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，在蛋白质动态平衡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动态平衡的关键。这些伴侣蛋白家族包括Hsp90、Hsp70、Hsp60、Hsp40、R2TP、CCT和prefoldin等。作为旨在发现和鉴定新的细胞伴侣的研究计划的一部分，我们将从结构和功能上表征新发现的URI1前折叠素样伴侣复合体(URI1C)。 包括我的小组在内的多个小组在下拉实验中发现URI1C伴侣复合体与Hsp90、Hsp70、CCT和R2TP相互作用。因此，它是一个中心伴侣复合体，与其他伴侣一起发挥作用，以确保蛋白质的动态平衡。URI1C由以下五个亚基组成：URI1、UXT、PDRG1、PFDN2和PFDN6。PFDN2和PFDN6也是典型的前折叠蛋白复合体(PFDN)的成员，PFDN由PFDN1至6组成。然而，与典型的PFDN伴侣相比，人们对URI1C的结构或功能知之甚少。到目前为止，URI1C只参与了一种细胞途径：RNA聚合酶II(RNAP II)的组装；然而，它可能还参与了许多其他途径。因此，拟议的项目有两个主要目标：(A)URI1C的重组、特征和结构阐明；(B)特定于URI1C的客户的识别和特征。 目的A.URI1C的生物物理和结构特征 URI1C分子伴侣亚基将利用pQLINK载体系统进行共表达和纯化。该系统能够实现His-Tag或GST-Tag融合蛋白以及未标记蛋白的共表达。随后，将对纯化的URI1C进行表征，以确定其寡聚状态和亚基化学计量比。然后将使用SAXS(小角X射线散射)、X射线结晶学和/或低温电子显微镜来确定该络合物的结构。 目标B.URI1C特定客户的识别和特征 为了全面研究URI1C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URI1C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，以鉴定复合体附近的蛋白质。在第二种方法中，我们将使用下拉标记进行常规亲和纯化，然后对所有URI1C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一份潜在URI1C客户端的命中列表，随后将在后续分析中进行验证。 总之，该项目的发现将第一次证明URI1C的细胞功能和结构，并确定其在细胞伴侣网络中的作用。我们的工作将为调节蛋白质动态平衡的新机制提供关键的见解。

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.