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起着至关重要的作用，是蛋白质质量控制系统的重要组成部分。胞质溶胶内的蛋白质降解是由atp依赖的蛋白酶进行的，它是由三个基本成分组成的多聚体复合物：一个与靶蛋白中的特定信号相互作用的识别结构域，一个atp驱动的蛋白质展开酶，它在结构上破坏结合蛋白并将其易位到第三个成分，以及一个紧密相关的自区隔蛋白酶。我们的研究包括对细菌和人类线粒体中atp依赖性Clp和Lon蛋白酶的结构和生化分析，以及它们的生物活性测定。研究主要集中在四个方面：ClpA、ClpX和Lon选择底物的基础；ClpP的结构动力学和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它们的活性；以及应激条件下人类ClpXP在线粒体功能和信号传导中的作用。一种普遍的蛋白质识别机制是通过控制蛋白质n端氨基酸子集（N-degrons）的暴露来操作的。通过降解机制（n -识别蛋白）的组件结合N-degrons允许这些蛋白质成为atp依赖性蛋白酶降解的目标。在细菌细胞中，ClpAP降解带有N-degrons的蛋白质，而接头蛋白ClpS是与N-degrons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六聚体只有一个ClpS分子可以递送底物。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具有两部分结合模式，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，并通过立体排斥其他ClpS分子来阻断后续ClpS分子的结合。为了了解含有N-degrons的蛋白质是如何在大肠杆菌中产生的，我们已经开始鉴定所有获得N-degrons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N-degrons的蛋白质，并鉴定出带有N-degrons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是从野生型细胞提取物中获得的20-50倍，这表明这些蛋白质是ClpAP/ClpS降解的底物。在下一阶段，我们将分离当系统的其他组分（如ph -氨基转移酶）发生突变时积累的蛋白质，并将开始确定靶向已确定的底物的调节和生理作用。ClpP的研究主要集中在允许底物进入降解室所需的结构变化上。低温电镜显示，ClpA结合后，ClpP的轴向孔扩大到直径大于18。为了研究ClpP n端环在这种结构重排过程中的构象，我们参与了一项合作，以获得开放通道状态下ClpP的晶体结构。酰基沉积肽抗生素（adep）诱导ClpP的开放通道构象，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表面的疏水沟槽结合，作为ClpA和ClpX的IGF/L环的对接位点。在结合ADEP的位置模拟了一个序列为IGF的短肽，证实了ADEP的结合状态模拟了ClpA或ClpX结合激活的状态。通过楔入七聚体环中的亚基之间，ADEP使亚基向上和向外旋转，从而允许n端环进入平行阵列，扩展轴向通道并从转运底物的路径中去除侧链。n端环的缺失允许ClpP降解未折叠的蛋白质，但破坏了七聚体环之间的相互作用。我们提出，亚基的取向既影响n端环的拥挤，也影响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柄区，以促进环之间的相互作用。由于ClpA和ClpX稳定了ClpP n端环的开放结构，一旦底物展开并通过ClpA或ClpX的轴向通道挤压，就不需要额外的能量来将底物转移到ClpP中。在Lon蛋白酶的结构分析方面取得了突破性进展。长链由一种亚基复合物组成，其中包含识别、伴侣/展开酶和蛋白酶结构域的串联排列。在一项合作研究中，解决了lon蛋白酶的核苷酸结合六聚体状态的晶体结构。该结构证实，Lon蛋白酶结构域面向伴侣结构域的内部腔室，形成一个大腔室，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ADP的闭合状态不能与具有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调的方式起作用。Lon具有独特的门控机制，其中从伴侣结构域伸出的环，受到核苷酸依赖性重组的影响，与n结构域或插入的跨膜结构域结合，形成识别和筛选适当底物的门控通道。这种结构为详细分析lonp蛋白酶的构象状态和功能残基开辟了道路，这是该酶重要生物学作用的基础，涉及一系列重要过程。人类ClpX和ClpP在线粒体基质中起作用，是线粒体完整性和细胞存活所必需的。siRNA处理后hClpP或hClpX的缺失也会导致细胞死亡。在DNA损伤、死亡受体结合和激酶抑制的作用下，hClpP的下调影响凋亡细胞死亡的时间和程度。对3种不同胁迫信号通路的相似反应表明，hClpP改变了线粒体的基本结构或生理。hClpP的下调使细胞对各种用于治疗癌症的药物敏感，并部分逆转多重耐药细胞的耐药性。我们已经开始研究线粒体蛋白质组在hClpP和HClpX缺失和过表达时的变化。用质谱法对2D凝胶进行分析，发现30多种蛋白质在hClpP耗尽后16小时内水平升高。许多已确定的蛋白质与氧化应激和其他应激反应有关。为了识别低丰度底物，未来的工作将集中在提高所用质谱方法的灵敏度上，并将特别关注通过捕获无活性形式的ClpP底物和使用ClpX复合物的下拉程序来富集潜在的生理目标。