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.

蛋白质生物化学组研究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的作用机制。Clp蛋白酶有三个组成部分：底物识别结构域（SspB、RssB或ClpS）、atp驱动的蛋白展开酶（ClpX或ClpA）和相关的自区隔蛋白酶ClpP。在过去的一年里，我们扩展了对ClpAP和接头蛋白ClpS进行的细胞内降解的理解，这是由一种称为n端规则的机制控制的。n端规则定义了一种机制，根据蛋白质n端氨基酸的特性，蛋白质被降解为目标。在大肠杆菌中，n端degron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复合物。在大肠杆菌细胞中，具有n端赖氨酸和精氨酸的蛋白也是靶标，因为它们通过Aat（一种氨基酰基tRNA蛋白转移酶）的作用获得Leu或Phe N-degron。我们报道了ClpS亲和柱可以捕获超过100种带有N-degrons的大肠杆菌蛋白。我们现在已经证明ClpS在从其他生物体中捕获n端规则蛋白方面具有普遍的效用。我们已经从细菌细胞（霍乱弧菌和枯草芽孢杆菌）以及真核细胞（包括酿酒酵母菌和智人）的提取物中分离出了数十种带有N-degrons的蛋白质。我们构建了一个ClpS突变体（M40A），它结合n端氨基酸，但失去了区分的能力。利用肽阵列，我们发现该突变体结合除天冬氨酸和谷氨酸外的所有n端氨基酸。哺乳动物细胞有几种不同种类的N-degron，但目前还没有分离特定N-degron蛋白的机制。我们将对ClpS进行诱变并筛选其与特定n -degron结合的能力，我们将利用它们从哺乳动物细胞中提取蛋白质，并在体内测试它们抑制不同n -degron降解蛋白质的能力。对大肠杆菌中n -末端规则降解的研究继续进行，试图确定在蛋白质中暴露n -降解的肽酶。我们克隆了YfbL，这是一种假定的蛋白酶，可以在细菌中的dna保护蛋白Dps中产生N-degron。Dps不再从yfbL发生突变的细胞中被拉下来。我们还克隆了最丰富的n端规则底物之一腐胺转氨酶（PATase）。PATase的独特之处在于它保留了n端蛋氨酸，并通过在n端添加亮氨酸和苯丙氨酸进行修饰。我们将在体外重建修饰反应，并确定负责调节修饰的因素。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打开轴向通道并激活不加区分的蛋白质降解。ADEP结合的位点也是ClpX和ClpA的对接位点，它们控制着底物向ClpP的传递。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ADEP结合所需的ClpP特征和打开通道的ClpP变构变化。我们随机诱变了ClpP，并发现了对ADEP不敏感但保留了与其同源atp酶的ClpP活性的突变体。我们在提供ClpP活性位点的轴向通道和影响对接位点形状的位点上发现了突变。我们已经纯化了几个突变体，正在研究它们的生化和酶性质。我们将纯化大量用于结晶，以确定改变其对ADEP结合反应的结构变化。这些突变体是罕见的，我们希望确定对接位点、活性位点和亚基接触位点之间参与变构通信的位点，所有这些位点都会影响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。形成七聚体环中亚基之间界面的两个关键残基用于螯合Zn。两个催化残基His122和Asp171也与Zn相互作用。我们已经观察到锌稳定了形成ClpP七聚环之间界面的手柄区域的坍塌形式。我们从北卡罗莱纳大学的Holden Thorp教授那里获得了一些可以增强锌与蛋白酶结合的苯并咪唑化合物。我们对这些化合物的初步筛选确定了一种化合物，它可以略微增强抑制作用。我们将要求我们的合作者制备类似的衍生苯并咪唑，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，获得了结核分枝杆菌ClpP活性形式的晶体结构。ClpP对结核分枝杆菌的生长至关重要，因此是潜在抗菌剂的一个有希望的靶点。我们现在有了一个3.0埃的活性晶体结构，它由一个七聚环ClpP1和一个七聚环ClpP2络合而成。只有这个杂络合物是活跃的。在一个络合物中存在两种形式的ClpP，通过允许组装只有一个环突变的四轴照相机，将有助于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，肽在这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，这些小分子抑制剂将作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。我们发现HClpP的过表达可以使接受抗癌药物顺铂治疗的细胞更好地存活。相反，当HClpP部分被敲除时，细胞对顺铂更敏感。当HClpP被敲低时，顺铂的积累增加，这表明可能需要HClpP活性来允许顺铂的快速吸收。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。我们发现顺铂优先结合到线粒体DNA中，HClpP对线粒体DNA中检测到的顺铂加合物水平有显著影响。HClpP与一种叫做佩诺特综合症的遗传性人类疾病有关。此外，纯合子敲除小鼠ClpP会导致严重的听力损失和不孕。这些结果表明，ClpP在哺乳动物细胞中起着重要甚至必不可少的作用。我们发现，在人类细胞培养中，siRNA处理导致hClpP或hClpX的急剧消耗导致细胞死亡。由于转染条件对培养细胞有压力，我们提出HClpP可能在压力条件下是必需的，这可以解释为什么纯合缺失CLPP的小鼠存活。蛋白质组学研究表明，在siRNA耗尽hClpP的16小时内，30种蛋白质增加，其中许多蛋白质参与应激反应。