Molecular Mechanisms for Regulation of Ubiquitin Metabolism

 描述(由申请人提供)：大多数细胞降解途径中的一个关键角色是泛素--一种76个氨基酸的多肽，它与蛋白质共价连接，以靶向它们的降解。尽管泛素在全球蛋白质稳定中起着核心作用，但人们对泛素的稳态调节及其在细胞中的稳定浓度知之甚少。重要的是，泛素动态平衡在人类疾病中经常被破坏--特别是与蛋白质错误折叠和神经退化相关的疾病。因此，迫切需要了解调节泛素代谢的基本生化机制，并确定维持适当泛素稳态的细胞机制。最近，我们在酵母中发现了一种信号机制，它通过控制泛素本身的磷酸化来调节细胞中的泛素代谢。我们的初步数据表明，泛素的磷酸化通过阻止去泛素化酶(DUB)沿着内吞途径识别，从而增加了它的降解速度和细胞内的转运速度。我们假设，泛素的动态平衡受到泛素通过内吞途径的速率以及决定泛素在液泡中是循环还是降解的信号通路的显着影响。这里概述的实验具有很强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更广泛的理解。目的1：剖析Ppz介导的泛素磷酸化调控机制。我们假设Ppz磷酸酶特异性地识别和去磷酸化连接到质膜上的蛋白质的Ser57磷酸化泛素。在这里，我们将利用体外生物化学、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机制。除了发现调节泛素磷酸化的新途径外，这一目标还具有揭示调节泛素代谢和动态平衡的生化机制的强大潜力。目的2：确定泛素的Ser57磷酸化如何调节脱泛素酶的活性。我们假设，Ser57泛素的磷酸化使其对DUB沿着内吞途径的去泛素化产生抵抗，从而促进了DUB检查点的绕过，阻止了泛素的循环。在这里，我们将结合生物物理方法、体外生物化学和活细胞成像来剖析Ser57磷酸化泛素的DUB旁路机制。这些实验将推动人们对泛素的磷酸化如何在泛素循环和内体分选的背景下改变其功能的新理解。这些实验还将详细阐明一项有助于微调体内泛素水平的调控决定。 牢房。

 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.