Biochemistry of Energy-Dependent (Intracellular) Protein Degradation

能量依赖性（细胞内）蛋白质降解的生物化学

• 批准号: 8157185
• 负责人: MICHAEL MAURIZI
• 金额: $ 121.63万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8157185
• 关键词: Biochemistry; protein degradation

Research in the Biochemistry of Proteins Section is focused on the function and control of protein degradation in bacterial and human cells. Intracellular protein degradation plays a critical part in controlling the levels of cellular regulatory proteins and is an essential element of protein quality control systems. Protein degradation within the cytosol is carried out by ATP-dependent proteases, which are multimeric complexes made up of three essential components: a recognition domain that interacts with specific signals in target proteins, an ATP-driven protein unfoldase that structurally disrupts the bound protein and translocates it to the third component, and a tightly associated self-compartmentalized protease. Our research encompasses structural and biochemical analysis of the ATP-dependent Clp and Lon proteases from bacteria and from human mitochondria and assays of their biological activities. Studies are focused on four major areas: the basis for substrate selection by ClpA, ClpX, and Lon; structural dynamics of ClpP and the mechanism by which unfolded proteins enter the degradation chamber; conformational changes in the AAA+ domains of Clps and Lon that contribute to their activities; and the role of human ClpXP in mitochondrial function and signaling under conditions of stress. One universal mechanism of protein recognition operates by controlled exposure of a subset of amino acids at the N-terminus of proteins (N-degrons). Binding of N-degrons by components of the degradative machinery (N-recognins) allows these proteins to be targeted for degradation by ATP-dependent proteases. In bacterial cells ClpAP degrades proteins with N-degrons, and the adaptor protein, ClpS, is the N-recognin that binds to N-degrons. We found that delivery of substrates occurs with only one molecule of ClpS per ClpA hexamer despite the presence of 6 equivalent N-domains capable of binding ClpS. Limiting the number of substrates trying to enter the narrow axial channels of ClpA avoids steric clashes between them. ClpS has a bipartite binding mode in which the globular domain interacts with a ClpA N-domain and the N-terminal 20 amino acids interact with the axial channel. The first molecule of ClpS binds to ClpA with high affinity and blocks binding of subsequent ClpS molecules by sterically excluding other ClpS molecules. To learn how proteins with N-degrons arise in E. coli, we have begun to identify all the proteins that acquire N-degrons. We used a ClpS affinity column to capture proteins with exposed N-degrons from cell extract and have identified unique proteins bearing N-degrons. We found that the number of proteins captured from ClpS or ClpA mutant cells is 20-50 times greater than that obtained from wild-type cell extracts, suggesting the proteins are substrates for degradation by ClpAP/ClpS. During the next phase, we will isolate proteins accumulating when other components of the system, such as the Phe-aminotransferase, are mutated and will begin to determine the regulatory and physiological effects of targeting the substrates that have been identified. Studies with ClpP have been focused on the structural changes that are needed to allow substrate entry into the degradation chamber. Cryo electron microscopy shows that the axial pore of ClpP expands to a diameter of greater than 18 when ClpA binds. To investigate the conformation of the ClpP N-terminal loops during this structural rearrangement, we engaged in a collaboration to get the crystal structure of ClpP in the open-channel state. Acyldepsipeptide antibiotics (ADEPs) induce an open-channel conformation of ClpP that can take up unfolded proteins and is highly activated for peptide degradation. The crystal structure of ADEP bound to ClpP showed that ADEP binds to the hydrophobic groove on the surface of ClpP that serves as the docking site for the IGF/L loops of ClpA and ClpX. A short peptide with the sequence IGF was modeled in the position of the bound ADEP and confirmed that the ADEP-bound state mimics the state activated by ClpA or ClpX binding. By wedging between the subunits in the heptameric ring, ADEP causes the subunits to rotate upward and outward, which allows the N-terminal loops to snap into a parallel array expanding the axial channel and removing side chains from the path translocating substrates use. Deletion of the N-terminal loops allows ClpP to degrade unfolded proteins but destabilizes the interactions between the heptameric rings. We propose that the orientation of subunits affects both crowding of the N-terminal loops and the handle regions that form the interface between heptamers. Binding of ClpX and ClpA (or ADEP) causes the subunits to rotate, opening the axial channel and reorienting the handle regions to favor interaction between the rings. Because ClpA and ClpX stabilize an open configuration of the ClpP N-terminal loops, there is no additional energy requirement for translocating substrates into ClpP once substrates are unfolded and extruded through the axial channels of ClpA or ClpX. A breakthrough has been obtained in the structural analysis of Lon proteases. Lons are composed of complexes of one type of subunit containing a tandem alignment of the recognition, chaperone/unfoldase, and protease domains. In a collaborative study, the crystal structure of nucleotide-bound hexameric state of Lon protease was solved. The structure confirmed that Lon protease domains are oriented facing the internal chamber of the chaperone domains, creating a large chamber in which unfolded or partially unfolded proteins are directly exposed to the proteolytic active sites. The structure also reveals that Lon subunits are in alternating conformational states around the ring. The closed state with tightly bound ADP cannot be adjacent to another subunit in the same state, indicating that the subunits within the hexamer function in a sequential rather than concerted manner. Lon has a unique gating mechanism in which loops protruding from the chaperone domain, which are subject to nucleotide-dependent reorganization, combine with either N-domains or inserted membrane-spanning domains to form a gated passageway for recognizing and screening appropriate substrates. This structure opens the way to detailed analysis of conformational states and functional residues in Lon proteases that underlie the important biological roles of this enzyme, which has been implicated in a range of vital processes. Human ClpX and ClpP function within the mitochondrial matrix and are needed for mitochondrial integrity and for cell survival. Depletion of hClpP or hClpX following treatment with siRNA also leads to cell death. Down regulation of hClpP affects the timing and extent of apoptotic cell death in response to DNA damage, death receptor binding, and kinase inhibition. The similarity in response to 3 divergent stress signaling pathways suggests that hClpP alters the basal structure or physiology of mitochondria. Down regulation of hClpP sensitizes cells to various drugs that have been used to treat cancer and partially reverse the drug resistance of multidrug resistant cells. We have begun to investigate the changes in the mitochondrial proteome in response to depletion and overexpression of hClpP and HClpX. Analysis of 2D gels followed by mass spectrometry has identified more than 30 proteins whose levels increased within 16 hours of depletion of hClpP. A number of the proteins identified are involved in the response to oxidative and other stress. To identify low abundance substrates, future efforts will be directed at improving the sensitivity of the mass spectroscopic methods used and particular focus will be placed on enriching for potential physiological targets by trapping substrates in inactive forms of ClpP and by pull-down procedures using ClpX complexes.

蛋白质生物化学部分的研究重点是细菌和人类细胞中蛋白质降解的功能和控制。细胞内蛋白质降解在控制细胞调节蛋白水平方面发挥着关键作用，并且是蛋白质质量控​​制系统的重要组成部分。细胞质内的蛋白质降解是由 ATP 依赖性蛋白酶进行的，这些蛋白酶是由三个基本成分组成的多聚体复合物：与目标蛋白中的特定信号相互作用的识别域、从结构上破坏结合蛋白并将其易位到第三个成分的 ATP 驱动的蛋白解折叠酶，以及紧密相关的自区室化蛋白酶。我们的研究包括对来自细菌和人类线粒体的 ATP 依赖性 Clp 和 Lon 蛋白酶进行结构和生化分析，并测定其生物活性。研究主要集中在四个主要领域：ClpA、ClpX 和 Lon 底物选择的基础； ClpP的结构动力学以及未折叠蛋白进入降解室的机制； Clps 和 Lon 的 AAA+ 结构域的构象变化有助于其活动；以及人 ClpXP 在应激条件下线粒体功能和信号传导中的作用。蛋白质识别的一种通用机制是通过控制蛋白质 N 末端氨基酸子集（N 降解决定子）的暴露来实现的。 N-降解决定子与降解机制（N-识别蛋白）成分的结合使得这些蛋白质成为 ATP 依赖性蛋白酶降解的目标。在细菌细胞中，ClpAP 使用 N-降解决定子降解蛋白质，而接头蛋白 ClpS 是与 N-降解决定子结合的 N-识别蛋白。我们发现，尽管存在 6 个能够结合 ClpS 的等效 N 结构域，但每个 ClpA 六聚体仅用一个 ClpS 分子进行底物递送。限制试图进入 ClpA 狭窄轴向通道的底物数量可以避免它们之间的空间冲突。 ClpS 具有二分结合模式，其中球状结构域与 ClpA N 结构域相互作用，N 端 20 个氨基酸与轴向通道相互作用。 ClpS 的第一个分子以高亲和力与 ClpA 结合，并通过空间排斥其他 ClpS 分子来阻止后续 ClpS 分子的结合。为了了解带有 N-降解决定子的蛋白质如何在大肠杆菌中产生，我们已经开始鉴定所有获得 N-降解决定子的蛋白质。我们使用 ClpS 亲和柱从细胞提取物中捕获带有暴露的 N-降解决定子的蛋白质，并鉴定出带有 N-降解决定子的独特蛋白质。我们发现从 ClpS 或 ClpA 突变细胞中捕获的蛋白质数量比从野生型细胞提取物中捕获的蛋白质数量多 20-50 倍，表明这些蛋白质是 ClpAP/ClpS 降解的底物。在下一阶段，我们将分离当系统的其他成分（例如苯丙氨酸转氨酶）发生突变时积累的蛋白质，并将开始确定针对已识别底物的调节和生理效应。 ClpP 的研究重点是允许底物进入降解室所需的结构变化。冷冻电镜显示，当 ClpA 结合时，ClpP 的轴向孔直径扩大到大于 18 。为了研究 ClpP N 端环在这种结构重排过程中的构象，我们进行了合作，以获得开通道状态下 ClpP 的晶体结构。酰基缩酚肽抗生素 (ADEP) 诱导 ClpP 形成开放通道构象，可以吸收未折叠的蛋白质，并高度激活肽降解。 ADEP 与 ClpP 结合的晶体结构表明，ADEP 与 ClpP 表面的疏水沟结合，该疏水沟作为 ClpA 和 ClpX 的 IGF/L 环的对接位点。在结合的 ADEP 的位置对具有序列 IGF 的短肽进行建模，并证实 ADEP 结合状态模拟了 ClpA 或 ClpX 结合激活的状态。通过楔入七聚环中的亚基之间，ADEP 导致亚基向上和向外旋转，这使得 N 端环卡入平行阵列，扩大轴向通道并从易位底物使用的路径中去除侧链。 N 末端环的删除使得 ClpP 能够降解未折叠的蛋白质，但会破坏七聚环之间的相互作用。我们提出亚基的方向影响 N 末端环的拥挤和形成七聚体之间界面的手柄区域。 ClpX 和 ClpA（或 ADEP）的结合导致亚基旋转，打开轴向通道并重新定向手柄区域以有利于环之间的相互作用。由于 ClpA 和 ClpX 稳定 ClpP N 端环的开放构型，因此一旦底物展开并通过 ClpA 或 ClpX 的轴向通道挤出，就不需要额外的能量将底物转移到 ClpP 中。 Lon蛋白酶结构分析取得突破。 Lons 由一种类型的亚基复合物组成，该亚基包含串联排列的识别、分子伴侣/解折叠酶和蛋白酶结构域。在一项合作研究中，Lon 蛋白酶的核苷酸结合六聚体状态的晶体结构得到了解决。该结构证实，Lon 蛋白酶结构域面向伴侣结构域的内室，形成一个大室，其中未折叠或部分未折叠的蛋白质直接暴露于蛋白水解活性位点。该结构还揭示了 Lon 亚基在环周围处于交替构象状态。具有紧密结合的 ADP 的闭合状态不能与处于相同状态的另一个亚基相邻，这表明六聚体内的亚基以顺序而不是协同的方式起作用。 Lon 具有独特的门控机制，其中从伴侣结构域突出的环（受到核苷酸依赖性重组）与 N 结构域或插入的跨膜结构域结合，形成门控通道，用于识别和筛选适当的底物。这种结构为详细分析 Lon 蛋白酶的构象状态和功能残基开辟了道路，这些构象状态和功能残基构成了该酶的重要生物学作用，与一系列重要过程有关。人类 ClpX 和 ClpP 在线粒体基质内发挥作用，是线粒体完整性和细胞存活所必需的。 siRNA 处理后 hClpP 或 hClpX 的消耗也会导致细胞死亡。 hClpP 的下调会影响因 DNA 损伤、死亡受体结合和激酶抑制而导致的凋亡细胞死亡的时间和程度。对 3 条不同应激信号通路的响应的相似性表明 hClpP 改变线粒体的基础结构或生理学。 hClpP 的下调使细胞对各种用于治疗癌症的药物敏感，并部分逆转多药耐药细胞的耐药性。我们已经开始研究线粒体蛋白质组因 hClpP 和 HClpX 的消耗和过度表达而发生的变化。对 2D 凝胶进行质谱分析，鉴定出超过 30 种蛋白质，其水平在 hClpP 耗尽后 16 小时内增加。已鉴定的许多蛋白质参与对氧化和其他应激的反应。为了识别低丰度底物，未来的努力将致力于提高所用质谱方法的灵敏度，并且特别关注通过以 ClpP 的非活性形式捕获底物以及使用 ClpX 复合物的下拉程序来富集潜在的生理目标。