Heme trafficking in prokaryotic cytochrome c biogenesis

项目概要 细胞色素 c 是高度保守的血红素蛋白，在细胞的电子传递链中发挥作用 呼吸、光合作用、解毒等功能。原核生物生存和繁衍的能力 在不同的、通常是敌对的环境中，这是其电子传输链的可塑性的直接结果， 其中细胞色素c是必需成分。我们投入了大量的精力来研究角色的作用 单个细胞色素 c，但对其生物发生了解甚少，这需要共价键 将血红素附着在保守的 CXXCH 基序上，以实现正确的折叠和功能。尽管他们的多样性，所有 细胞色素 c 由三种途径之一产生：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于我们的研究至关重要 了解生物能量学和细胞生存。虽然这三种途径的演变有所不同 完成生物发生的机制，都必须将血红素转运至全细胞色素 C 合成酶。血红素是一种 所有生物体中必需的辅助因子，不仅在呼吸的电子传递链中发挥作用，而且在呼吸的电子传递链中发挥作用 催化、调节和信号传导。然而，我们对血红素转运蛋白和血红素贩运的了解有限，因为 血红素的细胞毒性、运输的短暂性以及研究膜的技术挑战 蛋白质。因此，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研究 阐明血红素递送、运输和附着的一般机制的愿景，从 系统 I 途径。我们建议1）鉴定细胞质血红素受体和血红素机制 递送，2) 确定系统 I 血红素运输的路径，以及 3) 确定周质的要求 血红素附件。 System I 途径由八种整合膜蛋白 (CcmABCDEFGH) 和 提供了一个易于处理的模型系统来研究这些基本的生物学问题。建议 CcmABCD 运输血红素穿过细菌膜并将其附着到周质血红素伴侣 CcmE， 它将血红素运输至全细胞色素 c 合成酶 CcmFH。利用功能性重组大肠杆菌 系统中，系统 I 蛋白通过内源血红素进行纯化，消除了许多经常出现的技术障碍 与膜蛋白有关。重要的是，血红素附着反应发生在周质中，是 许多病原体的生存所需，并且血红素附着机制可能与 真核合成酶，因此 CcmFH 合成酶是新型抗菌药物的潜在靶点。我们提出的 对系统 I 的研究将同时提供对细胞色素 c 生物合成和一般知识的见解 血红素贩运机制，使我们能够研究两个基本的生物过程。一个 这项工作的自然延伸是将学到的一般原则和开发的方法应用于其他工作 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。

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.