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.

蛋白质片段生物化学的研究集中在细菌和人类细胞中蛋白质降解的功能和控制上。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，并且是蛋白质质量控​​制系统的重要元素。细胞质内的蛋白质降解是由ATP依赖性蛋白酶进行的，ATP依赖性蛋白酶是由三个基本成分组成的多聚体络合物：与靶蛋白中特定信号相互作用的识别域，ATP驱动的蛋白蛋白IFLOLDASE，一种结构上会破坏蛋白质并将其转化为蛋白质，并将其转化为第三组分的蛋白质，并与第三种相关的自我分解。我们的研究包括对ATP依赖性CLP和LON蛋白酶的结构和生化分析，来自人类的线粒体以及其生物学活性的测定。研究集中在四个主要领域：CLPA，CLPX和LON选择底物的基础； CLPP的结构动力学和展开的蛋白质进入降解室的机制； CLP和LON的AAA+领域的构象变化有助于其活动；以及在应力条件下人类ClPXP在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理是通过控制蛋白N末端（N-脱果龙）的一部分氨基酸的暴露来运行的。通过降解机制（N-识别剂）的成分结合N-脱绿素的结合使这些蛋白质可以通过ATP依赖性蛋白酶来降解。在细菌细胞中，CLPAP用N-脱果子降解蛋白质，而衔接蛋白Clps是与N-脱果子结合的N-识别蛋白。我们发现，尽管存在6个同等的N型域，但底物的递送仅以每个CLPA六聚体的CLP分子的形式出现。限制试图输入CLPA狭窄轴向通道的底物的数量避免了它们之间的空间冲突。 CLP具有两分结的模式，其中球状结构域与CLPA N域相互作用，而N末端20氨基酸与轴向通道相互作用。 CLP的第一个分子与CLPA结合具有高亲和力的CLPA，并通过在空间上排除其他CLPS分子来结合随后的CLPS分子的结合。为了了解大肠杆菌中蛋白质的蛋白质如何出现，我们已经开始识别所有获得N-脱果龙的蛋白质。我们使用CLPS亲和力柱从细胞提取物中捕获蛋白质，并鉴定出具有N-脱果龙的独特蛋白质。我们发现，从CLPS或CLPA突变细胞捕获的蛋白质数量比从野生型细胞提取物获得的蛋白质高20-50倍，这表明蛋白质是通过CLPAP/CLPS降解的底物。在下一阶段，当系统的其他组件（例如PHE-氨基转移酶）被突变时，我们将隔离蛋白质，并开始确定靶向已鉴定的底物的调节和生理效应。与CLPP的研究集中在允许底物进入降解室所需的结构变化上。冷冻电子显微镜表明，当CLPA结合时，CLPP的轴向孔扩展到大于18的直径。为了调查在此结构重排期间CLPP N末端环的构象，我们进行了协作，以使CLPP的晶体结构处于开放通道状态。阿素蛋白皮肽抗生素（EDEPS）诱导CLPP的开放通道构象，该构型可以吸收展开的蛋白质，并高度激活用于肽降解。与CLPP结合的ADEP的晶体结构表明，ADEP结合了CLPP表面上的疏水凹槽，该凹槽用作CLPA和CLPX的IGF/L环的对接位点。用序列IGF序列的短肽在结合EDEP的位置进行了建模，并确认EDEP结合状态模仿CLPA或CLPX结合激活的状态。通过在七聚环中的亚基之间楔入，ADEP会导致亚基向上和向外旋转，这使得N末端环可以将轴向通道的平行阵列捕捉到轴向通道的平行阵列，并从转换底物的路径上移除侧链使用。 N末端环的删除使CLPP降解了展开的蛋白质，但破坏了七聚环之间的相互作用。我们建议，亚基的方向既影响N端环的拥挤，又会影响形成七聚体之间界面的手柄区域。 CLPX和CLPA（或ADEP）的结合会导致亚基旋转，打开轴向通道并重新定位手柄区域以有利于环之间的相互作用。由于CLPA和CLPX稳定了CLPP N末端回路的开放构型，因此一旦展开底物并通过CLPA或Clpx的轴向通道挤出了底物，就没有其他能量要求将底物转移到CLPP中。在LON蛋白酶的结构分析中，已经获得了突破。 lons由一种类型的亚基的复合物组成，其中包含识别识别，伴侣/展开酶和蛋白酶结构域的串联比对。在一项协作研究中，解决了lon蛋白酶的六聚体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，形成一个大型腔室，其中展开或部分展开的蛋白质直接暴露于蛋白水解活性位点。该结构还表明，LON亚基处于环形周围的交替构象状态。具有紧密绑定ADP的封闭状态不能与同一状态的另一个亚基相邻，表明六聚体函数中的亚基是以顺序而不是协同的方式相邻的。 LON具有独特的门控机制，在该机制中，从伴侣结构域伸出的环将受核苷酸依赖性的重组约束，并与N域或插入的膜跨膜域结合，形成封闭式通道，以识别和筛选适当的底物。这种结构为详细分析构象状态和LON蛋白酶中功能残基的详细分析开辟了道路，这些蛋白酶是该酶的重要生物学作用的基础，该酶与一系列重要过程有关。线粒体基质中的人类CLPX和CLPP功能是线粒体完整性和细胞存活所需的。用siRNA处理后HCLPP或HCLPX的耗竭也导致细胞死亡。 HCLPP的下调会影响DNA损伤，死亡受体结合和激酶抑制作用的凋亡细胞死亡的时间和程度。响应3个发散应力信号通路的相似性表明HCLPP改变了线粒体的基础结构或生理学。下调HCLPP的下调将细胞敏感到用于治疗癌症并部分逆转多药耐药细胞的耐药性的各种药物。我们已经开始研究线粒体蛋白质组的变化，以响应HCLPP和HCLPX的耗竭和过表达。对2D凝胶的分析，然后是质谱法，已经确定了30多种蛋白质，其水平在HCLPP耗竭后的16小时内升高。鉴定出的许多蛋白质参与对氧化和其他应激的反应。为了确定低丰度底物，未来的努力将致力于提高所使用的质谱法的敏感性，并通过将CLPX复合物的下拉手术捕获不活跃的CLPP形式和下拉手术，将特定的重点放在对潜在生理目标的富集上。