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

热休克蛋白70(HSP70)家族的成员是依赖于ATP的分子伴侣，它们是细胞蛋白质稳态的中心枢纽。它们确保蛋白质达到并保持正确的三维折叠，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)家族的辅助伴侣的作用的变构调节，也被称为Hsp40。JDPs是一个蛋白质家族(在人类细胞中有40个成员)，它包含一个保守的J-结构域，它通过与连接HSP70核苷酸结合域和底物结合域的柔性连接子的远端相互作用来促进该结构域的ATP水解。A类和B类JDP成员与N-末端J-结构域、一个无序的富含GF连接子、2个C-末端底物结合域(CTD)和一个二聚结构域共享共同的结构。关键的区别特征是GF连接子的组成，在A类JDP的情况下，它被锌指结构域扩展。尽管它们在结构上高度相似，但A类和B类JDP参与了不同的细胞活动，A类JDP主要与单体错误折叠蛋白或小寡聚物种相互作用，而I类(DNAJB1/4)和II类(DNAJB6/8)B类蛋白优先与寡聚物种和较大聚集体相互作用。尽管对HSP70的伴侣机制进行了30年的研究，但JDP共同伴侣家族中的角色和多样性在很大程度上仍未从机制层面上进行探索。近年来，JDPs已经成为潜在的治疗靶点，用于选择性调节特定的HSP70活性，而不干扰(其他)基本的家务活动。因此，我们的目标是对JDP介导的人HSP70伴侣在细胞底物上的募集进行全面的分析，以回答基本的问题：规范的A类和B类JDPs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/描述推动JDP类和这些类内成员之间底物特异性的生化特性，2/确定B类JDP家族中调控开关的激活和差异，3/描述不同的调控机制如何决定执行不同细胞活动的HSP70复合体的组装，4/探索JDP类之间在调节HSP70功能方面的相互作用。总体而言，拟议的项目将产生互补的数据集，这些数据集结合在一起将建立对JDP介导的刺激HSP70伴侣活性的全面机制的理解。

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.