Assembly and characterization of recombinant COP9 Signalosome

描述(申请人提供)：COP9信号小体是一种从酵母到人类保存的多功能蛋白质复合体。它通过调节一个称为基于cullin的泛素连接酶的细胞机器大家族，在不同的细胞功能中发挥着重要的作用。基于cullin的泛素连接酶机制促进多泛素链与许多调节和信号蛋白底物的连接，以靶向蛋白酶体介导的降解。通过控制这些底物的蛋白质水平，基于cullin的泛素连接酶可以特异性地调节细胞周期进程、细胞内信号、DNA复制和修复以及基因转录等各种细胞事件。基于cullin的泛素连接酶的去调控与许多人类疾病有关，如肿瘤发生、HIV感染和神经系统疾病。最近的研究表明，COP9信号小体可能通过调节泛素样分子NeddS对cullin支架蛋白的翻译后修饰来协调基于cullin的泛素连接酶机制的组装。然而，要从机制上完整地理解COP9信号体的功能，需要对复合体进行详细的结构分析和基于结构的功能表征。这项建议旨在开发一种新的方法来生产大量重组COP9信号体，这是结晶所需的复合体。我们的初步研究表明，当COP9信号体亚单位在细菌中过度表达时，要么是不溶的，要么是不稳定的。为了解决这些问题，我们计划在细菌和昆虫细胞中同时共表达COP9信号小体复合体的所有八个亚基。这种方法已经使我们能够生产出可溶且稳定的COP9亚复合体。此外，我们还将对重组COP9信号小体复合体进行生化分析，以测试其与cullin结合和cullin脱氢酶的活性。本研究的长期目标是确定完整的COP9信号小体复合体的晶体结构，以揭示其功能的结构基础，特别是它在各种细胞通路中对基于cullin的泛素连接酶机制的调节。

DESCRIPTION (provided by applicant): COP9 Signalosome is a multi-functional protein complex conserved from yeast to humans. It plays an essential role in diverse cellular functions by regulating a large family of cellular machines named cullin- based ubiquitin ligases. The cullin-based ubiquitin ligase machinery promotes the attachment of a polyubiquitin chain to many regulatory and signaling protein substrates to target them for proteasome- mediated degradation. By controlling the protein level of these substrates, cullin-based ubiquitin ligases can specifically regulate various cellular events in cell cycle progression, intracellular signaling, DNA replication and repair, and gene transcription. De-regulations of cullin-based ubiquitin ligases have been implicated in a number of human diseases such as tumorigenesis, HIV infection, and neurological disorders. Recent studies have suggested that COP9 Signalosome might coordinate the assembly of the cullin-based ubiquitin ligase machinery through modulating the post-translational modification of the cullin scaffold proteins by a ubiquitin-like molecule, NeddS. A complete mechanistic understanding of the COP9 Signalosome's functions, however, requires detailed structural analyses and structure-based functional characterization of the complex. This proposal aims to develop a novel method for producing large quantities of recombinant COP9 Signalosome, which is required for crystallization of the complex. Our preliminary studies have shown that a number of the COP9 Signalosome subunits, when over-expressed in bacteria, are either insoluble or unstable. To tackle these problems, we plan to simultaneously co-express all eight subunits of the COP9 Signalosome complex in both bacteria and insect cells. This approach has already allowed us to produce a soluble and stable COP9 sub-complex. In addition, we will carry out biochemical analyses of the recombinant COP9 Signalosome complex to test its cullin-binding and cullin deneddylation activities. The long-term goal of this research is to determine the crystal structure of the full COP9 Signalosome complex in order to reveal the structural basis of its functions, in particular, its regulation of the cullin-based ubiquitin ligase machinery in the context of various cellular pathways.