Williams R Scott, Chasman Daniel I, Hau D Duong, Hui Benjamin, Lau Albert Y, Glover J N Mark
Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
J Biol Chem. 2003 Dec 26;278(52):53007-16. doi: 10.1074/jbc.M310182200. Epub 2003 Oct 8.
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.
迄今已鉴定出的大多数与癌症相关的BRCA1突变会导致该蛋白的翻译提前终止，这凸显了C末端的BRCT重复区域在介导BRCA1肿瘤抑制功能中的关键作用。然而，定位于BRCT区域的错义突变的分子和遗传效应在很大程度上仍不清楚。我们使用基于蛋白酶的检测方法，直接评估了BRCT结构域对源自乳腺癌筛查项目的一系列广泛的截短突变和单氨基酸取代的折叠敏感性。该蛋白能够耐受多达8个氨基酸的截短，但进一步缺失会导致BRCT折叠出现严重缺陷。这种分子表型可能与疾病易感性增加相关。对BRCT突变数据库进行交叉验证的计算评估表明，所有BRCT错义突变中多达一半通过蛋白质稳定性破坏效应导致BRCA1功能丧失和疾病发生。蛋白水平上蛋白水解方法和计算预测方法的联合使用来检测突变型BRCA1构象，将提高当前BRCA1筛查方案的效力，尤其是在缺乏可用于区分有害BRCT错义突变和良性多态性的临床数据的情况下。
