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如何由泛素（Ub）系统调节。Ub是一种丰富的76-残基蛋白，在真核生物中高度保守。泛素化- Ub与其他细胞内蛋白赖氨酸残基的共价结合-调节无数的细胞过程，包括细胞周期进程，DNA修复，转录，应激反应和信号转导。Ub是一种被称为蛋白酶体的多亚基ATP依赖蛋白酶破坏的靶蛋白信号。底物如何传递到蛋白酶体是该领域最具挑战性的问题之一。有人提出，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重要作用。我们将重点关注酿酒酵母Rad23，这是一个候选的接头分子，参与将泛素化的底物传递到蛋白酶体。Rad23有两个功能域：泛素样元件（UBL）和泛素相关基序（UBA）。UBL基序被证明直接结合蛋白酶体亚基Rpn1。包括我们在内的几个研究小组发现，UBA结构域优先结合泛素化底物。缺乏Rad23的酵母细胞缺乏蛋白水解能力。重要的是，Rad23在体内和体外都促进了蛋白酶体- ub缀合物的形成。最近，我们发现Rad23和Dsk2与Ufd2相互作用，Ufd2是一种对ub链组装很重要的E4酶。基于生物化学特性和遗传学证据，我们提出rad23样接头蛋白识别多泛素化底物，并通过各种结合伙伴将其传递给蛋白酶体。酵母Ufd2-Rad23复合物调节UFD底物、Hmg-CoA还原酶、转录因子Spt23和朊病毒蛋白的降解。我们提出以下目标，以努力破译接头分子在底物蛋白水解中的生物学作用。目的1是了解Rad23对底物选择性的机制。目的2是确定Rad23-Ufd2复合物的调控。目的3是明确rad23介导的蛋白水解在朊病毒生物发生中的作用。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.