Cytochrome c biogenesis

细胞色素c生物合成

• 批准号: 8962611
• 负责人: Robert G. Kranz
• 金额: $ 33.87万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-01 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8962611
• 关键词: Address; Aerobic; Antibiotics; Bacteria; Binding; Binding Sites; Biochemical; Bioenergetics; Biogenesis; Biological Assay; Cell membrane; Chemicals; Communicable Diseases; Complex; Cysteine; Cytochrome c Group; Cytochromes; Disease; Electron Transport; Engineering; Enzymes; Escherichia coli; Event; Figs - dietary; Goals; Growth; Heme; Hemeproteins; Hereditary Disease; Human; In Vitro; Inflammatory; Integral Membrane Protein; Intracellular Transport; Kinetics; Knowledge; Lead; Ligase; Location; Malignant Neoplasms; Mediating; Membrane; Membrane Proteins; Meningeal Tuberculosis; Mitochondria; Modeling; Molecular; Molecular Chaperones; Myopathy; Neurologic; One-Step dentin bonding system; Organism; Pathology; Pathway interactions; Penicillins; Plants; Preparation; Process; Prokaryotic Cells; Proteins; Proteolysis; Reaction; Recombinants; Respiratory Chain; Structure; System; Systems Analysis; Testing; Variant; antimicrobial; cofactor; crosslink; cytochrome c; ferrochelatase; frontier; fungus; heme 1; in vivo; pathogen; periplasm; polypeptide; public health relevance; reconstitution; respiratory; success; thioether; tool; trafficking

 DESCRIPTION (provided by applicant): Cytochromes are heme proteins essential for aerobic and anaerobic growth of most organisms, including human pathogens. Recently it has become clear that dedicated assembly factors are crucial for cytochrome biogenesis. The biogenesis of c-type cytochromes occurs by one of three pathways, systems I, II, or III. System I has eight (CcmABCDEFGH) and system II has two (CcsBA) dedicated assembly factors (membrane proteins), while system III of mitochondria uses a single enzyme called holocytochrome c synthase (HCCS). Because only prokaryotes use systems I and II and they function outside the cytoplasmic membrane, like the targets of penicillin, these pathways represent potential targets for new antimicrobial compounds. Moreover, human HCCS variants are responsible for the genetic disease MLS, and mitochondrial pathologies are implicated in cancer, myopathies, neurological, and other diseases. The c-type cytochromes possess heme that is covalently attached to the apocytochrome at two cysteines (at a CXXCH motif), a reaction carried out by the synthetase of each system. This study examines how proteins in systems I, II, and III attach heme to apocytochrome c, including where the CXXCH motif interacts on the synthetases (CcmF/H, CcsBA, HCCS), and how these synthetases function. Location of the CXXCH binding site on all synthetases represent major voids in our knowledge of the pathways, and in vitro reconstitution of synthetase functions are grand challenges for the field. The proposal takes advantage of recent success in purifying all proteins of systems I, II, and III from recombinant Escherichia coli. For most of these purified components, endogenous heme is co-purified, facilitating analyses of heme transport, red-ox control, and attachment mechanisms. Three aims are proposed, analyzing systems I (Aim 1), system II (Aim 2), and system III (Aim 3). System I is described in two steps. Step 1 is the CcmABCD-mediated synthesis and release of periplasmic holoCcmE (ie with heme). Aim 1A analyzes this step, establishing residues in CcmC that directly interact with heme for trafficking and testing our hypotheses on mechanisms of holoCcmE formation. In step 2, holoCcmE chaperones heme to the CcmF/H synthetase for attachment to apocytochrome c. Common goals are described for each of the three synthetases: establish the CXXCH binding site on CcmF/H (Aim 1B), CcsBA (Aim 2), and HCCS (Aim 3). This will be accomplished using in vivo and in vitro crosslinking approaches with purified synthetases. For each purified synthetase (Aim 1B, CcmF/H), CcsBA (Aim 2), HCCS (Aim 3), an in vitro attachment assay will be developed (ie attachment of heme to apocytochrome c). Additionally, Aim 3 will test and further elucidate our hypothesized four-step model of biogenesis by HCCS. Results here will unravel molecular mechanisms of biogenesis for all c-type cytochromes.

 描述（由适用提供）：细胞色素是大多数生物（包括人病原体）的有氧和厌氧生长必不可少的血红素蛋白。最近，很明显，专用组装因子对于细胞色素生物发生至关重要。 C型细胞色素的生物发生由三个途径之一，系统I，II或III发生。系统I具有八个（CCMABCDEFGH），并且系统II具有两个（CCSBA）专用组装因子（膜蛋白），而线粒体的系统III使用一种称为全胶质色素C合酶（HCC）的单个酶。由于只有原核生物使用系统I和II，并且它们像青霉素的靶标一样在细胞质膜之外起作用，因此这些途径代表了新的抗菌化合物的潜在靶标。此外，人类HCC的变体负责遗传疾病MLS，线粒体病理在癌症，肌病，神经系统和其他疾病中实施。 C型细胞色素具有血红素，该血红素在两个半胱氨酸（在CXXCH基序上）共价连接到载卵形上，这是由每个系统的合成酶进行的反应。这项研究研究了系统I，II和III中的蛋白质如何将血红素连接到启示色素C上，包括CXXCH基序在合成酶（CCMF/H，CCSBA，HCC，HCC）上相互作用以及这些合成酶的作用。 CXXCH结合位点在所有合成酶上的位置代表了我们对途径的知识中的主要空隙，并且合成酶功能的体外重构是该领域的巨大挑战。该提案利用了重组大肠杆菌I，II和III的所有蛋白质的最新成功。对于大多数这些纯化的组件，内源性血红素是共纯化的，支持血红素转运，红色氧化控制和附着机制的分析。提出了三个目标，分析的系统I（AIM 1），系统II（AIM 2）和System III（AIM 3）。系统I分为两个步骤。步骤1是CCMABCD介导的合成和外围圆度全球的释放（即具有血红素）。 AIM 1A分析了这一步骤，建立了CCMC中的残留物，该残留物与Heme直接相互作用，用于运输和检验我们关于Holoccme形成机制的假设。在步骤2中，将Holoccme链酮血红素血红素到CCMF/H合成酶，以附着在启示色素c上。三种合成酶中的每一个都描述了共同的目标：在CCMF/H上建立CXXCH结合位点（AIM 1B），CCSBA（AIM 2）和HCCS（AIM 3）。这将使用纯化合成酶的体内和体外交联方法来完成。对于每种纯化的合成酶（AIM 1B，CCMF/H），CCSBA（AIM 2），HCCS（AIM 3），将开发一个体外附着测定法（即血红素与启示色素C）的附着。此外，AIM 3将通过HCCS测试并进一步阐明我们假设的生物发生模型。这里的结果将揭示所有C型细胞色素生物发生的分子机制。