Detection of Protein Folding Defects Caused by BRCA1-BRCT Truncation and Missense Mutations
2003; Elsevier BV; Volume: 278; Issue: 52 Linguagem: Inglês
10.1074/jbc.m310182200
ISSN1083-351X
AutoresR. Scott Williams, Daniel I. Chasman, David Hau, Benjamin Hui, Albert Y. Lau, J. N. Mark Glover,
Tópico(s)CRISPR and Genetic Engineering
ResumoMost cancer-associated BRCA1 mutations identified to date result in the premature translational termination of the protein, highlighting a crucial role for the C-terminal, BRCT repeat region in mediating BRCA1 tumor suppressor function. However, the molecular and genetic effects of missense mutations that map to the BRCT region remain largely unknown. Using a protease-based assay, we directly assessed the sensitivity of the folding of the BRCT domain to an extensive set of truncation and single amino acid substitutions derived from breast cancer screening programs. The protein can tolerate truncations of up to 8 amino acids, but further deletion results in drastic BRCT folding defects. This molecular phenotype can be correlated with an increased susceptibility to disease. A cross-validated computational assessment of the BRCT mutation data base suggests that as much as half of all BRCT missense mutations contribute to BRCA1 loss of function and disease through protein-destabilizing effects. The coupled use of proteolytic methods and computational predictive methods to detect mutant BRCA1 conformations at the protein level will augment the efficacy of current BRCA1 screening protocols, especially in the absence of clinical data that can be used to discriminate deleterious BRCT missense mutations from benign polymorphisms. Most cancer-associated BRCA1 mutations identified to date result in the premature translational termination of the protein, highlighting a crucial role for the C-terminal, BRCT repeat region in mediating BRCA1 tumor suppressor function. However, the molecular and genetic effects of missense mutations that map to the BRCT region remain largely unknown. Using a protease-based assay, we directly assessed the sensitivity of the folding of the BRCT domain to an extensive set of truncation and single amino acid substitutions derived from breast cancer screening programs. The protein can tolerate truncations of up to 8 amino acids, but further deletion results in drastic BRCT folding defects. This molecular phenotype can be correlated with an increased susceptibility to disease. A cross-validated computational assessment of the BRCT mutation data base suggests that as much as half of all BRCT missense mutations contribute to BRCA1 loss of function and disease through protein-destabilizing effects. The coupled use of proteolytic methods and computational predictive methods to detect mutant BRCA1 conformations at the protein level will augment the efficacy of current BRCA1 screening protocols, especially in the absence of clinical data that can be used to discriminate deleterious BRCT missense mutations from benign polymorphisms. Germline mutations within the breast and ovarian cancer susceptibility gene BRCA1 predispose carriers to early-onset breast and breast-ovarian cancers (1Nathanson K.L. Wooster R. Weber B.L. Nathanson K.N. Nat. Med. 2001; 7: 552-556Crossref PubMed Scopus (376) Google Scholar). Accumulating evidence points to a role for the BRCA1 protein product in the regulation of multiple nuclear functions including transcription, recombination, DNA repair, and checkpoint control (2Monteiro A.N. Trends Biochem. Sci. 2000; 25: 469-474Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 3Scully R. Livingston D.M. Nature. 2000; 408: 429-432Crossref PubMed Scopus (561) Google Scholar, 4Venkitaraman A.R. Cell. 2002; 108: 171-182Abstract Full Text Full Text PDF PubMed Scopus (1435) Google Scholar). Tumor-associated mutations occur throughout the BRCA1 coding sequence, but cluster to sequences encoding the N-terminal RING finger domain and the two carboxy-terminal repeat BRCT 1The abbreviations used are: BRCTBRCA1 C-terminal domainBICBreast Cancer Information Core Database. domains (5Shattuck-Eidens D. McClure M. Simard J. Labrie F. Narod S. Couch F. Hoskins K. Weber B. Castilla L. Erdos M. Brody L. Friedman L. Ostermeyer E. Szabo C. King M.-C. Jhanwar S. Offit K. Norton L. Gilewski T. Lubin M. Osborne M. Black D. Boyd M. Steel M. Ingle S. Haile R. Lindblom A. Olsson H. Borg A. Bishop D.T. Solomon E. Radice P. Spatti G. Gayther S. Ponder B. Warren W. Stratton M. Liu Q. Fujimura F. Lewis C. Skolnick M.H. Goldgar D.E. JAMA. 1995; 273: 535-541Crossref PubMed Scopus (423) Google Scholar, 6Couch F.J. Weber B.L. Hum. Mutat. 1996; 8: 8-18Crossref PubMed Scopus (271) Google Scholar, 7Shen D. Vadgama J.V. Oncol. Res. 1999; 11: 63-69PubMed Google Scholar). BRCA1 C-terminal domain Breast Cancer Information Core Database. The molecular details of how BRCA1 mutations contribute to the pathogenesis of cancer remain largely unknown. The functional significance of the BRCT region is highlighted by the high degree of sequence conservation within the BRCT regions of among mammalian, Xenopus, and avian BRCA1 homologues (8Sharan S.K. Wims M. Bradley A. Hum. Mol. Genet. 1995; 4: 2275-2278Crossref PubMed Scopus (56) Google Scholar, 9Chen K.S. Shepel L.A. Haag J.D. Heil G.M. Gould M.N. Carcinogenesis. 1996; 17: 1561-1566Crossref PubMed Scopus (24) Google Scholar, 10Joukov V. Chen J. Fox E.A. Green J.B. Livingston D.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12078-12083Crossref PubMed Scopus (127) Google Scholar). Several lines of evidence reveal the BRCT is required for tumor suppressor function. A nonsense mutation, which removes 11 C-terminal residues of the second, BRCT (Tyr1853 → stop), is associated with early-onset breast cancer (11Friedman L.S. Ostermeyer E.A. Szabo C.I. Dowd P. Lynch E.D. Rowell S.E. King M.C. Nat. Genet. 1994; 8: 399-404Crossref PubMed Scopus (568) Google Scholar). Two cancer-linked BRCT missense mutations (12Miki Y. Swensen J. Shattuck-Eidens D. Futreal P.A. Harshman K. Tavtigian S. Liu Q. Cochran C. Bennett L.M. Ding W. Bell R. Rosenthal J. Hussey C. Tran T. McClure M. Frye C. Hattier T. Phelps R. Haugen-Strano A. Katcher H. Yakumo K. Gholami Z. Shaffer D. Stone S. Bayer S. Wray C. Bogden R. Dayananth P. Ward J. Tonin P. Narod S. Bristow P.K. Norris F.H. Helvering L. Morrison P. Rosteck P. Lai M. Barrett J.C. Lewis C. Neuhausen S. Cannon-Albright L. Goldgar D. Wiseman R. Kamb A. Skolnick M.H. Science. 1994; 266: 66-71Crossref PubMed Scopus (5390) Google Scholar) that destabilize the BRCT fold (13Williams R.S. Green R. Glover J.N. Nat. Struct. 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Cell. 2001; 105: 149-160Abstract Full Text Full Text PDF PubMed Scopus (561) Google Scholar), and the transcriptional co-repressor CtIP (19Yu X. Wu L.C. Bowcock A.M. Aronheim A. Baer R. J. Biol. Chem. 1998; 273: 25388-25392Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar, 20Li S. Chen P.L. Subramanian T. Chinnadurai G. Tomlinson G. Osborne C.K. Sharp Z.D. Lee W.H. J. Biol. Chem. 1999; 274: 11334-11338Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Furthermore, mice with homozygous targeted mutations removing the C-terminal half of BRCA1 are viable but develop tumors, suggesting the missing BRCT and/or other domains are expendable for survival, but not for tumor suppression (21Ludwig T. Fisher P. Ganesan S. Efstratiadis A. Genes Dev. 2001; 15: 1188-1193Crossref PubMed Scopus (113) Google Scholar). Although all frameshift or nonsense mutations recorded in the Breast cancer Information Core (BIC) resulting in BRCA1 protein truncation are viewed as functionally deleterious (6Couch F.J. Weber B.L. Hum. Mutat. 1996; 8: 8-18Crossref PubMed Scopus (271) Google Scholar, 7Shen D. Vadgama J.V. Oncol. Res. 1999; 11: 63-69PubMed Google Scholar), the physiological significance of the majority of missense variants has not been determined due to the absence of a distinctive functional assay for BRCA1. More than 70 missense substitutions have been recorded that alter the primary sequence of the tandem BRCT repeats, but pedigree analysis clarifying the disease linkage of these alleles is available for only eight of these variants (6Couch F.J. Weber B.L. Hum. Mutat. 1996; 8: 8-18Crossref PubMed Scopus (271) Google Scholar, 7Shen D. Vadgama J.V. Oncol. Res. 1999; 11: 63-69PubMed Google Scholar, 12Miki Y. Swensen J. Shattuck-Eidens D. Futreal P.A. Harshman K. Tavtigian S. Liu Q. Cochran C. Bennett L.M. Ding W. Bell R. Rosenthal J. Hussey C. Tran T. McClure M. Frye C. Hattier T. Phelps R. Haugen-Strano A. Katcher H. Yakumo K. Gholami Z. Shaffer D. Stone S. Bayer S. Wray C. Bogden R. Dayananth P. Ward J. Tonin P. Narod S. Bristow P.K. Norris F.H. Helvering L. Morrison P. Rosteck P. Lai M. Barrett J.C. Lewis C. Neuhausen S. Cannon-Albright L. Goldgar D. Wiseman R. Kamb A. Skolnick M.H. Science. 1994; 266: 66-71Crossref PubMed Scopus (5390) Google Scholar, 23Futreal P.A. Liu Q. Shattuck-Eidens D. Cochran C. Harshman K. Tavtigian S. Bennett L.M. Haugen-Strano A. Swensen J. Miki Y. et al.Science. 1994; 266: 120-122Crossref PubMed Scopus (1161) Google Scholar, 24Hayes F. Cayanan C. Barilla D. Monteiro A.N. Cancer Res. 2000; 60: 2411-2418PubMed Google Scholar, 25Vallon-Christersson J. Cayanan C. Haraldsson K. Loman N. Bergthorsson J.T. Brondum-Nielsen K. Gerdes A.M. Moller P. Kristoffersson U. Olsson H. Borg A. Monteiro A.N. Hum. Mol. Genet. 2001; 10: 353-360Crossref PubMed Scopus (151) Google Scholar, 26Carvalho M.A. Billack B. Chan E. Worley T. Cayanan C. Monteiro A.N. Cancer Biol. Ther. 2002; 1: 502-508Crossref PubMed Scopus (13) Google Scholar, 27Worley T. Vallon-Christersson J. Billack B. Borg A. Monteiro A.N. Cancer Biol. Ther. 2002; 1: 497-501Crossref PubMed Scopus (19) Google Scholar). Many of these amino acid substitutions may be linked with disease but remain as unclassified in the BIC, because the presence of the allele has not been tested in the general population, or the segregation of the allele with disease within a family is unclear (6Couch F.J. Weber B.L. Hum. Mutat. 1996; 8: 8-18Crossref PubMed Scopus (271) Google Scholar, 7Shen D. Vadgama J.V. Oncol. Res. 1999; 11: 63-69PubMed Google Scholar). The recent determination of the x-ray crystal structures of the rat and human BRCA1 BRCT repeat domains were important first steps toward understanding tumorigenic BRCT mutations and provide a novel platform for the interpretation of the effects of these alterations in the absence of clinical data (13Williams R.S. Green R. Glover J.N. Nat. Struct. Biol. 2001; 8: 838-842Crossref PubMed Scopus (239) Google Scholar, 15Williams R.S. Glover J.N. J. Biol. Chem. 2003; 278: 2630-2635Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 28Joo W.S. Jeffrey P.D. Cantor S.B. Finnin M.S. Livingston D.M. Pavletich N.P. Genes Dev. 2002; 16: 583-593Crossref PubMed Scopus (185) Google Scholar). In the present study we directly evaluate the consequences missense mutation on the structure of the human BRCA1 BRCT repeats. Using a proteolysis-based assay to probe the BRCT for non-native conformations, we show that the majority of the tested missense and truncations alter the folding state of the BRCT. Cross-validated computational analyses using the BRCT structure and the sequences of proteins homologous to the human BRCT from other organisms further suggest that many of the unclassified BRCT missense mutations are likely to be disease-predisposing and perturb BRCA1 structure/function through protein-destabilizing effects. Coding sequences for BRCT C-terminal truncations of human BRCA1 were amplified from the T7 promoter based expression vector for pLM1-BRCA1-(1646-1863) (13Williams R.S. Green R. Glover J.N. Nat. Struct. Biol. 2001; 8: 838-842Crossref PubMed Scopus (239) Google Scholar) using the following oligonucleotides: (fragment 1646-1859) FT7-5′-gga cga gaa ttc tta acc agg gag ctg att atg gtg aac aaa aga atg tcc atg-3′, CD6-5′-gat ctg gga tcc tca ggg gat ctg ggg tat cag-3′; (fragment 1646-1858) FT7, CD7-5′-gat ctg gga tcc tca gat ctg ggg tat cag gta-3′; (fragment 1646-1857) FT7, CD1-5′-gat ctg gga tcc tca ctg ggg tat cag gta ggt-3′; (fragment 1646-1855) FT7, CD5-5′-gat ctg gga tcc tca tat cag gta ggt gtc cag-3′; (fragment 1646-1853) FT7, CD4-5′-gat ctg gga tcc tca gta ggt gtc cag ctc ctg-3′; (fragment 1646-1852) FT7, 1853Ystop-5′-gat ctg gga tcc tca ggt gtc cag ctc ctg gca-3′; (fragment 1646-1851) FT7, CD3-5′-gat ctg gga tcc tca gtc cag ctc ctg gca ctg-3′; (fragment 1646-1849) FT7, CD2-5′-gat ctg gga tcc tca ctc ctg gca ctg gta gag-3′; (fragment 1646-1829) FT7, 1829stop-5′-gat ctg gga tcc tca aca cat ctg ccc aat tgc-3′; (fragment 1646-1805) FT7, 1805stop-5′-gat ctg gga tcc tca gac acc tgt gcc aag ggt-3′. The 5′ primer FT7 incorporates a ribosome binding site and an EcoRI site for cloning. The 3′ oligonucleotides include the relevant stop codons and a BamHI restriction site. Gel-purified PCR products were digested with EcoRI and BamHI and cloned into BamHI-EcoRI-digested pLM1 (29Sodeoka M. Larson C.J. Chen L. Leclair K.P. Verdine G.L.A. Bioorg. Med. Chem. Lett. 1993; 3: 1089Crossref Scopus (60) Google Scholar). All BRCT single amino acid substitutions were introduced into the BRCT fragment 1646-1859. For missense mutations A1708E, M1775R, and W1837R, mutated BRCA1 coding sequences were used as template for PCR amplification with the FT7 and CD6 primers. All other missense substitutions were engineered using PCR splicing methods (30Horton R.M. Ho S.N. Pullen J.K. Hunt H.D. Cai Z. Pease L.R. Methods Enzymol. 1993; 217: 270-279Crossref PubMed Scopus (435) Google Scholar). Primary PCR mutagenesis reactions used oligonucleotide FT7 with the appropriate reverse (R) mutagenesis oligonucleotide (see below) and CD6 with the appropriate foward (F) mutagenesis oligonucleotide. PCR products from the primary reactions were gel-purified from 1.5% agarose gels using a QIAEX2 kit (Qiagen) and mixed together with oligonucleotides FT7 and CD6 in the secondary PCR splicing reactions to generate mutated PCR products that were subsequently digested with EcoRI/BamHI and ligated to pLM1. The mutagenesis oligonucleotides used were: D1692Y, F-5′-gtt atg aaa aca tat gct gag ttt gtg-3′, R-5′-cac aaa ctc agc ata tgt ttt cat aac-3′; F1695L, F-5′-aca gat gct gag ctt gtg tgt gaa cgg-3′, R-5′-ccg ttc aca cac aag ctc agc atc tgt-3′; V1696L, F-5′-gat gct gag ttt ttg tgt gaa cgg aca-3′, R-5′-tgt ccg ttc aca caa aaa ctc agc atc-3′; C1697R, F-5′-gct gag ttt gtg cgt gaa cgg aca ctg-3′, R-5′-cag tgt ccg ttc acg cac aaa ctc agc-3′; R1699W, F-5′-ttt gtg tgt gaa tgg aca ctg aaa tat-3′, R-5′-ata ttt cag tgt cca ttc aca cac aaa-3′; R1699Q, F-5′-ttt gtg tgt gaa cag aca ctg aaa tat-3′, R-5′-ata ttt cag tgt ctg ttc aca cac aaa-3′; S1715R, F-5′-aaa tgg gta gtt aga tat ttc tgg gtg-3′, R-5′-cac cca gaa ata tct aac tac cca ttt-3′; W1718C, F-5′-gtt agc tat ttc tgt gtg acc cag tct-3′, R-5′-aga ctg ggt cac aca gaa ata gct aac-3′; T1720A, F-5′-tat ttc tgg gtg gcc cag tct att aaa-3′, R-5′-ttt aat aga ctg ggc cac cca gaa ata-3′; G1738E, F-5′-ttt gaa gtc aga gaa gat gtg gtc aat g-3′, R-5′-cat tga cca cat ctt ctc tga ctt caa a-3′; G1738R F-5′-ttt gaa gtc aga aga gat gtg gtc aat g-3′, R-5′-cat tga cca cat ctc ttc tga ctt caa a-3′; P1749R, F-5′-aac cac caa ggt cgt aag cga gca aga g-3′, R-5′-ctc ttg ctc gct tac gac ctt ggt ggt t-3′; R1751Q, F-5′-caa ggt cca aag caa gca aga gaa tcc-3′, R-5′-gga ttc tct tgc ttg ctt tgg acc ttg-3′; A1752P, F-5′-ggt cca aag cga cca aga gaa tcc cag-3′, R-5′-ctg gga ttc tct tgg tcg ctt tgg acc-3′; I1766S, F-5′-agg ggg cta gaa agc tgt tgc tat ggg-3′, R-5′-ccc ata gca aca gct ttc tag ccc cct-3′; M1783T, F-5′-caa ctg gaa tgg acc gta cag ctg tgt g-3′, R-5′-cac aca gct gta cgg tcc att cca ggt t; G1788V, F-5′-gta cag ctg tgt gtt gct tct gtg gtg-3′, R-5′-cac cac aga agc aac aca cag ctg tac-3′; V1804D, F-5′-ctt ggc aca ggt gac cac cca att gtg-3′, R-5′-cac aat tgg gtg gtc acc tgt gcc aag-3′; V1809F, F-5′-cac cca att gtg ttt gtg cag cca gat-3′, R-5′-atc tgg ctg cac aaa cac aat tgg gtg-3′; W1837G, F-5′-gtg acc cga gag ggg gtg ttg gac agt g-3′, R-5′-cac tgt cca aca ccc cct ctc ggg tca c-3′. All vectors were sequenced to confirm the success of the mutagenesis reactions. 0.2-0.5 μg of pLM1 plasmid encoding the BRCT variants were used directly as template for protein synthesis reactions with the TnT-Quick in vitro transcription/translation system (Promega). Immediately prior to proteolytic digestion, proteins were translated and labeled with [35S]methionine at 30 °C for 2 h. The reticulocyte lysates were then centrifuged for 2 min at 10,000 × g to remove insoluble material, and 3 μl of the lysate supernatants containing the labeled translation products were added to 12 μl of digestion buffer (150 mm NaCl, 50 mm potassium phosphate, pH 7.5) containing increasing concentrations of trypsin (Sigma) or 1-chloro-3-tosylamido-7-amino-2-heptanone or Nα-p-tosyl-l-lysine chloromethyl ketone-treated chymotrypsin (Sigma). After digestion at 20 °C for 12 min, the reactions were stopped with phenylmethylsulfonyl fluoride. Digestion products were electrophoresed on 15% SDS-PAGE gels and visualized with a phosphorimaging plate and a Molecular Dynamics Typhoon scanner. A local average background correction was used during quantification of the reaction products with ImageQuaNT (Amersham Biosciences). Structural diagrams were created with Bobscript (31Esnouf R.M. J. Mol. Graph Model. 1997; 15: 132-134Crossref PubMed Scopus (1796) Google Scholar, 32Esnouf R.M. Acta Crystallogr. D Biol. Crystallogr. 1999; 55: 938-940Crossref PubMed Scopus (851) Google Scholar) and rendered using Povray (www.povray.org). Method 1: Structure and Sequence-based Analysis—A probability of an effect on function for the missense mutations in BRCT was determined exactly as described using both feature set A and feature set B in Ref. 33Chasman D. Adams R.M. J. Mol. Biol. 2001; 307: 683-706Crossref PubMed Scopus (335) Google Scholar. Briefly, the crystal structure (PDB ID: 1JNX), the multiple sequence alignment for proteins homologous to human BRCT (see Fig. 3), and the chemical nature of the amino acid substitution are used to compute the values of features that are useful for predicting the effects of amino acid substitutions on protein function. For example, the quantitative estimate of solvent accessibility for a residue in a structure or its normalized phylogenetic entropy from a multiple sequence alignment are both features that can be viewed as having a quantitative relationship to the probability of an effect on function for the introduction of a mutant amino acid (33Chasman D. Adams R.M. J. Mol. Biol. 2001; 307: 683-706Crossref PubMed Scopus (335) Google Scholar). A probability of an effect on function for a test mutation is estimated by conditional probability as the fraction of training mutations derived from exhaustive mutagenesis of the Lac repressor (34Markiewicz P. Kleina L.G. Cruz C. Ehret S. Miller J.H. J. Mol. Biol. 1994; 240: 421-433Crossref PubMed Scopus (234) Google Scholar) and T4 lysozyme (35Rennell D. Bouvier S.E. Hardy L.W. Poteete A.R. J. Mol. Biol. 1991; 222: 67-88Crossref PubMed Scopus (291) Google Scholar) with an effect on function from among those with feature values are similar to the feature values of the test mutation. Method 2: Refined Sequence-based Analysis—The sequence-based procedure 2A. Y. Lau and D. I. Chasman, submitted for publication. for predicting the functional consequences of a mutation in a residue of human BRCT is an extension of the direct inspection of alternative amino acid existing at the corresponding residue in proteins homologous to the human BRCT. In essence, mutations that introduce amino acids observed at the corresponding residue of homologous proteins are judged to be tolerated by the human BRCT. The extension involves inferring, through the use of the Blocks9 mixture of Dirichlet priors (36Henikoff S. Henikoff J.G. Pietrokovski S. Bioinformatics. 1999; 15: 471-479Crossref PubMed Scopus (242) Google Scholar), the more likely of two hypotheses explaining why some amino acids are not observed at corresponding residues of homologous proteins. Either the sequences of the homologous proteins represent an incomplete sampling of all 20 amino acids at a mutated residue position or some of the 20 amino acids are incompatible with the structural and functional constraints on the mutated residue position. Inclusion of the alternative amino acid from a mutation in the inferred set of acceptable amino acids is evidence for its compatibility with biological function. Exclusion leads to a prediction of incompatibility. We previously demonstrated that the tandem BRCT repeat region of human BRCA1 forms a proteolytically resistant globular domain and that a cancer-linked mutation, Y1853ter, which removes the 11 C-terminal residues of the protein, reduces this proteolytic stability (13Williams R.S. Green R. Glover J.N. Nat. Struct. Biol. 2001; 8: 838-842Crossref PubMed Scopus (239) Google Scholar). To determine to what extent the BRCT fold could tolerate truncation mutations, we subjected a series BRCT deletion mutants to a proteolytic sensitivity assay (Fig. 1, see “Experimental Procedures”). The oncogenic mutation Y1853ter and all larger C-terminal deletions of the protein were degraded by the lowest concentrations of trypsin, whereas the full-length BRCT (aa 1646-1863) is highly resistant to cleavage (Fig. 1). Included with these mutations are the truncation protein products of two of the most common BIC frameshift mutants, 5382insC and IVS21-36del510, that result in stop codons at positions 1829 and 1805 of the BRCA1 coding sequence (7Shen D. Vadgama J.V. Oncol. Res. 1999; 11: 63-69PubMed Google Scholar) (Figs. 2 and 3). Thus, BRCT folding defects resulting from cancer-predisposing BRCA1 truncation mutations can be assayed for and detected at the protein level using a simple protease sensitivity assay.Fig. 2Structural effects of cancer-associated BRCA1 BRCT truncation-causing mutations.A, a stop codon at position 1805 results from frameshift IVS21-36del510, removing much of the C-terminal BRCT domain. B, frameshift 5382insC creates a stop codon at position 1829 in BRCA1 and is one of the most commonly recorded BIC mutations. C, a nonsense mutation 1853-ter results in the removal of the 11 C-terminal residues of the protein and is linked to disease. For A-C, red portions of the structures are deleted residues caused by truncation mutations. D, interaction of the C-terminal tail of BRCA1 with BRCT-C. Negative electrostatic potential is red and positive is blue. The C terminus of BRCA1 forms a 310 helix and an extended peptide that packs against α2′ and the β-sheet. C-terminal deletions beyond the hydrophobic residues Leu1854 and Ile1855 are destabilizing.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The deletion experiment also demonstrates the protein can tolerate removal of up to 8 residues, but further deletion from the C terminus greatly impairs the native folding of the domain, rendering it highly sensitive to proteolysis (Figs. 1 and 2D). Consistent with this finding, the transcriptional activation activity of the BRCT domains was abolished by C-terminal deletions that truncate beyond a hydrophobic pair of residues, Leu1854 and Ile1855 (24Hayes F. Cayanan C. Barilla D. Monteiro A.N. Cancer Res. 2000; 60: 2411-2418PubMed Google Scholar). These hydrophobes mark the C-terminal boundary for conservation of mammalian, avian, and Xenopus BRCA1 homologues (Fig. 3) and make critical aliphatic contacts to the β-sheet of the C-terminal BRCT in the structures of the human and rat BRCA1-BRCT repeats (Fig. 2D) (13Williams R.S. Green R. Glover J.N. Nat. Struct. Biol. 2001; 8: 838-842Crossref PubMed Scopus (239) Google Scholar, 28Joo W.S. Jeffrey P.D. Cantor S.B. Finnin M.S. Livingston D.M. Pavletich N.P. Genes Dev. 2002; 16: 583-593Crossref PubMed Scopus (185) Google Scholar). Hence, the transcriptional activation defects observed for BRCT deletion mutants likely result from destabilization of the protein. Similar to the truncation mutants, two cancer predisposing missense mutations, A1708E and M1775R, are destabilizing and exhibit altered BRCT protease susceptibility (13Williams R.S. Green R. Glover J.N. Nat. Struct. Biol. 2001; 8: 838-842Crossref PubMed Scopus (239) Google Scholar, 14Ekblad C.M. Wilkinson H.R. Schymkowitz J.W. Rousseau F. Freund S.M. Itzhaki L.S. J. Mol. Biol. 2002; 320: 431-442Crossref PubMed Scopus (40) Google Scholar, 15Williams R.S. Glover J.N. J. Biol. Chem. 2003; 278: 2630-2635Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). To gain insights into the effects of other patient-derived mutations recorded in the BIC, we generated 23 additional missense variants and tested these proteins for proteolytic sensitivity (see “Experimental Procedures,” Fig. 4). 20/25 of the missense mutations tested showed varying degrees of enhanced sensitivity to tryptic digestion at 20 °C (Fig. 4A). Five of six of the mutations that substitute an arginine into the protein (C1697R, S1715R, G1738R, P1749R, and W1837R) also show increased sensitivity to chymotryptic cleavage at 20 °C (Fig. 4B) suggesting that destabilizing effects, rather than the introduction of a new trypsin cleavage site, are responsible for the protease sensitivity. Mutant M1775R is also clearly destabilizing (14Ekblad C.M. Wilkinson H.R. Schymkowitz J.W. Rousseau F. Freund S.M. Itzhaki L.S. J. Mol. Biol. 2002; 320: 431-442Crossref PubMed Scopus (40) Google Scholar) and shows sensitivity to chymotrypsin at elevated temperatures (15Williams R.S. Glover J.N. J. Biol. Chem. 2003; 278: 2630-2635Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). The expression levels of the BRCT variants in the reticulocyte lysates typically range between 0.3- and 1.2-fold of wild type levels. Because the expressed variants constitute less than 5-10% of the total protein digested in the lysates and we are using logarithmic increases in trypsin concentrations, we can quantify the percentages of protein remaining following digestion at each level of protease and directly compare these values to establish a proteolysis-based hierarchy for the severity of the destabilizing effects (Fig. 5). Here we define highly destabilizing mutations as those mutants for which >60% of the protein is degraded at the lowest concentration (6 μg/ml) of trypsin. Intermediately destabilizing variants are >60% degraded at the intermediate trypsin concentration (60 μg/ml). Finally, the mutants showing wild type digestion profiles, with limited degradation until exposure to the highest trypsin concentration, are classified as having no destabilizing effect. Based on these criteria, the majority of the variants (13/25) are highly destabilizing, 7/25 are intermediately destabilizing, and 5/25 have no apparent effect. Homology models of the human BRCA1-BRCT repeats, built from the XRCC1 C-terminal BRCT structure (37Zhang X. Morera S. Bates P.A. Whitehead P.C. Coffer A.I. Hainbucher K. Nash R.A. Sternberg M.J. Lindahl T. Freemont P.S. EMBO J. 1998; 17: 6404-6411Crossref PubMed Scopus (224) Google Scholar), have been used to describe structural environments of BRCA1-BRCT missense variants (38Huyton T. Bates P.A. Zhang X. Sternberg M.J. Freemont P.S. Mutat. Res. 2000; 460: 319-332Crossref PubMed Scopus (133) Google Scholar). Because these descriptions are inaccurate in many respects, we have reclassified the BRCT missense mutations into the four following categories based on their distribution in the human BRCT repeat structure (13Williams R.S. Green R. Glover J.N. Nat. Struct. Biol. 2001; 8: 838-842Crossref PubMed Scopus (239) Google Scholar) (Fig. 5 and Table I) as follows.Table IStructure, function, and disease effects of BRCT missense mutationsMutantSecondary structureaSecondary structure is from the human BRCT domain structure (13).Mutant classProtease sensitivitybProtease sensitivity: (−), wild type, no effect; (+), intermediately destabilizing; and (++), highly destabilizing.Predictive method 1cPredictive method 1. Predicted effect on function is as described by Chasman and Adams (33). (+): The mutation is predicted to effect structure/function, the probability of an effect on function is >0.5. (−): The mutation is predicted to be a benign substitution, the probability of an effect on structure/function is <0.5 (see Supplementary Table I for calculated probabilities). For one mutation, G1788V, there were too few data points to estimate a probability.Predictive method 2dPredictive method 2, sequence based. (+), The mutation is predicted to effect structure/function; (−), The mutation is predicted to be a benign substitution.TranscriptioneTranscription effects are those reported by Monteiro et al. (45), Monteiro et al. (42), Hayes et al. (24), Vallon-Christersson et al. (25), and Worley et al. (27).Disease effectfDisease linkage data are from recorded entries in the BIC (Hayes et al. (24), Vallon-Christersson et al. (25), and T. S. Frank, personal communication). (+), linked to disease; (−), not linked; (?), unknown.Solubility
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