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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

基本信息

批准号：
10618929
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
项目类别：
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

项目摘要

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，其在细胞的电子传递链中起作用，呼吸、光合作用和解毒等功能。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，电子传递链的可塑性是直接结果，其中细胞色素C是必需成分。人们已经花了很大的精力来研究单个细胞色素c，但对它们的生物起源了解得少得多，这需要共价键。在保守的CXXCH基序处连接血红素以进行适当的折叠和功能。尽管他们的多样性，所有细胞色素c由三种途径之一产生，即系统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的研究至关重要。了解生物能量学和细胞存活。虽然这三种途径在进化过程中完成生物发生的机制都必须将血红素转运到全细胞色素C合成酶。血红素是一种所有生物体中的一种重要辅因子，不仅在呼吸的电子传递链中起作用，而且在催化、调节和信号传导。然而，我们的知识血红素转运和血红素贩运是有限的，血红素的细胞毒性，运输的瞬时性质和研究膜的技术挑战 proteins.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设想阐明血红素交付，运输和附件的一般机制，从系统I路径。我们的工作是：1）确定胞质血红素受体和血红素的作用机制递送，2）通过系统I确定血红素运输的路径，以及3）确定周质运输的要求。血红素附着系统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。建议使用CcmABCD 运输血红素穿过细菌膜并将其附着到CcmE，周质血红素伴侣，将血红素运输到全细胞色素C合成酶CcmFH。利用功能性重组E.杆菌系统，系统I蛋白质纯化与内源性血红素，消除了许多技术障碍，往往与膜蛋白有关。重要的是，血红素附着反应发生在周质中，许多病原体的生存所需的，并可能在血红素附着的机制不同，从因此，CcmFH合成酶是新型抗微生物剂的潜在靶标。我们提出的对系统I的研究将同时提供对细胞色素c生物发生和一般生物学的见解。血红素运输的机制，独特的定位我们研究两个基本的生物过程。一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领域，细胞色素c生物合成途径，以及其他原核和真核血红素转运蛋白。