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。细胞内蛋白质降解对控制细胞调节蛋白的水平至关重要，是蛋白质质量控制系统的关键要素。蛋白质降解是由多聚atp依赖的蛋白酶进行的，它有三个组成部分：底物识别域，atp驱动的蛋白质展开酶和紧密相关的自区隔化蛋白酶。我们的研究包括细菌和人类线粒体中atp依赖性Clp蛋白酶的结构和生化研究，以及它们的生物活性和功能分析。目前的研究主要集中在四个方面：ClpAP和ClpS在含有n -degron的蛋白质降解中的作用；ClpP的结构动力学和未折叠蛋白进入降解室的机制；人线粒体ClpX对底物的识别及其在线粒体功能和细胞生长中的作用以及ClpP在调节人体细胞对顺铂等抗癌药物的敏感性方面的作用。n端规则是一种普遍的机制，通过该机制，蛋白质通过n端氨基酸子集（称为N-degrons）被降解。n -degron被降解机制的组成部分（N-recognins）识别，使蛋白质成为atp依赖性蛋白酶降解的目标。在大肠杆菌中，ClpS结合N-degrons并将底物传递给ClpAP复合物。大肠杆菌中的n -degron是亮氨酸、苯丙氨酸、酪氨酸和色氨酸。具有n端赖氨酸和精氨酸的蛋白质通过Aat的作用获得N-degron， Aat是一种氨基转移酶，可将亮氨酸或苯丙氨酸添加到这些n端。由于新合成的蛋白质通常不含有这些n端氨基酸，因此n -degron必须作为翻译后修饰或蛋白质翻译错误的结果而产生。我们使用ClpS亲和柱捕获带有N-degrons的蛋白质，并回收了60种独特的蛋白质。许多蛋白质仅在具有功能的苯丙氨酸/亮氨酸转氨酶的细胞中可见，表明它们首先与n端赖氨酸或精氨酸一起产生，随后被Aat修饰。我们分析了几种具有缺陷蛋白起始因子的突变体，但没有显示出N-degrons数量或产量的增加。许多带有N-degrons的蛋白质是全长蛋白质的截断版本，似乎是部分蛋白质水解的结果。我们分析了50多个缺乏单一肽酶的大肠杆菌突变体，发现在缺乏特定肽酶的细胞中没有出现几种带有n -degron的蛋白质。我们正在继续对缺乏多种肽酶的细胞进行分析，以建立特定肽酶组与具有n -degron的特定蛋白质外观之间的联系。我们的假设是，蛋白质受到持续的监测，以评估其功能，反映在它们的折叠完整性或与功能伙伴的关联。ClpP的研究主要集中在由酰基沉积肽抗生素（ADEP）激活引起的细胞死亡机制，以及允许底物进入降解室所需的结构变化。ADEP与ClpP结合使其能够靶向新生多肽和功能蛋白的非结构化区域。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，或者当它们的非结构化区域由于与配体或相互作用伙伴的相互作用变化而可接近时。我们使用缺乏蛋白水解活性的ClpP突变体在体内捕获蛋白质，并将使用质谱法鉴定捕获的蛋白质，以确定在ADEP存在的情况下ClpP对蛋白质降解的总体程度。ClpP可以以两种状态存在，一种是手柄区域相互交错以扩大降解室并允许蛋白水解，另一种是手柄处于塌陷状态，这是一种潜在状态，或者是在降解周期中形成的一种瞬时中间物，允许肽产物的释放。我们发现Zn抑制ClpP，可能是通过稳定坍塌状态。我们在锌的存在下生长ClpP晶体，以获得结构数据，这将为环-环相互作用的动力学及其对活性的影响提供见解。最近，我们开始与哈佛医学院的Alfred Goldberg实验室合作，他为我们提供了从结核分枝杆菌中纯化的ClpP。结核分枝杆菌有两种ClpP同工酶，它们相互作用形成表达酶活性所需的混合四聚体。在一个复合体中存在两种形式的ClpP将有助于环相互作用的结构分析，例如，通过允许组装只有一个环突变的四面体。我们已经找到了形成Mbt-ClpP可衍射晶体的条件，并有望在今年获得天然蛋白质的结构。结核分枝杆菌ClpP是一个很有希望的目标，因为它对生长至关重要。晶体结构应该指导小分子抑制剂的设计，这些小分子抑制剂可能作为开发具有治疗潜力的化合物的先导。我们研究人类ClpX和ClpP的目的是确定它们在线粒体中的功能，并发现为什么它们对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。我们分析了线粒体蛋白质组随hClpP变化的变化。在hClpP耗竭的16小时内，超过30种蛋白质增加，其中许多与应激反应有关。ADEP诱导细胞应激并杀死人体细胞。过表达野生型而非失活的ClpP突变体使细胞对ADEP更敏感。暴露于ADEP后的细胞蛋白质组学分析显示，许多与应激反应相关的蛋白质升高。ADEP治疗后，一种主要阴离子转运蛋白的水平也发生了改变。ClpP在线粒体阴离子通量中的作用有待进一步研究。下调hClpP使细胞对顺铂敏感。值得注意的是，独立选择的顺铂耐药细胞发现ClpX和ClpP的表达水平升高。我们发现ClpP的水平影响顺铂在几种人类细胞系中的积累。当ClpP被敲低时，顺铂总蓄积增加，顺铂介导的线粒体DNA损伤明显增加。我们的研究结果表明，顺铂治疗后，线粒体DNA损伤在诱导细胞凋亡中起重要作用。为了研究ClpP和顺铂积累之间的联系，我们测量了在控制ClpP水平时铜转运体的水平。顺铂被认为是搭上铜转运体的便车进入和离开细胞。当ClpP过表达或敲低时，主要铜转运蛋白Ctr1未见变化。然而，ClpP水平与铜外排泵ATP7A水平之间存在显著相关性。ATP7A随hClpP的降低成比例降低，当hClpP过表达时，ATP7A升高。数据表明hClpP在影响ATP7A中起间接作用。我们假设hClpP影响线粒体和细胞质之间的金属离子通量，从而导致ATP7A上调或下调，并使细胞对顺铂的敏感性或高或低。我们正在进行全细胞测定，以测量hClpP敲低后的线粒体离子通量、线粒体膜电位和其他线粒体活动。

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 is essential to control the levels of cellular regulatory proteins and is a critical element of protein quality control systems. Protein degradation is performed by multimeric ATP-dependent proteases, which have three constituents: a substrate recognition domain, an ATP-driven protein unfoldase, and a tightly associated self-compartmentalized protease. Our research encompasses structural and biochemical studies of the ATP-dependent Clp proteases from bacteria and human mitochondria as well as analysis of their biological activities and functions. Current efforts focus on four major areas: the role of ClpAP and ClpS in degradation of proteins containing N-degrons; structural dynamics of ClpP and the mechanism by which unfolded proteins enter the degradation chamber; substrate recognition by human mitochondrial ClpX and the role of ClpXP in mitochondrial function and cell growth; and the role of ClpP in modulating the sensitivity of human cells to anti-cancer drugs such as cisplatin.The N-end rule is a universal mechanism by which proteins are targeted for degradation by virtue of a subset of N-terminal amino acids, referred to as N-degrons. N-degrons are recognized by components of the degradative machinery (N-recognins) enabling the proteins to be targeted for degradation by ATP-dependent proteases. In E. coli, ClpS binds N-degrons and delivers substrates to the ClpAP complex. The N-degrons in E. coli are leucine, phenylalanine, tyrosine, and tryptophan. Proteins with N-terminal lysine and arginine acquire an N-degron through the action of Aat, an amino transferase that adds leucine or phenylalanine to either of these N-termini. Because newly synthesized proteins do not as a rule contain these N-terminal amino acids, N-degrons must arise as a result of post-translational modification or through errors in protein translation. We used a ClpS affinity column to capture proteins with N-degrons and recovered >60 unique proteins. Many of the proteins were seen only in cells with a functional Phe/Leu-aminotransferase, indicating that they first arise with an N-terminal lysine or arginine, which is subsequently modified by Aat. We assayed several mutants with defective protein initiation factors, but none has shown an increase in the number or yield of N-degrons. Many proteins with N-degrons were truncated versions of full-length proteins and appear to result from partial proteolysis. We analyzed more than 50 E. coli mutants lacking single peptidases and found that several proteins with N-degrons did not appear in cells lacking specific peptidases. We are continuing the analysis with cells lacking multiple peptidases to establish links between specific sets of peptidases and the appearance of specific proteins with N-degrons. Our hypothesis is that proteins are subject to constant surveillance to assess their functionality as reflected by their folding integrity or their association with functional partners. Studies with ClpP are focused on the mechanism of cell death that results from activation by the acyldepsipeptide antibiotic, ADEP, and the structural changes that are needed to allow substrate entry into the degradation chamber. ADEP binding to ClpP allows it to target nascent polypeptides and unstructured regions of functional proteins. We hypothesize that cell death results from destruction of one or a few critical cellular proteins either before they are fully synthesized or when their unstructured regions are accessible as a result of changes in interactions with ligands or interacting partners. We are trapping proteins in vivo using a mutant of ClpP that lacks proteolytic activity and will identify the trapped proteins using mass spectrometry to determine the global extent of protein degradation by ClpP in the presence of ADEP.ClpP can exist in two states, one with the handle regions interlaced to expand the degradation chamber and allow proteolysis and another with the handles in a collapsed state, which is either a latent state or is a transient intermediate formed during the degradation cycle to allow release of peptide products. We found that Zn inhibits ClpP, possibly by stabilizing the collapsed state. We are growing crystals of ClpP in the presence of Zn to obtain structural data that will provide insight about the dynamics of ring-ring interactions and their affect on activity. Recently, we began collaboration with the laboratory of Alfred Goldberg at Harvard Medical School, who has provided us with purified ClpP from Mycobacterium tuberculosis. M. tuberculosis has two isozymes of ClpP, which interact with one another to form a mixed tetradecamer needed to express enzymatic activity. The presence of two forms of ClpP in one complex will facilitate structural analysis of the ring interactions, for example, by allowing assembly of tetradecamers in which only one ring is mutated. We have found conditions that give diffractable crystals of Mbt-ClpP and expect to have a structure of the native protein this year. M. tuberculosis ClpP is a promising target, because it is essential for growth. A crystal structure should guide the design of small molecules inhibitors that might serve as leads for the development of compounds with therapeutic potential.The goal of our studies of human ClpX and ClpP is to define their functions in mitochondria and to discover why they are needed for mitochondrial integrity and cell survival. Depletion of hClpP or hClpX following treatment with siRNA leads to cell death. We have analyzed the changes in the mitochondrial proteome in response to changes in hClpP. More than 30 proteins are increased within 16 hours of depletion of hClpP, many of which are involved in stress responses. ADEP induces cellular stress and kills human cells. Over expression of wild type but not inactive mutants of ClpP renders cells more sensitive to ADEP. Proteomics analysis of cells after exposure to ADEP revealed many proteins associated with stress responses that were elevated. Levels of a major anion transporter were also altered after ADEP treatment. The role ClpP plays in mitochondrial anion flux is under further study.Down regulation of hClpP sensitizes cells to cisplatin. Remarkably, cells independently selected for resistance to cisplatin have been found to have elevated levels of expression of ClpX and ClpP. We found that the levels of ClpP affect accumulation of cisplatin in several human cell lines. Total cisplatin accumulation increases when ClpP is knocked down, and there is a marked increase in cisplatin-mediated damage to mitochondrial DNA. Our results suggest that damage to mitochondrial DNA is important in inducing apoptosis following cisplatin treatment. To investigate the link between ClpP and cisplatin accumulation, we measured the levels of copper transporters when ClpP levels were manipulated. Cisplatin is believed to hitchhike on the copper transporters to enter and exit cells. No changes were observed in the major copper transporter, Ctr1, when ClpP was over-expressed or knocked down. However, there was a significant correlation between the levels of ClpP and the levels of the copper efflux pump, ATP7A. ATP7A decreased in proportion to the decrease in hClpP and increased when hClpP was over expressed. The data point to an indirect role for hClpP in affecting ATP7A. We hypothesize that hClpP affects metal ion flux between the mitochondria and the cytosol, which in turn leads to up or down regulation of ATP7A and renders the cell more or less sensitive to cisplatin. We are conducting whole cell assays to measure mitochondrial ion flux, mitochondrial membrane potential, and other mitochondrial activities following knock down of hClpP.