Functions and Substrates of COP9 Signalosome

描述(申请人提供)：多亚单位COP9信号小体(CSN)复合体广泛存在于从分裂酵母到人类的真核生物中，并参与了广泛的生物学过程，包括信号转导、转录调控和动植物发育。CSN促进泛素样蛋白Nedd8从cullin蛋白中裂解，我们认为CSN的这种脱氢酶活性是其不同生物学功能的基础。有趣的是，在CSN的Csn5/Jab1亚基中有一个保守的序列元件，我们称之为‘Jab1金属酶基序’(JAMM)，它是CSN脱氨酸酶活性所必需的。这项申请提出了解决这一观察结果产生的三个主要问题。首先，我们将使用生化和生物物理方法来评估JAMM是一类新的和保守的锌金属蛋白酶的活性部位的假说(目标1)。其次，通过构建和评估在JAMM中含有点突变的Csn5/Jab1分子的活性，我们将通过评估突变的Csn5/Jab1促进AP-1启动子元件c-jun依赖的转录的能力(目标2)来验证脱氧核酸酶活性是Csn5/Jab1最具特征性的功能之一的假设。第三，我们将寻求确定库林斯是否是CSN脱氢酶活性的唯一底物，或者未被发现的底物是否有助于解释CSN的多种生物学功能。这一目标将通过对积累在CSN缺陷突变体中的Nedd8修饰蛋白的纯化和肽序列来实现。鉴于CSN亚基与类固醇激素受体、细胞周期控制蛋白、转录调控蛋白和癌症相关蛋白之间的联系，详细了解CSN脱氨酸酶活性的生化特性、生理功能和生理靶点可能对正常和疾病人类细胞的生物学具有重要的意义。

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.