Biochemistry of Energy-Dependent (Intracellular) Protein Degradation

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和代谢调控途径的分析。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；以及人类ClpXP在细胞应激条件下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分子才能将底物递送到ClpAP复合物。一次只允许一个底物进入ClpA的轴向通道，避免了空间冲突和更有效的蛋白质易位。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 n端区域在限制结合化学计量中起作用，也在促进底物向ClpA的传递中起作用。利用低温电子显微镜和x射线晶体学对ClpS和蛋白质底物与ClpA六聚体结合的三元配合物进行结构研究，将有助于了解ClpS n端如何与ClpA结合，以及在atp驱动的底物转移过程中，这种结合是如何变化的。相关研究旨在了解含有N-degrons的蛋白质是如何产生的，并确定大肠杆菌中所有获得N-degrons的蛋白质。我们使用ClpS亲和柱捕获暴露N-degrons的蛋白质，并鉴定出50种独特的带有N-degrons的蛋白质。我们发现，分离的蛋白质数量取决于细胞中ClpS和ClpA的含量，证实了这些蛋白质是ClpAP/ClpS降解的底物。我们还发现一些蛋白质在一个依赖于酶的过程中获得n -degron，该酶修饰蛋白质的n端，使其易于被ClpS识别。我们正在测试的一个假设是，翻译起始的错误会引起标记蛋白质n端系统的活动，从而将其作为降解的目标。我们正在研究翻译起始有缺陷的菌株，并对培养物进行营养限制，以导致翻译错误增加，并研究获得新n -degron的蛋白质的数量和性质。我们发现，当细胞携带aat（转氨酶基因）突变时，当保真度所需的翻译起始因子之一也发生突变时，细胞生长非常差。这一发现表明转氨酶的活性与翻译的保真度之间存在联系。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。低温电镜观察显示，ClpP的轴向孔在与ClpA结合时，膨胀至直径大于18埃。我们与合作者获得了酰基沉积肽抗生素ADEP1结合诱导的开放状态下的ClpP晶体结构。开放通道形式的ClpP可以吸收未折叠的蛋白质，并对肽降解具有高度活性。当ADEP被添加到细胞中时，它与ClpP结合，并允许它在蛋白质合成过程中靶向新生多肽，并且在新合成的蛋白质折叠之前降解导致细胞死亡。我们假设细胞死亡是由于一个或几个关键的细胞蛋白质在完全合成之前被破坏，而不是蛋白质合成的整体下降。我们正在使用ClpP的突变形式在体内捕获新生蛋白质，我们将使用质谱法识别捕获的蛋白质，然后使用遗传方法确定必须不断合成以使细胞生长的蛋白质。我们已经开始寻找其他可以与ClpP结合并诱导ClpP开放状态的化合物。这些化合物将成为开发潜在新型抗生素的先导分子，并将作为体外研究的额外配体，旨在了解ClpP通过结构变化打开其底物进入通道的机制。我们研究人类ClpX和ClpP的主要目标是确定它们在线粒体中的功能，并发现为什么它们对于线粒体完整性和细胞存活是必需的。siRNA处理后hClpP或hClpX的缺失导致细胞死亡，至少部分原因是诱导细胞凋亡。在DNA损伤、死亡受体结合和激酶抑制的反应中，hClpP的下调使细胞对凋亡细胞死亡敏感。对3种不同的应激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。我们发现选择顺铂耐药的细胞ClpX和ClpP的表达水平升高。我们目前的工作主要集中在描述线粒体蛋白质组在hClpP和hClpX耗尽和过表达时的变化。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，其中许多与应激反应有关。我们还研究了抗生素抗坏血氯素衍生物诱导内质网应激对线粒体ClpP和Lon蛋白酶水平的影响。其中一种衍生物AS-6可导致内质网应激，诱导自噬并导致细胞凋亡。有趣的是，在AS-6处理后，ClpP水平短暂上升然后下降，我们正在研究在这些条件下ClpP的缺失是否导致了细胞凋亡。我们发现ADEP在体外也能激活人ClpP，诱导细胞应激并杀死细胞。过表达野生型而非失活的ClpP突变体使细胞对ADEP更敏感。我们正在分离adep激活的ClpP的潜在靶点，方法是使用无活性的ClpP突变体捕获它们，拉下捕获的底物/ClpP复合物，并通过消化和串联质谱法鉴定捕获的蛋白质。

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 and the metabolic and regulatory pathways in which they function. 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 cellular 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. We found that delivery of substrates to the ClpAP complex occurs with only one molecule of ClpS per ClpA hexamer despite the presence of 6 equivalent N-domains capable of binding ClpS. Allowing only one substrate at a time to enter the axial channels of ClpA avoids steric clashes and more efficient protein translocation. 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 ClpS N-terminal region plays a role in limiting the binding stoichiometry and also in facilitating hand-off of substrates to ClpA. Structural studies of the ternary complex of ClpS and protein substrate bound to ClpA hexamers using cryo electron microscopy and x-ray crystallography will provide insight as to how the ClpS N-terminus binds to ClpA and how the binding changes upon ATP-driven substrate transfer. Related studies are designed to learn how proteins with N-degrons arise and to identify all the proteins that acquire N-degrons in E. coli. We have used a ClpS affinity column to capture proteins with exposed N-degrons and have identified >50 unique proteins bearing N-degrons. We found that the number of proteins isolated depends on how much ClpS and ClpA are present in the cells confirming that the proteins are substrates for degradation by ClpAP/ClpS. We have also found some proteins acquire N-degrons in a process dependent on the enzyme, Phe-aminotransferase, which modifies the N-terminus of proteins to make them easily recognized by ClpS. One hypothesis we are testing is that errors in translation initiation evoke the activity of a system for tagging the N-terminus of the protein, thereby targeting it for degradation. We are examining strains defective in translation initiation and are subjecting cultures to nutrient limitation to cause increased errors in translation and are examining the number and nature of the proteins that acquire novel N-degrons. We found that when cells carry a mutation in aat, the gene for the aminotransferase, they grow very poorly when one of the translation initiation factors required for fidelity is also mutated. This finding suggests a link between the activity of the aminotransferase and fidelity of translation. 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 angstroms when ClpA binds. With our collaborators, we obtained the crystal structure of ClpP in the open state induced when the acyldepsipeptide antibiotic, ADEP1, is bound. The open-channel form of ClpP can take up unfolded proteins and is highly activated for peptide degradation. When ADEP is added to cells it binds to ClpP and allows it to target nascent polypeptides during protein synthesis, and degradation of the newly synthesized proteins before they can fold leads to cell death. We hypothesize that cell death results from destruction of one or a few critical cellular proteins before they can be fully synthesized rather then a global drop in protein synthesis. We are trapping nascent proteins in vivo using a mutant form of ClpP, and we will identify the trapped proteins using mass spectrometry and then use genetic methods to define the ones that must be constantly synthesized to allow cells to grow. We have begun to search for other compounds that can bind to ClpP and induce the open state of ClpP. These compounds will be lead molecules for the development of potential novel antibiotics and will serve as additional ligands for in vitro studies aimed at understanding the mechanism by which ClpP undergoes the structural changes that open its substrate access channel. The major goal of our studies of human ClpX and ClpP is to define their functions within mitochondria and to discover why they are needed for mitochondrial integrity and for cell survival. Depletion of hClpP or hClpX following treatment with siRNA leads to cell death in part at least to induction of apoptosis. Down regulation of hClpP sensitizes cells to 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 found that cells selected for resistance to cisplatin have elevated levels of expression of ClpX and ClpP. Out current efforts are focused on describing the changes in the mitochondrial proteome in response to depletion and over expression 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, many of which are involved in the stress responses. We have also examined the effects of derivatives of the antibiotic ascochlorin that induce endoplasmic reticulum stress on the levels of mitochondrial ClpP and Lon proteases. One derivative, AS-6, leads to ER stress, induces autophagy and leads to apoptotic cell death. Interestingly, ClpP levels transiently increase and then decline after AS-6 treatment, and we are investigating whether the loss of ClpP is responsible for induction of apoptosis under these conditions. We have found that ADEP, which also activates human ClpP in vitro, induces cellular stress and kills cells. Over expression of wild type but not inactive mutants of ClpP renders cells more sensitive to ADEP. We are in the process of isolating potential targets of ADEP-activated ClpP by trapping them using an inactive ClpP mutant, pulling down the trapped substrate/ClpP complex, and identifying the trapped proteins by digestion followed by tandem MS/MS.