Cytochrome C biogenesis

项目概要（从当前获奖期-GM 47909，第20-23年） 细胞色素是大多数生物体需氧和厌氧生长所必需的血红素蛋白， 包括人类病原体。最近，人们已经清楚地认识到，专门的组装因素至关重要 细胞色素的生物合成。c型细胞色素的生物合成通过三种途径之一发生， 系统I、II或III。系统I有8个（CcmABCDEFGH），系统II有2个（CcsBA）专用 组装因子（膜蛋白），而线粒体的系统III使用一种称为 全细胞色素c合酶（HCCS）。因为只有原核生物使用系统I和II， 在细胞质膜外，就像青霉素的靶点一样，这些途径代表了潜在的 新的抗菌化合物的目标。此外，人类HCCS变体负责 遗传疾病MLS和线粒体病理与癌症，肌病， 神经和其他疾病。 C型细胞色素具有血红素，其共价连接到脱辅基细胞色素的两个 半胱氨酸（在CXXCH基序），由每个系统的合成酶进行的反应。本研究 研究了系统I、II和III中的蛋白质如何将血红素连接到脱辅基细胞色素c上，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 功能CXXCH结合位点在所有合成酶上的位置代表了我们的研究中的主要空白。 途径的知识，以及合成酶功能的体外重建， 外地 该提案利用了最近在纯化系统I、II和III的所有蛋白质方面的成功 从重组大肠杆菌。对于大多数这些纯化的组分，内源性血红素是共- 纯化，促进血红素运输，red-ox控制和附着机制的分析。三 提出了目标，分析系统I（目标1），系统II（目标2），系统III（目标3）。第一系统 分两步描述。第一步是CcmABCD介导的周质合成和释放 holoCcmE（含血红素）。目的1A分析这一步骤，在CcmC中建立直接 与血红素相互作用的运输和测试我们的假设的机制holoCcmE形成。在 步骤2，全CcmE伴侣血红素与CcmF/H合成酶连接，用于连接脱辅基细胞色素c。 三种合成酶的共同目标是：建立CXXCH结合位点， CcmF/H（目标1B）、CcsBA（目标2）和HCCS（目标3）。这将使用体内和体内 用纯化的合成酶进行体外交联。对于每种纯化的合成酶（Aim 1B， CcmF/H）、CcsBA（目标2）、HCCS（目标3），将开发体外附着试验（即附着 血红素到脱辅基细胞色素c）。此外，目标3将测试和进一步阐明我们假设的四个- HCCS生物发生分步模型 这里的结果将解开所有c型细胞色素的生物发生的分子机制。

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.