire. Elle concerne également des séquences polynucléotidiques codant ces nouveaux polypeptides. D'autres aspects de l'invention portent sur des sondes polynucléotidiques, sur des anticorps se rapportant à ces nouveaux polypeptides, et sur des méthodes diagnostiques et thérapeutiques.
PCT/US1999/025285 1998-11-02 1999-10-28 Gene et proteine regulant la mort cellulaire WO2000026228A1 (fr)

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WO2002064790A2 (fr) * 2000-12-19 2002-08-22 The Johns Hopkins University Proteine jfy induisant l'apoptose rapide
JP2008509882A (ja) * 2004-06-15 2008-04-03 セライト リミテッド 癌治療
WO2017137761A1 (fr) 2016-02-10 2017-08-17 Hilmar M Warenius Compositions et leurs utilisations

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US5763223A (en) * 1995-06-29 1998-06-09 Immunex Corporation DNA encoding a cytokine that induces apoptosis

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002064790A2 (fr) * 2000-12-19 2002-08-22 The Johns Hopkins University Proteine jfy induisant l'apoptose rapide
WO2002064790A3 (fr) * 2000-12-19 2003-05-30 Univ Johns Hopkins Proteine jfy induisant l'apoptose rapide
JP2008509882A (ja) * 2004-06-15 2008-04-03 セライト リミテッド 癌治療
EP1781780B1 (fr) * 2004-06-15 2012-05-23 Theryte Limited Traitement du cancer
WO2017137761A1 (fr) 2016-02-10 2017-08-17 Hilmar M Warenius Compositions et leurs utilisations

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