The Role of Ubiquitin Phosphorylation in Cellular Aging

项目摘要 衰老和神经变性的细胞特征之一是蛋白质的普遍下降。 细胞的降解能力，导致受损和错误折叠的蛋白质积累，从而可能 威胁到细胞的完整性和生存能力。在大多数细胞降解途径中起关键作用的是泛素-a76 与蛋白质共价连接的氨基酸多肽，目的是针对蛋白质的降解。尽管它的 在全球蛋白质稳定中的核心作用我们对泛素本身的调节知之甚少。因此，有一个 迫切需要了解调节泛素代谢的基本生化机制 探讨泛素稳态与细胞衰老的关系。最近，我们发现了一个 酵母中一种新的信号机制，它通过控制细胞内的泛素代谢来调节泛素的代谢 泛素本身的磷酸化。重要的是，我们发现酵母细胞表达仿磷蛋白 泛素具有显著延长有丝分裂后寿命的作用。根据我们的初步结果，我们假设 泛素的磷酸化不仅改变了它的新陈代谢，而且通常加速了全球蛋白质 这样做可以延长细胞有丝分裂后的寿命。这篇文章中概述的实验 Proposal将从以下具体目标探讨这一假设： 目的1.确定泛素磷酸化如何延长酵母寿命。我们假设生命 与泛素磷酸化相关的跨度延长是由于全球蛋白质降解的增加。至 对此进行测试，我们将进行遗传分析，以确定延长寿命的退化途径 磷酸泛素的存在。我们还将利用新开发的试剂来定义和量化 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微调 泛素-蛋白酶体系统在衰老过程中的活动。这些实验将阐明 与泛素磷酸化相关的寿命延长的生化机制。 目的2.鉴定泛素蛋白激酶并检测其调节酵母衰老的能力。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 按时间顺序老化。为了鉴定酵母泛素蛋白激酶(S)，我们将采用遗传学和生化相结合的方法 筛选方法。候选激酶将被测试膜运输和细胞老化 表型。酵母泛素蛋白激酶(S)的鉴定将极大地促进我们对 泛素代谢及其与膜运输调节和有丝分裂后衰老的关系。 总之，这项提案中概述的实验具有很强的潜力来定义这种关系 泛素动态平衡、全球蛋白质稳定性和细胞衰老之间的关系。最终，这将有助于我们的 了解有丝分裂后细胞的寿命，并可能导致识别新的途径 通常会改变全球蛋白质的稳定性。

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.