The Role of Ubiquitin Phosphorylation in Cellular Aging

项目摘要 衰老和神经退化的细胞标志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的积累， 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的关键参与者是泛素- a 76 与蛋白质共价结合以靶向其降解的氨基酸肽。尽管 在全球蛋白质稳定性的核心作用，我们知道很少关于泛素本身的调节。因此，有一个 迫切需要了解负责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 酵母中的一种新的信号传导机制，通过控制细胞内的泛素代谢来调节细胞内的泛素代谢。 泛素自身的磷酸化。重要的是，我们发现表达磷酸化模拟物的酵母细胞 泛素显示出显著延长的有丝分裂后寿命。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， 降解，并且这样做延长了细胞的有丝分裂后寿命。本文中概述的实验 本提案将探讨这一假设，具体目标如下： 目标1.确定泛素磷酸化如何延长酵母寿命。我们假设 与泛素磷酸化相关跨度延伸是由于蛋白质降解的整体增加。到 测试这一点，我们将进行遗传分析，以确定降解途径，赋予寿命延长， 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，以及这些变化如何微调 泛素-蛋白酶体系统在衰老过程中的活性。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。 目标2.确定泛素激酶和测试其调节酵母老化的能力。我们假设 负责泛素Ser 57磷酸化的激酶活性也将在酵母中起重要作用 时间老化为了鉴定酵母泛素激酶，我们将采用遗传和生物化学方法， 筛选方法。将检测候选激酶的膜运输和细胞老化 表型。酵母泛素激酶的鉴定将大大促进我们对 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，本提案中概述的实验具有很强的潜力来定义这种关系 泛素稳态、整体蛋白质稳定性和细胞衰老之间的联系。最终，这将有助于我们 了解有丝分裂后细胞的寿命，并可能导致识别新的途径， 通常会改变整体蛋白质的稳定性。

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.