Function and Regulation of a Novel Membrane-Associated Deubiquitinating Enzyme

项目总结/摘要 泛在化是调节几乎所有生物过程的重要机制，并且 泛蛋白系统的缺陷可导致诸如癌症和神经变性的疾病。由于 它们在逆转和调节泛素化、去泛素化酶中的作用已经成为关键 细胞调节剂和有前途的药物靶点。因此，必须彻底了解 去泛素化酶的功能和调节。 本研究的目的是阐明MINDY的功能和调节， K48链特异性去泛素化酶家族，生物学功能尚不清楚。在我们 初步研究，我们发现酵母中的一个MINDY池（称为Miy 1）与血浆有关， 膜的有趣的是，破坏MIY 1基因会导致质膜蛋白质含量明显增加， 蛋白质泛素化和跨膜蛋白的显著不稳定。我们假设 MINDY家族酶是膜蛋白泛素化的关键调节因子。来测试我们 假设和实现我们的目标，提出了三个具体目标。 目的1：Miy 1的生物学功能是什么？我们假设Miy 1维持着正常的 通过逆转错误形成的K48连接的细胞膜蛋白的单泛素化， 这些蛋白质上的泛素链。为了验证这一点，我们将研究Miy 1如何影响泛素化的良好- 建立了质膜蛋白底物。我们还将鉴定那些泛素化的蛋白质 在MIY 1-破坏性突变体中模式改变。对于任何确定的候选人，我们将使用体外和 体内方法来确定Miy 1如何影响它们的水平、稳定性和亚细胞定位。 目标2：Miy 1是如何调节的？在我们的初步研究中，我们发现Miy 1经历了 磷酸化和类小泛素化。我们的假设是Miy 1受这些翻译后蛋白的调控， 修改.为了验证这一点，我们将确定修饰位点和负责的酶， 突变体，阻断修饰，并检查阻断这些修饰对细胞的影响。 Miy 1的活性和功能。或者，Miy 1可以通过其结合伴侣进行调节。为了验证这个， 我们将鉴定与Miy 1相互作用的蛋白质，并研究它们在调节Miy 1功能中的作用。 目的3：Miy 1的功能和调控是否保守？Miy 1的人类同源物是 MINDY-1.鉴于细胞调节在进化过程中的高度保守性， 我们从酵母中的Miy 1所学到的东西可以扩展到人类的MINDY-1。为了验证这一点，我们 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SUMO化的调节， 以及其相互作用蛋白质，以类似于Miy 1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.