Mechanisms of Hsp100 chaperones

Hsp100 分子伴侣的机制

• 批准号: 496815431
• 负责人: Privatdozent Dr. Axel Mogk
• 金额: --
• 依托单位: Zentrum für Molekulare Biologie (ZMBH)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/496815431?language=en
• 关键词: Mechanisms; Hsp100; chaperones

Bacterial Hsp100 chaperones are central components of protein quality control systems. They form hexameric ring structures and thread protein substrates in an ATP-fueled process through their central channel. Hsp100s come in two flavors. Some (e.g. ClpC, ClpE) associate with peptidases (e.g. ClpP) to form proteolytic complexes targeting misfolded substrates to degradation. Others (e.g. ClpB, ClpG) function as disaggregases reactivating aggregated proteins. The ATPase and threading activities of Hsp100 are tightly controlled to protect cells from deleterious unfolding events. This can be achieved by partner proteins, which target Hsp100s to their substrates while concurrently stimulating ATPase activities. We previously dissected the regulation of the partner-dependent ClpB and ClpC chaperones. M-domains (MDs) function as molecular switches, repressing ClpB/ClpC ATPase activities in absence of partners, while also enabling for activation by additionally serving as partner binding sites. In the former funding period we identified ClpG, ClpL and ClpE as partner-independent Hsp100 family members, raising the question how their ATPase and threading activities are regulated. We observe that these Hsp100s form diverse, large assemblies consisting of individual rings. Ring interactions are mediated via MDs. MD mutants that only form hexamers exhibit toxicity in vivo, while mutants that enhance assembly formation exhibit reduced activity. These findings underline the functional relevance of ring assemblies and point to a novel mode of Hsp100 regulation. We plan to:- Determine the dynamics of ring interactions and study how interventions that stabilize or destabilize the assemblies affect Hsp100 activities- Dissect how the assembly pathway is altered by substrate proteins- Determine the architecture of Hsp100 ring assemblies by cryo EM tomographyWe additionally identified ClpG and ClpL as standalone disaggregases providing enhanced resistance to Gram-negative (ClpG) and Gram-positive bacteria (ClpL), against thermal-based sterilization protocols applied in food industry and hospitals. Both disaggregases are encoded on plasmids or mobile genomic islands allowing for spreading of these persistence factors among bacterial communities. ClpG and ClpL target aggregated but not soluble misfolded proteins, protecting nascent polypeptides from the potent unfoldases. This specificity is provided by distinct N-terminal domains (NDs), which are essential and sufficient for aggregate binding. We plan to:- Determine the structures of the aggregate-binding NDs of ClpG and ClpL and identify and validate substrate binding sites- Determine how many NDs must be present in a ring to provide selectivity for protein aggregates- Study how both disaggregases cooperate with sHsp chaperones that organize protein aggregation- Transfer enhanced heat resistance to higher eukaryotes (e.g. plants) by heterologous expression of clpG and clpL genes

细菌Hsp 100分子伴侣是蛋白质质量控制系统的核心组成部分。它们通过中央通道在ATP驱动的过程中形成六聚环结构和螺旋蛋白底物。HSP 100有两种类型。一些（例如ClpC、ClpE）与肽酶（例如ClpP）缔合以形成靶向错误折叠的底物降解的蛋白水解复合物。其他（例如ClpB、ClpG）作为解聚剂再活化聚集蛋白。Hsp 100的ATP酶和穿线活性受到严格控制，以保护细胞免受有害的解折叠事件的影响。这可以通过伴侣蛋白来实现，其将Hsp 100靶向其底物，同时刺激ATP酶活性。我们以前解剖的合作伙伴依赖的ClpB和ClpC伴侣的调节。M-结构域（MD）作为分子开关起作用，在不存在配偶体的情况下抑制ClpB/ClpC ATP酶活性，同时还能够通过另外作为配偶体结合位点而激活。在前一个资助期，我们确定ClpG，ClpL和ClpE作为合作伙伴独立的Hsp 100家族成员，提出了一个问题，他们的ATP酶和线程活动是如何调节的。我们观察到，这些Hsp 100形成不同的，由单个环组成的大型组件。环相互作用通过MD介导。仅形成六聚体的MD突变体在体内表现出毒性，而增强组装体形成的突变体表现出降低的活性。这些发现强调了环组件的功能相关性，并指出了一种新的Hsp 100调节模式。我们计划： 确定环相互作用的动力学，并研究稳定或不稳定组装的干预措施如何影响Hsp 100活性- 剖析组装途径如何被底物蛋白质改变- 通过冷冻EM断层扫描确定Hsp 100环组装体的结构我们还将ClpG和ClpL鉴定为独立的解聚剂，其提供对革兰氏阴性（ClpG）和革兰氏阳性细菌（ClpL）的增强的抵抗力，对抗食品工业和医院中应用的基于热的灭菌方案。这两种解聚剂都在质粒或移动的基因组岛上编码，允许这些持久性因子在细菌群落中传播。ClpG和ClpL靶向聚集但不可溶的错误折叠蛋白，保护新生多肽免受有效的解折叠酶的影响。这种特异性由不同的N-末端结构域（ND）提供，其对于聚集体结合是必需的且足够的。我们计划： 确定ClpG和ClpL的聚集体结合ND的结构，并鉴定和验证底物结合位点- 确定环中必须存在多少个ND以提供蛋白质聚集体的选择性- 研究这两种解聚剂如何与组织蛋白质聚集的sHsp分子伴侣合作- 通过clpG和clpL基因的异源表达将增强的耐热性转移到高等真核生物（例如植物）