Structural dynamics and function of the COP9 signalosome

COP9信号体的结构动力学和功能

• 批准号: 10256020
• 负责人: Lan Huang
• 金额: $ 30.9万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-15 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10256020
• 关键词: Address; Adopted; Affinity Chromatography; Animals; Architecture; Binding; Biological; Biological Process; Biology; Cell physiology; Cells; Chemistry; Cleaved cell; Complement; Complex; Coupled; Crystallization; Cullin Proteins; Data; Development; Developmental Process; Evolution; Family; Family member; GPS1 gene; Goals; Health; Human; In Vitro; Lead; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Medicine; Methodology; Modification; Molecular; Molecular Conformation; Plants; Polyubiquitination; Protein Complex Subunit; Proteins; Reagent; Regulation; Reporting; Reproducibility; Research Personnel; Resolution; Role; Structure; System; Technology; Testing; Ubiquitin; Ubiquitin Like Proteins; Ubiquitination; Work; base; cancer therapy; crosslink; dynamic system; flexibility; human disease; improved; in vivo; inhibitor/antagonist; member; multicatalytic endopeptidase complex; novel; novel strategies; polypeptide; preservation; protein complex; protein degradation; protein protein interaction; receptor; tool; tumor; ubiquitin-protein ligase

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.

COP9 信号体 (CSN) 是一种进化上保守的、必需的多亚基蛋白质复合物，对于 控制动物和植物的多种细胞和发育过程。具有 8 个亚基 (CSN1-CSN8)， CSN 作为 Deneddylase 起作用，负责将泛素样蛋白 Nedd8 修饰从 neddylated 底物，包括 cullin 蛋白，Cullin–RING 泛素 E3 连接酶的关键成分 (CRL) 代表最大的多亚基 E3 超家族，拥有 240 多个成员。 协调泛素蛋白酶体系统 (UPS) 中约 20% 的蛋白质降解。 CSN在真核生物学中的作用体现在其控制CRL活性和CRL介导的蛋白质的功能 CSN 失调与许多人类疾病有关，包括癌症和 CSN。 抑制在癌症治疗方面显示出巨大的潜力。全面了解 CSN 如何发挥作用。 细胞对于揭示 CSN 生物学的分子细节并确定其在人类健康中的作用至关重要 最近的结构分析揭示了中等分辨率的 CSN 结构，并发现了这一点。 自由CSN以非活动状态存在。已经表明，CSN自动抑制只能被释放。 通过与 neddylated cullin 结合诱导 CSN 激活所需的实质性构象变化。 由于许多可互换底物受体 (SR) 的结构差异，人类 CRL 尽管进行了广泛的结构研究，但该家族内部的结构多样性仍然未知。 CSN如何与细胞中CRL的不同结构相互作用以释放其自动抑制并启用其功能 在监管 CRL 方面，我们一直在努力解决 CSN 可能使用动态拓扑来识别和适应的问题。 由于传统结构工具的限制，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 需要大型异质蛋白质复合物的拓扑来检验这一假设。 表明交联质谱 (XL-MS) 最适合验证我们的假设，因为它 因此，我们建议开发和使用新型 XL-MS。 有效剖析全方位结构动力学和相关构象变化的技术 为此，我们的具体目标是 1) 探索结构。 使用组合 XL-MS 方法的 CSN 动力学；2) 将 CSN 的构象变化映射到 拟议的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，同时也推动定量 XL-MS 研究 体外和体内研究动力学和异质蛋白质复合物的新水平。