Mechanisms of small heat shock protein sequestrases

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的蛋白质种类的积累，这与细胞毒性有关。一个复杂的蛋白质静止网络通过它们的再折叠、降解或隔离到细胞内包涵体中来去除错误折叠的蛋白质。固存是一种最近才被认识到的蛋白质质量控制策略，其涉及的细胞因素、潜在机制和生理功能才刚刚开始揭示。在酿酒酵母中，蛋白质的分离是由Hsp42和Btn2分离酶完成的。Hsp42是小热休克蛋白（sHsp）家族的成员，与Btn2具有共同的特征，包括存在大的无序区域、自组装能力和将结合底物引导到Hsp70/ hsp100依赖的重折叠途径。在之前的工作中，我们研究了Hsp42隔离酶活性的基本机制，包括结合底物的朊病毒样结构域和调节隔离酶活性的带电无序结构域。Hsp42和Btn2都能在有限的Hsp70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内含物中，从而释放了Hsp70的容量。这种独特的隔离酶突变体表型使我们能够建立一种筛选秀丽隐杆线虫隔离酶的方法，该方法可以恢复缺乏隔离酶和限制Hsp70活性的酵母突变体的生长和蛋白质隔离。我们的初步实验确定了秀丽隐杆线虫的sHsps作为隔离酶，有助于延长动物的寿命。其他sHsps没有表现出这种活性，表明后生动物中sHsps的功能特化。在这里，我们计划确定sHsp隔离酶的机制，它们在后生动物中的生理相关性，以及隔离酶如何将结合底物分类到重折叠途径。我们的主要研究目标是：(1)通过在酵母sequestrase突变体筛选中测试来自不同生命领域的sHsps，探索封存是否代表sHsps的保守活性；(2)明确sHsp固溶酶活性背后的生化和结构特征；(3)确定sequestrase阳性sHsps在秀丽隐杆线虫中的生理作用；(4)以Hsp42为模型，分析隔离酶活性如何受到环境因素的调节；(5)通过低温电子断层扫描确定固酯酶/底物复合物的结构，以及Hsp70/Hsp100分解气体如何调节它们。通过这些实验，我们希望揭示驱动sHsps蛋白质隔离功能的关键机制，这构成了细胞抵抗蛋白质毒性应激的主要防线。

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.