Mechanisms of Protein Disaggregation and Turnover by AAA+ Chaperones

项目摘要/摘要 蛋白质的解聚和周转对蛋白质的稳态(蛋白质稳态)和细胞活力是必不可少的。 在细胞应激和衰老过程中会发生功能障碍，加速有害的蛋白质聚集和淀粉样蛋白 队形。改进的机械学理解对于确定蛋白平衡通路如何失效和 确定预防神经退行性疾病和其他蛋白质错误折叠疾病的治疗目标。 热休克蛋白(HSP)100是保守的AAA家族成员，作为蛋白质在所有生命中发挥重要功能 展开酶和解聚酶。它们形成六聚体，由三磷酸腺苷驱动，催化转位 多肽底物通过中央通道。未折叠的蛋白质随后被热休克蛋白分子重折叠。 伴侣或被相关的酶降解，例如在蛋白酶体的情况下。 在实现功能状态结构方面的挑战导致了相互冲突的机械模型 AAA超级家族。重点研究保守的Hsp100成员、酵母Hsp104和细菌CLP蛋白， 我们已经克服了这些挑战，通过使用低温电子显微镜来确定生物化学结构 已定义的功能复合体。我们确定了AAA解聚酶的第一个底物结合结构 (HSP104)处于不同的移位状态，并发现这些机器通过旋转机制运行，包括 精确的底物抓取和释放状态以及两个氨基酸的转位步骤。自从我们上次提交以来 在这一应用中，我们已经确定了ClpAP aaa蛋白酶的多种结构。 底物的展开和蛋白质的共同降解我们的发现揭示了一种新的范式，即氨基酸如何分解S 以机械方式展开衬底。下一个要解决的主要问题是：移位机制是如何 (现在被认为在AAA S中高度保守)与特定的细胞功能相结合？我们的长期合作 目标是确定易位和展开是如何针对不同的蛋白平衡和细胞压力进行精确调整的 响应函数。该应用程序目标是识别耦合的关键变构控制机制 ATP驱动的转位到底物的识别、展开和降解。在这里我们将：(SA1)确定 ClpAP“细菌蛋白酶体”复合体的蛋白质去折叠和蛋白质分解机制；(SA2)测定 Hsp104如何与天然底物和淀粉样蛋白相互作用和解聚；以及(SA3)决定 HSP70分子伴侣与HSP104协同促进底物负载。在这项工作完成后，我们将 识别构象网络和蛋白质：定义核心转位周期的蛋白质相互作用 变构连接以指定这些AAA机器的不同细胞功能。

PROJECT SUMMARY/ABSTRACT Protein disaggregation and turnover are essential for protein homeostasis (proteostasis) and cell viability. Malfunction occurs during cell stress and aging, accelerating deleterious protein aggregation and amyloid formation. Improved mechanistic understanding is critical for determining how proteostasis pathways fail and for identifying therapeutic targets in preventing neurodegenerative disease and other protein mis-folding diseases. Heat shock protein (Hsp) 100 members of the conserved AAA+ family serve critical functions in all life as protein unfoldases and disaggregases. They form hexameric, ATP-driven machines that catalyze the translocation of polypeptide substrates through a central channel. The unfolded proteins are then refolded by Hsp molecular chaperones or degraded by an associated protease, such as in the case of the proteasome. Challenges in achieving structures of functional states have led to conflicting mechanistic models across the AAA+ superfamily. Focusing on conserved Hsp100 members, yeast Hsp104 and the bacterial Clp proteins, we have overcome these challenges by using cryo-electron microscopy to determine structures of biochemically defined, functional complexes. We determined the first substrate-bound structures of a AAA+ disaggregase (Hsp104) in distinct translocation states and discovered these machines operate by a rotary mechanism involving precise substrate gripping and release states and a two amino acid translocation step. Since our last submission of this application, we have determined multiple structures of the ClpAP AAA+ protease undergoing active substrate unfolding and proteolysis Together our discoveries reveal a new paradigm for how AAA+s mechanically unfold substrates. The next major question to address is: How is the translocation mechanism (which is now considered highly conserved among AAA+s) coupled to specific cellular functions? Our long-term goal is to determine how translocation and unfolding are precisely tuned for different proteostasis and cell stress response functions. The objective for this application is to identify key allosteric control mechanisms that couple ATP-driven translocation to substrate recognition, unfolding and degradation. Here we will: (SA1) Determine mechanisms of protein unfolding and proteolysis by the ClpAP “bacterial proteasome” complex; (SA2) Determine how Hsp104 interacts with and disaggregates native substrates and amyloids; and (SA3) Determine how the Hsp70 chaperone collaborates with Hsp104 to promote substrate loading. At the completion of this work we will identify conformational networks and protein:protein interactions that define how the core translocation cycle connects allosterically to specify distinct cellular functions of these AAA+ machines.