Solution structure and dynamics of polyubiquitin chains

描述（由申请人提供）：该提案旨在表征聚泛蛋白（polyUb）链的溶液结构、动力学和相互作用，聚泛蛋白（polyUb）链在调节许多细胞过程（从细胞周期的进展到转录）中充当信号分子。激活、抗原加工和蛋白质的囊泡运输。底物与不同连接的polyUb链的缀合使靶蛋白在细胞中具有不同的命运。特别是，Lys 48连接的polyUb在泛素-蛋白酶体蛋白水解途径中充当通用信号，这是影响各种重要细胞事件的短寿命蛋白质周转的主要调节机制。了解polyUb链如何被26 S蛋白酶体和其他下游效应分子识别是我们理解调控机制的核心。尽管越来越多的信息，由多聚泛素化和众多的Ub结合蛋白的下游事件的关系，多聚Ub信号调节的细胞过程中的识别，在Ub介导的信号的多样性的分子基础仍然不清楚。在了解polyUb信号传导的特异性起源之前，不同polyUb链发出不同结果信号的能力的潜在机制仍有待阐明。获得这些信息是绝对必要的，以发展一个分子的理解，不同的链是如何能够作为特定的信号。拟议工作的目的是表征各种类型的polyUb链的结构和识别特性，以便在分子水平上了解Ub作为多功能细胞信号的能力的结构基础。我们将关注所谓的非典型链：通过Lys 48或Lys 63以外的赖氨酸连接（例如，Lys 11）、具有异质键的链（例如，Lys 11，Lys 48，Lys 63，头-尾），线性和分支，以及由Ub和Ub样蛋白组成的异源链（Rub 1/Nedd 8）。我们还将分析典型的Lys 48连接的polyUb链与模型底物蛋白缀合，以检查缀合对polyUb标签和底物的影响。此外，我们将表征（聚）Ub复合物与ubistatin，小分子抑制剂，阻止polyUb信号识别的蛋白酶体，以协助设计下一代ubistatin衍生物。我们将使用现代NMR方法来确定这些链在溶液中的三维结构，并表征其结合特性和与受体的复合物。这将使用来自自旋弛豫和残余偶极耦合测量的长程取向约束的组合进行，并补充来自NOE的距离信息和顺磁弛豫增强以及顺磁标签引入的伪接触位移。NMR数据将与小角X射线和中子散射（SAXS，SANS）数据相结合，以更好地表征polyUb链的结构和构象系综。 公共卫生相关性：泛素介导的信号传导参与调节多种生物过程，包括细胞发育和分裂、免疫应答和信号转导。泛素-蛋白酶体蛋白水解途径是短寿命蛋白质周转的主要机制，一直是广泛药物设计工作的目标，因为该途径中的缺陷与多种疾病相关，包括癌症、神经退行性疾病、发育障碍、免疫和炎症性疾病以及肌肉萎缩。这项研究将扩展我们对泛素介导的信号通路中识别和调节的分子机制的理解，并有助于设计控制泛素依赖性蛋白质降解的新型抑制剂。

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. PUBLIC HEALTH RELEVANCE: Ubiquitin-mediated signaling is involved in the regulation of a wide variety of biological processes including cell development and division, the immune response, and signal transduction. The ubiquitin-proteasome proteolytic pathway, the principal mechanism for turnover of short-lived proteins, has been the target for extensive drug design efforts, as defects in this pathway are associated with a variety of diseases, including cancers, neurodegenerative disorders, developmental disorders, immune and inflammatory disorders, and muscle wasting. This research will extend our understanding of the molecular mechanisms of recognition and regulation in ubiquitin-mediated signaling pathways and assist in the design of novel inhibitors that control ubiquitin-dependent protein degradation.