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

基本信息

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
项目类别：
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

项目摘要

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）：本研究将探讨泛素连接酶的底物识别机制以及泛素修饰在转录调控中的功能。酵母 Mat?2 转录阻遏蛋白 (?2) 的泛素化对于 Cdc48 依赖性从 DNA 中去除以及被蛋白酶体降解是必要的。泛素与α2的附着是由两条不同的途径介导的，其中之一涉及异二聚体E3泛素连接酶Slx57Slx8。我的假设是，Slx57Slx8 识别需要 α2 蛋白的一个或多个特定区域。因此，本提案旨在识别Slx57Slx8识别的α2中的退化信号（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 蛋白中产生单个氨基酸变化，从而损害其被 Slx57Slx8 的识别。 Slx57Slx8 对底物的识别引起了人们极大的兴趣，因为虽然 ?2 是 Slx57Slx8 仅有的两种已确认的内源底物之一，但越来越多的证据表明 Slx57Slx8 参与 SUMO 化蛋白质（或具有 SUMO 样结构域的蛋白质）的降解，以确保正确的 DNA 损伤修复和细胞周期进展。 Slx57Slx8 是 E3 泛素连接酶的进化上保守的 SUMO 靶向泛素连接酶 (STUbL) 家族的成员，该家族包括人类蛋白 RNF4。因此，该项目期间阐明的机制将提供对其他泛素连接酶和其他细胞过程的深入了解。该提案还将研究 β2 的突变体 (β2 -3A)，该突变体与 DNA 的亲和力比野生型 β2 降低，半衰期更长。该项目的目的是确定 DNA 结合对泛素介导的 β2 降解的影响。该项目将在遗传相互作用、蛋白质.DNA 相互作用和蛋白质.蛋白质相互作用的测定中比较野生型α2 和α2 -3A。我的主要假设是 12-3A 是蛋白酶体的不良底物，因为它无法与 AAA ATPase Cdc48 和/或 Cdc48 辅因子正确相互作用。该假设包括这样的假设：Cdc48（和辅助因子）对 ?2 的活性仅限于 DNA 相关的 12。虽然 Cdc48 及其辅助因子在染色质重塑中的确切功能尚不清楚，但这些因子很可能有能力以泛素依赖性方式改变几种 DNA 相关蛋白复合物的组成。由于已知许多转录调节因子和其他 DNA 结合蛋白被泛素化，因此该项目的影响可能超出 ?2 系统。公共健康相关性：人体细胞的正常生长和功能需要及时将一种称为泛素的小蛋白质与其他蛋白质结合和去除。已知控制泛素附着或去除的酶的缺陷会导致人类发育异常、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将泛素连接到其他蛋白质的酶的理解，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。