Functions and Substrates of COP9 Signalosome

描述（由申请人提供）：多亚基COP9信号体（CSN）复合体存在于从裂变酵母到人类的真核生物中，并且涉及广泛的生物过程，包括信号转导，转录调节以及动植物发育。CSN促进cullin蛋白中泛素样蛋白Nedd8的裂解，我们认为CSN的这种“去eddylase”活性是其多种生物学功能的基础。有趣的是，CSN的Csn5/Jab1亚基中的一个保守序列元件，我们称之为“Jab1金属酶基元”（JAMM），是CSN去eddylase活性所必需的。这个应用程序建议解决从这个观察中产生的三个主要问题。首先，我们将采用生物化学和生物物理方法来评估JAMM作为一种新的、保守的锌金属蛋白酶活性位点的假设（目的1）。其次，通过构建和评估在JAMM中携带点突变的Csn5/Jab1分子的活性，我们将通过评估突变体Csn5/Jab1促进AP-1启动子元件的c- jun依赖性转录的能力，来验证去eddylase活性是Csn5/Jab1最具特征的功能之一的假设（Aim 2）。第三，我们将试图确定cullins是否是CSN依赖性酶活性的唯一底物，或者未发现的底物是否有助于解释CSN的多种生物学功能。这一目标将通过纯化和对csn缺陷突变体中积累的nedd8修饰蛋白的肽测序来实现。鉴于CSN亚基与类固醇激素受体、细胞周期控制蛋白、转录调节蛋白和癌症相关蛋白之间的联系，详细了解CSN去eddylase活性的生化特性、生理功能和生理靶点可能对人类正常和患病细胞的生物学有重要意义。

DESCRIPTION (provided by applicant): The multisubunit COP9 signalosome (CSN) complex is found across eukaryotes ranging from fission yeast to humans, and has been implicated in a broad range of biological processes including signal transduction, transcriptional regulation, and plant and animal development. CSN promotes cleavage of the ubiquitin-like protein Nedd8 from cullin proteins, and we propose that this 'deneddylase' activity of CSN underlies its diverse biological functions. Interestingly, a conserved sequence element in the Csn5/Jab1 subunit of CSN that we refer to as the 'Jab1 metalloenzyme motif' (JAMM) is required for the deneddylase activity of CSN. This application proposes to address three major questions that arise from this observation. First, we will employ biochemical and biophysical methods to evaluate the hypothesis that JAMM serves as the active site for a new and conserved class of zinc metalloproteases (Aim 1). Second, by constructing and evaluating the activity of Csn5/Jab1 molecules that harbor point mutations in JAMM, we will test the hypothesis that deneddylase activity underlies one of the best-characterized functions of Csn5/Jab1 by evaluating the ability of mutant Csn5/Jab1 to promote c-Jun-dependent transcription from AP-1 promoter elements (Aim 2). Third, we will seek to determine whether cullins are the exclusive substrates of CSN deneddylase activity, or whether undiscovered substrates may help account for the diverse biological functions of CSN. This objective will be pursued by purification and peptide sequencing of Nedd8-modified proteins that accumulate in CSN-deficient mutants. Given the connections that have been forged between CSN subunits and steroid hormone receptors, cell cycle control proteins, transcriptional regulatory proteins, and cancer-related proteins, a detailed understanding of the the biochemical properties, physiological functions, and physiological targets of CSN's deneddylase activity is likely to shed important light on the biology of normal and diseased human cells.