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。蛋白质降解对于控制细胞调节蛋白的水平至关重要，并且是蛋白质质量控​​制系统的关键部分。蛋白质降解由 ATP 依赖性蛋白酶完成，该蛋白酶具有三个组成部分：底物识别结构域、ATP 驱动的蛋白质解折叠酶和相关的自区室化蛋白酶。我们的研究包括对细菌和人类线粒体中的 Clp 蛋白酶进行结构和生化研究，并分析其生物活性。去年，我们在了解 ClpAP 和接头蛋白 ClpS 进行的细胞内降解方面取得了实质性进展。 N 端规则是一种根据蛋白质 N 端氨基酸的特性来靶向降解蛋白质的机制。不同的 N 端降解决定子被降解机制的组件识别，从而允许 ATP 依赖性蛋白酶靶向蛋白质。在大肠杆菌中，接头 ClpS 结合 N-降解决定子 Leu、Ph、Tyr 和 Trp，并将蛋白质递送至 ClpAP 复合物。 N 末端 Lys 和 Arg 的蛋白质通过 Aat（一种氨酰 tRNA 蛋白质转移酶）的作用获得 Leu 或 Phe N 降解决定子。我们捕获了 100 多种带有与 ClpS 结合的 N 降解决定子的蛋白质。几乎所有蛋白质的 N 末端均被截短。许多蛋白质的 N 末端赖氨酸或精氨酸已被亮氨酸/苯丙氨酸氨酰基转移酶修饰。通过筛选 50 多个被注释为蛋白酶或肽酶的基因发生突变的菌株，我们鉴定了负责切割特定蛋白质的肽酶。 N-降解决定子周围的序列揭示了似乎充当内切蛋白酶识别位点的基序。一种假设是，细胞蛋白存在一些被蛋白酶靶向的内在位点，产生带有 N 端降解决定子的裂解产物。这种切割可能是改变蛋白质功能并改变蛋白质参与的复合物的生物活性的细胞机制。第二个假设是，内切蛋白酶与 ClpSAP 相结合是蛋白质质量控​​制中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。 ClpP 的研究重点是结合酰基缩肽抗生素 ADEP 导致的细胞死亡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制成的抗生素。当与 ClpP 结合时，ADEP 会激活部分未折叠蛋白质的无差别降解。 ADEP 正在被开发为针对人类病原体的新型抗生素。目前的研究重点是参与结合 ADEP 的 ClpP 的主要序列和结构特征以及打开轴向通道的 ClpP 变构变化。该项目变得更加重要，因为 ADEP 结合位点也是 ClpX 和 ClpA 的对接位点。我们筛选了随机诱变的 ClpP 文库，以鉴定对 ADEP 不敏感但保留 ClpP 及其同源 ATP 酶活性的 ClpP 突变体。这些突变体应该非常罕见，并且将识别 ClpP 中用于 ADEP 或 ClpX 结合的位点或参与功能位点之间的变构通讯。为了确保诱变的 ClpP 保留活性，我们设计了一种质粒，该质粒有条件地表达有毒蛋白质，该蛋白质必须被 ClpXP 降解才能保证细胞存活。我们分离了 8 个具有所需特性的不同 ClpP 突变体，并对突变体进行了表征。所有这些都包含多个突变，我们正在分离各个突变，以确定哪些等位基因显示出观察到的表型。这些研究将深入了解 ClpP 的作用及其与不同激活配体的相互作用。由于缺乏可添加到细胞培养物中抑制 ClpP 的抑制剂，对 Clp 功能的研究受到阻碍。二价 Zn 抑制 ClpP，我们获得了 ClpP 的晶体结构并确定了 Zn 的结合位点。锌被两个关键残基螯合，这两个残基形成七聚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，一种是手柄交错以扩大降解室，另一种是手柄处于折叠状态。后者在允许产物释放的降解周期期间是潜伏状态或瞬时中间体。 Zn 促进或稳定 ClpP 的塌陷状态。我们将从北卡罗来纳大学的 Holden Thorp 教授那里获得一组双（苯并咪唑）化合物，这些化合物可以增强 Zn 与特定丝氨酸蛋白酶的结合。化合物核心上附着的取代基可以大大增强结合亲和力和特异性，我们将筛选大量此类化合物，寻找对ClpP具有高亲和力的抑制剂。 ClpP 对于许多人类病原体（包括结核分枝杆菌）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十四聚体。一种复合物中存在两种形式的 ClpP 将有助于环相互作用的结构分析，例如，通过允许组装其中只有一个环突变的十四聚体。我们已经结晶了 Mbt-ClpP 并获得了约 3.6 埃的密度图。我们已经证实该结构包含由 ClpP1 和 ClpP2 环组成的混合十四聚体。我们预计今年将获得天然蛋白质的结构。晶体结构应该指导小分子抑制剂的设计，这些抑制剂将成为开发具有治疗潜力的化合物的先导。我们对人类 ClpX 和 ClpP 研究的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 的消耗会导致细胞死亡。使用 siRNA 去除 hClpP 后 16 小时内，超过 30 种蛋白质增加。许多蛋白质参与应激反应。 ADEP 会引起细胞应激并杀死人体细胞。 ClpP 野生型而非失活突变体的过度表达使细胞对 ADEP 更加敏感。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 治疗后主要阴离子转运蛋白的水平也发生了变化。当 ClpP 被敲低时，顺铂的积累会增加，顺铂介导的线粒体 DNA 损伤也会显着增加。我们发现线粒体 DNA 的损伤对于顺铂治疗后诱导细胞凋亡很重要。为了研究 ClpP 和顺铂积累之间的联系，我们测量了顺铂用来进入和离开细胞的铜转运蛋白的水平。当 ClpP 过度表达或敲低时，铜转运蛋白 Ctr1 没有观察到变化，但 ClpP 的水平与铜外排泵 ATP7A 的水平之间存在相关性。数据表明 hClpP 对 ATP7A 的影响具有间接作用。我们假设 hClpP 影响线粒体和细胞质之间的金属离子通量，进而导致 ATP7A 的上调或下调，并使细胞对顺铂或多或少敏感。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性。