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: 8762996
• 负责人: MICHAEL MAURIZI
• 金额: $ 80.96万
• 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/8762996
• 关键词: 26S proteasome; ATP Hydrolysis; ATP phosphohydrolase; ATP-Dependent Proteases; Active Sites; Adaptor Signaling Protein; Affect; Affinity; Alleles; Amino Acids; Amino Acyl Transfer RNA; Anions; Antibiotics; Antineoplastic Agents; Apoptosis; Bacteria; Binding; Biochemical; Biochemistry; Biological; Biological Assay; Cell Culture Techniques; Cell Death; Cell Death Signaling Process; Cell Nucleus; Cell Survival; Cells; Cellular Stress; Cellular Stress Response; Cisplatin; Cleaved cell; Communication; Complex; Copper; Coupled; Cytosol; Data; Development; Docking; Down-Regulation; Ensure; Enzymes; Escherichia coli; Eukaryotic Cell; Exposure to; Genes; Goals; Growth; Growth and Development function; Hour; Human; Individual; Investigation; Ions; Isoenzymes; Laboratories; Libraries; Ligand Binding; Ligands; Link; Maps; Measures; Mediating; Membrane Potentials; Metabolic; Metals; Mitochondria; Mitochondrial DNA; Molecular; Molecular Chaperones; Molecular Machines; Molecular Models; Mutate; Mutation; Mycobacterium tuberculosis; N-terminal; North Carolina; Organelles; Participant; Pathway interactions; Peptide Hydrolases; Peptides; Phenotype; Plasmids; Play; Property; Protein Biochemistry; Protein translocation; Proteins; Proteolysis; Proteomics; Quality Control; Research; Research Project Grants; Role; Serine Protease; Site; Small Interfering RNA; Specificity; Streptococcus; Stress; Structure; System; Therapeutic; Therapeutic Agents; Transferase; Universities; Up-Regulation; Virulence; Work; antimicrobial; arginyllysine; base; biological adaptation to stress; bis-benzimidazole; cell killing; density; design; efflux pump; endopeptidase Clp; endopeptidase La; genetic regulatory protein; in vivo; inhibitor/antagonist; insight; killings; knock-down; leucyl-phenylalanine; medical schools; mitochondrial membrane; molecular modeling; mutant; novel; novel diagnostics; novel therapeutics; pathogen; protein degradation; protein function; rapid growth; response; screening; small molecule; tryptophyltyrosine; unfoldase; uptake

Research in the Biochemistry of Proteins Section is focused on the function and control of protein degradation in bacterial and human cells. Protein degradation is essential to control the levels of cellular regulatory proteins and is a critical part of protein quality control systems. Protein degradation is performed by ATP-dependent proteases, which have three constituents: a substrate recognition domain, an ATP-driven protein unfoldase, and an associated self-compartmentalized protease. Our research includes structural and biochemical studies of the Clp proteases from bacteria and human mitochondria and analysis of their biological activities. We have made substantial progress in the past year in understanding intracellular degradation carried out by ClpAP and the adaptor protein, ClpS. The N-end rule is a mechanism by which proteins are targeted for degradation based on the identity of their N-terminal amino acids. Different N-end degrons are recognized by components of the degradative machinery allowing the proteins to be targeted by ATP-dependent proteases. In E. coli, the adaptor ClpS binds the N-degrons Leu, Ph, Tyr, and Trp and delivers proteins to the ClpAP complex. Proteins with N-terminal Lys and Arg acquire a Leu or Phe N-degron by the action of Aat, an aminoacyl tRNA protein transferase. We captured more than 100 proteins with N-degrons bound to ClpS. Virtually all of the proteins were N-terminally truncated. Many of the proteins had N-terminal Lys or Arg that had been modified by Leu/Phe aminoacyl transferase. By screening strains with mutations in over 50 genes annotated as proteases or peptidases, we identified peptidases responsible for cleavage of specific proteins. The sequences surrounding the N-degrons revealed motifs that appear to act as recognition sites for endoproteases. One hypothesis is that there are intrinsic sites cellular proteins that are targeted by proteases, generating cleaved products with N-end degrons. Such cleavage might be a cellular mechanism to alter the function of proteins and modify the biological activity of complexes in which the proteins participate. A second hypothesis is that endoproteases combined with ClpSAP are previously unappreciated participants in protein quality control and conduct constant surveillance of proteins to assess their functionality structural integrity. Studies with ClpP are focused on the mechanism of cell death that results from binding the acyldepsipeptide antibiotic ADEP and the structural changes needed for substrate entry into the degradation chamber. ADEP is an antibiotics made by Streptococcus hawaiiensis. When bound to ClpP ADEP activates indiscriminate degradation of partially unfolded proteins. ADEPs are being developed as novel antibiotics to target human pathogens. Current research is focused on the primary sequence and structural features of ClpP involved in binding ADEP and in the allosteric changes in ClpP that open the axial channel. The project has added importance because the site of ADEP binding is also the docking site for ClpX and ClpA. We screened a library of randomly mutagenized ClpP to identify mutants of ClpP that are insensitive to ADEP but retain activity of ClpP with its cognate ATPases. These mutants should be very rare and will identify sites in ClpP for ADEP or ClpX binding or involved in allosteric communication between functional sites. To ensure that the mutagenized ClpP retains activity, we designed a plasmid that conditionally expresses a toxic protein that must be degraded by ClpXP for cell survival. We isolated 8 different mutants of ClpP with the desired properties and are characterizing the mutants. All contain multiple mutations, and we are separating individual mutations to identify which of the alleles displays the observed phenotypes. These studies will provide insight onto the workings of ClpP and its interactions with different activating ligands. Studies of Clp function have been hindered by the lack of inhibitors that can be added to cell cultures to inhibit ClpP. Divalent Zn inhibits ClpP, and we have obtained a crystal structure of ClpP and identified the sites at which Zn binds. Zn is chelated by two critical residues that form the interface between subunits in the heptameric ring. Two catalytic residues, His122 and Asp171, also interact with the Zn. ClpP exists in two states, one with the handles interlaced to expand the degradation chamber and another with the handles in a collapsed state. The latter is either a latent state or a transient intermediate during the degradation cycle that allows product release. Zn promotes or stabilizes a collapsed state of ClpP. We will obtain a set of bis(benzimidazole) compounds from Prof. Holden Thorp at the University of North Carolina that can enhance Zn binding to specific serine proteases. Substituents attached to the core of the compound can greatly enhanced binding affinity and specificity, and we will screen a large number of such compounds to find an inhibitor with high affinity for ClpP. ClpP is essential for growth or for virulence for a number of human pathogens, including Mycobacterium tuberculosis. We are collaborating with the laboratory of Alfred Goldberg at Harvard Medical School, who has provided us with purified ClpP from M. 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 crystallized Mbt-ClpP and obtained a density map at about 3.6 Angstroms. We have confirmed that the structure contains mixed tetradecamers made up of ClpP1 and ClpP2 rings. We expect to have a structure of the native protein this year. The crystal structure should guide the design of small molecule inhibitors that will 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. More than 30 proteins are increased within 16 hours of depletion of hClpP with siRNA. Many of the proteins 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. Cisplatin accumulation increases when ClpP is knocked down, and there is a marked increase in cisplatin-mediated damage to mitochondrial DNA. We find 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, which are used by cisplatin to enter and exit cells. No changes were observed in the copper transporter Ctr1 when ClpP was over-expressed or knocked down, but there was a correlation between the levels of ClpP and the levels of the copper efflux pump, ATP7A. 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.

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。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部分。蛋白质降解是由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相关的自区隔蛋白酶。我们的研究包括从细菌和人类线粒体中提取的Clp蛋白酶的结构和生化研究以及它们的生物活性分析。在过去的一年中，我们在了解ClpAP及其接头蛋白ClpS进行的细胞内降解方面取得了实质性进展。n端规则是蛋白质根据其n端氨基酸的特性而被降解的一种机制。不同的n端降解被降解机制的组成部分识别，从而使蛋白质成为atp依赖性蛋白酶的靶标。在大肠杆菌中，接头ClpS结合N-degrons Leu、Ph、Tyr和Trp，并将蛋白质传递给ClpAP复合物。具有n端赖氨酸和精氨酸的蛋白质通过Aat（一种氨基tRNA蛋白转移酶）的作用获得亮氨酸或亮氨酸N-degron。我们捕获了100多个与ClpS结合的N-degrons蛋白。几乎所有的蛋白质都被n端截断。许多蛋白质的n端赖氨酸或精氨酸被亮氨酸/亮氨酸氨基酰基转移酶修饰。通过筛选超过50个被标记为蛋白酶或肽酶的基因突变的菌株，我们确定了负责切割特定蛋白质的肽酶。围绕N-degrons的序列揭示了似乎作为内源性蛋白酶识别位点的基序。一种假设是，细胞蛋白存在被蛋白酶靶向的内在位点，产生具有n端退化的裂解产物。这种分裂可能是一种改变蛋白质功能和改变蛋白质参与的复合物生物活性的细胞机制。第二种假设是，内源性蛋白酶与ClpSAP联合在蛋白质质量控制中是以前未被重视的参与者，并对蛋白质进行持续监测以评估其功能结构完整性。对ClpP的研究主要集中在结合酰基沉积肽抗生素ADEP导致细胞死亡的机制以及底物进入降解室所需的结构变化。ADEP是一种由夏威夷链球菌产生的抗生素。当与ClpP结合时，ADEP激活部分未折叠蛋白的不加区分的降解。人们正在开发adep作为针对人类病原体的新型抗生素。目前的研究主要集中在参与结合ADEP的ClpP的主要序列和结构特征，以及ClpP打开轴向通道的变构变化。由于ADEP结合的位点也是ClpX和ClpA的对接位点，因此该项目具有更大的重要性。我们筛选了一个随机突变的ClpP库，以鉴定对ADEP不敏感但保留与其同源atp酶活性的ClpP突变体。这些突变体应该是非常罕见的，并且将识别ClpP中与ADEP或ClpX结合或参与功能位点之间的变构通信的位点。为了确保诱变的ClpP保持活性，我们设计了一种质粒，可以有条件地表达一种有毒蛋白，这种蛋白必须被ClpXP降解才能使细胞存活。我们分离了8种具有所需特性的ClpP突变体，并对这些突变体进行了表征。所有这些都包含多个突变，我们正在分离单个突变，以确定哪些等位基因表现出观察到的表型。这些研究将深入了解ClpP的工作原理及其与不同激活配体的相互作用。由于缺乏可以添加到细胞培养物中抑制ClpP的抑制剂，Clp功能的研究一直受到阻碍。二价锌抑制ClpP，我们获得了ClpP的晶体结构，并确定了锌的结合位点。锌被两个关键残基螯合，形成七聚体环中亚基之间的界面。两个催化残基His122和Asp171也与Zn相互作用。ClpP以两种状态存在，一种是把手交错以扩展降解室，另一种是把手处于折叠状态。后者要么是潜在状态，要么是降解周期中允许产物释放的瞬态中间产物。Zn促进或稳定了ClpP的坍塌状态。我们将从北卡罗莱纳大学的Holden Thorp教授那里获得一组可以增强锌与特定丝氨酸蛋白酶结合的苯并咪唑（benzimidazole）化合物。取代基附着在化合物核心上可以大大增强结合亲和力和特异性，我们将大量筛选此类化合物，寻找对ClpP具有高亲和力的抑制剂。ClpP对包括结核分枝杆菌在内的许多人类病原体的生长或毒力至关重要。我们正在与哈佛医学院的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。我们对Mbt-ClpP进行了结晶，得到了约3.6埃的密度图。我们已经证实该结构包含由ClpP1和ClpP2环组成的混合四层照相机。我们希望今年能有一个天然蛋白质的结构。晶体结构应该指导小分子抑制剂的设计，这些小分子抑制剂将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。在用siRNA去除hClpP的16小时内，有30多种蛋白质增加。许多蛋白质都与应激反应有关。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。当ClpP被敲低时，顺铂积累增加，顺铂介导的线粒体DNA损伤显著增加。我们发现顺铂治疗后线粒体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，我们测量了铜转运体的水平，铜转运体是顺铂进出细胞的必经之路。当ClpP过表达或敲低时，铜转运体Ctr1没有变化，但ClpP的水平与铜外排泵ATP7A的水平存在相关性。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。