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: 8937640
• 负责人: MICHAEL MAURIZI
• 金额: $ 78.1万
• 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/8937640
• 关键词: 26S proteasome; ATP phosphohydrolase; ATP-Dependent Proteases; Active Sites; Adaptor Signaling Protein; Affect; Affinity; Amino Acids; Amino Acyl Transfer RNA; Antibiotics; Antineoplastic Agents; Aspartate; Automobile Driving; Bacillus subtilis; Bacteria; Benzimidazoles; Binding; Biochemical; Biochemistry; Biological; Cell Culture Techniques; Cell Death; Cell Death Signaling Process; Cell Nucleus; Cell Survival; Cells; Cisplatin; Cleaved cell; Collaborations; Communication; Complex; Coupled; Crystallization; Cultured Cells; DNA; Dependence; Development; Docking; Enzymes; Equilibrium; Escherichia coli; Escherichia coli Proteins; Eukaryotic Cell; Foundations; Glutamates; Goals; Growth; Hand; Homo sapiens; Homologous Gene; Hour; Human; In Vitro; Infertility; Inherited; Investigation; Knock-out; Knockout Mice; Ligand Binding; Ligands; Malignant Neoplasms; Mammalian Cell; Metabolic; Methionine; Mitochondria; Mitochondrial DNA; Modification; Molecular; Multiple Myeloma; Mus; Mutate; Mutation; Mycobacterium tuberculosis; N Domain; N-terminal; Normal Cell; North Carolina; Organelles; Organism; Pathway interactions; Peptide Hydrolases; Peptides; Perrault syndrome; Pharmaceutical Preparations; Physiological; Play; Property; Protein Biochemistry; Proteins; Proteomics; Putrescine; Quality Control; Reaction; Reporting; Research; Role; Saccharomyces cerevisiae; Shapes; Site; Small Interfering RNA; Streptococcus; Stress; Structure; Testing; Transaminases; Transfection; Transferase; Universities; Vibrio cholerae; Virulence; adduct; antimicrobial; arginyllysine; base; benzimidazole; biological adaptation to stress; cell growth; cell injury; cell killing; cell transformation; deafness; design; endopeptidase Clp; endopeptidase La; hearing impairment; human disease; in vivo; inhibitor/antagonist; knock-down; leucyl-phenylalanine; medical schools; multicatalytic endopeptidase complex; mutant; novel; pathogen; protein degradation; protein function; response; small molecule; unfoldase; uptake

The Biochemistry of Proteins Section conducts research on the function and control of protein degradation in bacterial and human cells and on the mechanism of action of the ATP-dependent proteases ClpAP and ClpXP. Clp proteases have three constituents: a substrate recognition domain (SspB, RssB, or ClpS), an ATP-driven protein unfoldase (ClpX or ClpA), and an associated self-compartmentalized protease, ClpP. In the past year we have extended our understanding of intracellular degradation carried out by ClpAP and the adaptor protein, ClpS, which is governed by a mechanism called the N-end rule. The N-end rule defines a mechanism by which proteins are targeted for degradation based on the identity of their N-terminal amino acids. In E. coli, N-end degrons are recognized by ClpS, which binds the N-terminal Leu, Phe, Tyr, and Trp. ClpS interacts with the N-domain of ClpA and hands off the N-end rule substrates to the ClpAP complex. In E. coli cells, proteins with N-terminal Lys and Arg are also targeted, because they acquire a Leu or Phe N-degron through the action of Aat, an aminoacyl tRNA protein transferase. We reported that a ClpS affinity column could capture more than 100 E. coli proteins with N-degrons. We have now shown that ClpS has general utility for capturing N-end rule proteins from other organisms. We have isolated scores proteins with N-degrons from extracts of bacterial cells (Vibrio cholerae and Bacillus subtilis), as well as from extracts of eukaryotic cells, including Saccharomyces cerevisiae and Homo sapiens. We have constructed a mutant of ClpS (M40A) that binds N-terminal amino acids but has lost the ability to discriminate. Using a peptide array we found that this mutant binds all N-terminal amino acids except aspartate and glutamate. Mammalian cells have several different classes of N-degrons but currently there is no mechanism for isolation of proteins bearing a specific N-degron. We will mutagenize ClpS and screen for the ability to bind specific classes of N-degrons and we will use them to pull out proteins from mammalian cells and test their ability to inhibit degradation of proteins with different N-degrons in vivo. Studies of N-end rule degradation in E. coli continue with attempts to identify the peptidase that expose N-degrons in proteins. We cloned YfbL, a putative protease that generates an N-degron in Dps, a DNA-protecting protein in bacteria. Dps is no longer pulled down from cells in which yfbL has been mutated. We also cloned putrescine aminotransferase (PATase), one of the most abundant N-end rule substrates. PATase is unique in that the N-terminal methionine is retained and is modified by addition of Leu and Phe to the N-terminus. We will reconstruct the modification reaction in vitro and identify factors that are responsible for regulating the modification. 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 antibiotic made by Streptococcus hawaiiensis. When bound to ClpP ADEP opens the axial channel and activates indiscriminate protein degradation. The site of ADEP binding is also the docking site for ClpX and ClpA, which govern delivery of substrates to ClpP. ADEPs are being developed as novel antibiotics to target human pathogens. Current research is focused on the features of ClpP needed for ADEP binding and for the allosteric changes in ClpP that open the channel. We randomly mutagenized ClpP and identified mutants that are insensitive to ADEP but retain ClpP activity with its cognate ATPases. We found mutations in the axial channel that provides access to the ClpP active site and in sites that affect the shape of the docking site. We have purified several of the mutants and are studying their biochemical and enzymatic properties. We will purify larger quantities for crystallization in order to identify the structural changes that alter their response to ADEP binding. These mutants are rare and we expect to identify sites involved in allosteric communication between the docking site, the active site, and the subunit contact sites, all of which affect ClpP activity. Until recently, studies of Clp function have been hindered by the lack of compounds 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. Two critical residues that form the interface between subunits in the heptameric ring serve to chelate the Zn. Two catalytic residues, His122 and Asp171, also interact with the Zn. We have observed that Zn stabilizes a collapsed form of the handle region that forms the interface between the ClpP heptameric rings. We obtained a number of bis (benzimidazole) compounds from Prof. Holden Thorp at the University of North Carolina that can enhance Zn binding to proteases. Our preliminary screen of these compounds identified one compound that gave a slight enhancement of inhibition. We will ask our collaborators to prepare similar derivatized bis(benzimidazoles) and test them for their efficacy as co-inhibitors. We have made substantial progress in our collaboration with Alfred Goldberg at Harvard Medical School to obtain a crystal structure of the active form of ClpP from Mycobacterium tuberculosis. ClpP is essential for growth of M. tuberculosis and thus is a promising target for potential antimicrobials. We now have a 3.0 Angstrom crystal structure of the active form, which consists of a heptameric ring of ClpP1 complexed with a heptameric ring of ClpP2. Only this hetero-complex is active. The presence of two forms of ClpP in one complex will facilitate structural analysis of the ring interactions by allowing assembly of tetradecamers in which only one ring is mutated. We observe the activating peptide in the ClpP1 and ClpP2 active sites, but interestingly the peptide binds in opposite orientations in the two sites. The crystal structure should guide the design of small molecule inhibitors that will serve as leads for the development of compounds that can block the growth of M. tuberculosis and other pathogens. 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. We found that over expression of HClpP allows better survival of cells treated with the anti-cancer drug, cisplatin. Conversely, cells were more sensitive to cisplatin when HClpP was partially knocked down. Cisplatin accumulation increased when HClpP was knocked down, suggesting that HClpP activity might be needed to allow rapid uptake of cisplatin. Alternatively, HClpP activity could affect one or more enzymes that metabolize cisplatin or cisplatin adducts in the cell. We find that cisplatin is incorporated preferentially into mitochondrial DNA and that HClpP has a dramatic effect on the level of cisplatin adducts detected in mitochondrial DNA. HClpP has been implicated in a hereditary human disease called Perrault's syndrome. In addition, homozygous knockout of ClpP in mice leads to profound hearing loss and infertility. These results indicate that ClpP plays some important or even essential role in mammalian cells. We find that in human cell culture drastic depletion of hClpP or hClpX by treatment with siRNA leads to cell death. Because the conditions for transfection are stressful to cultured cells we propose that HClpP might be essential under conditions of stress, which would explain why mice with homozygous deletion of CLPP survive. Proteomics studies reveal that 30 proteins are increased within 16 hours of depletion of hClpP with siRNA and that many of the proteins are involved in stress responses.

蛋白质生物化学部分研究细菌和人体细胞中蛋白质降解的功能和控制，以及 ATP 依赖性蛋白酶 ClpAP 和 ClpXP 的作用机制。 Clp 蛋白酶具有三个组成部分：底物识别结构域（SspB、RssB 或 ClpS）、ATP 驱动的蛋白质解折叠酶（ClpX 或 ClpA）以及相关的自区室化蛋白酶 ClpP。在过去的一年中，我们扩展了对 ClpAP 和接头蛋白 ClpS 进行的细胞内降解的理解，该降解受称为 N 端规则的机制控制。 N 端规则定义了一种机制，通过该机制，蛋白质可以根据其 N 端氨基酸的特性进行降解。在大肠杆菌中，N 端降解决定子被 ClpS 识别，ClpS 结合 N 端 Leu、Phe、Tyr 和 Trp。 ClpS 与 ClpA 的 N 结构域相互作用，并将 N 端规则底物交给 ClpAP 复合体。在大肠杆菌细胞中，N 末端 Lys 和 Arg 的蛋白质也成为目标，因为它们通过 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。我们报道 ClpS 亲和柱可以捕获 100 多种带有 N-降解决定子的大肠杆菌蛋白。我们现在已经证明 ClpS 具有从其他生物体捕获 N 端规则蛋白的通用性。我们从细菌细胞（霍乱弧菌和枯草芽孢杆菌）提取物以及真核细胞（包括酿酒酵母和智人）提取物中分离出多种带有 N 降解决定子的蛋白质。我们构建了 ClpS (M40A) 的突变体，它结合 N 端氨基酸，但失去了区分能力。使用肽阵列，我们发现该突变体结合除天冬氨酸和谷氨酸之外的所有 N 端氨基酸。哺乳动物细胞有几种不同类别的 N-降解决定子，但目前还没有分离带有特定 N-降解决定子的蛋白质的机制。我们将诱变 ClpS 并筛选结合特定类别 N-降解决定子的能力，我们将使用它们从哺乳动物细胞中提取蛋白质，并测试它们在体内抑制不同 N-降解决定子蛋白质降解的能力。对大肠杆菌中 N 端规则降解的研究仍在继续，试图鉴定暴露蛋白质中 N 端降解决定子的肽酶。我们克隆了 YfbL，一种假定的蛋白酶，可在 Dps（细菌中的一种 DNA 保护蛋白）中产生 N-降解决定子。 yfbL 突变的细胞中的 Dps 不再被拉低。我们还克隆了腐胺转氨酶 (PATase)，它是最丰富的 N 端规则底物之一。 PATase 的独特之处在于保留了 N 末端甲硫氨酸，并通过在 N 末端添加 Leu 和 Phe 进行修饰。我们将在体外重建修饰反应并确定负责调节修饰的因素。 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产生的抗生素。当 ADEP 与 ClpP 结合时，会打开轴向通道并激活不加区别的蛋白质降解。 ADEP 结合位点也是 ClpX 和 ClpA 的对接位点，控制底物向 ClpP 的递送。 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 ADEP 结合所需的 ClpP 特征以及打开通道的 ClpP 变构变化。我们随机诱变 ClpP 并鉴定出对 ADEP 不敏感但保留 ClpP 与其同源 ATP 酶活性的突变体。我们在提供进入 ClpP 活性位点的轴向通道和影响对接位点形状的位点中发现了突变。我们已经纯化了几种突变体，并正在研究它们的生化和酶学特性。我们将纯化大量样品进行结晶，以确定改变其对 ADEP 结合反应的结构变化。这些突变体很罕见，我们希望鉴定出参与对接位点、活性位点和亚基接触位点之间变构通讯的位点，所有这些都会影响 ClpP 活性。直到最近，由于缺乏可以添加到细胞培养物中抑制 ClpP 的化合物，对 Clp 功能的研究一直受到阻碍。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。形成七聚环中亚基之间的界面的两个关键残基用于螯合锌。两个催化残基 His122 和 Asp171 也与 Zn 相互作用。我们观察到 Zn 稳定了手柄区域的塌陷形式，该手柄区域形成 ClpP 七聚环之间的界面。我们从北卡罗来纳大学的 Holden Thorp 教授那里获得了许多双（苯并咪唑）化合物，它们可以增强锌与蛋白酶的结合。我们对这些化合物的初步筛选确定了一种能够略微增强抑制作用的化合物。我们将要求我们的合作者制备类似的衍生化双（苯并咪唑）并测试它们作为共抑制剂的功效。我们与哈佛医学院的 Alfred Goldberg 的合作取得了实质性进展，从结核分枝杆菌中获得了 ClpP 活性形式的晶体结构。 ClpP 对于结核分枝杆菌的生长至关重要，因此是潜在抗菌药物的一个有希望的靶标。我们现在得到了活性形式的 3.0 埃晶体结构，它由 ClpP1 的七聚环与 ClpP2 的七聚环复合组成。只有该异质复合物具有活性。一种复合物中存在两种形式的 ClpP 将通过允许组装其中仅一个环发生突变的十四聚体来促进环相互作用的结构分析。我们在 ClpP1 和 ClpP2 活性位点观察到激活肽，但有趣的是，肽在两个位点以相反的方向结合。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能够阻止结核分枝杆菌和其他病原体生长的化合物的先导。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。我们发现 HClpP 的过度表达可以使用抗癌药物顺铂治疗的细胞更好地存活。相反，当 HClpP 部分被抑制时，细胞对顺铂更加敏感。当 HClpP 被敲低时，顺铂的积累增加，这表明可能需要 HClpP 活性才能快速摄取顺铂。或者，HClpP 活性可能影响细胞中代谢顺铂或顺铂加合物的一种或多种酶。我们发现顺铂优先掺入线粒体 DNA 中，并且 HClpP 对线粒体 DNA 中检测到的顺铂加合物水平具有显着影响。 HClpP 与一种称为佩罗综合征的遗传性人类疾病有关。此外，小鼠 ClpP 纯合敲除会导致严重的听力损失和不育。这些结果表明ClpP在哺乳动物细胞中发挥着一些重要甚至必不可少的作用。我们发现，在人类细胞培养物中，通过 siRNA 处理，hClpP 或 hClpX 的急剧消耗会导致细胞死亡。由于转染条件对培养细胞有压力，我们认为 HClpP 在压力条件下可能是必需的，这可以解释为什么 CLPP 纯合缺失的小鼠能够存活。蛋白质组学研究表明，用 siRNA 去除 hClpP 后 16 小时内，30 种蛋白质增加，其中许多蛋白质与应激反应有关。