Functional specification of HSP70 by J-domain protein co-chaperones

热休克蛋白70 （HSP70）家族的成员是atp依赖的分子伴侣，是细胞蛋白稳态的中心枢纽。它们确保蛋白质达到并保持正确的三维折叠，在聚集时被分解，或被细胞降解途径清除。ATP水解促进hsp70捕获底物，因此是启动功能性伴侣循环的关键步骤。它受底物结合和j结构域蛋白（JDP）家族的共伴侣蛋白（也称为Hsp40）的作用的变构调节。jdp是一个扩展的蛋白家族（人类细胞中的bbb40成员），包含一个保守的j结构域，通过与连接该结构域和底物结合域的柔性连接体的远端相互作用，促进HSP70核苷酸结合域的ATP水解。A类和B类JDP成员具有相同的结构，包括n端j结构域、一个无序富GF连接子、2个c端底物结合结构域（CTDs）和一个二聚化结构域。关键的区别特征是GF连接器的组成，在A类jdp的情况下，它被锌指域扩展。尽管具有高度的结构相似性，A类和B类JDPs与不同的细胞活动有关，A类JDPs主要与单体错误折叠蛋白或小寡聚物相互作用，而I类（DNAJB1/4）和II类（DNAJB6/8）的B类蛋白优先与寡聚物和较大的聚集体相互作用。尽管对HSP70伴侣机制进行了三十年的研究，但在机制层面上，JDP共同伴侣家族的作用和多样性仍未得到很大程度的探索。近年来，jdp作为选择性调节特定HSP70活性而不干扰（其他）基本家务活动的潜在治疗靶点已成为前景。因此，我们的目标是对jdp介导的人类HSP70伴侣向细胞底物的募集进行全面分析，以回答一个基本问题：规范的a类和B类jdp如何使HSP70在人类细胞中如此多不同的折叠过程中发挥作用？我们的具体目标是：1/描述驱动JDP类和这些类成员之间底物特异性的生化特性；2/确定B类JDP家族中激活的触发因素和调节开关的差异；3/描述不同的调节机制如何决定执行不同细胞活动的HSP70复合物的组装；4/探测JDP类之间在调节HSP70功能方面的相互作用。总的来说，拟议的项目将产生互补的数据集，结合起来将建立JDP介导的HSP70伴侣活性刺激的全面机制理解。

Members of the heat shock protein 70 (HSP70) family are ATP-dependent molecular chaperones that act as central hub of cellular protein homeostasis. They ensure proteins attain and maintain their correct three-dimensional fold, become disassembled when aggregated, or cleared by cellular degradation pathways. ATP hydrolysis promotes substrate trapping by Hsp70s and therefore is the critical step that initiates the functional chaperone cycle. It is allosterically regulated both by substrate binding and the action of co-chaperones of the J-domain protein (JDP) family, also referred to as Hsp40. JDPs are an extended family of proteins (>40 members in human cells) that contain a conserved J-domain which promotes ATP hydrolysis in the HSP70 nucleotide binding domain via distal interactions with the flexible linker connecting this domain with the substrate binding domain. Class A and B JDP members share a common architecture with the N-terminal J-domain, a disordered GF rich linker, 2 C-terminal substrate binding domains (CTDs) and a dimerisation domain. The key differentiating feature is the composition of the GF linker, which in the case of class A JDPs is extended by a zinc finger domain. Despite their high structural similarities, class A and class B JDPs have been implicated in distinct cellular activities, with class A JDPS primarily interacting with monomeric misfolded proteins or small oligomeric species, while class B proteins of both type I (DNAJB1/4) and type II (DNAJB6/8) preferentially interact with oligomeric species and larger aggregates. Despite three decades of research into the HSP70 chaperone machinery, the roles and diversity within the JDP co-chaperone family remains largely unexplored on a mechanistic level. In recent years, JDPs have come to the foreground as potential therapeutic targets for the selective tuning of specific HSP70 activities without perturbing (other) essential housekeeping activities. We therefore aim to perform a comprehensive analysis of JDP-mediated recruitment of the human HSP70 chaperone to cellular substrates to answer the fundamental question: how do canonical class A and class B JDPs get HSP70 to act in so many different folding processes in the human cell? Our specific aims are: 1/ Describe biochemical properties that drive substrate specificity between JDP classes and member within these classes, 2/ Identify the trigger for activation and differences in regulatory switches within the class B JDP family, 3/ Characterise how different regulatory mechanisms determine the assembly of HSP70 complexes that perform different cellular activities and, 4/ Probe the interplay between JDP classes in tuning HSP70 function.Overall, the proposed project will generate complementary datasets that combined will build a comprehensive mechanistic understanding of JDP mediated stimulation of HSP70 chaperone activity.