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.

蛋白质部分的生物化学对细菌和人类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的作用机理进行了研究。 CLP蛋白酶具有三个成分：底物识别结构域（SSPB，RSSB或CLP），ATP驱动的蛋白IFOLDASE（CLPX或CLPA），以及相关的自身分类的蛋白酶CLPP。在过去的一年中，我们扩展了对由CLPAP和适配器蛋白CLPS进行的细胞内降解的理解，该蛋白质受称为N-End规则的机制的控制。 N末端规则定义了一种机制，该机制通过其基于其N末端氨基酸的身份将蛋白质靶向降解。在大肠杆菌中，N端degrons被CLP识别，该clps结合了N端Leu，Phe，Tyr和Trp。 CLPS与CLPA的N域相互作用，并将N端规则基板移交给CLPAP复合物。在大肠杆菌细胞中，还针对具有N末端LYS和ARG的蛋白质，因为它们通过AAT（氨基酰基TRNA蛋白转移酶）的作用获得了Leu或Phe n-Degron。我们报告说，CLPS亲和力柱可以捕获具有N-脱粒龙的100多个大肠杆菌蛋白。我们现在已经表明，CLP具有从其他生物体中捕获N端规则蛋白的一般效用。我们具有来自细菌细胞提取物（纤维状霍乱和枯草芽孢杆菌）的提取物以及来自真核细胞提取物的N-脱果素的分离蛋白，包括酿酒酵母和同性恋剂。我们已经构建了结合N末端氨基酸但失去区分能力的CLP（M40A）突变体。使用肽阵列，我们发现该突变体结合除天冬氨酸和谷氨酸以外的所有N末端氨基酸。哺乳动物细胞具有几种不同类别的N-脱果龙，但目前尚无隔离特定N-脱果龙的蛋白质的机制。我们将诱变Clps和筛选，以结合特定类别的N-脱果龙的能力，我们将使用它们从哺乳动物细胞中拔出蛋白质，并测试其在体内使用不同N-degrons抑制蛋白质降解的能力。大肠杆菌中N末端规则降解的研究继续尝试鉴定暴露于蛋白质中N-脱绿素的肽酶。我们克隆了YFBL，这是一种推定的蛋白酶，在DPS中产生N-脱核酸酶，DPS是细菌中DNA的蛋白质。 DP不再从YFBL突变的细胞中撤下。我们还克隆了腐烂氨基转移酶（PATase），这是最丰富的N端规则底物之一。 patase是独一无二的，因为N端蛋氨酸被保留，并通过向N末端添加LEU和PHE来改变。我们将在体外重建修饰反应，并确定负责调节修饰的因素。使用CLPP的研究集中在结合脂肪肽抗生素EDEP和底物进入降解室所需的结构变化所产生的细胞死亡机理上。 ADEP是夏威夷链球菌生产的一种抗生素。当绑定到CLPP时，EDEP打开轴向通道并激活不加选择的蛋白质降解。 ADEP结合的位点也是CLPX和CLPA的对接位点，它们控制了底物向CLPP的递送。 ADEP正在作为靶向人类病原体的新型抗生素开发。当前的研究集中在ADEP结合所需的CLPP的特征以及打开通道的CLPP的变构变化所需的特征上。我们随机诱变的CLPP并鉴定出对EDEP不敏感但使用其同源性ATPases的CLPP活性不敏感的突变体。我们在轴向通道中发现了突变，该突变可访问CLPP活动位点以及影响对接位点形状的站点。我们已经纯化了几个突变体，并正在研究它们的生化和酶促特性。我们将净化大量的结晶数量，以确定改变其对ADEP结合的响应的结构变化。这些突变体很少见，我们希望确定对接站点，活动地点和亚基触点站点之间涉及变构通信的地点，所有这些位点都会影响CLPP活动。直到最近，由于缺乏可以添加到细胞培养物抑制CLPP的化合物的缺乏，对CLP功能的研究一直受到阻碍。二价锌抑制了CLPP，我们获得了CLPP的晶体结构，并鉴定了Zn结合的位点。两个关键残基形成了七聚环中亚基之间的界面可用于螯合Zn。两个催化残基His122和ASP171也与Zn相互作用。我们已经观察到Zn稳定了手柄区域的折叠形式，该形式形成了clpp七聚环之间的界面。我们从北卡罗来纳大学的霍尔顿·索普教授那里获得了许多BIS（苯咪唑）化合物，可以增强Zn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化合物，从而略有抑制作用。我们将要求我们的合作者准备类似的衍生化合物（Benzimidazoles），并测试其作为共抑制剂的功效。我们在哈佛医学院与阿尔弗雷德·戈德堡（Alfred Goldberg）的合作中取得了重大进展，以从结核分枝杆菌中获得活跃形式的晶体结构。 CLPP对于结核分枝杆菌的生长至关重要，因此是潜在抗菌剂的有希望的靶标。现在，我们有一个3.0埃的活性形式晶体结构，该结构由与Clpp2的七聚体环组成的Clpp1的七聚体环。只有此异质复合物活跃。一种复合物中的两种形式的CLPP的存在将通过允许组装四成员的组装来促进对环相互作用的结构分析，而四成员只有一个环被突变。我们观察到Clpp1和Clpp2活性位点中的激活肽，但有趣的是，肽在两个位点的相反方向上结合。晶体结构应指导小分子抑制剂的设计，这些抑制剂将作为开发可以阻止结核分枝杆菌和其他病原体生长的化合物的铅。我们对人类CLPX和CLPP的研究的目的是定义它们在线粒体中的功能，并发现为什么需要它们才能使线粒体完整性和细胞存活需要它们。我们发现，HCLPP的过度表达允许用抗癌药物顺铂治疗的细胞更好地生存。相反，当将HCLPP部分击倒时，细胞对顺铂更敏感。当HCLPP被击倒时，顺铂积累增加，这表明可能需要HCLPP活性以快速摄取顺铂。另外，HCLPP活性可能会影响一种或多种代谢顺铂或顺铂加合物中的酶。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的水平具有巨大的作用。 HCLPP与称为佩罗综合症的遗传性人类疾病有关。此外，小鼠CLPP的纯合敲除导致听力丧失和不育。这些结果表明，CLPP在哺乳动物细胞中起重要甚至重要的作用。我们发现，在人类细胞培养中，通过用siRNA处理，HCLPP或HCLPX急剧耗竭会导致细胞死亡。由于转染的条件对培养的细胞压力很大，因此我们建议HCLPP在压力条件下可能是必不可少的，这可以解释为什么CLPP纯合缺失的小鼠生存。蛋白质组学研究表明，HCLPP用siRNA耗尽16小时内，有30种蛋白质增加，并且许多蛋白质参与胁迫反应。