Hai Rao - US grants

[unreadable] DESCRIPTION (provided by applicant): Our long-term goal is to understand how the fate of proteins is regulated by the ubiquitin (Ub) system. Ub, an abundant 76-residue protein, is highly conserved among eukaryotes. Ubiquitylation - the covalent conjugation of Ub to lysine residues on other intracellular proteins - regulates a myriad of cellular processes, including cell cycle progression, DNA repair, transcription, stress responses and signal transduction. Ub is best known as a signal to target proteins for destruction by a multisubunit, ATP dependent protease termed the proteasome. How the substrates are delivered to the proteasome is one of the most challenging issues in the field. It is proposed that adaptor molecules, which selectively recognize ubiquitylated substrates, perform this vital function in deciding the final destination of substrates. We will focus on S. cerevisiae Rad23, a candidate adaptor molecule involved in delivering ubiquitylated substrates to the proteasome. Rad23 has two functional domains: a ubiquitin-like element (UBL), and a ubiquitin-associated motif (UBA). The UBL motif was shown to directly bind the proteasome subunit Rpn1. Several groups including ours found that the UBA domain preferentially binds ubiquitylated substrates. And yeast cells lacking Rad23 are deficient in proteolysis. Importantly, Rad23 promotes the formation of the proteasome-Ub conjugates complex in vivo and in vitro. More recently, we found that Rad23 and Dsk2 interact with Ufd2, an E4 enzyme important for Ub-chain assembly. Based on biochemical properties and genetic evidence, we propose that Rad23-like adaptor proteins recognize multi-ubiquitylated substrates and deliver them to the proteasome through various binding partners. The yeast Ufd2-Rad23 complex regulates the degradation of UFD substrates, Hmg-CoA reductase, and the transcription factor Spt23, and prion protein. We propose the following aims in an effort to decipher the biological role of the adaptor molecules in substrate proteolysis. Aim 1 is to understand the mechanism underlying the substrate selectivity of Rad23. Aim 2 is to determine the regulation of the Rad23-Ufd2 complex. Aim 3 is to define the function of Rad23-mediated proteolysis in prion biogenesis. These studies should reveal novel insights into the mechanisms and functions of the Ub system, and provide defined molecular targets for future intervention in human diseases. [unreadable] [unreadable] [unreadable]

? DESCRIPTION (provided by applicant): Our long-term goal is to understand the mechanism and function of the ubiquitin- proteasome system in health and disease. This proposal focuses on the function of the XPC-Rad23 complex in DNA damage response and proteolysis. Mutations in XPC protein lead to a genetic disorder Xeroderma pigmentosum (XP) that exhibits extreme sunlight sensitivity and a strong propensity for skin cancer. XPC was known previously as a key DNA repair factor that cells employ to protect from sunlight-inflicted DNA injury. Specifically, XPC forms a complex with Rad23 and together scan the genome for DNA lesions, which can trigger nucleotide excision repair. However, not all of the phenotypes in XPC patients can be easily explained by DNA repair defects, suggesting that XPC may have other cellular roles. Others and we have previously found that its cofactor Rad23 participates in ubiquitin-mediated proteolysis. Rad23 specifically promotes the transfer of ubiquitylated substrates to the proteasome, which remains one of the most challenging issues in the ubiquitin field. Why do cells employ the same XPC-Rad23 complex in two distinct processes: DNA repair and proteolysis? We hypothesize that XPC and Rad23 functionally coordinate proteolysis and DNA damage response, and XPC mechanistically assists Rad23 in substrate selection. We have carved out unique niches and established solid footings to unravel the detailed mechanisms underlying the biological function of Rad23 and XPC in yeast and mammalian cells. We will tackle the elusive mechanism governing the dual role of XPC and Rad23 in proteolysis and DNA damage response via various state-of-art approaches including molecular biology, biochemistry, biophysics, genomics and proteomics. We propose the following aims in an effort to decipher the biological role of the XPC-Rad23 complex in substrate proteolysis. Aim 1 is to define the physiological role of Rad23 and XPC in mammalian cells. Aim 2 is to determine the biological function of Rad23 and Rad4 (yeast counterpart of XPC) in yeast. These studies should reveal novel insights into the mechanisms and functions of the ubiquitin system in DNA damage response. As ubiquitin plays pivotal regulatory functions in nearly every area of cell biology, the results will strongly impact other biological studies and provide defined molecular targets for future intervention in human diseases.