权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Molecular Mechanisms for Regulation of Ubiquitin Metabolism

泛素代谢调节的分子机制

基本信息

批准号：
9252906
负责人：
Jason A MacGurn
金额：
$ 3.24万
依托单位：
VANDERBILT UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-03-01 至 2021-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9252906
关键词：
Amino Acids Amyotrophic Lateral Sclerosis Binding Biochemical Biochemistry Biogenesis Bypass Cell membrane Cells Cellular Stress Critical Pathways Data Degradation Pathway Disease Endosomes Enzymes Epitopes Genetic Goals Half-Life Homeostasis Human Huntington Disease In Vitro Maps Mediating Metabolism Modification Molecular Nerve Degeneration Neurodegenerative Disorders Pathway interactions Peptides Phosphoric Monoester Hydrolases Phosphorylation Polyubiquitin Positioning Attribute Proteins Recycling Regulation Resistance Role Route Signal Pathway Signal Transduction Sorting - Cell Movement Testing Ubiquitin Ubiquitination Vacuole Yeasts biophysical techniques human disease improved in vitro testing live cell imaging prevent protein degradation protein misfolding public health relevance reconstitution research study response trafficking

项目摘要

DESCRIPTION (provided by applicant): A key player in most cellular degradation pathways is ubiquitin - a 76 amino acid peptide that is covalently conjugated to proteins in order to target their degradation. Despite its central role in global protein stability very little is known about he regulation of ubiquitin homeostasis and its steady state concentration in the cell. Importantly, ubiquitin homeostasis is often disrupted in human diseases - particularly diseases associated with protein misfolding and neurodegeneration. Thus, there is a critical need to understand the basic biochemical mechanisms responsible for regulating ubiquitin metabolism and to define cellular mechanisms that maintain proper ubiquitin homeostasis. Recently, we identified a signaling mechanism in yeast which regulates ubiquitin metabolism in the cell by controlling the phosphorylation of ubiquitin itself. Our preliminary data indicates that phosphorylation of ubiquitin increases its rate of degradation and the rate of endocytic trafficking in the cell by preventing recognition by deubiquitylating enzymes (DUBs) along the endocytic route. We hypothesize that ubiquitin homeostasis is significantly impacted by the rate of ubiquitin flux through the endocytic pathway and by signaling pathways that determine whether ubiquitin is recycled or degraded in the vacuole. The experiments outlined here have strong potential to define mechanisms that regulate ubiquitin metabolism in the cell, which will likely improve our broader understanding of degradation pathways and global protein stability. Aim 1: Dissect the mechanism of Ppz-mediated regulation of ubiquitin phosphorylation. We hypothesize that Ppz phosphatases specifically recognize and de-phosphorylate Ser57 phospho-ubiquitin that is conjugated to proteins at the plasma membrane. Here, we will use in vitro biochemistry, genetics and live cell imaging experiments to dissect the mechanism of Ppz-mediated regulation of ubiquitin phosphorylation. In addition to uncovering new pathways that regulate ubiquitin phosphorylation, this aim also has strong potential to reveal biochemical mechanisms that regulate ubiquitin metabolism and homeostasis. Aim 2: Determine how Ser57 phosphorylation of ubiquitin regulates deubiquitylase activities. We hypothesize that Ser57 phosphorylation of ubiquitin confers resistance to deubiquitiylation by DUBs along the endocytic pathway, thereby facilitating bypass of DUB checkpoints and preventing the recycling of ubiquitin. Here, we will use a combination of biophysical methodologies, in vitro biochemistry, and live cell imaging to dissect the mechanism of DUB bypass by Ser57 phosphorylated ubiquitin. These experiments will drive new understanding of how phosphorylation of ubiquitin can alter its function in the context of ubiquitin recycling and endosomal sorting. These experiments will also elucidate in biochemical detail a regulatory decision that contributes to the fine tuning of ubiquitin levels in the cell.