Targeting ubiquitylated proteins to the proteasome

描述(由申请人提供)：我们的长期目标是了解泛素(Ub)系统如何调节蛋白质的命运。Ub是一种富含76个残基的蛋白质，在真核生物中高度保守。泛素化-Ub与其他细胞内蛋白质上赖氨酸残基的共价偶联-调节着无数的细胞过程，包括细胞周期进程、DNA修复、转录、应激反应和信号转导。最广为人知的是，UB是一种被称为蛋白酶体的多亚单位、ATP依赖的蛋白酶体破坏的靶蛋白的信号。如何将底物输送到蛋白酶体是该领域最具挑战性的问题之一。有人认为，选择性识别泛素化底物的接头分子，在决定底物的最终目的地方面发挥着至关重要的作用。我们将重点研究酿酒酵母RAD23，这是一个候选的接头分子，参与将泛素化底物输送到蛋白酶体。RAD23有两个功能结构域：泛素样元件(UBL)和泛素相关基序(UBA)。Ubl基序与蛋白酶体亚基Rpn1直接结合。包括我们在内的几个研究小组发现，UBA结构域优先与泛素化底物结合。缺乏RAD23的酵母细胞蛋白降解能力不足。重要的是，RAD23在体内和体外都能促进蛋白酶体-Ub结合物的形成。最近，我们发现RAD23和Dsk2与Ufd2相互作用，Ufd2是一种对Ub链组装至关重要的E4酶。基于生化特性和遗传证据，我们认为RAD23样适配子蛋白识别多泛素化底物，并通过不同的结合伙伴将它们运送到蛋白酶体。酵母Ufd2-RAD23复合体调节UFD底物、HMG-CoA还原酶、转录因子Spt23和Pron蛋白的降解。我们提出了以下目标，以努力破译接头分子在底物蛋白分解中的生物学作用。目的1是了解RAD23底物选择性的机制。目的2是确定RAD23-Ufd2复合体的调节。目的3确定RAD23介导的蛋白分解在蛋白生物发生中的作用。这些研究将揭示Ub系统的机制和功能的新见解，并为未来对人类疾病的干预提供明确的分子靶点。

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.