Mechanisms of Hsp100 chaperones

细菌Hsp100伴侣是蛋白质质量控制系统的核心组成部分。它们形成六聚体环结构，并通过其中心通道在atp燃料过程中连接蛋白质底物。hsp100有两种口味。一些（如ClpC， ClpE）与肽酶（如ClpP）结合形成蛋白水解复合物，靶向错误折叠的底物进行降解。其他的（如ClpB， ClpG）作为分解气体的功能，使聚集的蛋白质重新激活。Hsp100的atp酶和线程活性受到严格控制，以保护细胞免受有害的展开事件。这可以通过伴侣蛋白来实现，伴侣蛋白将hsp100靶向到它们的底物上，同时刺激atp酶的活性。我们之前剖析了依赖伴侣的ClpB和ClpC伴侣的调控。m结构域（MDs）作为分子开关，在缺少伴侣的情况下抑制ClpB/ClpC atp酶的活性，同时也通过作为伴侣结合位点激活。在之前的融资期内，我们将ClpG、ClpL和ClpE确定为独立于合作伙伴的Hsp100家族成员，提出了它们的atp酶和线程活动是如何调节的问题。我们观察到这些hsp100形成了不同的，由单个环组成的大型组合。环相互作用是通过MDs介导的。仅形成六聚体的MD突变体在体内表现出毒性，而增强组装形成的突变体表现出活性降低。这些发现强调了环组件的功能相关性，并指出了Hsp100调控的新模式。我们计划：-确定环相互作用的动力学，并研究稳定或破坏组装的干预措施如何影响Hsp100的活性-剖析组装途径如何被底物蛋白改变-通过低温电子显微镜层析成像确定Hsp100环组装的结构。我们还确定ClpG和ClpL是独立的分解气体，可增强对革兰氏阴性菌（ClpG）和革兰氏阳性菌（ClpL）的抗性。反对食品工业和医院采用的基于热的灭菌方案。这两种分解气体都被编码在质粒或移动的基因组岛上，允许这些持久性因子在细菌群落中传播。ClpG和ClpL靶向聚集但不溶的错误折叠蛋白，保护新生多肽免受强效展开酶的影响。这种特异性是由不同的n端结构域（NDs）提供的，这是聚合结合所必需的和充分的。我们计划：-确定ClpG和ClpL的聚集结合NDs的结构，并鉴定和验证底物结合位点-确定在一个环中必须存在多少个NDs才能提供蛋白质聚集的选择性-研究这两种解聚气体如何与组织蛋白质聚集的sHsp伴侣蛋白合作-通过异源表达ClpG和ClpL基因将增强的耐热性转移到高级真核生物（例如植物）

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