Analysis of HuI5, a proteasome-associated ubiquitin ligase

HuI5（一种蛋白酶体相关泛素连接酶）的分析

基本信息

批准号：
7926999
负责人：
BRADLEY PEARSE
金额：
$ 4.76万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-01 至 2012-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7926999
关键词：
26S proteasome Affect Animal Model Binding Binding Sites Biological Models Catalytic Domain Cell physiology Clinical Trials Complex Coupling Cyclins Data Dependence Deubiquitinating Enzyme Development Disease Docking Ensure Enzymes Exhibits Family Future In Vitro Lead Ligase Link Malignant Neoplasms Maps Mass Spectrum Analysis Measures Molecular Machines Mutation Neurodegenerative Disorders Pathway interactions Phenotype Play Polyubiquitin Proteasome Inhibitor Proteins Public Health Regulation Research Design Role Saccharomyces cerevisiae Screening procedure Signal Transduction TP53 gene Testing Ubiquitin Work Yeasts cancer prevention cancer therapy cell growth genetic regulatory protein in vivo member multicatalytic endopeptidase complex mutant novel particle protein degradation protein misfolding reconstitution targeted delivery ubiquitin ligase

项目摘要

DESCRIPTION (provided by applicant): Objective: To investigate the mechanism of Hul5 noncatalytic activity and its functional coupling to Ubp6 in protein degradation at the proteasome. Hypothesis: I hypothesize that there is a discrete noncatalytic function of Hul5 and that it inhibits the deubiquitinating activity of UbpG to ensure commitment to and accelerate substrate degradation. Specific Aims: (1) To characterize the functional regions of HulS. (2) To investigate the link between Ubp6 and the noncatalytic function of Hul5. (3) To examine the in vivo dependence of substrate degradation by the proteasome on the noncatalytic activity of Hul5. Research design: The model organism utilized in these studies will be S. cerevisiae. The initial studies will be focused on identifying the specific regions of Hul5 that are responsible for its many functions. Truncations and mutations in Hul5 will be generated and screened for phenotypes in yeast. Mutants will be isolated that are deficient in noncatalytic function, proteasome docking, and ubiquitin chain binding, respectively. Next, the mutants generated in Hul5 will be combined with mutants in Ubp6 to elucidate the interplay between Hul5 and Ubp6 through phenotypic screening. Following this, the functional coupling of Hul5 and Ubp6 will be reconstituted in vitro by altering the composition of the proteasome and measuring degradation of a characterized substrate. Finally, mass spectrometry will be utlized to identify endogenous substrates affected by Hul5 noncatalytic activity. Detailed analysis of the degradation rates of these identified substrates will reveal the importance and contribution of Hul5 to global protein degradation. In addition, combinations of Hul5 and Ubp6 mutants will be analyzed by mass spectrometry to investigate how the coupling of their antagonizing functions controls proteasome function and how imbalance in Hul5 and Ubp6 activity can affect substrate degradation. Relevance to public health: This proposal aims to investigate the fundamentals of the commitment of substrates to proteasomal degradation. The improper disposal of proteins that regulate cell growth and division is a well documented instigant of cancer. A large number of proteasome inhibitors are currently in clinical trials for cancer treatment. Therefore, a better understanding of the regulation of the proteasome will aid in the development of future approaches to cancer prevention and treatment.

描述（申请人提供）：目的：研究HUL5非催化活性的机理及其功能偶联与蛋白质降解中蛋白质降解的功能偶联。假设：我假设HUL5具有离散的非催化功能，并且它抑制了UBPG的去泛素化活性，以确保对底物降解的承诺和加速。具体目的：（1）表征HULS的功能区域。（2）研究UBP6与HUL5的非催化功能之间的联系。（3）检查蛋白酶体对HUL5非催化活性的底物降解的体内依赖性。研究设计：这些研究中使用的模型有机体将是酿酒酵母。最初的研究将集中在确定负责其许多功能的HUL5的特定区域。 HUL5中的截短和突变将产生并筛选酵母中的表型。突变体将分别孤立，分别缺乏催化功能，蛋白酶体对接和泛素链结合。接下来，在HUL5中产生的突变体将与UBP6中的突变体结合，以通过表型筛选阐明HUL5和UBP6之间的相互作用。此后，将通过改变蛋白酶体的组成并测量特征性底物的降解来在体外重新构成HUL5和UBP6的功能耦合。最后，将对质谱法进行UTLED，以鉴定受HUL5非催化活性影响的内源性底物。对这些已确定的底物的降解率的详细分析将揭示HUL5对全球蛋白质降解的重要性和贡献。此外，将通过质谱法分析HUL5和UBP6突变体的组合，以研究其拮抗功能的耦合如何控制蛋白酶体功能以及HUL5和UBP6活性中的失衡如何影响底物降解。与公共卫生的相关性：该提案旨在调查底物对蛋白酶体降解的承诺的基本面。调节细胞生长和分裂的蛋白质的处置不当是癌症的启动。目前，许多蛋白酶体抑制剂正在癌症治疗中进行临床试验。因此，更好地了解蛋白酶体的调节将有助于开发未来的预防癌症预防和治疗方法。