Solution structure and dynamics of polyubiquitin chains

项目摘要 这项工作将表征多聚泛素（polyUb）链的溶液结构、动力学和相互作用， 其作为信号分子在调节许多细胞过程中起作用， 通过细胞周期的进展，转录激活，抗原加工和囊泡运输， proteins.底物与不同连接类型的polyUb链的偶联使靶蛋白与不同连接类型的polyUb链结合。 不同的命运特别是，Lys 48连接的polyUb在泛素-蛋白质中充当通用信号。 蛋白酶体蛋白水解途径，是短寿命蛋白质周转的主要调节机制， 影响着各种重要的细胞活动了解26 S如何识别polyUb链 蛋白酶体和其他下游效应分子是我们理解 调控尽管越来越多的信息对细胞过程的调控， 多聚泛素化和许多泛素结合蛋白的鉴定，这些蛋白将polyUb信号与 尽管泛素介导的信号转导在下游事件中具有多样性，但其分子基础仍不清楚。的 不同polyUb链发出不同结果信号的能力的潜在机制仍有待进一步研究。 在理解polyUb信号传导中特异性的起源之前阐明。获取这些信息是 这是绝对必要的，以便发展一个分子的理解，不同的链是如何能够作为 具体信号。所提出的工作的目标是表征的结构和识别性能 为了在分子水平上理解泛素的结构基础， 作为一个通用的能力，但具体的细胞信号。这些研究将集中在所谓的非典型 泛素链：通过除Lys 48或Lys 63以外的赖氨酸连接（例如，Lys 6、Lys 27）和含有 异质连接（例如，Lys 11和Lys 48），分支和未分支。我们还将确定 典型的，Lys 48连接的tetraUb链在溶液中的构象，以获得对 它们被细胞受体识别的机制。最后，这些研究将描述 并确定与最近设计的结合polyUb的环肽的polyUb复合物的结构 以便于进一步开发和优化这种完全新颖的 类调节剂和潜在的治疗泛素介导的信号通路。我们将使用现代 NMR方法结合小角X射线和中子散射（SAXS，SANS）来确定 polyUb链在溶液中的三维结构和构象系综， 表征它们与受体的结合偏好和复合物。

Project Summary This work will 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 linkage types 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 ubiquitin-binding proteins that tie the polyUb signal to downstream events, the molecular basis of diversity in ubiquitin-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 ubiquitin's ability to serve as a versatile yet specific cellular signal. These studies will focus on the so-called non-canonical ubiquitin chains: linked via lysines other than Lys48 or Lys63 (e.g., Lys6, Lys27) and chains containing heterogeneous linkages (e.g., Lys11 & Lys48), branched and unbranched. We will also determine the conformations of the canonical, Lys48-linked tetraUb chains in solution in order to gain insights into the mechanisms of their recognition by cellular receptors. Finally, these studies will characterize the interactions and determine the structures of polyUb complexes with the recently designed cyclic peptides that bind polyUb with high affinity and specificity in order to facilitate further development and optimization of this entirely novel class of modulators and potential therapeutics for ubiquitin-mediated signaling pathways. We will use modern NMR approaches in combination with small-angle X-ray and neutron scattering (SAXS, SANS) to determine the three-dimensional structures and conformational ensembles of polyUb chains in solution and to characterize their binding preferences and complexes with receptors.