Function and Regulation of a Novel Membrane-Associated Deubiquitinating Enzyme

项目摘要/摘要 泛素化是调节几乎每一个生物过程的重要机制，并且 泛素系统的缺陷可能会导致癌症和神经退化等疾病。由于 它们在逆转和调节泛素化、去泛素化酶方面的作用已成为关键 细胞调节器和有希望的药物靶点。因此，必须彻底了解 脱泛素酶的功能和调节。 本研究的目的是阐明新发现的明迪蛋白的功能和调控。 脱泛素酶家族，具有K48链专一性，生物学功能不明确。在我们的 初步研究发现，酵母中的Mindy池(称为Miy1)与血浆有关 薄膜。有趣的是，破坏MIY1基因会导致质膜明显增加 蛋白质泛素化和跨膜蛋白质的显著不稳定。我们假设 Mindy家族是膜蛋白泛素化的关键调节因子。测试我们的 为了实现我们的目标，我们提出了三个具体目标。 目的1：Miy1的生物学功能是什么？我们假设Miy1保持适当的 通过逆转错误形成的K48连接的质膜蛋白的单一泛素化 这些蛋白质上的泛素链。为了测试这一点，我们将研究Miy1如何影响Well的泛素化- 建立了质膜蛋白底物。我们还将识别其泛素化的蛋白质 在MIY1干扰突变体中，模式发生了变化。对于任何确定的候选人，我们将使用体外和 体内方法，以确定Miy1如何影响其水平、稳定性和亚细胞定位。 目标2：Miy1是如何调控的？在我们的初步研究中，我们发现Miy1经历了这两种情况 磷酸化和相穆化。我们的假设是Miy1受这些翻译后 修改。为了测试这一点，我们将确定修饰位置和负责的酶，生成 阻止这些修改的突变体，并检查阻止这些修改对 Miy1的活性和功能。或者，Miy1可以通过它的结合伙伴来调节。为了测试这一点， 我们将鉴定与Miy1相互作用的蛋白，并研究它们在调节Miy1功能中的作用。 目的3：Miy1的功能和调控是否保守？Miy1的人类同源物是 明迪-1。鉴于细胞调控在进化过程中高度保守的性质，很有可能 我们从酵母中的Miy1中学到的东西可以推广到人类的Mindy-1中。为了测试这一点，我们将 确定Mindy-1是否类似地调节质膜蛋白的泛素化；我们还将 研究Mindy-1的活性和功能是否受磷酸化和苏莫化的调节 以及它的相互作用蛋白，以类似于Miy1的方式。

Project Summary/Abstract Ubiquitination is an important mechanism that regulates nearly every biological process, and defects in the ubiquitin system can lead to diseases such as cancer and neurodegeneration. Due to their role in reversing and regulating ubiquitination, deubiquitinating enzymes have emerged as critical cell regulators and promising drug targets. Thus, it is imperative to gain a thorough understanding of the function and regulation of deubiquitinating enzymes. The goal of this study is to elucidate the function and regulation of MINDY, a newly discovered family of deubiquitinating enzymes that are K48-chain specific with unclear biological functions. In our preliminary studies, we find that a pool of MINDY in yeast (dubbed Miy1) is associated with plasma membrane. Interestingly, disrupting the MIY1 gene leads to a clear increase in plasma membrane protein ubiquitination and a marked destabilization of a transmembrane protein. We hypothesize that MINDY family of enzymes are key regulators for the ubiquitination of membrane proteins. To test our hypothesis and to achieve our objectives, three specific aims are proposed. Aim 1: What is the biological function of Miy1? We hypothesize that Miy1 maintains proper mono-ubiquitination of plasma membrane proteins by reversing the erroneously formed K48-linked ubiquitin chain on these proteins. To test this, we will examine how Miy1 affects ubiquitination of well- established plasma membrane protein substrates. We will also identify proteins whose ubiquitination pattern is altered in the MIY1-disruptive mutants. For any identified candidate, we will use in vitro and in vivo methods to determine how Miy1 affects their level, stability, and subcellular localization. Aim 2: How is Miy1 regulated? In our preliminary studies, we find that Miy1 undergoes both phosphorylation and sumoylation. Our hypothesis is that Miy1 is regulated by these post-translational modifications. To test this, we will identify the modification sites and responsible enzymes, generate mutants that block the modifications, and examine the effects of blocking these modifications on the activity and function of Miy1. Alternatively, Miy1 may be regulated via its binding partners. To test this, we will identify Miy1-interacting proteins and examine their roles in regulating the function of Miy1. Aim 3: Are the function and regulation of Miy1 conserved? The human homolog of Miy1 is MINDY-1. Given the highly conserved nature of cell regulation across evolution, it is highly likely that what we have learned from Miy1 in yeast can be extended to MINDY-1 in humans. To test this, we will determine if MINDY-1 similarly modulates ubiquitination of plasma membrane proteins; we will also investigate if the activity and function of MINDY-1 is regulated by phosphorylation and sumoylation as well as its interacting proteins, in a manner similar to Miy1.