Structural and functional analysis of the URI1 prefoldin-like chaperone complex

URI1 前折叠蛋白样伴侣复合物的结构和功能分析

• 批准号: RGPIN-2020-04074
• 负责人: Houry, Walid
• 金额: $ 3.06万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=716432
• 关键词: Structural; functional; analysis; URI1; prefoldin

Molecular chaperones are a highly interactive group of proteins that fulfill central roles in all aspects of protein homeostasis. They are key to maintaining protein homeostasis under normal and stress conditions. These chaperone families include Hsp90, Hsp70, Hsp60, Hsp40, R2TP, CCT, and prefoldin among others. As part of a research program aimed at discovering and characterizing novel cellular chaperones, we will structurally and functionally characterize the newly discovered URI1 prefoldin-like chaperone complex (URI1C). The URI1C chaperone complex was identified to interact with Hsp90, Hsp70, CCT and R2TP in pulldown assays by multiple groups including my group. Hence, it is a central chaperone complex that functions with other chaperones to ensure protein homeostasis. URI1C consists of the following five subunits: URI1, UXT, PDRG1, PFDN2, and PFDN6. PFDN2 and PFDN6 are also members of the canonical prefoldin complex (PFDN), which consists of PFDN1 to 6. However, in contrast to the canonical PFDN chaperone, very little is known about the structure or function URI1C. So far, URI1C has been implicated in one cellular pathway: RNA polymerase II (RNAP II) assembly; however, it is likely involved in many other pathways. Hence, the proposed project has two main aims: (A) URI1C reconstitution, characterization, and structural elucidation; (B) Identification and characterization of URI1C-specific clients. Aim A. Biophysical and structural characterization of URI1C Subunits of the URI1C chaperone will be co-expressed and purified using the pQLINK vector system. This system enables the co-expression of His-tag or GST-tag fusion proteins as well as untagged proteins. Subsequently, purified URI1C will be characterized to determine its oligomeric state and subunit stoichiometry. The structure of this complex will then be determined using SAXS (small angle X-ray scattering), X-ray crystallography, and/or cryoEM. Aim B. Identification and characterization of URI1C-specific clients To globally examine the function of URI1C in the cell, we will use two different proteomic approaches. In the first approach, Biotin-proximity labeling identification (BioID) followed by mass spectrometry will be performed using URI1C subunits as bait to identify proteins within the vicinity of the complex. In the second approach, we will carry out regular affinity purification using the FLAG-tag for pulldown followed by mass spectrometry (AP-MS) of all the URI1C proteins as well as of their individual domains. These experiments will provide us with a hit list of potential URI1C clients that will subsequently be verified in follow up assays. In conclusion, the findings from this project will be the first to demonstrate the cellular function and structure for URI1C and establish its role within the cellular chaperone network. Our work will provide critical insights into novel mechanisms regulating protein homeostasis.

分子伴侣是一组高度相互作用的蛋白质，在蛋白质稳态的各个方面发挥核心作用。它们是在正常和应激条件下维持蛋白质稳态的关键。这些伴侣家族包括Hsp90、Hsp70、Hsp60、Hsp40、R2TP、CCT和prefoldin等。作为旨在发现和表征新型细胞伴侣的研究项目的一部分，我们将对新发现的URI1前折叠蛋白样伴侣复合物（URI1C）进行结构和功能表征。