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

基本信息

批准号：
7732887
负责人：
MICHAEL MAURIZI
金额：
$ 92.69万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
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/7732887
关键词：
26S proteasome ATP Hydrolysis ATP-Dependent Proteases ATPase Domain Active Sites Adaptor Signaling Protein Affect Aftercare Amino Acids Antibodies Antineoplastic Agents Apoptotic Articular Range of Motion Bacteria Binding Binding Sites Biochemical Biochemical Genetics Biochemistry Biological Biological Assay C-terminal Cell Death Cell Survival Cells Cisplatin Collaborations Communication Complex Condition Coupled Coupling Cultured Cells Cytosol DNA Damage Diagnostic E coli Lon protein Electron Microscopy Endopeptidases Enzymes Escherichia coli Eukaryota Eukaryotic Cell Event Gel Goals Green Fluorescent Proteins Growth and Development function Heart Helper-Inducer T-Lymphocyte Homologous Gene Human Human Activities Indium Laboratories Learning Life Mammalian Cell Maps Measurable Membrane Methionine Minor Mitochondria Molecular Molecular Chaperones Molecular Conformation Molecular Machines Motion N Domain N-terminal Nature New Agents Operating System Peptide Hydrolases Peptides Physiological Play Pliability Positioning Attribute Process Property Protein Binding Protein Biochemistry Protein translocation Proteins Quality Control Range of motion exercise Reaction Recombinants Recovery Regulatory Pathway Research Research Project Grants Resistance Resolution Ribosomes Role Serine Site Small Interfering RNA Specificity Stress Structure Substrate Interaction System TNFSF10 gene Therapeutic Agents Time Translations Ultracentrifugation antimicrobial base cell type design endopeptidase La genetic regulatory protein in vivo inhibitor/antagonist killings molecular modeling multicatalytic endopeptidase complex mutant novel polypeptide protein degradation rapid growth receptor response sedimentation coefficient size tandem mass spectrometry tmRNA two-dimensional unfoldase

项目摘要

Research conducted in the Biochemistry of Proteins Section is focused on the function and control of protein degradation in bacterial and human cells. Intracellular protein degradation plays a critical part in controlling the levels of important cellular regulatory proteins and is an essential component of the protein quality control system. Most protein degradation within the cytosol is carried out by ATP-dependent proteases, which are multi-component molecular machines. The heart of the machine is an ATP-driven protein unfoldase that binds a specific protein target, disrupts its structure, and translocates the unfolded protein into the proteolytic chamber of a tightly associated self-compartmentalized endopeptidase. Our studies encompass structural and biochemical analysis of the ATP-dependent Clp and Lon proteases from E. coli and from human mitochondria and assay of their biological activities in cultured cells. In studies of human ClpXP, we found that hClpP and hClpX are required for cell viability. Human cells in culture die during the first 48 h after treatment with siRNA directed against either HCLPP or HCLPX. Intracellular hClpP protein levels decrease rapidly when synthesis is shut off, indicating that hClpP is unstable and must be continuous replenished to maintain ClpP activity in mitochondria. The decrease in hClpX protein is relatively modest but cell death occurs in nearly the same time as with HCLPP knockdown, suggesting that newly synthesized ClpX is needed for survival of cells. The decrease in enzymatic activity in cells treated with siRNA was shown with a fluorescent substrate whose levels respond to changes in hClpP or hClpX amounts. Green fluorescent protein (GFP) bearing a degradation tag (SsrA) for the bacterial ClpXP is degraded and undetectable in wild type cells. Knockdown of either hClpP or hClpX led to measurable GFP and more than 4-fold stabilization of the protein. Since, purified hClpXP cannot degrade GFP-SsrA, we propose that factors in mitochondria can stimulate the activity of human ClpXP or act as adaptor proteins to help deliver substrates to hClpXP. Short term treatment with HCLPP or HCLPX siRNA sensitizes the cells to apoptotic cell death in the presence of cisplatin or staurosporin. Over expression of human ClpP confers resistance to killing by the DNA-damaging agent, cisplatin, and by an inducer of the cell death receptor, TRAIL. Protection requires hClpP proteolytic activity and also the C-terminal domain of hClpP, a unique feature of mammalian ClpP proteins. We extended our studies of degradation of endogenous SsrA-tagged proteins in E. coli, which are produced when the tmRNA system detects stalled ribosomes and releases incomplete C-terminally tagged polypeptide chains. Using an anti-SsrA antibody prepared in our laboratory, we established that ClpXP plays the major role in degradation of SsrA-tagged proteins and that Lon and ClpAP have minor roles in degrading them. We showed that the tagging system operates at a high level when proteins are over expressed in cells and we were able to detect significant amounts of tagged over expressed proteins even in wild type cells. We are in the process of isolating endogenous SsrA-tagged proteins using the ability of mutant forms of ClpP to trap protein substrates in vivo. We have found by two-dimensional gels that there are fewer than 30-40 proteins that appear to be SsrA-tagged, suggesting that tagging does not occur equally for all translation reaction. We will isolate and identify SsrA-tagged substrates to determine what proteins are preferentially SsrA-tagged and whether tagging serves a regulatory function. We are conducting a study to define the parameters for the selective degradation of proteins bearing N-degrons in E. coli. We showed that proteins with non-canonical N-terminal amino acids (those not naturally exposed by activity of methionine amino peptidase) require ClpS, a small adaptor proteins that binds to the N-domain of ClpA, for degradation. We find that peptides with N-degrons are competitive inhibitors of protein degradation by ClpAP/ClpS and that adding methionine or serine to the N-terminus blocks the ability to inhibit ClpS activity. We are collaborating with Dr. Di Xia to crystallize N-degron peptide complexes with ClpS and ClpS bound to ClpA. We are mutagenizing conserved residues in the putative N-degron binding site of ClpS and will isolate and characterize the mutant proteins. To learn how proteins with N-degrons arise in cells, we have constructed mutants lacking other proteases that will retain the ability to specifically target N-degron proteins to ClpAP. We will express inactive ClpP to trap N-degron proteins, identify them by tandem mass spectrometry, and analyze for normal or abnormal N-terminal residues. Recoveries in the presence and absence of ClpS will be compared to determine if ClpS is needed for all N-degron targeting. Our structural studies are focused on novel aspects pertaining to substrate interaction and the response of the Clp complexes to substrate binding. ClpA undergoes a large conformational change upon binding of peptide substrates. In ultracentrifugation studies (done in collaboration with Dr. Grzegorz Piszczek, NHLBI) we have shown that binding of a decapeptide slightly reduces the sedimentation coefficient of ClpA and causes a dramatic narrowing of the distribution of structurally distinct species. Narrowed distribution indicates that peptide binding stabilizes a uniform conformation of ClpA and supports the hypothesis that peptides bind in the central channel of the hexamer. Peptide binding has the same effect on ClpA lacking the N-domain, indicating that the conformational flexibility is not due to the mobile N-domains. With ClpA lacking the C-terminal ATPase domain (D2), we found that the distribution was already narrow and not further narrowed by peptide binding. These results establish that peptides can migrate to ClpA-D2 and further suggest that the conformational variability of ClpA is due to motion in ClpA-D2. We purified mutants of ClpA with shorter linkers between the N-domain and ClpA-D1. ClpA with deletions of 10, 15, and 20 amino acids in the linker had activities similar to wild type. By electron microscopy (done in collaboration with Alasdair Seven, NIAMS) the ClpA N-domains were more visible when linkers were shorter, indicating that their range of motion was restricted. We will use ClpA hexamers with less mobile N-domain to map the position of ClpS bound to ClpA and to visualize substrates bound to the ClpS/N-domain complex. The N-terminal peptides of ClpP affect activity and interact with ClpA and ClpX. We expressed ClpP lacking a portion of the N-terminal loop and thus has a larger axial channel. This form of ClpP has greatly enhanced peptidase activity in the absence of activation by ClpX or ClpA. Activity is nearly 40% of fully activated wild-type ClpP. We do not know whether increased activity is due to the increased size of the channel , allowing peptides into the chamber of ClpP more rapidly, or to increased activity at the proteolytic site due to allosteric communication between the N-terminus and the active site. Surprisingly, ClpX and ClpA inhibit the activity of ClpP when the N-terminal loop is deleted. Inhibition s [summary truncated at 7800 characters]

在蛋白质生物化学部分进行的研究集中在细菌和人类细胞中蛋白质降解的功能和控制上。细胞内蛋白质降解在控制重要细胞调节蛋白水平方面起着至关重要的作用，并且是蛋白质质量控制系统的重要组成部分。细胞质内的大多数蛋白质降解是由ATP依赖性蛋白酶（是多组分分子机器）进行的。机器的核心是ATP驱动的蛋白展览酶，它结合特定的蛋白质靶标，破坏其结构，并将展开的蛋白质转移到紧密相关的自隔离式内肽酶的蛋白水解腔中。我们的研究涵盖了来自大肠杆菌和人类线粒体的ATP依赖性CLP和LON蛋白酶的结构和生化分析，以及其在培养细胞中生物学活性的测定。在人类CLPXP的研究中，我们发现细胞活力需要HCLPP和HCLPX。培养物中的人类细胞在针对HCLPP或HCLPX的siRNA处理后的最初48小时死亡。关闭合成时，细胞内HCLPP蛋白水平迅速降低，表明HCLPP不稳定，必须连续补充以维持线粒体中的CLPP活性。 HCLPX蛋白的降低相对适度，但细胞死亡几乎与HCLPP敲低同一时间发生，这表明细胞存活需要新合成的CLPX。用荧光底物显示了用siRNA处理的细胞中酶活性的降低，其水平响应HCLPP或HCLPX量的变化。在野生型细胞中降解并且无法检测到具有降解标签（SSRA）的绿色荧光蛋白（GFP）。 HCLPP或HCLPX的敲低导致可测量的GFP和蛋白质的4倍以上。由于纯化的HCLPXP无法降解GFP-SSRA，因此我们建议线粒体中的因素可以刺激人ClPXP的活性或充当衔接蛋白，以帮助将底物传递给HCLPXP。用HCLPP或HCLPX siRNA的短期治疗在存在顺铂或星孢子蛋白的存在下将细胞敏感到凋亡细胞死亡。人类CLPP的表达赋予了对DNA破坏剂顺铂的抗性，以及细胞死亡受体的诱导剂Trail。保护需要HCLPP蛋白水解活性以及HCLPP的C末端结构域，HCLPP是哺乳动物CLPP蛋白的独特特征。我们扩展了对大肠杆菌中内源性SSRA标签蛋白降解的研究，当TMRNA系统检测到失速的核糖体并释放不完整的C末端标记的多肽链时会产生。使用在我们的实验室中制备的抗SSRA抗体，我们确定CLPXP在降解SSRA标记的蛋白质中起主要作用，而LON和CLPAP在降解它们方面具有较小的作用。我们表明，当蛋白质在细胞中过度表达时，标记系统在高水平上运行，即使在野生型细胞中，我们也能够检测到大量标记的蛋白质上。我们正在使用突变形式的CLPP捕获体内蛋白质底物的能力来分离内源性SSRA标记的蛋白质。我们已经通过二维凝胶发现了似乎被SSRA标记的蛋白质少于30-40个蛋白质，这表明所有翻译反应的标记并非同样发生。我们将隔离并鉴定标有SSRA标签的底物，以确定哪些蛋白质优先使用SSRA标签，以及标记是否具有调节功能。我们正在进行一项研究，以定义在大肠杆菌中携带N-脱脂龙的蛋白质选择性降解的参数。我们表明，具有非典型N末端氨基酸的蛋白质（非自然暴露于蛋氨酸氨基肽酶的活性的蛋白质）需要CLP，这是一种与CLPA的N-域结合的小衔接蛋白，以降解。我们发现，具有N-脱脂龙的肽是CLPAP/CLPS蛋白质降解的竞争抑制剂，并且在N末端添加蛋氨酸或丝氨酸会阻止抑制CLPS活性的能力。我们正在与Di Xia博士合作，以与Clps结晶的N-Degron肽配合物结晶，并与CLPA结合。我们正在将CLPS假定的N-Degron结合位点中的保守残基化，并将分离并表征突变蛋白。为了了解细胞中蛋白质的蛋白质是如何出现的，我们构建了缺乏其他蛋白酶的突变体，这些突变体将保留特异性靶向N-脱绿蛋白蛋白的能力。我们将表达不活跃的CLPP来捕获N-Degron蛋白，通过串联质谱识别它们，并分析正常或异常的N末端残基。将在存在和不存在CLP的情况下进行回收，以确定是否需要所有N-Degron靶向的CLP。我们的结构研究集中在与底物相互作用有关的新方面以及CLP复合物对底物结合的反应。 CLPA在肽底物结合后经历了巨大的构象变化。在超速离心研究中（与NHLBI的Grzegorz Piszczek合作进行），我们已经表明，脱肽的结合略微降低了CLPA的沉积系数，并导致结构上不同物种的分布的巨大狭窄。狭窄的分布表明肽结合稳定了CLPA的均匀构象，并支持肽在六聚体中央通道中结合的假设。肽结合对缺乏N域的CLPA具有相同的影响，表明构象柔韧性不是由于移动N域引起的。由于CLPA缺乏C末端ATPase结构域（D2），我们发现该分布已经狭窄，并且不会因肽结合而进一步缩小。这些结果表明，肽可以迁移到CLPA-D2，并进一步表明CLPA的构象变异性是由于CLPA-D2中的运动引起的。我们纯化了CLPA的突变体，并在N域和CLPA-D1之间具有较短的接头。连接器中缺失10、15和20个氨基酸的CLPA具有类似于野生型的活动。通过电子显微镜（与Alasdair Seven，Niams合作完成），当接头较短时，CLPA n域更为明显，表明它们的运动范围受到限制。我们将使用具有较少移动N域的CLPA六聚体来绘制与CLPA结合的CLP的位置，并可视化与Clps/N-N-domain复合物结合的底物。 CLPP的N末端肽会影响活性并与CLPA和CLPX相互作用。我们表达了缺少N末端环的一部分的CLPP，因此具有较大的轴向通道。在没有CLPX或CLPA激活的情况下，这种形式的CLPP具有大大增强的肽酶活性。活性几乎是完全活化的野生型CLPP的40％。我们不知道活动的增加是由于通道的大小增加，使肽更快地进入CLPP的腔室，还是由于N末端和活性位点之间的变构通信而导致蛋白水解位点的活性增加。令人惊讶的是，当删除N末端环时，CLPX和CLPA抑制CLPP的活性。抑制s [摘要以7800个字符截断]