The Role of Ubiquitin Phosphorylation in Cellular Aging

泛素磷酸化在细胞衰老中的作用

• 批准号: 9165414
• 负责人: Jason A MacGurn
• 金额: $ 22.13万
• 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: 2016-08-01 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9165414
• 关键词: Address; Adopted; Affect; Aging; Amino Acids; Amyotrophic Lateral Sclerosis; Biochemical; Cell Aging; Cell physiology; Cells; Critical Pathways; Data; Decision Making; Degradation Pathway; Development; Exhibits; Foundations; Functional disorder; Genetic; Genetic Screening; Goals; Homeostasis; Human; Huntington Disease; In Vitro; Lead; Longevity; Mediating; Membrane Protein Traffic; Metabolism; Mitotic; Nerve Degeneration; Neurodegenerative Disorders; Pathway interactions; Peptides; Phenotype; Phosphorylation; Phosphotransferases; Play; Positioning Attribute; Post-Translational Protein Processing; Proteins; Proteome; Proteomics; Reagent; Regulation; Research; Role; Serine; Signal Pathway; Signal Transduction; System; Testing; Therapeutic; Time; Ubiquitin; Ubiquitination; Work; Yeasts; cell age; genetic analysis; improved; multicatalytic endopeptidase complex; novel; protein degradation; protein misfolding; research study; screening; tool

Project Summary One of the cellular hallmarks of aging and neurodegeneration is a general decline in the protein degradation capacity of the cell, resulting in the accumulation of damaged and misfolded proteins which can threaten cellular integrity and viability. 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 we know very little about the regulation of ubiquitin itself. Thus, there is a critical need to understand the basic biochemical mechanisms responsible for regulating ubiquitin metabolism and to explore the relationship between ubiquitin homeostasis and cellular aging. Recently, we identified a novel signaling mechanism in yeast which regulates ubiquitin metabolism in the cell by controlling the phosphorylation of ubiquitin itself. Importantly, we have found that yeast cells expressing phosphomimetic ubiquitin exhibit a significantly extended post-mitotic lifespan. Given our preliminary results, we hypothesize that phosphorylation of ubiquitin not only alters its metabolism but generally accelerates global protein degradation and in doing so extends the post-mitotic life span of the cell. The experiments outlined in this proposal will explore this hypothesis in the following specific aims: Aim 1. Determine how ubiquitin phosphorylation extends yeast life span. We hypothesize that the life span extension associated with ubiquitin phosphorylation is due to a global increase in protein degradation. To test this, we will perform genetic analysis to define the degradation pathway that confers life span extension in the presence of phospho-ubiquitin. We will also leverage newly-developed reagents to define and quantify how ubiquitin phosphorylation affects the ubiquitin-modified proteome and how such changes may finely tune the activity of the ubiquitin-proteasome system during an aging time course. These experiments will elucidate the biochemical mechanism of life span extension associated with ubiquitin phosphorylation. Aim 2. Identify ubiquitin kinases and test their ability to modulate yeast aging. We hypothesize that the kinase activity responsible for Ser57 phosphorylation of ubiquitin will also play an important role in yeast chronological aging. To identify yeast ubiquitin kinase(s), we will adopt both genetic and biochemical screening approaches. Candidate kinases will be tested for membrane trafficking and cellular aging phenotypes. Identification of the yeast ubiquitin kinase(s) will significantly advance our understanding of ubiquitin metabolism and its relationship to the regulation of membrane traffic and post-mitotic aging. Together, the experiments outlined in this proposal have a strong potential to define the relationship between ubiquitin homeostasis, global protein stability, and cellular aging. Ultimately, this will contribute to our understanding of longevity in post-mitotic cells and may lead to the identification of new pathways that generally alter global protein stability.

项目摘要 衰老和神经变性的细胞标志之一是蛋白质的总体下降 细胞的降解能力，导致受损和错误折叠的蛋白的积累 威胁细胞完整性和生存能力。大多数细胞降解途径中的关键参与者是泛素 - A 76 共价为蛋白质以靶向其降解的氨基酸胡椒粉。尽管有它 在全球蛋白质稳定性中的核心作用我们对泛素本身的调节知之甚少。那有一个 了解负责控制泛素代谢的基本生化机制的批判性需求 并探索泛素稳态与细胞衰老之间的关系。最近，我们确定了 酵母中的新型信号传导机制，通过控制细胞中泛素代谢的调节 泛素本身的磷酸化。重要的是，我们发现表达磷酸化的酵母细胞 泛素暴露了有效的有丝分裂后寿命。鉴于我们的初步结果，我们假设 泛素的磷酸化不仅会改变其代谢，而且通常会加速全球蛋白质 降解并这样做会扩大细胞的有丝分裂后寿命。在此概述的实验 提案将在以下具体目的中探讨这一假设： 目标1。确定泛素磷酸化如何延长酵母寿命。我们假设生活 与泛素磷酸化相关的跨度延伸是由于蛋白质降解的全球增加所致。到 对此进行测试，我们将执行遗传分析以定义赋予生命跨度延长的降解途径 磷酸泛素的存在。我们还将利用新开发的试剂定义和量化 泛素磷酸化如何影响泛素修饰的蛋白质组以及这种变化如何细微调整 在老化的时间课程中，泛素 - 蛋白酶体系统的活动。这些实验将阐明 与泛素磷酸化相关的生命跨度延伸的生化机制。 目标2。识别泛素激酶并测试其调节酵母老化的能力。我们假设 负责Ser57泛素磷酸化的激酶活性也将在酵母中发挥重要作用 年代老化。为了鉴定酵母菌素激酶，我们将采用遗传和生化 筛选方法。候选激酶将测试膜运输和细胞衰老 表型。酵母泛素激酶（S）的识别将大大提高我们对 泛素代谢及其与膜交通和有丝分裂后衰老的调节的关系。 总之，本提案中概述的实验具有很强的潜力来定义关系 在泛素稳态，全球蛋白质稳定性和细胞衰老之间。最终，这将有助于我们 了解后有丝分裂细胞中的寿命，并可能导致对新途径的识别 通常会改变全球蛋白质稳定性。