Cytochrome c biogenesis

 描述（由申请人提供）：细胞色素是大多数生物（包括人类病原体）需氧和厌氧生长所必需的血红素蛋白。最近人们已经清楚，专门的组装因子对于细胞色素生物合成至关重要。C型细胞色素的生物发生通过三种途径之一，系统I，II或III。系统I有八个（CcmABCDEFGH）和系统II有两个（CcsBA）专用的组装因子（膜蛋白），而线粒体系统III使用一种称为全细胞色素c合成酶（HCCS）的酶。因为只有原核生物使用系统I和II，并且它们在细胞质膜外发挥作用，就像青霉素的靶标一样，这些途径代表了新抗菌化合物的潜在靶标。此外，人类HCCS变体是遗传疾病MLS的原因，并且线粒体病理与癌症、肌病、神经系统疾病和其他疾病有关。 C型细胞色素具有血红素，其在两个半胱氨酸处（在CXXCH基序处）共价连接到脱辅基细胞色素，这是由每个系统的合成酶进行的反应。本研究探讨了如何在系统I，II和III中的蛋白质连接血红素脱辅基细胞色素c，包括CXXCH基序相互作用的合成酶（CcmF/H，CcsBA，HCCS），以及这些合成酶的功能。所有合成酶上的CXXCH结合位点的位置代表了我们对途径的知识中的主要空白，并且合成酶功能的体外重建是该领域的巨大挑战。 该提案利用了最近成功纯化重组大肠杆菌中系统I、II和III的所有蛋白质。对于大多数这些纯化的成分，内源性血红素共纯化，促进血红素运输，red-ox控制和附着机制的分析。提出了三个目标，分析系统I（目标1），系统II（目标2）和系统III（目标3）。系统I分两步描述。第一步是CcmABCD介导的周质holoCcmE（即血红素）的合成和释放。目的1A分析了这一步骤，建立CcmC中的残基，直接与血红素相互作用，用于运输，并测试我们对holoCcmE形成机制的假设。在步骤2中，holoCcmE伴侣血红素与CcmF/H合成酶连接，以连接到脱辅基细胞色素c。描述了三种合成酶的共同目标：在CcmF/H（目标1B）、CcsBA（目标2）和HCCS（目标3）上建立CXXCH结合位点。这将通过使用纯化的合成酶的体内和体外交联方法来实现。对于每种纯化的合成酶（Aim 1B、CcmF/H）、CcsBA（Aim 2）、HCCS（Aim 3），将开发体外附着试验（即血红素与脱辅基细胞色素c的附着）。此外，目标3将测试和进一步阐明我们假设的四步模型的生物合成的HCCS。 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。

 DESCRIPTION (provided by applicant): 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.