MOLECULAR ANALYSIS OF FANCONIS ANEMIA C PROTEIN

FANCONIS 贫血 C 蛋白的分子分析

• 批准号: 2445267
• 负责人: HAGOP YOUSSOUFIAN
• 金额: $ 18.22万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-07-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2445267
• 关键词: DNA damage; DNA repair; binding proteins; biological signal transduction; cell cycle; chemical association; chimeric proteins; chromosomes; congenital aplastic anemia; crosslink; gel electrophoresis; gene complementation; gene expression; gene targeting; intermolecular interaction; molecular pathology; phosphorylation; protein structure function; proteins; southern blotting; stress proteins; tissue /cell culture

DESCRIPTION (Adapted from investigator's abstract): Fanconi anemia is an inherited disorder characterized by chromosomal instability, cancer susceptibility, and a presumed defect in DNA repair. The long-term objectives of this proposal are to understand the biochemical function and cellular interactions of FACC, a protein that is deficient in Fanconi anemia complement group C patients. The cloning of the FACC gene by its ability to rescue lymphoblastoid cell lines derived from Fanconi anemia complement group C patients from the toxicity of bi- functional DNA cross-linking reagents (i.e., mitomycin-C and diepoxybutane [DEB]) has clearly established the role of this protein in the pathobiology of Fanconi anemia. However, fundamental questions concerning its mechanism of action, regulation, and biological function remain. The Principal Investigator has generated a polyclonal antibody directed against FACC and has been able to localize this polypeptide to the cytoplasm and perinuclear structures in a large number of mammalian cells, both under steady state conditions, with induction of cell cycle, and after exposure to DNA-damaging agents. He has also demonstrated that a chimeric FACC molecule that contains the constant region of human IgG1 heavy chain at its carboxy-terminus (FACC gamma 1) is able to correct the hypersensitivity of Fanconi anemia complement group C lymphoblastoid cells to mitomycin-C induced cytotoxicity and yet remains exclusively in the cytoplasm. Interestingly, when the nuclear localization signal of SV40 large T-antigen is subcloned upstream of FACC and this construct transfected into patient-derived lymphoblastoid cells, FACC localized to the nucleus, but was unable to correct mitomycin-C induced cytotoxicity. Taken together, these data (recently published in PNAS) clearly demonstrate the cytoplasmic localization of FACC, and thus suggest that its role in DNA repair must be indirect. The PI has also developed an in vitro assay for protein-protein interactions and demonstrated that FACC binds specifically to at least three ubiquitous cytoplasmic proteins of molecular weights 65 kD, 50 kD, and 35 kD (called the FACC-binding proteins, or FABPs). This has led him to propose that FACC forms a multimeric complex which could play a role in genomic stability, perhaps as a "gatekeeper", regulating the access of genotoxic agents to the nucleus, or recruiting DNA repair proteins. He further hypothesizes that other complementation group abnormalities in Fanconi anemia could result from structural defects in other components of this multimeric complex. Specific Aim 1 of this proposal is to study the subcellular targeting of FACC to cytoplasmic and perinuclear structures and its metabolic fate during stress states and passage through the cell cycle. In Specific Aim 2, the Principal Investigator will elucidate the functional domains of FACC and its association with internal membranes, and will attempt to identify potential targeting signals and phosphorylation sites that may regulate its localization and function. In Specific Aim 3, using a sensitive Southern blotting assay, the relationship of FACC to DNA cross-linking will be analyzed in an effort to identify specific molecular targets. Finally, in Specific Aim 4, structure of the FACC macromolecular complex will be elucidated by isolation of cDNA clones for the FABPs, development of assays to detect in vivo interactions of FACC and FABPs, and the analysis of the structure and expression of FABPs in both Fanconi anemia and non-Fanconi anemia cells. It is anticipated that these studies will provide fundamental insights into the regulation of genomic stability by cytoplasmic activities, and should also provide insight into the molecular pathobiology of Fanconi anemia.

描述（根据调查员的摘要改编）：Fanconi贫血是 以染色体不稳定性，癌症为特征的遗传疾病 敏感性和DNA修复中假定缺陷。长期 该建议的目标是了解生化功能 FACC的细胞相互作用，一种缺乏的蛋白质 Fanconi贫血补体C患者。 FACC的克隆 基因通过其营救淋巴细胞细胞系的能力 Fanconi贫血补体C组患者来自Bi-的毒性 功能性DNA交联试剂（即丝裂霉素C和 DiePoxybutane [DEB]）清楚地确定了该蛋白的作用 在法科尼贫血的病理学中。但是，基本问题 关于其作用，调节和生物学功能机理 保持。主要研究者已经产生了多克隆抗体 针对FACC，已经能够将此多肽定位到 大量哺乳动物中的细胞质和核周结构 细胞，均在稳态条件下，诱导细胞周期， 并在暴露于DNA伤害剂后。他也证明了 包含人类恒定区域的嵌合FACC分子 IgG1重链在其羧基末端（FACC伽马1）能够 纠正法科尼贫血补体组C的超敏反应C 淋巴母细胞对丝裂霉素-C诱导的细胞毒性，但仍然存在 仅在细胞质中。有趣的是，当核 SV40大型T-抗原的定位信号在上游 FACC和该构造转染到患者衍生的淋巴母细胞中 细胞，FACC位于原子核，但无法纠正 丝霉素C诱导的细胞毒性。综上所述，这些数据（最近 在PNA中发表）清楚地证明了 FACC，因此表明其在DNA修复中的作用必须间接。 PI还开发了用于蛋白质蛋白质的体外测定法 相互作用并证明FACC至少与 分子量的三种无处不在的细胞质蛋白65 kd，50 kd， 和35 kD（称为FACC结合蛋白或Fabps）。这已经引起了 他建议FACC形成一个多聚体综合体，可以玩 在基因组稳定性中的作用，也许是“看门人”，调节 将遗传毒性剂访问核或募集DNA修复 蛋白质。他进一步假设其他互补小组 范科尼贫血异常可能是由于结构缺陷导致的 该多聚体复合物的其他组成部分。 该提案的具体目的1是研究 FACC至细胞质和核周结构及其代谢命运 在应力状态和通过细胞周期中通过。具体 AIM 2，主要研究人员将阐明功能域 FACC及其与内部膜的关联，并将尝试 确定潜在的靶向信号和磷酸化位点 可以调节其本地化和功能。在特定的目标3中，使用 敏感的南部印迹测定法，FACC与DNA的关系 交联将分析以确定特定 分子靶标。最后，在特定的目标4中，FACC的结构 大分子复合物将通过分离cDNA克隆来阐明 对于Fabps，开发测定以检测体内相互作用 FACC和FABP，以及对结构和表达的分析 Fanconi贫血和非壮大性贫血细胞中的Fabps。这是 预计这些研究将为您提供基本见解 通过细胞质活性调节基因组稳定性， 还应洞悉范科尼的分子病理学 贫血。