Cytochrome C biogenesis

细胞色素C生物合成

• 批准号: 9983433
• 负责人: Robert G. Kranz
• 金额: $ 23.56万
• 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 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9983433
• 关键词: Aerobic; Anaerobic Bacteria; Antibiotics; Award; Bacteria; Binding Sites; Biogenesis; Biological Assay; Cell membrane; Chemicals; Communicable Diseases; Cysteine; Cytochrome c Group; Cytochromes; Disease; Enzymes; Escherichia coli; Genetic Diseases; Goals; Growth; Heme; Hemeproteins; Human; In Vitro; Inflammatory; Knowledge; Ligase; Location; Malignant Neoplasms; Mediating; Membrane Proteins; Meningeal Tuberculosis; Mitochondria; Modeling; Molecular; Molecular Chaperones; Myopathy; Neurologic; Organism; Pathology; Pathway interactions; Penicillins; Prokaryotic Cells; Proteins; Reaction; Recombinants; Respiratory Chain; System; Systems Analysis; Testing; Variant; antimicrobial; crosslink; cytochrome c; human pathogen; in vivo; periplasm; reconstitution; respiratory; success; trafficking

Project Summary (from current Period of award—GM47909, years 20-23) 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.

项目摘要（来自当前获奖期间 - GM47909，20-23 年） 细胞色素是大多数生物体有氧和无氧生长所必需的血红素蛋白， 最近已经清楚，专用的组装因子至关重要。 c 型细胞色素的生物发生通过以下三种途径之一发生， 系统 I、II 或 III 系统 I 有 8 个 (CcmABCDEFGH)，系统 II 有两个 (CcsBA) 专用。 组装因子（膜蛋白），而线粒体系统 III 使用一种称为 因为只有原核生物使用系统 I 和 II 并且它们发挥作用。 在细胞质膜外，就像青霉素的靶标一样，这些途径代表了潜在的 此外，人类 HCCS 变体也是新抗菌化合物的靶点。 遗传病 MLS 和线粒体病理学与癌症、肌病、 神经系统疾病和其他疾病。 c 型细胞色素具有在两个位置共价连接到脱辅基细胞色素的血红素。 半胱氨酸（位于 CXXCH 基序），由每个系统的合成酶进行的反应。 检查系统 I、II 和 III 中的蛋白质如何将血红素附着到脱细胞色素 c，包括 CXXCH 基序与合成酶（CcmF/H、CcsBA、HCCS）相互作用，以及这些合成酶如何相互作用 所有合成酶上的 CXXCH 结合位点的位置代表了我们的主要空白。 对途径的了解以及合成酶功能的体外重建是对 领域。 该提案利用了最近在纯化系统 I、II 和 III 的所有蛋白质方面取得的成功 对于大多数这些纯化成分，内源血红素是来自重组大肠杆菌。 纯化，有利于血红素运输、氧化还原控制和附着机制的分析。 提出了目标，分析系统 I（目标 1）、系统 II（目标 2）和系统 III（目标 3）。 分两步描述，步骤 1 是 CcmABCD 介导的周质合成和释放。 HoloCcmE（即使用血红素）。目标 1A 在此步骤中建立直接在 CcmC 中的残基。 与血红素相互作用进行运输并测试我们对 HoloCcmE 形成机制的假设。 步骤 2，holoCcmE 将血红素陪伴至 CcmF/H 合成酶，以附着脱辅基细胞色素 c。 描述了三种合成酶中每一种的共同目标：在 CcmF/H（目标 1B）、CcsBA（目标 2）和 HCCS（目标 3） 这将在体内和体内完成。 对于每种纯化的合成酶（目标 1B， CcmF/H)、CcsBA (目标 2)、HCCS (目标 3)，将开发体外附着测定（即附着 此外，Aim 3 将测试并进一步阐明我们捕获的四种- HCCS 生物发生的步骤模型。 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。

项目成果

Transmembrane heme delivery systems.

跨膜血红素递送系统。

• DOI: 10.1073/pnas.95.9.5003
• 发表时间: 1998-04-28
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Barry S. Goldman;David L. Beck;E. M. Monika;R. Kranz
• 通讯作者: R. Kranz

Heme Trafficking and Modifications during System I Cytochrome c Biogenesis: Insights from Heme Redox Potentials of Ccm Proteins.

系统 I 细胞色素 c 生物发生过程中的血红素运输和修饰：Ccm 蛋白血红素氧化还原潜力的见解。

• 发表时间: 2016-06-07
• 影响因子: 2.9
• 作者: Sutherland, Molly C;Rankin, Joel A;Kranz, Robert G
• 通讯作者: Kranz, Robert G

Engineered holocytochrome c synthases that biosynthesize new cytochromes c.

工程化的全细胞色素 c 合酶可生物合成新的细胞色素 c。

• 发表时间: 2017-02-28
• 影响因子: 11.1
• 作者: Mendez, Deanna L;Babbitt, Shalon E;King, Jeremy D;D'Alessandro, John;Watson, Michael B;Blankenship, Robert E;Mirica, Liviu M;Kranz, Robert G
• 通讯作者: Kranz, Robert G

Analysis of the fnrL gene and its function in Rhodobacter capsulatus.

荚膜红杆菌fnrL基因及其功能分析

• 发表时间: 1997-12
• 影响因子: 3.2
• 作者: Zeilstra;Gabbert, K;Mouncey, N J;Kaplan, S;Kranz, R G
• 通讯作者: Kranz, R G

Four genes are required for the system II cytochrome c biogenesis pathway in Bordetella pertussis, a unique bacterial model.

百日咳博德特氏菌（一种独特的细菌模型）的系统 II 细胞色素 c 生物发生途径需要四个基因。

• 发表时间: 2000-11
• 影响因子: 3.6
• 作者: Beckett, C S;Loughman, J A;Karberg, K A;Donato, G M;Goldman, W E;Kranz, R G
• 通讯作者: Kranz, R G