Solution structure and dynamics of polyubiquitin chains

多聚泛素链的溶液结构和动力学

• 批准号: 8588939
• 负责人: DAVID FUSHMAN
• 金额: $ 29.52万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-03-01 至 2015-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8588939
• 关键词: 26S proteasome; Address; Amaze; Binding; Binding Proteins; Biochemical; Biological Process; Cell Cycle; Cell physiology; Cells; Chemicals; Commit; Complement; Complex; Computer Simulation; Computing Methodologies; Coupling; Data; Defect; Development; Disease; Drug Design; Enzymes; Event; Head; Immune; Immune response; Immunity; Inflammation; Inflammatory; Label; Length; Life; Ligand Binding; Link; Lysine; Malignant - descriptor; Malignant Neoplasms; Measurement; Mediating; Methods; Modeling; Molecular; Molecular Conformation; Myopathy; Neurodegenerative Disorders; Neutrons; Outcome; Pathway interactions; Polyubiquitin; Polyubiquitination; Property; Proteins; Regulation; Relaxation; Research; Residual state; Roentgen Rays; Signal Pathway; Signal Transduction; Signaling Molecule; Solutions; Specificity; Structure; Structure-Activity Relationship; System; Tail; Testing; Transcriptional Activation; Ubiquitin; Work; antigen processing; base; design; developmental disease; flexibility; inhibitor/antagonist; multicatalytic endopeptidase complex; next generation; novel; novel strategies; protein degradation; protein transport; receptor; receptor binding; response; small molecule; three dimensional structure; wasting

DESCRIPTION (provided by applicant): This proposal is to characterize the solution structure, dynamics, and interactions of polyubiquitin (polyUb) chains, which function as signaling molecules in the regulation of a host of cellular processes, ranging from progression through the cell cycle, to transcriptional activation, antigen processing and vesicular trafficking of proteins. Conjugation of substrates to polyUb chains of different linkages commits the target protein to distinct fates in the cell. In particular, Lys48-linked polyUb acts as a universal signal in the ubiquitin-proteasome proteolytic pathway, the principal regulatory mechanism for the turnover of short-lived proteins that influences a variety of vital cellular events. Understanding how polyUb chains are recognized by the 26S proteasome and other downstream effector molecules is central to our understanding of the mechanisms of regulation. Despite an increasing wealth of information on the cellular processes regulated by polyubiquitination and the identification of numerous Ub-binding proteins that tie the polyUb signal to downstream events, the molecular basis of diversity in Ub-mediated signaling remains unclear. The mechanisms underlying the ability of different polyUb chains to signal for distinct outcomes remain to be elucidated before the origin of specificity in polyUb signaling is understood. Obtaining such information is absolutely necessary in order to develop a molecular understanding of how different chains are able to act as specific signals. The objective of the proposed work is to characterize the structure and recognition properties of various types of polyUb chains, in order to understand at a molecular level the structural basis for Ub's ability to serve as a versatile cellular signal. We will focus on the so-called non-canonical chains: linked via lysines other than Lys48 or Lys63 (e.g., Lys11), chains with heterogeneous linkages (e.g., Lys11, Lys48, Lys63, head-to-tail), both linear and branched, as well as heterologous chains composed of Ub and Ub-like proteins (Rub1/Nedd8). We will also analyze the canonical, Lys48-linked polyUb chain conjugated to a model substrate protein, in order to examine the effect of the conjugation on both the polyUb tag and the substrate. In addition, we will characterize (poly)Ub complexes with ubistatins, small molecule inhibitors that block polyUb signal recognition by the proteasome, in order to assist in designing the next generation of ubistatin derivatives. We will use modern NMR approaches to determine the three-dimensional structure of these chains in solution and characterize their binding properties and complexes with receptors. This will be performed using a combination of long-range, orientational constraints derived from spin-relaxation and residual dipolar coupling measurements, complemented with distance information from NOEs and paramagnetic relaxation enhancements and pseudocontact shifts introduced by paramagnetic labels. NMR data will be combined with small-angle X-ray and neutron scattering (SAXS, SANS) data, to better characterize the structure and the conformational ensemble of polyUb chains.

描述（由申请人提供）：该建议是为了表征溶液结构，动力学和多泛素（polyub）链的相互作用，这些链在调节大量细胞过程的调节中起着信号分子的作用，范围从细胞周期到细胞周期，到转录激活，抗原加工，抗原处理，抗原处理和蛋白质的囊泡。底物与不同链接的多蛋白链的结合将目标蛋白归于细胞中不同的命运。特别是，Lys48链接的多UB充当泛素 - 蛋白酶体蛋白水解途径中的通用信号，这是短寿命蛋白周转的主要调节机制，影响了各种重要的细胞事件。了解26S蛋白酶体和其他下游效应分子如何识别多元链对于我们对调节机制的理解至关重要。尽管关于多泛素化调节的细胞过程的信息越来越多，并且鉴定了将polyub信号与下游事件联系起来的许多UB结合蛋白，但UB介导的信号传导中多样性的分子基础仍不清楚。在理解polyub信号传导中特异性的起源之前，尚待阐明不同多蛋白链发信号的能力的基础机制。获得此类信息是绝对必要的，以发展分子了解不同链如何充当特定信号的理解。拟议工作的目的是表征各种多类链链的结构和识别特性，以便在分子级别理解UB作为多功能细胞信号的能力的结构基础。 We will focus on the so-called non-canonical chains: linked via lysines other than Lys48 or Lys63 (e.g., Lys11), chains with heterogeneous linkages (e.g., Lys11, Lys48, Lys63, head-to-tail), both linear and branched, as well as heterologous chains composed of Ub and Ub-like proteins (Rub1/Nedd8).我们还将分析与模型底物蛋白结合的规范，LYS48连接的多核链，以检查结合对Polyub TAG和底物的影响。此外，我们还将表征（poly）UB复合物与ubistatins，小分子抑制剂，这些抑制剂可以通过蛋白酶体阻断polyub信号识别，以帮助设计下一代的ubistatin衍生物。我们将使用现代的NMR方法来确定溶液中这些链的三维结构，并表征它们与受体的结合特性和复合物。这将使用由自旋 - 浮肿和残留的偶极耦合测量得出的远程，定向约束的组合进行，并与NOES的距离信息以及顺磁性弛豫的增强和伪触觉偏移相互补充。 NMR数据将与小角度X射线和中子散射（SAX，SANS）数据结合使用，以更好地表征Polyub链的结构和构象合奏。