Mechanisms of Protein Disaggregation and Turnover by AAA+ Chaperones

项目总结/摘要 蛋白质解聚和周转对于蛋白质稳态（蛋白质稳态）和细胞活力是必不可少的。 在细胞应激和老化过程中发生功能障碍，加速有害的蛋白质聚集和淀粉样蛋白 阵提高机制的理解是至关重要的，以确定如何蛋白质代谢途径失败， 鉴定预防神经变性疾病和其它蛋白质错误折叠疾病的治疗靶点。 热休克蛋白（Hsp）100保守的AAA+家族成员作为蛋白质在所有生命中发挥关键作用 解折叠酶和解聚酶。它们形成六聚体，ATP驱动的机器，催化 多肽底物通过中央通道。然后，未折叠的蛋白质被热休克蛋白分子重折叠， 蛋白酶体可以是伴随分子或被相关蛋白酶降解的，例如在蛋白酶体的情况下。 实现功能状态结构的挑战导致了相互冲突的机械模型， AAA+超级家族重点关注保守的Hsp 100成员、酵母Hsp 104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的结构， 功能性的复合物。我们确定了AAA+解聚酶的第一个底物结合结构 （Hsp 104）在不同的易位状态，并发现这些机器通过旋转机制， 精确的底物夹持和释放状态以及两个氨基酸的移位步骤。自从我们上次提交 在本申请中，我们已经确定了ClpAP AAA+蛋白酶的多种结构， 底物解折叠和蛋白质水解我们的发现揭示了AAA+ 机械展开基板。下一个要解决的主要问题是： （现在被认为是高度保守的AAA+s）耦合到特定的细胞功能？我们的长期 我们的目标是确定易位和解折叠是如何针对不同的蛋白质稳态和细胞应激进行精确调节的 响应函数。本申请的目的是确定关键的变构控制机制， ATP驱动的易位到底物识别、解折叠和降解。在这里，我们将：（SA 1）确定 通过ClpAP“细菌蛋白酶体”复合物的蛋白质解折叠和蛋白质水解的机制;（SA 2）确定 Hsp 104如何与天然底物和淀粉样蛋白相互作用并解聚;以及（SA 3）确定Hsp 104如何与天然底物和淀粉样蛋白相互作用并解聚。 Hsp 70分子伴侣与Hsp 104协同促进底物负载。在这项工作完成后，我们将 确定构象网络和蛋白质：蛋白质相互作用，定义核心易位循环 连接变构以指定这些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.