Structural dynamics and function of the COP9 signalosome

COP 9信号体（CSN）是一种进化上保守的必需多亚基蛋白复合物，对于 控制着动植物的多种细胞和发育过程。具有8个亚基（CSN 1-CSN 8）， CSN作为一种去泛素化酶，负责切割泛素样蛋白Nedd 8修饰， neddylated底物，包括Cullin蛋白，Cullin-RING泛素E3连接酶的关键组分 （CRL）。CRL代表了最大的多亚基E3超家族，有超过240个成员， 在泛素-蛋白酶体系统（UPS）中协调约20%的蛋白质降解。的生物学意义 CSN在真核生物学中的作用主要表现在对CRL活性的调控和CRL介导的蛋白质表达 降解CSN失调与包括癌症在内的许多人类疾病有关， 抑制已经显示出癌症治疗巨大潜力。全面了解CSN如何在 细胞对于揭示CSN生物学基础的分子细节和确定其在人类健康中的作用至关重要 和医药最近的结构分析揭示了中等分辨率的CSN结构，并发现 空闲CSN处于非活动状态。研究表明，CSN自抑制只有在 通过与neddylated cullin结合，诱导CSN活化所需的大量构象变化。 由于许多可互换底物受体（SR）的结构差异，人CRL具有以下特征： 在家庭中有着极高的结构性障碍。尽管进行了广泛的结构研究， CSN如何与细胞中CRL的不同结构相互作用以释放其自抑制并使其发挥功能 在管理CRL方面。我们假设CSN可能使用动态拓扑来识别和适应 不同的CRL。由于传统结构工具的局限性，可以探测高度动态的新方法 结构，比较广泛的构象变化，以及表征体内结构 大的异质蛋白质复合物的拓扑结构，需要测试这一假设。最近的工作 表明交联质谱法（XL-MS）最适合验证我们的假设，因为它 拥有所有必要的能力。因此，我们建议开发和使用新的XL-MS 技术，以有效地剖析全方位的结构动力学和构象变化相关的 CSN在体外和细胞中的活化和功能。为此，我们的具体目标是：1）探索结构 使用组合XL-MS方法测定CSN的动力学; 2）绘制CSN在 在体外和体内与CRL结合。该项目不仅有助于解决重要但尚未解决的问题， 与CSN激活和功能相关的生物学问题，但也推动了定量XL-MS研究， 为研究体内外动力学和异质蛋白质复合物提供了新的水平。

The COP9 signalosome (CSN) is an evolutionally conserved, essential multi-subunit protein complex critical for controlling diverse cellular and developmental processes in animals and plants. With 8 subunits (CSN1-CSN8), CSN functions as a deneddylase responsible for cleaving ubiquitin-like protein Nedd8 modification from neddylated substrates including cullin proteins, the key components of Cullin–RING ubiquitin E3 ligases (CRLs). CRLs represent the largest superfamily of multi-subunit E3s with more than 240 members, which orchestrate ~20% of protein degradation in the ubiquitin-proteasome system (UPS). The biological significance of CSN in eukaryotic biology is manifested by its function in controlling CRL activity and CRL-mediated protein degradation. CSN dysregulation has been implicated in many human diseases including cancers, and CSN inhibition has shown great potential for cancer therapy. A comprehensive understanding of how CSN works in cells is essential to uncovering molecular details underlying CSN biology and defining its role in human health and medicine. Recent structural analysis has revealed CSN architecture at medium resolution, and discovered that free CSN exists in an inactive state. It has been shown that CSN auto-inhibition can only be released through binding to neddylated cullins to induce substantial conformational changes required for CSN activation. Due to structural differences in numerous interchangeable substrate receptors (SRs), human CRLs have extremely high structural diversities within the family. Despite extensive structural studies, it remains unknown how CSN interacts with diverse structures of CRLs in cells to release its auto-inhibition and enable its function in regulating CRLs. We hypothesize that CSN might use dynamic topology to recognize and accommodate different CRLs. Due to limitations in traditional structural tools, novel approaches that can probe highly dynamic structures, compare a wide spectrum of conformational changes, as well as characterize in vivo structural topologies of large heterogeneous protein complexes, are needed to test this hypothesis. Recent work suggests that cross-linking mass spectrometry (XL-MS) is best suited for validating our hypothesis as it possesses all of the required capabilities. Therefore, we propose to develop and employ novel XL-MS technologies to effectively dissect the full-range of structural dynamics and conformational changes associated with CSN activation and function in vitro and in cells. To this end, our specific aims are 1) probing the structural dynamics of CSN using a combinatory XL-MS approach; 2) mapping conformational changes of CSN upon binding to CRLs in vitro and in vivo. The proposed project will not only help address important yet unresolved biological questions associated with CSN activation and function, but also propel quantitative XL-MS studies to a new level for studying dynamics and heterogeneous protein complexes in vitro and in vivo.