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

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。这些分子伴侣家族包括Hsp 90、Hsp 70、Hsp 60、Hsp 40、R2 TP、CCT和前折叠蛋白等。作为旨在发现和表征新型细胞伴侣的研究计划的一部分，我们将在结构和功能上表征新发现的URI 1前折叠蛋白样伴侣复合物（URI 1C）。 URI 1C分子伴侣复合物被鉴定为与Hsp 90，Hsp 70，CCT和R2 TP在pulldown试验中相互作用。因此，它是一个中央伴侣复合物，与其他伴侣一起发挥作用，以确保蛋白质稳态。URI 1C由以下五个亚基组成：URI 1、UXT、PDRG 1、PFDN 2和PFDN 6。PFDN 2和PFDN 6也是典型前折叠蛋白复合物（PFDN）的成员，其由PFDN 1至6组成。然而，与典型的PFDN分子伴侣相反，对URI 1C的结构或功能知之甚少。到目前为止，URI 1C参与了一种细胞途径：RNA聚合酶II（RNAP II）组装;然而，它可能参与了许多其他途径。因此，拟议的项目有两个主要目标：（A）URI 1C重建，表征和结构阐明;（B）URI 1C特定客户端的识别和表征。 目标A。URI 1C的生物物理和结构表征 URI 1C分子伴侣的亚基将使用pQLINK载体系统共表达和纯化。该系统能够共表达His标签或GST标签融合蛋白以及未标记的蛋白。随后，将对纯化的URI 1C进行表征，以确定其低聚状态和亚基化学计量。然后使用SAXS（小角X射线散射）、X射线晶体学和/或cryoEM确定该复合物的结构。 瞄准B。URI 1C特定客户端的识别和表征 为了全面检查URI 1C在细胞中的功能，我们将使用两种不同的蛋白质组学方法。在第一种方法中，将使用URI 1C亚基作为诱饵进行生物素邻近标记鉴定（BioID），然后进行质谱分析，以鉴定复合物附近的蛋白质。在第二种方法中，我们将使用FLAG标签进行常规亲和纯化，然后对所有URI 1C蛋白及其单个结构域进行质谱分析（AP-MS）。这些实验将为我们提供潜在URI 1C客户端的命中列表，随后将在后续分析中进行验证。 总之，该项目的发现将首次证明URI 1C的细胞功能和结构，并确定其在细胞伴侣蛋白网络中的作用。我们的工作将提供重要的见解，新的机制调节蛋白质稳态。

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.