Heme trafficking in prokaryotic cytochrome c biogenesis

原核细胞色素 C 生物发生中的血红素运输

• 批准号: 10434154
• 负责人: Molly Cuddy Sutherland
• 金额: $ 40万
• 依托单位: UNIVERSITY OF DELAWARE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10434154
• 关键词: Address; Aerobic; Anaerobic Bacteria; Bioenergetics; Biogenesis; Biological; Biological Models; Biological Process; Catalysis; Cell physiology; Drug Metabolic Detoxication; Electron Transport; Environment; Escherichia coli; Eukaryota; Foundations; Future; Heme; Hemeproteins; Individual; Integral Membrane Protein; Investigation; Knowledge; Ligase; Membrane; Membrane Proteins; Molecular; Molecular Chaperones; Nature; Organism; Pathway interactions; Photosynthesis; Play; Positioning Attribute; Prokaryotic Cells; Proteins; Reaction; Recombinants; Regulation; Respiration; Role; Signal Transduction; System; Vision; Work; antimicrobial; cytochrome c; cytotoxicity; heme receptor; insight; novel; pathogen; periplasm; protein function; trafficking

Project Summary Cytochromes c are highly conserved heme proteins that function in electron transport chains for cellular functions such as respiration, photosynthesis and detoxification. The ability of prokaryotes to survive and thrive in diverse, often hostile environments is a direct result of the plasticity of their electron transport chains, of which cytochrome c is an essential component. Much effort has been devoted to studying the roles of individual cytochromes c, but much less is understood about their biogenesis, which requires the covalent attachment of heme at a conserved CXXCH motif for proper folding and function. Despite their diversity, all cytochromes c are made by one of three pathways, System I (prokaryotes), System II (prokaryotes) and System III (eukaryotes), thus elucidation of the molecular mechanisms of these pathways is critical to our understanding of bioenergetics and cellular survival. While the three pathways have evolved different mechanisms to accomplish biogenesis, all must transport heme to a holocytochrome c synthetase. Heme is an essential co-factor in all organisms, functioning not only in electron transport chains for respiration, but also for catalysis, regulation and signaling. Yet our knowledge of heme transporters and heme trafficking is limited due to heme’s cytotoxicity, the transient nature of trafficking and the technical challenges of studying membrane proteins. Thus, we must also address the mechanisms of heme trafficking and here we describe our long-term vision to elucidate the general mechanisms of heme delivery, transport and attachment, beginning with the System I pathway. We propose to 1) identify the cytoplasmic heme receptor and mechanisms of heme delivery, 2) determine the path of heme trafficking by System I, and 3) identify the requirements for periplasmic heme attachment. The System I pathway consists of eight integral membrane proteins (CcmABCDEFGH) and provides a tractable model system to study these fundamental biological questions. CcmABCD are proposed to transport heme across the bacterial membrane and attach it to CcmE, the periplasmic heme chaperone, which trafficks heme to the holocytochrome c synthetase, CcmFH. Utilizing a functional, recombinant E. coli system, the System I proteins purify with endogenous heme, removing many of the technical barriers often associated with membrane proteins. Importantly, the heme attachment reaction occurs in the periplasm, is required for the survival of many pathogens, and likely differs in mechanisms of heme attachment from the eukaryotic synthetase, thus the CcmFH synthetase is a potential target for novel antimicrobials. Our proposed studies on System I will simultaneously provide insights into cytochrome c biogenesis and general mechanisms of heme trafficking, uniquely positioning us to study two fundamental biological processes. A natural extension of this work is to apply the general principles learned and approaches developed to the other cytochrome c biogenesis pathways, as well as to other prokaryotic and eukaryotic heme transporters.

项目摘要 细胞色素C是高度保守的血红素蛋白，可在电子传输链中起作用用于细胞 呼吸，光合作用和排毒等功能。原核生物生存和壮成长的能力 在潜水员中，通常敌对的环境是其电子传输链的可塑性的直接结果 哪个细胞色素C是必不可少的组成部分。已经大力研究了 单个细胞色素c，但对其生物发生的理解少得多，这需要共价 血红素在组成的CXXCH基序上的附着，以进行适当的折叠和功能。尽管它们的多样性 细胞色素C由三个途径之一，系统I（原核生物），系统II（原核生物）和 系统III（真核生物），因此阐明这些途径的分子机制对我们来说至关重要 了解生物能学和细胞存活。而三个途径的发展不同 实现生物发生的机制，所有人都必须将血红素转运到全染色组C-合成酶。血红素是一个 所有生物中的必需副因素，不仅在电子传输链中进行呼吸，还可以在 催化，调节和信号传导。然而，我们对血红素转运者和血红素贩运的了解有限 对于血红素的细胞毒性，贩运的短暂性和研究膜的技术挑战 蛋白质。那就是我们还必须解决血红素贩运的机制，在这里我们描述了我们的长期 阐明血红素传递，运输和依恋的一般机制的愿景，从 系统I路径。我们提出1）确定血红素的细胞质血红素受体和机制 交付，2）确定系统i的血红素贩运路径，3）确定对周质的要求 血红素附件。系统I途径由八种整体膜蛋白（CCMABCDEFGH）和 提供了一个可拖动的模型系统来研究这些基本的生物学问题。提出了CCMABCD 为了将血红素跨过细菌膜并将其连接到CCME，Parlasmic血红素链酮， 哪些流动性血红素与全染色型C合成酶CCMFH。利用功能性重组大肠杆菌 系统，系统I蛋白质用内源性血红素纯化，通常消除许多技术障碍 与膜蛋白有关。重要的是，血红素的附着反应发生在pariplasm中， 许多病原体生存所必需的，以及可能从 真核合成酶，因此CCMFH合成酶是新型抗菌剂的潜在靶标。我们提出的 关于系统的研究，我将简单地提供有关细胞色素C生物发生和一般性的见解 血红素贩运的机制，独特地定位了我们研究两个基本生物学过程。一个 这项工作的自然扩展是将所学的一般原则和开发的方法应用于另一个 细胞色素C生物发生途径以及其他原核和真核血红素转运蛋白。