Function and Regulation of a Novel Membrane-Associated Deubiquitinating Enzyme

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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.