Targeting ubiquitylated proteins to the proteasome

描述（由申请人提供）：我们的长期目标是了解蛋白质的命运是如何由泛素（Ub）系统调节的。Ub是一种含有76个氨基酸残基的蛋白质，在真核生物中高度保守。泛素化（Ub与其他细胞内蛋白质上的赖氨酸残基的共价结合）调节无数细胞过程，包括细胞周期进程、DNA修复、转录、应激反应和信号转导。Ub最为人所知的是作为靶蛋白的信号，用于被称为蛋白酶体的多亚基、ATP依赖性蛋白酶破坏。底物如何被递送到蛋白酶体是该领域中最具挑战性的问题之一。有人建议，适配器分子，它选择性地识别泛素化的底物，执行这一重要功能，在决定底物的最终目的地。我们将专注于S。cerevisiae Rad 23是一种参与将泛素化底物递送至蛋白酶体的候选衔接分子。Rad 23具有两个功能结构域：泛素样元件（UBL）和泛素相关基序（乌巴）。UBL基序显示直接结合蛋白酶体亚基Rpn 1。包括我们在内的几个研究小组发现，乌巴结构域优先结合泛素化底物。缺乏Rad 23的酵母细胞缺乏蛋白质水解。重要的是，Rad 23在体内和体外促进蛋白酶体-Ub缀合物复合物的形成。最近，我们发现Rad 23和Dsk 2与Ufd 2相互作用，Ufd 2是一种对Ub链组装很重要的E4酶。基于生物化学特性和遗传学证据，我们提出Rad 23样衔接蛋白识别多个泛素化底物，并通过各种结合伴侣将其传递到蛋白酶体。酵母Ufd 2-Rad 23复合物调节UFD底物、Hmg-CoA还原酶、转录因子Spt 23和朊病毒蛋白的降解。我们提出以下目标，努力破译的衔接分子在底物蛋白水解的生物学作用。目的1是了解Rad 23的底物选择性的机制。目的2是确定Rad 23-Ufd 2复合物的调节。目的3：明确Rad 23介导的蛋白水解在朊病毒生物合成中的作用。这些研究将揭示Ub系统的机制和功能的新见解，并为未来干预人类疾病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.