Functions and Substrates of COP9 Signalosome

描述（由申请人提供）：多亚基COP 9信号体（CSN）复合物在从裂殖酵母到人类的真核生物中发现，并涉及广泛的生物过程，包括信号转导、转录调控以及植物和动物发育。CSN促进切割的泛素样蛋白Nedd 8从cullin蛋白，我们建议，这种“deneddylase”的CSN活动的基础上，其多样的生物学功能。有趣的是，CSN的Csn 5/Jab 1亚基中的保守序列元件，我们称之为“Jab 1金属酶基序”（JAMM）是CSN的去核酶活性所必需的。本申请提出解决由该观察引起的三个主要问题。首先，我们将采用生物化学和生物物理学方法来评估JAMM作为一类新的和保守的锌金属蛋白酶（目的1）的活性位点的假设。第二，通过构建和评估JAMM中含有点突变的Csn 5/Jab 1分子的活性，我们将通过评估突变体Csn 5/Jab 1促进AP-1启动子元件的c-Jun依赖性转录的能力来测试去核酶活性是Csn 5/Jab 1最具特征的功能之一的假设（目的2）。第三，我们将寻求确定cullin是否是唯一的CSN deneddylase活性的底物，或是否未发现的底物可能有助于解释CSN的多种生物学功能。这一目标将通过纯化和肽测序Nedd 8修饰的蛋白质，在CSN缺陷突变体中积累。鉴于CSN亚基和类固醇激素受体，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，CSN的去核酶活性的生化特性，生理功能和生理目标的详细了解可能会对正常和患病的人类细胞的生物学产生重要影响。

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.