WO2002100897A2 - Complexes moleculaires brca1/acc alpha, applications diagnostiques et therapeutiques - Google Patents
Complexes moleculaires brca1/acc alpha, applications diagnostiques et therapeutiques Download PDFInfo
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- WO2002100897A2 WO2002100897A2 PCT/FR2002/002016 FR0202016W WO02100897A2 WO 2002100897 A2 WO2002100897 A2 WO 2002100897A2 FR 0202016 W FR0202016 W FR 0202016W WO 02100897 A2 WO02100897 A2 WO 02100897A2
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57415—Specifically defined cancers of breast
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57449—Specifically defined cancers of ovaries
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
Definitions
- the invention relates to a protein-protein complex involved in the predisposition to breast and ovarian cancer. More particularly, this protein-protein interaction has applications for the screening of molecules having a therapeutic activity in the treatment and prevention of cancer, as well as diagnostic applications.
- Breast cancer is a very common condition, affecting almost 10% of women in the western world. There are two forms of this cancer: a sporadic majority form and a familial form affecting 5 to 10% of cases.
- risk transmission takes place in an autosomal dominant mode (Ford D. et al., 1998), and mammary tumors are frequently associated with other cancers, notably ovarian cancer.
- BRCA1 Breast Cancer 1
- BRCA2 located in 17q21 (Miki Y. et al., 1994)
- BRCA2 located in 13q12-13 ( Wooster R. et al., 1995).
- the BRCA1 gene spans almost 81 kb (Smith TM et al., 1996) and codes for a ubiquitous transcript of 7.8 kb translated into a protein of 1,863 amino acids.
- the BRCA1 gene is very conserved, its murine counterpart codes for a protein of 1812 amino acids identical for 58% to the human BRCA1 protein (Bennett LM et al., 1995).
- the function of the BRCA1 protein is still poorly understood and the study of its cellular localization has been the subject of controversy.
- BRCA1 is a nuclear protein of 220 kDa (Scully R. et al., 1997a).
- the shortened spliced form of exon 1 is a cytoplasmic protein of 1 10 kDa.
- a zinc finger domain at the amino-terminal end (Miki Y. et al., 1994)
- a BRCT domain BRCA1 C- terminus at the carboxy-terminal end
- BRCA1 is a transcription activator (Chapman MS and Verma IM, 1996; Monteiro A. ⁇ .A. et al., 1996) and is associated with AR ⁇ polymerase II (Scully R. et al., 1997b) ;
- BRCA1 is implicated in AD ⁇ repair pathways (Scully R. et al., 1997c; Hakem R. et al., 1997; Ouchi T. et al., 1998) and cell cycle control (Larson JS et al., 1997);
- BRCA1 (Wu LC et al., 1996), p53 (Zhang H. et al., 1998a), c-Myc (Wang Q. et al. ., 1998), Rad 51 (Scully R. et al., 1997a), BRCA2 (Chen J. et al., 1998), CtlP (Yu X. et al., 1998), AR ⁇ helicase A within the AR ⁇ polymerase II holoenzyme complex (Anderson SF et al., 1998), histone deacetylase complex (Yarden R. et al., 1999), or the BACH1 protein (Cantor SB et al., 2001); their interaction regions are distributed over the entire BRCAL protein
- the BRCT module seems to play a major role in mediating protein interactions (complex
- BRCA1 protein partners interacting specifically with its C-terminal end containing the BRCT domain have already been identified: BRCA2, CtlP, RNA helicase A and the histone deacetylase complex, the protein BACH1.
- the inventors carried out a co-sedimentation test in a murine fibroblast lysate, with a GST fusion protein containing amino acids 1583 to 1812 of Murine brcal, named GST-BRCT. Analysis of the protein co-sedimentation complexes showed that a major band of 210 kDa was visible only in the GST-BRCT sample. The inventors have shown that the tandem of the two BRCT domains is necessary and sufficient for training of this complex. In addition, the inventors have shown that the formation of this complex is conserved in human cells, using a GST-BRCThuman fusion protein in co-sedimentation experiments similar to those mentioned above. The inventors' work on the functional role of
- BRCA1 thus made it possible to highlight a new protein-protein interaction involving on the one hand the protein BRCA1 and more particularly the BRCT domain located at the carboxy-terminal end thereof and, on the other hand, a known protein , acetyl-Coenzyme A-carboxylase, form ⁇ (ACC- ⁇ ).
- the BRCA1 protein and the gene coding for this protein are described in particular in patent applications EP 699 754 and EP 705 902.
- the sequence SEQ ID No. 13 corresponds to the cDNA of the murine Brcal gene (Genebank: MMU31625), SEQ ID No. 14 to the amino acid sequence of murine Brcal, SEQ ID No. 15 corresponds to the cDNA of the human BRCA1 gene (Genebank: HSU14680), and SEQ ID No. 16 to the amino acid sequence of human BRCA1 .
- ACC- ⁇ is a 265 kDa cytoplasmic protein whose enzymatic activity plays a key role in the synthesis pathway of long chain fatty acids.
- the human ACC- ⁇ cDNA (SEQ ID No. 17) was cloned in 1995 by
- Abu-Elheiga et al. Its sequence is very conserved in humans, rats, chicken and yeast, in particular at the sites of binding to biotin, ATP and coenzyme A.
- the protein sequence of human ACC- ⁇ is indicated by SEQ ID No. 18.
- Co-immunoprecipitation experiments, with an anti-Brcal antibody, on Bosc cell lysates transfected with the wild form of the murine Brcal protein or its spliced form ⁇ 1 1 were carried out. They have made it possible to confirm that the two forms of the murine protein associate with ACC- ⁇ in vivo. Similarly, the entire human BRCA1 protein coprecipitates with the endogenous ACC- ⁇ of transfected Bosc cells.
- a polyclonal antibody directed against the amino-terminal ends (MDEPSPLAQPLELNQ) (SEQ ID n ° 1) and carboxy-terminal (AEVIRILSTMDSPST) (SEQ ID No. 2) of the human ACC- ⁇ protein was prepared in rabbits and immunopurified against these two peptide sequences.
- the mutations A1708E, P1749R, and M1775R creating an amino acid substitution in the BRCT A domain, in the binding sequence of the two BRCT domains and in the BRCT B domain respectively, annihilate the binding of human BRCA1 to ACC- ⁇ .
- the interaction BRCA1 / ACC- ⁇ can be advantageously used for the development of molecules for therapeutic purposes .
- the subject of the invention is therefore a molecular complex comprising:
- a first polypeptide comprising the amino acid sequence 1640 to 1863 of the human BRCA1 protein or the same amino acid sequence of the BRCA1 protein in another animal species, and
- a second polypeptide comprising a fragment of the ACC- ⁇ protein capable of binding said first polypeptide.
- the region defined by amino acid residues 1640 to 1863 of the human BRCA1 protein is called "minimal domain of BRCA1".
- the same amino acid sequence of the protein is used
- BRCA1 in another animal species an ortholog of the domain defined by amino acids 1640 to 1863 of the human BRCA1 protein.
- said same amino acid sequence of the BRCA1 protein in another animal species is a sequence from a non-human mammal, and in particular the amino acid sequence 1583-1812 of the murine BRCA1 protein.
- the domain defined by amino acids 1583 to 1812 of the murine BRCA1 protein thus constitutes an ortholog of the minimal domain of the human BRCA1 protein.
- ACC- ⁇ protein is meant the human ACC- ⁇ protein or the corresponding protein of an animal species, in particular of a non-human mammal, in particular the mouse.
- a fragment of the ACC- ⁇ protein capable of binding the minimal domain of human BRCA1 or the corresponding domain of an animal species, or a polypeptide comprising said domain constitutes a "minimal domain of the ACC- ⁇ protein".
- the identification of the minimum domain of the ACC- ⁇ protein is part of the routine work of a person skilled in the art. This domain can be identified by conventional techniques such as the production of truncated forms of the ACC- ⁇ protein and co-immunoprecipitation experiments, for example in the presence of the amino acid domain 1640 to 1863 of the human BRCA1 protein.
- a minimal domain of the ACC- ⁇ protein notably includes the entire ACC- ⁇ protein.
- the subject of the invention is also a method for screening molecules capable of modulating the interaction between BRCA1 and ACC- ⁇ , that is to say preventing or promoting the formation of the complex, or even dissociating, in whole or in part, the complex form.
- the invention therefore relates to a method of screening, in particular in vitro, of molecules useful for the prevention or treatment of breast and / or ovarian cancer in which the molecules are tested for their ability to modulate the interaction between BRCA1 and ACC- ⁇ proteins.
- a screening method comprises: a) bringing, in any order, two partners or three different partners selected from the group consisting of a first polypeptide comprising the amino acid sequence 1640 to 1863 of human BRCA1 protein or the same amino acid sequence of the BRCA1 protein, a second polypeptide comprising a fragment of the ACC- ⁇ protein capable of binding said first polypeptide and a candidate molecule; b) the incubation of said partners for a sufficient time to allow their possible interaction; c) in the case where only two different partners have been selected in step a), the addition of the third partner selected from said group and the incubation for a time sufficient to allow possible interaction; d) determining the capacity of the candidate molecule to modulate the interaction between BRCA1 and ACC- ⁇ . More particularly, such a method makes it possible to identify molecules capable of modulating the interaction between BRCA1 and ACC- ⁇ .
- the determination of the capacity of the candidate molecule to modulate the interaction between BRCA1 and ACC- ⁇ can be carried out using appropriate separation and / or detection means, well known to those skilled in the art.
- detectable markers such as fluorescent, isotopic or chromogenic markers.
- a polypeptide comprising the minimum domain of BRCA1 a polypeptide comprising the minimum domain of ACC- ⁇ and at least one candidate molecule can be brought into contact and incubated for a sufficient time to allow their interaction, including possibly the formation or dissociation of the BRCA1 / ACC- ⁇ complex.
- At least one candidate molecule and a polypeptide comprising the minimum domain of BRCA1 or ACC- ⁇ are brought into contact and incubated for a time sufficient to allow their possible interaction.
- the missing minimum domain of BRCA1 or ACC- ⁇ is then added and the whole is incubated for a time sufficient to allow the interaction of all the elements, including possibly the formation of the BRCA1 / ACC- ⁇ complex.
- a polypeptide comprising the minimum domain of BRCA1 and a polypeptide comprising the minimum domain of ACC- ⁇ are preincubated so as to allow the formation of the BRCA1 / ACC- ⁇ complex before the addition of at least one molecule candidate and incubation of the whole for a time sufficient to allow their interaction and possibly the dissociation of the BRCA1 / ACC- ⁇ complex.
- a preferred embodiment of this method comprises the following steps: a) bringing into contact a first polypeptide comprising the amino acid sequence 1640 to 1863 of the human BRCA1 protein or the same amino acid sequence of the protein BRCA1 with a second polypeptide comprising a fragment of the ACC- ⁇ protein capable of binding said first polypeptide, at least one of the polypeptides being detectably labeled; b) adding a candidate molecule capable of modulating the interaction; c) incubating said polypeptides in the presence of the candidate molecule under conditions and for a period of time sufficient for the bond between said polypeptides to take place; d) quantifying the number of labeled molecules bound in the presence of increasing concentrations of the candidate molecule.
- the present invention therefore provides a method for screening molecules capable of modulating the interaction between BRCA1 and ACC- ⁇ , characterized in that said molecule is an antagonist of the interaction BRCA1 / ACC- ⁇ .
- the present invention provides a method for screening molecules capable of modulating the interaction between BRCA1 and ACC- ⁇ , characterized in that said molecule is an agonist of the BRCA1 / ACC- ⁇ interaction.
- antagonist is meant a molecule whose action is opposed to the formation or maintenance of the BRCA1 / ACC- ⁇ complex.
- the antagonistic molecules according to the invention include, for example, inhibitors capable of preventing or limiting the interaction between BRCA1 and ACC- ⁇ or molecules capable of dissociating the complex when it is formed.
- agonist is meant a molecule whose action contributes to, or increases the formation of the BRCA1 / ACC- ⁇ complex.
- the present invention relates to a method of ex vivo identification of molecules constituting endogenous ligands of the BRCA1 / ACC- ⁇ complex.
- the term ligand has the common sense of a molecule capable of interacting with the BRCA1 / ACC- ⁇ complex. More particularly, a ligand can constitute an effector of the biological activity of the BRCA1 / ACC- ⁇ complex. In this sense, a cellular receptor of the BRCA1 / ACC- ⁇ complex constitutes an example of an endogenous ligand for the complex.
- the identification of ligands of the complex can be carried out by conventional methods well known to those skilled in the art, such as immunoprecipitation, the double hybrid method, immunohistochemistry.
- the identification method according to the invention comprises: a) bringing the BRCA1 / ACC- ⁇ complex into contact with a biological sample under conditions allowing the formation of interactions between the complex and possible ligands; b) the detection of interactions between the BRCA1 / ACC- ⁇ complex and said ligands; and c) identifying the ligands interacting with the complex.
- the BRCA1 / ACC- ⁇ complex brought into contact with the biological sample can advantageously be marked using detectable markers, such as fluorescent markers or by a radioisotopic.
- the candidate molecules or ligands of the screening or identification methods described above include peptide compounds, peptidomimetics or other organic compounds. These molecules can be endogenous, natural or synthetic or even be mixtures of compounds.
- the candidate molecules can be structurally defined or not, as is generally the case when it comes to extracts.
- peptidomimetics is meant here an organic molecule which mimics certain properties of the peptides, for example their specificity of binding and / or their physiological activity. Peptidomimetics are generally obtained by modifying the structure of a peptide. These modifications tend in particular to improve the resistance to enzymatic degradation, the bioavailability of the compound or even its pharmacokinetic properties.
- antibodies or aptamers constitute examples of candidate molecules.
- the aptamers constitute a class of molecules which represent an alternative to antibodies in terms of molecular recognition.
- Aptamers are sequences of oligonucleotides with the ability to recognize virtually any class of target molecules with high affinity and specificity.
- Such ligands can be isolated by a screening called SELEX (Systematic Evolution of Ligands by EXponential enrichment) from a bank of random sequences, as described in Tuerk and Gold (1990).
- SELEX Systematic Evolution of Ligands by EXponential enrichment
- the random sequence library can be obtained by DNA synthesis by combinatorial chemistry. In such a library, each member is a linear oligomer, optionally chemically modified, corresponding to a unique sequence.
- Candidate molecules thus identified capable of modulating the interaction between BRCA1 and ACC- ⁇ or of constituting a ligand of the complex, can be used for the treatment and / or prevention of breast and / or ovarian cancers, in particular familial forms of breast and / or ovarian cancer.
- a subject of the invention is also a method of in vitro diagnosis of a predisposition to breast and / or ovarian cancer, comprising the determination of a modification of the BRCA1 / ACC- ⁇ interaction in a subject. compared to a control population, the modification of the BRCA1 / ACC- ⁇ interaction being associated with a variation in the risk of developing breast and / or ovarian cancer.
- modification of the BRCA1 / ACC- ⁇ interaction is meant in particular the absence or the presence of detectable BRCA1 / ACC- ⁇ complex in said subject, or else a quantitative or qualitative difference of the BRCA1 / ACC- ⁇ complex by compared to a control population.
- the presence of the BRCA1 / ACC- ⁇ complex may for example be detected by a method such as co-immunoprecipitation, for example using the antibody D16 (Santa Cruz Biotechnology, California, USA) directed against BRCA1, and d a polyclonal rabbit antibody directed against the amino-terminal (MEDPSPLAQPLELNQ, SEQ ID No. 1) and carboxy-terminal (AEVIRILSTMDSPST, SEQ ID No. 2) ends of the human ACC- ⁇ protein.
- Quantitative difference is meant a variation in the amount of BRCA1 / ACC- ⁇ complexes detectable.
- qualifying difference is meant in particular a variation capable of modifying the biological activity of the complex, such as for example increased or decreased stability, or a modification of the specificity of binding of the complex.
- the invention relates to an anti-human ACC- ⁇ antibody, directed against the peptides of sequence SEQ ID No. 1 and SEQ ID No. 2 of the human ACC- ⁇ protein.
- Such an antibody is particularly useful for detecting the presence of a BRCA1 / ACC- ⁇ complex, for example by immunoprecipitation.
- the use of such an anti-ACC- ⁇ antibody can also allow the cellular localization of the ACC- ⁇ protein as well as the localization of the ACC- ⁇ -BRCA1 complex in the same cells, by immunochemical technique or by immunofluorescence for example.
- a diagnostic application may include observation of cells under the microscope in order to detect the existence of this complex and the expected localization of this complex.
- the present invention therefore also provides a method of ex vivo detection of a BRCA1 / ACC- ⁇ complex comprising bringing an antibody according to the invention into contact with a biological sample under conditions allowing the immunoprecipitation of ACC- ⁇ , and the detection of BRCA1 in the precipitate, the presence of BRCA1 in the precipitate being indicative of the formation of a BRCA1 / ACC- ⁇ complex.
- the present invention also relates to antibodies directed against the molecular complex BRCA1 / ACC- ⁇ , characterized in that they do not interact with the proteins BRCA1 or ACC- ⁇ alone.
- the selectivity of such antibodies with respect to the BRCA1 / ACC- ⁇ complex can in particular be obtained by producing antibodies recognizing the BRCA1 / ACC- ⁇ complex, by immunization in animals for example, and by carrying out a negative selection step, that is to say by eliminating the antibodies produced having cross-reactivity with the isolated BRCA1 protein and the isolated ACC- ⁇ protein.
- Such negative selection can be made by immunoaffinity chromatography.
- They may be poly- or monoclonal antibodies or their fragments, chimeric antibodies, in particular humanized or immunoconjugated.
- Polyclonal antibodies can be obtained from the serum of an animal immunized against a protein according to the usual procedures.
- an appropriate peptide complex can be used as the antigen, such as a complex associating the minimum domain of BRCA1 and ACC- ⁇ , which can be coupled via a reactive residue to a protein (such as keyhole limpet hemocyanin KLH) or another peptide.
- Rabbits are immunized with the equivalent of 1 mg of the peptide antigen according to the procedure described by Benoit et al. (1982). At four-week intervals, the animals are treated with injections of 200 ⁇ g of antigen and bled 10-14 days later.
- the antiserum is examined to determine its ability to bind to the radiolabelled iodine antigen peptide, prepared by the method chloramine-T and is then purified by chromatography on a column of carboxymethyl cellulose ion exchange (CMC).
- CMC carboxymethyl cellulose ion exchange
- the antibody molecules are then collected in mammals and isolated to the desired concentration by methods well known to those skilled in the art, for example, using DEAE Sephadex to obtain the IgG fraction.
- the antibodies can be purified by immunoaffinity chromatography using solid phase immunizing polypeptides.
- the antibody is brought into contact with the immunizing polypeptide in solid phase for a sufficient time so as to make the polypeptide immunoreact with the antibody molecule in order to form an immunological complex in solid phase.
- the monoclonal antibodies can be obtained according to the conventional method of culture of hybridomas described by Kôhler and Milstein (1975).
- the antibodies or antibody fragments of the invention can be, for example, chimeric antibodies, humanized antibodies, Fab and F (ab ') 2 fragments. They can also be in the form of immunoconjugates or labeled antibodies.
- the antibodies of the invention in particular the monoclonal antibodies, can in particular be used for the ex vivo detection by immunochemistry of the BRCA1 / ACC- ⁇ complex, for example by immunofluorescence, gold labeling, immunoperoxidase, in particular in the methods of screening described above.
- the pUHD 10.3 plasmids expressing either wild-type Brcal or the ⁇ 1 1 form were prepared as described in Bachelier, R et al. (2000).
- the plasmid pcDNA3 ⁇ -BRCA1wt expressing the human form of BRCA1 has been previously described (6).
- the plasmid pGEX-BRCT was obtained by amplification by polymerase chain reaction (PCR) of Brcal nucleotides 4747 to 5436 using the cloned murine Brcal cDNA as a template, and the following primers: 5'-GCGAATTCACATCTTCAGAAGAAAGAGC-3 '(SEQ ID No. 3) and 5'-GCGTCGACTTAATCATTGGAGTCTTGTGG-3' (SEQ ID No. 4).
- the 0.7 kb chain polymerization reaction product was cloned into EcoRI / Sa / l sites of pGEX-4T-1 (Amersham Pharmacia Biotech). Shorter constructions with the first or second modules of the BRCT tandem were obtained with the following primers 5'-
- a longer form pGEX-BRCT L containing the nucleotides 4534 to 5436 of Brcal was also prepared using the following primers: 5'- GCGAATTCGAAGGAACCCCATACCTG-3 '(SEQ ID No. 9) and ⁇ '-GCGTCGACTTAATCATTGGAGTCTTGTGG-S' (SEQ ID No. 10).
- a pGEX-BRCT (M) mutant was produced with the same primers as those used for wild-type GST-BRCT using a Brcal cDNA having the nonsense mutation 1777X, as a template.
- the plasmid pGEX- BRCTh expressing the human form of the BRCT tandem was obtained by amplification of nucleotides 4917 to 5592 using the cDNA of BRCA1 inserted in pCDNA3 ⁇ , and the following primers: 5'-
- PGEX-BRCTh mutants were produced with the same primers as those used for wild type GST-BRCTh using a BRCA1 cDNA having the nonsense mutation R1835X or Y1853 X, or the missense mutations A1708E, P1749R, or M1775R , as a matrix.
- BRCA1 cDNA having the nonsense mutation R1835X or Y1853 X, or the missense mutations A1708E, P1749R, or M1775R , as a matrix.
- the cells were cultured in DMEM medium supplemented with 10% fetal calf serum. MCF7 cells were grown in the same medium with 5 mg / ml insulin. For radiolabelling, the cells were cultured for one hour in a DMEM medium without methionine, then for three hours in a DMEM medium without methionine containing 250 ⁇ Ci of [ 35 S] labeled methionine.
- the standard method of precipitation with calcium phosphate was used (Gibco BRL) with 5 ⁇ g of total plasmid DNA. Cells were harvested 48 hours after transfection.
- the fusion proteins with GST were synthesized in Escherichia coli, strain JM 109 (Promega) transformed with pGEX-4T-1, pGEX-BRCT, pGEX-BRCT A, pGEX-BRCT B, pGEX-BRCT L, pGEX-BRCT M or pGEX-BRCTh as well as the mutated forms of BRCTh (A1708E, P1749R, M1775R, R1835X and Y1853X). They were purified by affinity chromatography on Glutathione-Sepharose.
- the co-sedimentation binding test was carried out in vitro by incubating cell lysates, in the presence of non-recombinant GST proteins or of fusion proteins-GST and of Glutathione-Sepharose beads. 10 ⁇ g of GST or GST-fusion were used in each binding test. The protein complexes were carefully washed (50 mM Tris HCl pH 7.6, 100 mKCI, 0.05% Tween 20, 1 mM DTT, 0.2 mM PMSF) to remove non-specific protein interactions and released by heating to 100 ° C in a loading buffer 5X SDS-PAGE and 100 mM DTT. The proteins were analyzed by SDS-PAGE and visualized by autoradiography, staining with silver (Bio-Rad SilverStain) or staining with Bleu de Comassie depending on the nature of the lysate used.
- Immunoprecipitations of endogenous or overexpressed proteins in vivo were carried out in a lysis buffer with 1 ⁇ g of specific antibody (anti-BRCA1 OP92, Oncogene Research Products; anti-Brcal D16, Santa Cruz Biotechnology ; anti-ACC- ⁇ L3J74) and the protein G-sepharose for two hours at 4 ° C. After precipitation, the beads were washed three times with the lysis buffer and the proteins were eluted by heating for five minutes in the SDS loading buffer.
- specific antibody anti-BRCA1 OP92, Oncogene Research Products; anti-Brcal D16, Santa Cruz Biotechnology ; anti-ACC- ⁇ L3J74
- the proteins were separated on 6 to 10% polyacrylamide gels containing SDS (SDS-PAGE) and loaded on poly (vinylidene difluoride) (PVDF) membranes (Immobilon-P, Millipore).
- the membranes were saturated in TBS containing 0.05% Tween 20 and 5% milk powder.
- Incubations with primary and secondary antibodies were carried out in TBS containing 0.05% Tween 20 and 2% milk powder, and the proteins were detected by chemiluminescence (ECL, Amersham Pharmacia Biotech or Super Signal West Dura Extended , Pierce).
- the cytosolic fraction was obtained after incubation of the cells for 10 minutes on ice in RYMO buffer (10 mM Tris HCI pH 7.6, 1 mM MgCI 2 , 0.5 mM CaCl 2 , 0.25M sucrose, 0, 6% NP40), and centrifugation (2,000 rpm, 5 minutes).
- the nuclear pellet was then clarified for 10 minutes on ice in RIPA buffer (137 mM NaCl, 2.7 mM KCI, 25 mM Tris pH 8, 1% NP 40) and centrifuged at 13,000 rpm for five minutes to remove insoluble matter.
- the 210 kDa protein of interest stained with Comassie Blue was excised from the SDS-PAGE gel and then washed with 50 mM ammonium acetate buffer, pH 7 for one hour. The supernatant was discarded and the gel slice washed in a 50/50% (v / v) solution of acetonitrile / 25 mM ammonium acetate, pH 7.5 for one hour and finally with water pure before complete dehydration in a vacuum centrifuge. The gel pieces were rehydrated with a minimum amount of a modified porcine trypsin solution (PROMEGA Madison, Wl, USA) containing 0.25 ⁇ g of protease. If necessary, additional buffer was added until the gel patch was completely rehydrated. Digestion took place at 37 ° C for four hours.
- a modified porcine trypsin solution PROMEGA Madison, Wl, USA
- SM-MALDI The mass spectra of the tryptic digestion products were obtained on a MALDI-TOF Voyager Elite X1 mass spectrometer (Perspective Biosystems), equipped with delayed extraction. The instrument was used in reflectron mode. 1 ⁇ l of the digestion product was deposited directly on the sample support and mixed with 1 ⁇ l of a saturated solution of 2,5-dihydrobenzoic acid. A list of peptide masses was obtained for each protein digestion product. This peptide mass profile was then processed with appropriate software in order to identify the proteins (MS-
- the tryptic peptides of the 210 kDa protein were identified from the mass map of the tryptic fragments.
- the tryptic digestion products were extracted twice with a 50/50% (v / v) solution of acetonitrile / 25 mM ammonium acetate pH 7.5.
- the digestion solution and the extracts were pooled, dried in a vacuum centrifuge and desalted with ZipTip C18 (Millipore, Bedford, Ma, USA) before SM / SM analysis.
- a Q-TOF instrument (Micromass, Manchester, United Kingdom) was used with a Z-Spray ion source operating in "nanospray" mode. About 3 to 5 ⁇ l of each desalinated sample was introduced into a needle (medium sampling needle, PROTAN Inc., Odense, Denmark) for carrying out the SM / SM experiments. The capillary voltage was set to an average voltage of 1000 volts and the sample cone to 50 volts. Glufibrinopeptide was used to calibrate the instrument for SM / SM mode.
- the SM / SM spectra were transformed using MaxEnt3 (MassLynx, Micromass Ltd), and the amino acid sequences were analyzed using PepSeq (BioLynx, Micromass Ltd). Amino acid sequences, sequence fragments or ionic peptide fragments that could be determined were used to screen protein and TSE libraries using appropriate Pepfrag software
- the endolytic peptides were separated by HPLC on a DEAE-C18 column with a 0.1% acetonitrile / TFA gradient.
- the sequencing of the isolated peptides was carried out in accordance with the Dman degradation on a Procise sequencer from Applied Biosystems. Amino acid sequences were used to screen the protein database for the search for BLAST homologies
- GST-BRCT A GST fusion protein containing amino acids 1583 to 1812 from murine Brcal, named GST-BRCT, was constructed, expressed in Escherichia coli, and purified on glutathione sepharose beads.
- Whole cell lysates were prepared from 7.10 6 NIH3T3 cells labeled with 35 S methionine. The lysates were pre-purified using glutathione-Sepharose beads.
- NIH3T3 cells were separated into nuclear and cytoplasmic fractions. A co-sedimentation test carried out on these two fractions was analyzed by SDS-PAGE followed by silver staining. The results showed that the interaction protein was predominantly cytoplasmic. The correct fractionation of these cells was checked using antibodies directed against nuclear p300 and cytoplasmic ⁇ -tubulin.
- the protein was digested into several peptides enzymatically by trypsin, then the mixture was analyzed by MALDI-SM using the reflectron mode.
- the mono-isotopic masses of peptides from proteolytic digestion were compared with the masses of a peptide database, calculated from the protein database nr of NCBI with mass tolerances of ⁇ 0.2 Da and with partially oxidized methionine residues (m / z values listed in deep and sm-fit programs (see experimental procedure): 920.47, 931.52, 938.49, 948.51, 1034.51, 1045.62, 1065.63, 1084.51, 1087.66, 1092.59, 1097.66, 1165.73, 1179.76, 1199.73, 1232.77, 1235.68, 1267.70, 1300.64, 1308.75, 1322.72, 1365.71, 1438.79, 1493.77, 1571.72, 1791.77).
- the other parameters were as follows: range of variation of the protein mass of 100-300 kDa; unmodified cysteines, maximum of three cleavage sites not taken into account.
- the leading protein was Rattus norvegicus acetyl-CoA carboxylase (EC 6.4.1.2; swiss prot access code P 11497) with 16 possible peptides out of the 25 peptides listed had a probability close to 1 (9.9e-1 ) and was clearly distinguished from the candidate coming in second position found during the research (homo sapiens protein KIAA 1286; probability of 4.0e-3).
- a microsequencing of the peptide was also carried out.
- a preparative co-sedimentation test of GST using 5.10 7 NIH3T3 cells was carried out to obtain 25 pmol of protein intended to be digested and sequenced.
- a slice of polyacrylamide gel containing 5 ⁇ g of the 210 kDa band of interest (approximately 25 pmol) was excised.
- the protein was digested enzymatically into peptides by endolysin, then the peptides were separated by chromatography on DEAE-C18 and sequenced.
- the sequences 298 KAAEEVGYP 306 AND 2267 KDLVEWLEK 2275 were obtained. As expected, they showed 100% homology with Rattus norvegicus acetyl-CoA carboxylase (EC 6.4.1.2; swiss prot access number P 11497).
- ACC- ⁇ of 265 kDa and ACC- ⁇ of 280 kDa Two major forms of ACC of animal origin have been described so far: ACC- ⁇ of 265 kDa and ACC- ⁇ of 280 kDa.
- the 5 peptide sequences obtained here have 100% homology with the ACC- ⁇ of the rat.
- the murine ACC- ⁇ gene has not been cloned.
- the coding sequence of the ⁇ gene is well conserved in the different species.
- the rat ACC- ⁇ cDNA sequence (Lopez-Casillas et al., 1988) is highly similar (90%) to the human ACC- ⁇ sequence (Abu-Elheiga L. et al. ., 1995).
- the highly conserved biotin binding site and the putative ATP and coenzyme A binding sites are identical in humans, rats and yeast.
- the inventors have demonstrated that the BRCT tandem of Brcal binds to endogenous ACC- ⁇ in murine fibroblasts. Since the BRCA1 gene is associated with a familial form of breast cancer, the inventors have sought the interaction between the BRCT and ACC- ⁇ tandem in the mammary gland.
- the GST co-sedimentation tests were carried out with four mammary murine epithelial cell lines, namely Be4a, I3G2, Tac-2 and J3B1, and the bound proteins were detected using peroxidase coupled to streptavidin. The ability of the BRCT tandem to bind ACC- ⁇ is conserved in all these cell lines.
- the rat ACC- ⁇ cDNA sequence (Lopez-Casillas et al., 1988) is highly similar (90%) with the human ACC- ⁇ sequence (Abu-Elheiga L. et al. ., 1995).
- the results represented here indicate that the murine tandem BRCT binds as well to the human ACC- ⁇ form as to the murine form. Consequently, to further characterize this interaction, the inventors sought the capacity of the human BRCT tandem to bind the murine and human forms of ACC- ⁇ . Co-sedimentation tests have been carried out on murine NIH3T3 fibroblast cell lines and the human mammary epithelial line HBL 100.
- the GST-BRCT A fragment which lacks the sequences of the B domain of BRCT is incapable of associating with ACC- ⁇ .
- the GST-BRCT B fragment which lacks the sequences of the BRCT A domain does not bind ACC- ⁇ .
- GST-Brca1 N containing the 220 N-terminal residues of Brcal, as well as GST alone, were not able to capture ACC- ⁇ , which demonstrates the specificity of the interaction between ACC- ⁇ and the carboxy region -terminal of Brcal.
- the corresponding truncated GST-BRCTh fusion protein does not bind ACC- ⁇ .
- the study of other family mutations such as the A1708E, P1749R, or M1775R mutations, which create an amino acid substitution in the BRCT A domain, in the binding sequence of the two BRCT domains or in the BRCT B domain respectively, or the nonsense mutation Y1853X which leads to a truncated BRCT B domain, shows that these mutations also abolish the binding of BRCA1 to ACC- ⁇ .
- Bosc cells were transfected with expression plasmids coding for full-length Brcal or the short form Brca1- ⁇ 1 1.
- the lysates prepared from these transfected cells were immunoprecipitated with the antibody D16 (Santa Cruz Biotechnology) directed against BRCA1 and each immunoprecipitate was fractionated by SDS-PAGE. The presence of endogenous ACC- ⁇ in these immuno-precipitates was then determined by Western blot using peroxidase coupled to streptavidin.
- the endogenous ACC- ⁇ was co-immunoprecipitated from cells expressing Brcal and cells expressing the short form Brcal - ⁇ 1 1.
- Bosc cells were transfected with a plasmid expressing the BRCAL protein Lysates were immunoprecipitated with the antibody OP92 (Oncogene Research Products) directed against BRCA1 and the presence of endogenous ACC- ⁇ in the co-immunoprecipitates was analyzed by Western blot using peroxidase coupled with streptavidin. As expected, human BRCA1 co-precipitates with endogenous ACC- ⁇ .
- BRCA1 is mainly a nuclear protein
- the results obtained in co-sedimentation tests and the results concerning the cytoplasmic localization of ACC- ⁇ raise the question of the physiological significance of the interaction between BRCA1 and ACC- ⁇ .
- the inventors have re-examined the subcellular localization of BRCA1 and of the murine isoform Brca1- ⁇ l 1 transiently expressed in Bosc cells.
- fractionation techniques have been used to study the sub-cellular localization of BRCA1 and of the murine Brcal - ⁇ 1 1 form. (Bachelor, R et al., 2000). After cell fractionation, nuclear and cytoplasmic extracts from the transfected cells were separated by SDS-PAGE and subjected to analysis by Western Blot.
- L3J74 The specificity of this anti-ACC- ⁇ antibody called L3J74, was tested by Western Blot technique in several cell lines (human lines: HBL100, MCF7, BT20, Bosc, HCC1937; murine line: NIH3T3) and several tissue lysates (breast , ovary, pancreas), under experimental conditions making it possible to clearly distinguish the ⁇ and ⁇ forms of ACC.
- the results showed that L3J74 was specific for the ⁇ form of ACC, and that its affinity for the human form was much higher than that observed for the murine form of the protein.
- This antibody was then tested by immunoprecipitation technique on different cell lysates (HBL100, Bosc, NIH3T3).
- BRCA1 is required for embryonic cellular proliferation in the mouse.
- the tumor suppressor BRCA 1 gene is required for embryonic cellular proliferation in the mouse.
- BRCA1 is a component of the R ⁇ A polymerase II holoenzyme. Proc. Natl. Acad. Sci. USA. 94: 5605-5610. Scully R. et al. (1997c). Dynamic changes of BRCA1 subnuclear location and phosphorylation state are initiated by DNA damage. Cell. 90: 425-435.
- BRCA1 interacts with components of the histone deacetylase complex. Proc. Natl. Acad. Sci. USA. 96: 4983-4988.
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Priority Applications (4)
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AU2002317247A AU2002317247A1 (en) | 2001-06-13 | 2002-06-12 | Brca1/acc alpha molecular complexes, diagnostic and therapeutic applications |
CA002450663A CA2450663A1 (fr) | 2001-06-13 | 2002-06-12 | Complexes moleculaires brca1/acc alpha, applications diagnostiques et therapeutiques |
EP02745520A EP1430081A2 (fr) | 2001-06-13 | 2002-06-12 | Complexes moleculaires brca1/acc alpha, applications diagnostiques et therapeutiques |
US10/480,434 US20060105403A1 (en) | 2001-06-13 | 2002-06-12 | Braca1/acc alpha molecular complexes, diagnostic and therapeutic applications |
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FR01/07740 | 2001-06-13 | ||
FR0107740A FR2826012B1 (fr) | 2001-06-13 | 2001-06-13 | Complexes moleculaires brca1/accalpha, applications diagnostiques et therapeutiques |
FR02/02789 | 2002-03-05 | ||
FR0202789A FR2836919B1 (fr) | 2002-03-05 | 2002-03-05 | Complexes moleculaires brca1/accalpha, applications diagnostiques et therapeutiques |
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WO2002100897A2 true WO2002100897A2 (fr) | 2002-12-19 |
WO2002100897A3 WO2002100897A3 (fr) | 2004-04-22 |
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EP (1) | EP1430081A2 (fr) |
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0785216B2 (fr) † | 1995-12-18 | 2006-06-07 | The University of Utah Research Foundation | Gène BRCA2 lié au chromosome 13, de la susceptibilité au cancer du sein |
EP1759020A2 (fr) * | 2004-06-21 | 2007-03-07 | Exelixis, Inc. | Acacs utilises comme genes modificateurs de la voie igf et leurs procedes d'utilisation |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0699754A1 (fr) * | 1994-08-12 | 1996-03-06 | Myriad Genetics, Inc. | Méthode de diagnose pour la prédisposition au cancer du sein et des ovaires |
-
2002
- 2002-06-12 CA CA002450663A patent/CA2450663A1/fr not_active Abandoned
- 2002-06-12 US US10/480,434 patent/US20060105403A1/en not_active Abandoned
- 2002-06-12 AU AU2002317247A patent/AU2002317247A1/en not_active Abandoned
- 2002-06-12 EP EP02745520A patent/EP1430081A2/fr not_active Withdrawn
- 2002-06-12 WO PCT/FR2002/002016 patent/WO2002100897A2/fr not_active Application Discontinuation
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
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EP0699754A1 (fr) * | 1994-08-12 | 1996-03-06 | Myriad Genetics, Inc. | Méthode de diagnose pour la prédisposition au cancer du sein et des ovaires |
Non-Patent Citations (4)
Title |
---|
MAGNARD CLÉMENCE ET AL: "BRCA1 interacts with acetyl-CoA carboxylase through its tandem of BRCT domains." ONCOGENE, vol. 21, no. 44, 3 octobre 2002 (2002-10-03), pages 6729-6739, XP002216279 ISSN: 0950-9232 * |
MILGRAUM LZ. ET AL.: "Enzymes of the fatty acid synthesis pathway are highly expressed in in situ breast carcinoma." CLIN CANCER RES 1997 NOV;3(11):2115-20., XP001053474 * |
YARDEN RI. ET AL.: "Identification of proteins that interact with BRCA1 by Far-Western library screening." J CELL BIOCHEM 2001;83(4):521-31., XP002216278 * |
YARDEN RONIT I ET AL: "BRCA1 interacts with components of the histone deacetylase complex." PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES, vol. 96, no. 9, 27 avril 1999 (1999-04-27), pages 4983-4988, XP002189865 ISSN: 0027-8424 cité dans la demande * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0785216B2 (fr) † | 1995-12-18 | 2006-06-07 | The University of Utah Research Foundation | Gène BRCA2 lié au chromosome 13, de la susceptibilité au cancer du sein |
EP1759020A2 (fr) * | 2004-06-21 | 2007-03-07 | Exelixis, Inc. | Acacs utilises comme genes modificateurs de la voie igf et leurs procedes d'utilisation |
JP2008503239A (ja) * | 2004-06-21 | 2008-02-07 | エクセリクシス, インク. | IGF経路のモディファイヤーとしてのACACsおよび使用方法 |
EP1759020A4 (fr) * | 2004-06-21 | 2008-04-16 | Exelixis Inc | Acacs utilises comme genes modificateurs de la voie igf et leurs procedes d'utilisation |
Also Published As
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US20060105403A1 (en) | 2006-05-18 |
CA2450663A1 (fr) | 2002-12-19 |
AU2002317247A1 (en) | 2002-12-23 |
EP1430081A2 (fr) | 2004-06-23 |
WO2002100897A3 (fr) | 2004-04-22 |
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