Cytochrome C Biogenesis

细胞色素C生物发生

• 批准号: 7267433
• 负责人: Robert G. Kranz
• 金额: $ 29.15万
• 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 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7267433
• 关键词: ATP Hydrolysis; ATP-Binding Cassette Transporters; Address; Aerobic; Affinity; Arabidopsis; Archaeoglobus; Avidity; Bacillus (bacterium); Binding; Binding Sites; Biochemical Genetics; Bioenergetics; Biogenesis; C-terminal; Caenorhabditis; Cell membrane; Cells; Chimeric Proteins; Chlamydomonas; Chloroplasts; Complex; Cryptosporidium; Cysteine; Cytochrome c Group; Cytochromes; Cytoplasm; Defect; Deinococcus; Dependence; Engineering; Enzymes; Escherichia; Escherichia coli; Euglena; Genes; Genus Mycobacterium; Gram-Positive Bacteria; Growth; Hela Cells; Helicobacter; Heme; Hemeproteins; Hereditary Disease; Human; Invertebrates; Life; Ligase; Ligation; Literature; Membrane; Membrane Proteins; Metals; Mitochondria; Molecular; Molecular Chaperones; Mus; Myxococcus; N-terminal; Nature; Nomenclature; Object Attachment; Organism; Oxidation-Reduction; Paramecium; Pathway interactions; Plants; Plasmodium; Porphyrins; Process; Production; Prokaryotic Cells; Property; Proteins; Proteobacteria; Protoporphyrins; Protozoa; Recombinants; Research Personnel; Rhodobacter; Role; Saccharomyces; Side; Signal Transduction; Surface; Synechocystis; System; Testing; Tetrahymena; Time; Triticum; Volvox; analog; antimicrobial drug; base; cofactor; cytochrome C synthetase; cytochrome c; ferrochelatase; frontier; heme a; in vivo; inhibitor/antagonist; pathogen; periplasm; porin; protein function; reconstitution; success; thioether

DESCRIPTION (provided by applicant): Cytochromes are heme proteins essential for the aerobic and anaerobic growth of most organisms, including human pathogens. Relatively recently it has become clear that dedicated assembly pathways and factors are crucial for cytochrome biogenesis. The assembly of c-type cytochromes occurs by one of three pathways, called systems I, II, and III. Systems I and II have nine and four assembly factors (membrane proteins) respectively, while system III uses a single enzyme called cytochrome c heme lyase. Defects in system III result in certain human genetic diseases. Because only prokaryotes, plants, and protozoa use systems I or II, these pathways represent potential targets for antimicrobial agents. The c-type cytochromes possess heme that is covalently ligated to the apocytochrome at two cysteines, which must be reduced for attachment to the heme. Since assembly of c-type cytochromes occurs at the outer surface of the inner membrane, cells must deliver reduced heme, synthesized inside the cell, to the secreted unfolded apocytochrome. In this application we take advantage of our previous success for reconstituting all three systems in recombinant Escherichia coli to study the in vivo concentrations and features of heme that are optimal for each system. The heme delivery pathways will be established. Furthermore, the protein(s) required for the final cytochrome c synthetase (ligase) activity of each system will be purified and characterized for heme and apocytochrome c ligation properties, binding, and mechanisms. Based on our findings that certain metal porphyrins are specific inhibitors of the systems I and II pathways, other porphyrin analogs will be tested for their ability to be ligated to apocytochrome c (or to inhibit biogenesis), further delineating the properties of heme that are critical for ligation. In summary, this study will address which protein(s) constitute the cytochrome c synthetases, how heme is delivered, what concentrations and features of heme are required for each system. Results will help unravel why three very different pathways have emerged in nature and mechanisms to target them.

描述（由申请人提供）：细胞色素是血红素蛋白，对于大多数生物体（包括人类病原体）的需氧和厌氧生长至关重要。最近，人们已经清楚，专用的组装途径和因子对于细胞色素的生物发生至关重要。 c 型细胞色素的组装通过称为系统 I、II 和 III 的三种途径之一进行。系统 I 和 II 分别具有 9 个和 4 个组装因子（膜蛋白），而系统 III 使用一种称为细胞色素 c 血红素裂解酶的酶。 III 系统的缺陷会导致某些人类遗传疾病。由于只有原核生物、植物和原生动物使用系统 I 或 II，因此这些途径代表了抗菌药物的潜在靶标。 c型细胞色素具有在两个半胱氨酸处与脱辅基细胞色素共价连接的血红素，必须将其还原以附着到血红素上。由于 c 型细胞色素的组装发生在内膜的外表面，因此细胞必须将细胞内合成的还原血红素递送至分泌的未折叠脱辅基细胞色素。在此应用中，我们利用之前在重组大肠杆菌中重建所有三个系统的成功来研究每个系统的最佳血红素体内浓度和特征。将建立血红素输送途径。此外，每个系统的最终细胞色素 c 合成酶（连接酶）活性所需的蛋白质将被纯化并表征其血红素和脱辅基细胞色素 c 连接特性、结合和机制。基于我们的发现，某些金属卟啉是系统 I 和 II 途径的特异性抑制剂，将测试其他卟啉类似物与脱辅基细胞色素 c 连接（或抑制生物发生）的能力，进一步描述对连接至关重要的血红素特性。总之，本研究将探讨哪些蛋白质构成细胞色素 C 合成酶、如何递送血红素、每个系统需要什么浓度和特征的血红素。结果将有助于揭示为什么自然界中出现了三种截然不同的途径以及针对它们的机制。