The F-box Protein FBXO44 Mediates BRCA1 Ubiquitination and Degradation

BRCA1 mutations account for a significant proportion of familial breast and ovarian cancers. In addition, reduced BRCA1 protein is associated with sporadic cancer cases in these tissues. At the cellular level, BRCA1 plays a critical role in multiple cellular functions such as DNA repair and cell cycle checkpoint control. Its protein level is regulated in a cell cycle-dependent manner. However, regulation of BRCA1 protein stability is not fully understood. Our earlier study showed that the amino terminus of BRCA1 harbors a degron sequence that is sufficient and necessary for conferring BRCA1 degradation. In the current study, we used mass spectrometry to identify Skp1 that regulates BRCA1 protein stability. Small interfering RNA screening that targets all human F-box proteins uncovered FBXO44 as an important protein that influences BRCA1 protein level. The Skp1-Cul1-F-box-protein44 (SCF^FBXO44) complex ubiquitinates full-length BRCA1 in vitro. Furthermore, the N terminus of BRCA1 mediates the interaction between BRCA1 and FBXO44. Overexpression of SCF^FBXO44 reduces BRCA1 protein level. Taken together, our work strongly suggests that SCF^FBXO44 is an E3 ubiquitin ligase responsible for BRCA1 degradation. In addition, FBXO44 expression pattern in breast carcinomas suggests that SCF^FBXO44-mediated BRCA1 degradation might contribute to sporadic breast tumor development.     

HUWE1 interacts with BRCA1 and promotes its degradation in the ubiquitin–proteasome pathway

The cellular BRCA1 protein level is essential for its tumor suppression activity and is tightly regulated through multiple mechanisms including ubiquitn–proteasome system. E3 ligases are involved to promote BRCA1 for ubiquitination and degradation. Here, we identified HUWE1/Mule/ARF-BP1 as a novel BRCA1-interacting protein involved in the control of BRCA1 protein level. HUWE1binds BRCA1 through its N-terminus degron domain. Depletion of HUWE1 by siRNA-mediated interference significantly increases BRCA1 protein levels and prolongs the half-life of BRCA1. Moreover, exogenous expression of HUWE1 promotes BRCA1 degradation through the ubiquitin–proteasome pathway, which could explain an inverse correlation between HUWE1 and BRCA1 levels in MCF10F, MCF7 and MDA-MB-231 breast cancer cells. Consistent with a functional role for HUWE1 in regulating BRCA1-mediated cellular response to DNA damage, depletion of HUWE1 by siRNA confers increased resistance to ionizing radiation and mitomycin. These data indicate that HUWE1 is a critical negative regulator of BRCA1 and suggest a new molecular mechanism for breast cancer pathogenesis.     

Localization of BRCA1 and a splice variant identifies the nuclear localization signal.

Inherited mutations in BRCA1 confer susceptibility to breast and ovarian neoplasms. However, the function of BRCA1 and the role of BRCA1 in noninherited cancer remain unknown. Characterization of alternately spliced forms of BRCA1 may identify functional regions; thus, we constructed expression vectors of BRCA1 and a splice variant lacking exon 11, designated BRCA1 delta 672-4095. Immunofluorescence studies indicate nuclear localization of BRCA1 but cytoplasmic localization of BRCA1 delta 672-4095. Two putative nuclear localization signals (designated NLS1 and NLS2) were identified in exon 11; immunofluorescence studies indicate that only NLS1 is required for nuclear localization. RNA analysis indicates the expression of multiple, tissue-specific forms of BRCA1 RNAs; protein analysis with multiple antibodies suggests that at least three BRCA1 isoforms are expressed, including those lacking exon 11. The results suggest that BRCA1 is a nuclear protein and raise the possibility that splicing is one form of regulation of BRCA1 function by alteration of the subcellular localization of expressed proteins.     

BRCA1蛋白的序列及空间结构的分析

查询人的BRCA1蛋白的氨基酸序列,利用生物信息学的方法进行相似性搜索,获得一系列BRCA1蛋白的氨基酸序列。选择了其中的11条序列,对BRCA1蛋白进行了多重序列分析和进化分析,对BRCA1蛋白的BRCT结构域进行三维同源模型的构建与比较分析。分析结果表明:BRCA1中某些特定部位的氨基酸序列高度保守;确定氨基酸的保守位点并联合进化分析可对基因错义突变的致病性做初步地猜测;相近物种来源的BRCA1具有较近的亲缘关系,而且具有极其相似的三维空间结构。这些为研究BRCA1蛋白的结构与功能关系提供指导意义。     

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.     

The BRCA1 Tumor Suppressor Binds to Inositol 1,4,5-Trisphosphate Receptors to Stimulate Apoptotic Calcium Release

The inositol 1,4,5-trisphosphate receptor (IP₃R) is a ubiquitously expressed endoplasmic reticulum (ER)-resident calcium channel. Calcium release mediated by IP₃Rs influences many signaling pathways, including those regulating apoptosis. IP₃R activity is regulated by protein-protein interactions, including binding to proto-oncogenes and tumor suppressors to regulate cell death. Here we show that the IP₃R binds to the tumor suppressor BRCA1. BRCA1 binding directly sensitizes the IP₃R to its ligand, IP₃. BRCA1 is recruited to the ER during apoptosis in an IP₃R-dependent manner, and, in addition, a pool of BRCA1 protein is constitutively associated with the ER under non-apoptotic conditions. This is likely mediated by a novel lipid binding activity of the first BRCA1 C terminus domain of BRCA1. These findings provide a mechanistic explanation by which BRCA1 can act as a proapoptotic protein.     

BRCA1 Haploinsufficiency,but not Heterozygosity for a BRCA1-truncating Mutation,Deregulates Homologous Recombination

Humans heterozygous for BRCA1 mutations have a high risk of losing the remaining wild-type BRCA1 allele and developing breast/ovarian cancer, but a molecular basis for this has not yet been determined. It is thought that heterozygosity status — reduced wild-type BRCA1 protein dosage (haploinsufficiency) and/or the presence of a mutant BRCA1 protein — may affect BRCA1 functions and heighten the risk of cancer promoting mutations. BRCA1 maintains genome stability, at least in part, by regulating homologous recombination according to the type of DNA damage. To investigate whether this BRCA1 function is affected by heterozygosity status, we employed, as recombination reporters, human breast cancer MCF-7 cells known to have a single wild-type BRCA1 allele and reduced BRCA1 protein dosage. These cells revealed: 1) a spontaneous hyper-recombination phenotype; 2) reduced efficiency in homologous recombination repair of DNA double-strand breaks (DSBs); and 3) sensitivity to the DSB-inducing chemotherapeutic agent mitomycin C. Correction of BRCA1 protein dosage to the wild-type level reversed all these phenotypes, whereas physiological expression of the cancer-eliciting BRCA1 5382insC mutant allele had no effect on either phenotype. These findings implicate BRCA1 C-terminal domain in recombination control, and indicate that BRCA1 haploinsufficiency alone, which is also a feature of sporadic breast/ovarian cancer, is sufficient to compromise genome stability by triggering spontaneous recombination events that are likely to account for the loss of the remaining wild-type BRCA1 allele and increased cancer risk. Our observations may also have implications for the medical management of cancer patients and cancer prevention.     

Analysis of Loss of heterozygosity and immunohistochemistry in BRCA1 gene in sporadic breast cancers

BRCA1 is a tumour suppressor gene (TSG), which predisposes cancer to both breast and ovary. The primary objective of the present study is to ascertain the involvement of BRCA1 gene in the pathogenesis of sporadic breast cancer women in Chennai (South India) by analysing its protein expression by immunohistochemistry (IHC) and loss of heterozygosity (LOH) for confirmation of the involvement of TSG in the study population. We found down regulation of BRCA1 protein (54%) in IHC and it was correlated with the clinicopathological parameters of the patients. We found near significant correlation (P < 0.063) between BRCA1 protein expression and clinicopathological parameters. We found 30% LOH in our study and it was also correlated with the clinicopathological parameters. No correlation was found between LOH and clinicopathological parameters. Though we found no correlation, the results revealed in this study support the involvement of BRCA1 TSG in the pathogenesis of sporadic breast cancer women in Chennai (South India).     

Cancer-causing BRCA2 missense mutations disrupt an intracellular protein assembly mechanism to disable genome maintenance

Cancer-causing missense mutations in the 3418 amino acid BRCA2 breast and ovarian cancer suppressor protein frequently affect a short (∼340 residue) segment in its carboxyl-terminal domain (DBD). Here, we identify a shared molecular mechanism underlying their pathogenicity. Pathogenic BRCA2 missense mutations cluster in the DBD’s helical domain (HD) and OB1-fold motifs, which engage the partner protein DSS1. Pathogenic - but not benign – DBD mutations weaken or abolish DSS1-BRCA2 assembly, provoking mutant BRCA2 oligomers that are excluded from the cell nucleus, and disable DNA repair by homologous DNA recombination (HDR). DSS1 inhibits the intracellular oligomerization of wildtype, but not mutant, forms of BRCA2. Remarkably, DSS1 expression corrects defective HDR in cells bearing pathogenic BRCA2 missense mutants with weakened, but not absent, DSS1 binding. Our findings identify a DSS1-mediated intracellular protein assembly mechanism that is disrupted by cancer-causing BRCA2 missense mutations, and suggest an approach for its therapeutic correction.     

Role of MERIT40 in stabilization of BRCA1 complex: A protein–protein interaction study

MERIT40 is a novel associate of the BRCA1-complex, thus play an essential role in DNA damage repair mechanism. It is the least implicit protein and its structural and functional aspects of regulating the stability of BRCA1–MERIT40 complex remain equivocal. Analysis of protein–protein interactions between BRCA1 and its cellular binding partners like ABRAXAS, RAP80 and MERIT40 would help to understand the role of protein complex integrity in DNA repair mechanism. The recombinant proteins were purified and their structural aspects were elucidated by spectroscopic methods. Interaction analysis was carried out to determine binding partners of MERIT40. MERIT40 showed interaction with bridging molecule, called ABRAXAS, thus generate a scaffold among various members which further stabilizes the entire complex. It acts as an adapter molecule by interacting with BRCA1-BRCT in non-phosphorylation dependent manner. The feature enlighten on structural and interaction profile of BRCA1-complex member to elucidate their role in complex stability and DNA repair process.