乳腺癌易感蛋白BRCA1的BRCT1结构域染色质伸展活性的定位

乳腺癌易感基因BRCA1（Breast cancer susceptibility gene 1）在乳腺癌的发生、发展中起重要作用。BRCA1 C末端含有2个BRCT结构域（BRCT1和BRCT2）,许多乳腺癌易感突变发生在BRCA1的BRCT结构域中。利用染色质结构检测技术表明,BRCT结构域具有染色质伸展活性。本文利用缺失突变技术构建了6种BRCT1结构域（1642-1736 aa）缺失突变体并将BRCT1结构域中与染色质伸展相关的重要区域定位到1691-1721之间的氨基酸残基;用丙氨酸扫描技术构建了10种BRCT1结构域丙氨酸扫描突变体并将重要氨基酸残基序列定位到1707-1711之间的IAGGK。利用定位的重要区域进行Blast分析,结果找到一新型同源蛋白质。BRCT1结构域的定位有助于预测BRCT1结构域突变后发生乳腺癌的风险,也为进一步研究BRCT1结构域的功能机制提供了有用的材料。     

乳腺癌易感蛋白BRCA1的BRCT2结构域染色质伸展活性的定位

40 %～ 5 0 %的遗传性乳腺癌和至少 80 %的既有乳腺癌又有卵巢癌家族史的患者是由BRCA1突变引起的 .BRCA1C末端含有 2个BRCT结构域 (BRCT1和BRCT2 ) ,它们与BRCA1的重要功能密切相关 .许多乳腺癌易感突变发生在BRCA1的BRCT结构域中 .利用染色质结构检测技术表明 ,BRCT结构域具有染色质伸展活性 .利用缺失突变技术构建了 6种BRCT2结构域 (175 6～ 185 2位氨基酸残基 )缺失突变体并将BRCT2结构域中与染色质伸展相关的重要区域定位到 175 6～ 180 8之间的氨基酸残基 ;用丙氨酸扫描技术构建了 6种BRCT2结构域丙氨酸扫描突变体并将重要氨基酸残基序列定位到 1784～ 1788之间的VQLCG .BRCT2结构域的定位有助于预测BRCT2结构域突变后发生乳腺癌的风险 ,也为进一步研究BRCT2结构域的功能机制提供了有用的材料 .     

The functional impact of BRCA1 BRCT domain variants using multiplexed DNA double-strand break repair assays

Pathogenic variants in BRCA1 are associated with a greatly increased risk of hereditary breast and ovarian cancer (HBOC). With the increased availability and affordability of genetic testing, many individuals have been identified with BRCA1 variants of uncertain significance (VUSs), which are individually detected in the population too infrequently to ascertain a clinical risk. Functional assays can be used to experimentally assess the effects of these variants. In this study, we used multiplexed DNA repair assays of variants in the BRCA1 carboxyl terminus to functionally characterize 2,271 variants for homology-directed repair function (HDR) and 1,427 variants for cisplatin resistance (CR). We found a high level of consistent results (Pearson’s r = 0.74) in the two multiplexed functional assays with non-functional variants located within regions of the BRCA1 protein necessary for its tumor suppression activity. In addition, functional categorizations of variants tested in the multiplex HDR and CR assays correlated with known clinical significance and with other functional assays for BRCA1 (Pearson’s r = 0.53 to 0.71). The results of the multiplex HDR and CR assays are useful resources for characterizing large numbers of BRCA1 VUSs.     

BRCA1在人类肿瘤细胞中的表达

人类乳癌易感基因1(BRCA1)是乳癌,卵巢癌和前列腺癌的危险因素之一,而且表现出许多的生物功能.采用WesternBlotting和半定量RT-PCR的方法,我们检测了内源性BRCA1蛋白质和mR-NA在从十一种人类肿瘤组织中建立的四十三种肿瘤细胞系的表达水平.在不同的肿瘤细胞中BR-CA1的表达水平是各不一样的.而且并没有发现BRCA1的表达和细胞的内源性p53基因状况有明显的相关性.通过采用细胞转染乳头状瘤病毒-E6致癌基因或采用畸变的p53基因(143Ala→Va1)而导致的p53基因功能失活并不对内源性BRCA1本底表达水平产生任何的影响,但两种与p53功能有关p21(-/-)和Gadd45基因剔除则轻微地增加BRCA1蛋白质的表达.因此,虽然我们目前还不清楚BRCA1在人类肿瘤细胞中不同表达的功能意义,但本文的结果为进一步研究BRCA1在不同肿瘤细胞系的生物功能提供了有价值的背景资料.     

A BRCA1-interacting lncRNA regulates homologous recombination

Long non-coding RNAs (lncRNAs) are important players in diverse biological processes. Upon DNA damage, cells activate a complex signaling cascade referred to as the DNA damage response (DDR). Using a microarray screen, we identify here a novel lncRNA, DDSR1 (DNA damage-sensitive RNA1), which is induced upon DNA damage. DDSR1 induction is triggered in an ATM-NF-κB pathway-dependent manner by several DNA double-strand break (DSB) agents. Loss of DDSR1 impairs cell proliferation and DDR signaling and reduces DNA repair capacity by homologous recombination (HR). The HR defect in the absence of DDSR1 is marked by aberrant accumulation of BRCA1 and RAP80 at DSB sites. In line with a role in regulating HR, DDSR1 interacts with BRCA1 and hnRNPUL1, an RNA-binding protein involved in DNA end resection. Our results suggest a role for the lncRNA DDSR1 in modulating DNA repair by HR.     

Characterisation of the BRCT domains of the breast cancer susceptibility gene product BRCA1

The breast cancer susceptibility gene product BRCA1 is a tumour suppressor but the biochemical and biological functions that underlie its role in carcinogenesis remain to be determined. Here, we characterise the solution properties of the highly conserved C terminus of BRCA1, consisting of a tandem repeat of the BRCT domain (BRCT-tan), that plays a critical role in BRCA1-mediated tumour suppression. The overall free energy of unfolding of BRCT-tan is high (14.2 kcal mol(-1) at 20 degrees C in water) but unfolding occurs via an aggregation-prone, partly folded intermediate. A representative set of cancer-associated sequence variants was constructed and the effects on protein stability were measured. All of the mutations were highly destabilising and they would be expected to cause loss of function for this reason. Over half could not be purified in a soluble form, indicating that these residues are critical for maintaining structural integrity. The remaining mutants exhibited much greater aggregation propensities than the wild-type, which is most likely a consequence of their reduced thermodynamic stability relative to the partly folded intermediate. The mutations characterised here are located at different sites in the BRCT-tan structure that do not explain fully their effects on the protein's stability. Thus, the results indicate an important role for biophysical studies in assessing the significance of sequence variants and in determining how they cause disease.     

Reproductive factors and breast cancer risk among BRCA1 or BRCA2 mutation carriers: Results from ten studies

Although reproductive factors are among the most well-established risk factors for breast cancer in the general population, it is still a matter for debate whether these factors act as risk modifiers among BRCA1 or BRCA2 mutation carriers. This meta-analysis is the first to be performed to determine the relationship between reproductive factors and breast cancer risk among BRCA1 and BRCA2 mutation carriers. We searched the PubMed database up to February 2013. A total of ten studies met the inclusion criteria. The results showed that the reproductive factors may be associated with breast cancer risk only among BRCA1 mutation carriers. No association was found between parity and breast cancer risk. Compared with women at the youngest age in the first-birth category, women in the oldest age category were at a 38% lower risk of breast cancer (RR = 0.62, 95%CI = 0.45–0.85). Breastfeeding for at least 1 or 2 years was associated with a 37% reduction in breast cancer risk (RR = 0.63, 95%CI = 0.46–0.86). Women at the oldest age in the menarche category were at a 34% lower risk of breast cancer (RR = 0.66, 95%CI = 0.53–0.81) than women in the youngest age category. However, none of the reproductive factors were associated with breast cancer risk among BRCA2 mutation carriers. In conclusion, late age at first birth, breastfeeding, and late age at menarche protect against breast cancer in BRCA1 mutation carriers only. Further studies are needed to explore the mechanisms.     

Characterization of cancer‐linked BRCA1‐BRCT missense variants and their interaction with phosphoprotein targets

The breast cancer tumor suppressor protein BRCA1 is involved in DNA repair and cell cycle control. Mutations at the two C‐terminal tandem (BRCT) repeats of BRCA1 detected in breast tumor patients were identified either to lower the stability of the BRCT domain and/or to disrupt the interaction of BRCT with phoshpopeptides. The aim of this study was to analyze five BRCT pathogenic mutations for their effect on structural integrity and protein stability. For this purpose, the five cancer‐associated BRCT mutants: V1696L, M1775K, M1783T, V1809F, and P1812A were cloned in suitable prokaryotic protein production vectors, and the recombinant proteins were purified in soluble and stable form for further biophysical studies. The biophysical analysis of the secondary structure and the thermodynamic stability of the wild‐type, wt, and the five mutants of the BRCT domain were performed by Circular Dichroism Spectroscopy (CD) and Differential Scanning Microcalorimetry (DSC), respectively. The binding capacity of the wt and mutant BRCT with (pBACH1/BRIP1) and pCtIP were measured by Isothermal Titration Calorimetry (ITC). The experimental results demonstrated that the five mutations of the BRCT domain: (i) affected the thermal unfolding temperature as well as the unfolding enthalpy of the domain, to a varying degree depending upon the induced destabilization and (ii) altered and/or abolished their affinity to synthetic pBACH1/BRIP1 and pCtIP phosphopeptides by affecting the structural integrity of the BRCT active sites. The presented experimental results are one step towards the elucidation of the effect of various missense mutations on the structure and function of BRCA1‐BRCT. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Role of BRCA1 and BRCA2 gene mutations in epithelial ovarian cancer in Indian population: a pilot study

Ovarian cancer is a silent killer as most patients have non-specific symptoms and usually present in advanced stage of the disease. It occurs due to certain genetic alterations and mutations namely founder mutations, 187delAG and 5385insC in BRCA1 and 6174delT in BRCA2 which are associated with specific family histories. These highly penetrant susceptibility genes responsible for approximately half of families containing 2 or more ovarian cancer cases account for less than 40% of the familial excess malignancy risk. The remaining risk may be due to single nucleotide polymorphisms (SNPs) which are single base change in a DNA sequence with usual alternatives of two possible nucleotides at a given position. Preliminary study involving 30 women with histologically proven epithelial ovarian cancer was conducted and their detailed genetic analysis was carried out. Regions of founder mutations on BRCA1 and BRCA2 were amplified and sequenced using primers designed based on 200 bp upstream and downstream regions of the mutation sites. Five sequence variants in BRCA1 were identified of which three novel sequence variants were found in 23 patients while in BRCA2, one novel sequence variant was found. The three founder mutations 187delAG, 5385insC in BRCA1 and 6174delT in BRCA2 were not seen in any of the subjects.     

Identification and functional characterization of a PP1-binding site in BRCA1

The phosphorylation state of the tumor suppressor protein BRCA1 is tightly associated with its functions including cell cycle control and DNA repair. Protein kinases involved in the DNA damage checkpoint control, such as ATM, ATR, and hCds1/Chk2, have been shown to phosphorylate and activate BRCA1 upon DNA damage. We reported previously that protein phosphatase 1alpha (PP1alpha) interacts with and dephosphorylates hCds1/Chk2-phosphorylated BRCA1. This study demonstrates the identification of a PP1-binding motif 898KVTF901 in BRCA1. Mutation or deletion of critical residues in this PP1-binding motif substantially reduces the interaction between BRCA1 and PP1alpha. PP1alpha can also dephosphorylate ATM and ATR phosphorylation sites in BRCA1 and may serve as a general regulator for BRCA1 phosphorylation. Unlike wild-type BRCA1, expression of the PP1 non-binding mutant BRCA1 protein in BRCA1-deficient cells failed to enhance survival after DNA damage. Taken together, these results suggest that interaction with PP1alpha is important for BRCA1 function.     

PRIMA-1 cytotoxicity correlates with nucleolar localization and degradation of mutant p53 in breast cancer cells

PRIMA-1 has been identified as a compound that restores the transactivation function to mutant p53 and induces apoptosis in cells expressing mutant p53. Studies on subcellular distribution of the mutant p53 protein upon treatment with PRIMA-1^Met, a methylated form of PRIMA-1, have suggested that redistribution of mutant p53 to nucleoli may play a role in PRIMA-1 induced apoptosis. Here, we specifically investigated the influence of PRIMA-1 on cellular localization of mutated p53-R280K endogenously expressed in tumour cells. By using immunofluorescence staining, we found a strong nucleolar redistribution of mutant p53 following PRIMA-1 treatment. This subcellular localization was associated to p53 degradation via ubiquitylation. When cells were treated with adriamycin, neither nucleolar redistribution nor mutant p53 down modulation and degradation were observed. Interestingly, cells where p53-R280K was silenced were more sensitive to PRIMA-1 than the parental ones. These results indicate that in some cellular context, the cell sensitivity to PRIMA-1 could depend on the abolition of a gain-of-function activity of the mutated p53, through a protein degradation pathway specifically induced by this compound.     

Deciphering the BRCA1 Tumor Suppressor Network

The BRCA1 tumor suppressor protein is a central constituent of several distinct macromolecular protein complexes that execute homology-directed DNA damage repair and cell cycle checkpoints. Recent years have borne witness to an exciting phase of discovery at the basic molecular level for how this network of DNA repair proteins acts to maintain genome stability and suppress cancer. The clinical dividends of this investment are now being realized with the approval of first-in-class BRCA-targeted therapies for ovarian cancer and identification of molecular events that determine responsiveness to these agents. Further delineation of the basic science underlying BRCA network function holds promise to maximally exploit genome instability for hereditary and sporadic cancer therapy.