Mechanisms of small heat shock protein sequestrases

小热休克蛋白多价螯合酶的机制

• 批准号: 461328467
• 负责人: Professor Dr. Bernd Bukau
• 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/461328467?language=en
• 关键词: Mechanisms; small; heat; shock; protein

The imbalance of protein homeostasis caused by stress conditions or cellular ageing results in the accumulation of misfolded protein species, which is linked to cell toxicity. A sophisticated proteostasis network removes misfolded proteins by their refolding, degradation or sequestration into intracellular inclusions. Sequestration is a protein quality control strategy that has been recognized only recently, and the cellular factors involved, underlying mechanisms and physiological functions of sequestration are just beginning to unfold.Protein sequestration is best characterized in Saccharomyces cerevisiae where it is executed by the Hsp42 and Btn2 sequestrases. Hsp42 is member of the small heat shock protein (sHsp) family and shares common features with Btn2, including the presence of large disordered regions, the ability to self-assemble and to direct bound substrates to Hsp70/Hsp100-dependent refolding pathways. In previous work we investigated the basic mechanism of Hsp42 sequestrase activity, which involves a prion-like domain that binds substrates and a charged, disordered domain that regulates sequestrase activity. Both Hsp42 and Btn2 promote cell survival under conditions of limited Hsp70 availability, presumably because misfolded proteins become secluded into inclusions which frees Hsp70 capacity. This unique phenotype of sequestrase mutants enabled us to set up a screen for C. elegans sequestrases that restore growth and protein sequestration in yeast mutants with lacking sequestrase and limiting Hsp70 activities. Our preliminary experiments identified selected C. elegans sHsps as sequestrases, which contribute to life span of the animals. Other sHsps do not show such activity, indicating functional specialization among sHsps in metazoa. Here, we plan to determine the mechanism of sHsp sequestrases, their physiological relevance in a metazoan and how sequestrases are triaging bound substrates to refolding pathways. Our major research aims are: (1) explore whether sequestration represents a conserved activity of sHsps by testing sHsps from different kingdoms of life in our yeast sequestrase mutant screen; (2) define the biochemical and structural features that underlie sHsp sequestrase activity; (3) determine the physiological role of sequestrase positive sHsps in C. elegans; (4) dissect how sequestrase activity is regulated by environmental cues using Hsp42 as model; (5) determine the structures of sequestrase/substrate complexes by cryo electron tomography and how they are modulated by Hsp70/Hsp100 disaggregases. With these experiments we expect to reveal key mechanisms driving the protein sequestration function of sHsps, which constitutes a prime line of defense of cells against proteotoxic stress.

由应激条件或细胞老化引起的蛋白质稳态失衡导致错误折叠蛋白质种类的积累，这与细胞毒性有关。复杂的蛋白质稳定网络通过其重折叠、降解或螯合到细胞内内含物中来去除错误折叠的蛋白质。螯合是一种蛋白质质量控制策略，直到最近才被认识到，螯合的细胞因素，潜在的机制和生理功能才刚刚开始展开。热休克蛋白42是小热休克蛋白（sHsp）家族的成员，并与Btn 2共享共同的特征，包括存在大的无序区域，自组装能力和指导结合底物的热休克蛋白70/热休克蛋白100依赖性重折叠途径。在以前的工作中，我们研究了热休克蛋白42螯合酶活性的基本机制，其中涉及朊病毒样结构域结合底物和带电的，无序的结构域，调节螯合酶活性。Hsp 42和Btn 2在有限的Hsp 70可用性条件下促进细胞存活，可能是因为错误折叠的蛋白质被隔离到释放Hsp 70能力的内含物中。这种独特的螯合酶突变体表型使我们能够建立一个筛选C。在缺乏螯合酶和限制Hsp 70活性的酵母突变体中恢复生长和蛋白质螯合的线虫螯合酶。我们的初步实验确定了选定的C。线虫sHsps作为螯合酶，有助于动物的寿命。其他sHsps不显示这样的活动，表明在后生动物中的sHsps的功能专业化。在这里，我们计划确定sHsp螯合酶的机制，它们在后生动物中的生理相关性，以及螯合酶如何将结合底物筛选到重折叠途径。我们的主要研究目标是：（1）通过在我们的酵母螯合酶突变体筛选中测试来自不同生命界的sHsps来探索螯合作用是否代表sHsps的保守活性;（2）确定sHsps螯合酶活性的生化和结构特征;（3）确定螯合酶阳性sHsps在C.优雅的;（4）以热休克蛋白42为模型，研究了环境因素对螯合酶活性的影响，（5）利用低温电子断层扫描技术研究了螯合酶/底物复合物的结构，以及它们如何受热休克蛋白70/热休克蛋白100分解气体的调节。通过这些实验，我们期望揭示驱动sHsps的蛋白螯合功能的关键机制，sHsps构成了细胞对抗蛋白毒性应激的主要防线。