Mechanisms of protein degradation and transcriptional regulation by ubiquitin mod

泛素mod的蛋白质降解和转录调控机制

• 批准号: 8122699
• 负责人: CHRISTOPHER HICKEY
• 金额: $ 4.84万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8122699
• 关键词: ATP phosphohydrolase; Address; Affinity; Amino Acid Substitution; Amino Acids; Binding Proteins; Biological Assay; C-terminal; Cell Cycle; Cell Cycle Progression; Cell physiology; Cells; Complex; DNA; DNA Binding; DNA Repair; DNA-Binding Proteins; DNA-Protein Interaction; Defect; Development; Ensure; Enzymes; Excision; Family; Gene Fusion; Genes; Genetic; Goals; Growth; Half-Life; Human; Life; Malignant Neoplasms; Mediating; Modification; Mutagenesis; Mutation; N-terminal; Neurodegenerative Disorders; Pathway interactions; Polyubiquitin; Post-Translational Protein Processing; Proteins; Resistance; Role; Signal Transduction; System; Transcription Repressor/Corepressor; Transcriptional Regulation; Ubiquitin; Ubiquitination; Work; Yeasts; chromatin remodeling; cofactor; homeodomain; human disease; insight; interest; member; multicatalytic endopeptidase complex; mutant; nervous system disorder; novel; protein complex; protein degradation; therapy development; transcription factor; ubiquitin ligase; ubiquitin-protein ligase

DESCRIPTION (provided by applicant): This study will address mechanisms of substrate recognition by ubiquitin ligases and functions of ubiquitin modifications in transcriptional regulation. Ubiquitination of the yeast Mat?2 transcriptional repressor (?2) is necessary for its Cdc48-dependent removal from DNA as well as its degradation by the proteasome. The attachment of ubiquitin to ?2 is mediated by two distinct pathways, one of which involves the heterodimeric E3 ubiquitin ligase Slx57Slx8. My hypothesis is that a specific region or regions of the ?2 protein is/are required for its recognition by Slx57Slx8. Therefore, this proposal aims to identify a degradation signal (degron) in ?2 that is recognized by Slx57Slx8. The main experimental approach that will be employed for this project is random mutagenesis of the MAT??2 gene to generate single amino acid changes in the ?2 protein that impair its recognition by Slx57Slx8. The recognition of substrates by Slx57Slx8 is of great interest because while ?2 is one of only two confirmed endogenous substrates for Slx57Slx8, accumulating evidence implicates Slx57Slx8 in the degradation of SUMOylated proteins (or proteins with SUMO-like domains) to ensure proper DNA damage repair and cell cycle progression. Slx57Slx8 is a member of the evolutionarily conserved SUMO-targeted ubiquitin ligase (STUbL) family of E3 ubiquitin ligases that includes the human protein RNF4. Thus, the mechanisms elucidated during this project will offer insight into other ubiquitin ligases and other cellular processes. This proposal will also investigate a mutant of ?2 (?2 -3A) that has reduced affinity for DNA and a longer half-life than wild-type ?2. The aim of this project is to determine the effect of DNA binding on the ubiquitin-mediated degradation of ?2. This project will compare wild-type ?2 to ?2 -3A in assays of genetic interaction, protein.DNA interaction, and protein.protein interaction. My main hypothesis is that 12-3A is a poor substrate for the proteasome because it fails to properly interact with the AAA ATPase Cdc48 and/or Cdc48 cofactors. This hypothesis includes the postulate that the activity of Cdc48 (and cofactors) on ?2 is restricted to DNA-associated 12. While the exact functions of Cdc48 and its cofactors in chromatin remodeling are unclear, it is probable that these factors have the ability to alter the composition of several DNA-associated protein complexes in a ubiquitin-dependent manner. Since many transcriptional regulators and other DNA-binding proteins are known to be ubiquitinated, this project is likely to have implications beyond the ?2 system. PUBLIC HEALTH RELEVANCE: The proper growth and function of human cells requires the timely attachment and removal of a small protein called ubiquitin to and from other proteins. Defects in the enzymes that control ubiquitin attachment or removal are known to cause human developmental abnormalities, neurodegenerative disorders, and many different forms of cancer. This project aims to strengthen our understanding of the enzymes that attach ubiquitin to other proteins, with the long-term goal of developing therapies to treat cancers, neurological disorders, and other human diseases.

描述（由申请人提供）：本研究将解决通过泛素连接酶和转录调节中泛素修饰的功能的底物识别机制。酵母垫的泛素化？2转录阻遏物（？2）对于cdc48依赖性从DNA中的去除以及蛋白酶体降解是必需的。泛素对2的附着是由两种不同的途径介导的，其中一种涉及异二聚体E3泛素连接酶SLX57SLX8。我的假设是，SLX57SLX8识别其识别的特定区域或区域是/所需的区域。因此，该建议旨在确定SLX57SLX8认可的2中的降解信号（DEGRON）。该项目将采用的主要实验方法是MAT ?? 2基因的随机诱变，以在？2蛋白质中产生单个氨基酸变化，从而损害了SLX57SLX8的识别。 SLX57SLX8对底物的识别引起了人们的极大兴趣，因为虽然2是SLX57SLX8的两个确认的内源性底物之一，积累证据表明SLX57SLX8在Sumoylated蛋白质（或Sumo-like tim like tim ti-Sumo样域）降解中，以确保适当的DNA损害损伤和细胞损伤的损害。 SLX57SLX8是E3泛素连接酶的进化保守的Sumo靶向泛素连接酶（Stubl）的成员，其中包括人蛋白RNF4。因此，在本项目中阐明的机制将洞悉其他泛素连接酶和其他细胞过程。该提案还将调查一个突变体的突变体（？2 -3a），该突变体对DNA的亲和力降低，半衰期比野生型？2更长。该项目的目的是确定DNA结合对泛素介导的降解的影响？2。该项目将在遗传相互作用，蛋白质相互作用和蛋白质相互作用的测定中比较野生型？2至？2 -3a。我的主要假设是12-3a对于蛋白酶体而言是较差的底物，因为它无法与AAA AAA ATPase CDC48和/或CDC48辅助因子正确相互作用。该假设包括一个假设，即Cdc48（和辅因子）对2的活性仅限于DNA相关12。虽然CDC48的确切功能及其在染色质重塑中的辅助因子的确切功能尚不清楚，但可能会改变这些因素能够改变几种DNA与蛋白质复合物中的蛋白质复合物的成分。由于已知许多转录调节剂和其他DNA结合蛋白是泛素化的，因此该项目可能具有超出“ 2系统”的影响。 公共卫生相关性：人类细胞的适当生长和功能需要及时的附着和去除一种称为泛素的小蛋白质与其他蛋白质。已知控制泛素附着或去除的酶缺陷会引起人类发育异常，神经退行性疾病和许多不同形式的癌症。该项目旨在加强我们对附加泛素与其他蛋白质相关的酶的理解，其长期目标是开发治疗癌症，神经系统疾病和其他人类疾病的疗法。