Analysis of BRCA1 E3 Ubiquitin Ligase & Phosphopeptide Binding Activity

BRCA1 E3 泛素连接酶分析

基本信息

批准号：
7409835
负责人：
Latarsha Juanita Reid
金额：
$ 3.46万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-01-15 至 2012-01-14
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7409835
关键词：
Address Adult Alanine Alleles Amino Acids Animals Area Arginine BARD1 gene BRCA1 Associated RING Domain 1 Protein BRCA1 Mutation BRCA1 Protein BRCA1 gene BRCT Domain Binding Biochemical Biological Biological Process Breast Carcinoma C-terminal Cancer Patient Cause of Death Cell Cycle Checkpoint Cell Line Cells Chromosome abnormality Comparative Study Complex DNA Damage DNA Double Strand Break DNA Repair DNA repair protein Defect Development Disease Double Strand Break Repair Embryo Enzymes Family Gene Expression Gene Targeting Genes Genetic Genome Stability Germ-Line Mutation Healthcare Hereditary Breast Carcinoma Heterodimerization Histopathology Human In Vitro Individual Isoleucine Knock-in Mouse Knockout Mice Lead Left Localized Maintenance Malignant Neoplasms Mammary Neoplasms Mammary Tumorigenesis Mammary gland Measures Mediating Methods Mission Modeling Modification Molecular Molecular Profiling Mouse Strains Mus Mutant Strains Mice Mutation N-terminal National Cancer Institute Phenotype Phosphopeptides Play Point Mutation Positioning Attribute Process Property Proteins Public Health Publications Research Design Role Serine System Testing Tissues Transcriptional Regulation Tumor Suppression Ubiquitin United States Woman base established cell line functional loss helicase improved in vivo inorganic phosphate malignant breast neoplasm mutant mutant mouse model null mutation polymerization protein structure repaired response tumor tumorigenesis ubiquitin-protein ligase

项目摘要

DESCRIPTION (provided by applicant): Although germline mutations of the BRCA1 gene are a major cause of familial breast cancer, the molecular mechanisms by which BRCA1 suppresses tumorigenesis are still poorly understood. My dissertation project is to analyze the biological function of the ring domain and the BRCT domain of BRCA1. In vivo, BRCA1 exists as a heterodimer with the BARD1 protein and has potent E3 ligase activity within its ring domains. Ubiquitylation is a stepwise process involving E1, E2 and E3 enzymes that mediate the covalent attachment of ubiquitin to a target protein. The E3 ubiquitin ligase catalyzes the transfer of an ubiquitin molecule from the E2 enzyme to the substrate. BRCA1 also has two BRCT repeats that have phosphopeptide binding activity. Studies indicate that this domain is important for the role BRCA1 plays in DNA repair and transcriptional regulation. SPECIFIC AIMS: To determine whether the E3 ubiquitin ligase and phosphopeptide binding activity of BRCA1 is required for 1) normal animal development, 2) BRCA1- mediated mammary tumor suppression, or 3) Brcal function in DNA double strand break repair. Study Design & Methods: To address my first aim, I am using a knock-in gene targeting approach to generate mice with specific point mutations within the BRCA1 gene that eliminate either the E3 ubiquitin ligase activity or the phosphopeptide binding activity. The phenotype of these Brcal mutant mice will then be compared to those of wild type and other existing Brcal mutant mouse models. To address my second aim, a cre/lox gene targeting system will be used to generate mice strains that express, in a mammary-specific fashion, the BRCA1 mutant alleles. If the E3 ubiquitin ligase or phosphopeptide binding activity is required for mammary tumor suppression, then these mice should develop breast carcinomas with a latency and histopathology similar to mice that harbor mammary-specific Brcal- or Bard1-null mutations. To test the third hypothesis, I will establish cell lines that only express the functionally-inactive BRCA mutant alleles and will determine whether these activities are required for double strand DNA break repair and the various cell cycle checkpoints in which BRCA1 has been implicated. These finding are important in terms of public health because cancer is the second leading cause of death and specifically breast cancer is the second leading cause of death for women in the United States. Many cancer predisposing mutations have been identified within the ring domain and BRCT repeats of BRCA1 in human breast cancer cases. These findings could also lead to more specialized treatments and overall improved health care for cancer patients, which supports the mission of the National Cancer Institute.

描述（由申请人提供）：尽管BRCA1基因的生殖线突变是家族性乳腺癌的主要原因，但BRCA1抑制肿瘤发生的分子机制仍然很少了解。我的论文项目是分析环域和BRCA1的BRCT结构域的生物学功能。在体内，BRCA1作为与Bard1蛋白的异二聚体存在，并且在其环域内具有有效的E3连接酶活性。泛素化是涉及E1，E2和E3酶的逐步过程，可介导泛素与靶蛋白的共价附着。 E3泛素连接酶催化泛素分子从E2酶转移到底物。 BRCA1还具有两个具有磷酸肽结合活性的BRCT重复序列。研究表明，该结构域对于BRCA1在DNA修复和转录调控中的作用很重要。具体目的：确定BRCA1的E3泛素连接酶和磷酸肽结合活性是否需要1）正常动物发育，2）BRCA1- BRCA1-介导的乳腺肿瘤抑制，还是3）DNA双链中断的BRCAL功能。研究设计和方法：为了解决我的第一个目标，我使用的是一种基因靶向方法来生成具有BRCA1基因内特定点突变的小鼠，从而消除了E3泛素连接酶活性或磷酸肽结合活性。然后将将这些BRCAL突变小鼠的表型与野生型和其他现有Brcal突变小鼠模型的表型进行比较。为了解决我的第二个目标，将使用CRE/LOX基因靶向系统来产生小鼠菌株，该菌株以乳腺特异性的方式表达BRCA1突变等位基因。如果乳腺肿瘤抑制需要E3泛素连接酶或磷酸肽结合活性，那么这些小鼠应形成具有潜伏期和组织病理学的乳腺癌，类似于具有乳腺特异性brcal-或bard1-null突变的小鼠。为了检验第三个假设，我将建立仅表达功能性的BRCA突变等位基因的细胞系，并确定是否需要这些活性进行双链DNA断裂修复以及与BRCA1有关的各种细胞周期检查点。这些发现在公共卫生方面很重要，因为癌症是死亡的第二大原因，尤其是乳腺癌是美国妇女的第二大死亡原因。在人类乳腺癌病例中，在环形结构域和BRCT重复序列中已经确定了许多癌症诱发突变。这些发现还可能导致更专业的治疗方法，并为癌症患者提供总体改善的医疗保健，这支持了国家癌症研究所的任务。