Molecular Mechanisms for Regulation of Ubiquitin Metabolism

泛素代谢调节的分子机制

• 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
• 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

 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.

 描述（由适用提供）：大多数细胞降解途径中的关键参与者是泛素 - 一种76个氨基酸肽，与蛋白质共轭以靶向其降解。尽管在整体蛋白质稳定性中具有核心作用，但对他调节泛素稳态及其在细胞中的稳态浓度知之甚少。重要的是，泛素稳态通常会因人类疾病而破坏，尤其是与蛋白质错误折叠和神经变性有关的疾病。这是迫切需要了解负责确定泛素代谢的基本生化机制，并定义维持适当泛素稳态的细胞机制。最近，我们通过控制泛素本身的磷酸化来确定酵母中的信号传导机制，该机制通过控制细胞中的泛素代谢。我们的初步数据表明，泛素的磷酸化增加了其降解率和通过防止沿着内吞途径的去泛素化酶（DUB）识别的细胞中内吞交通率。我们假设泛素稳态受到泛素通量通过内吞途径的速率以及确定泛素是否在真空中回收还是降解的信号通路。此处概述的实验具有强大的潜力来定义调节细胞中泛素代谢的机制，这可能会提高我们对降解途径和全球蛋白质稳定性的广泛理解。目标1：剖析PPZ介导的泛素磷酸化调节的机理。我们假设PPZ磷酸酶特异性识别和去磷酸化的Ser57磷酸泛素蛋白，该磷酸 - 泛素蛋白在质膜上偶联。在这里，我们将使用体外生物化学，遗传学和活细胞成像实验来剖析PPZ介导的泛素磷酸化调节的机理。除了发现调节泛素磷酸化的新途径外，该目标还具有强大的潜力来揭示调节泛素代谢和稳态的生化机制。 AIM 2：确定Ser57泛素的磷酸化如何调节去偶联酶活性。我们假设泛素的Ser57磷酸化沿沿着内吞途径的配音赋予对去泛素的抗性，从而支持DUB检查点的旁路并防止泛素的回收利用。在这里，我们将使用生物物理方法，体外生物化学和活细胞成像的组合来剖析Ser57磷酸化的泛素的DUB绕道机制。这些实验将推动对泛素磷酸化如何在泛素回收和内体分类的背景下改变其功能的新理解。这些实验还将在生化细节中阐明一个调节决策，有助于对泛素水平进行微调 细胞。