Cytochrome c biogenesis

 描述(申请人提供)：细胞色素是对大多数生物体，包括人类病原体的需氧和厌氧生长必不可少的血红素蛋白。最近，细胞色素生物发生的关键是有专门的组装因子。C型细胞色素的生物发生有三个途径之一，系统I、系统II或系统III。系统I有八个(CcmABCDEFGH)，系统II有两个(CcsBA)专用组装因子(膜蛋白)，而系统III线粒体使用单一的酶称为全细胞色素c合成酶(HCCS)。因为只有原核生物使用系统I和II，并且它们像青霉素的靶标一样在细胞质膜外发挥作用，所以这些途径是新抗菌化合物的潜在靶点。此外，人类HCCS变异是遗传性疾病MLS的罪魁祸首，线粒体病理与癌症、肌病、神经和其他疾病有关。C型细胞色素具有血红素，它以两个半胱氨酸(在CXXCH基序)共价连接到凋亡素上，这一反应由每个系统的合成酶进行。本研究考察了系统I、II和III中的蛋白质如何将血红素结合到细胞色素c上，包括CXXCH基序在哪里与合成酶(CCMF/H、CcsBA、HCCS)相互作用，以及这些合成酶是如何发挥作用的。CXXCH结合位点在所有合成酶上的定位代表着我们对这些途径的了解的主要空白，而合成酶功能的体外重建是该领域的重大挑战。该方案利用了最近从重组大肠杆菌中纯化系统I、II和III的所有蛋白质的成功。对于这些纯化的大部分成分，内源血红素是共纯化的，这有助于分析血红素的运输、红牛控制和附着机制。提出了三个目标，分析系统一(目标1)、系统二(目标2)和系统三(目标3)。系统I分两个步骤进行描述。第一步是CcmABCD介导的周质HoloCcmE的合成和释放(即与血红素)。目的1A分析这一步骤，建立CCMC中直接与血红素相互作用进行贩运的残留物，并检验我们关于holoCcmE形成机制的假设。在步骤2中，HoloCcmE陪伴CCMF/H合成酶的血红素与细胞色素c连接。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。这将通过体内和体外用纯化的合成酶进行交联的方法来完成。对于每个纯化的合成酶(Aim 1B，CCMF/H)，CcsBA(Aim 2)，HCCS(Aim 3)，将建立体外附着试验(即血红素与细胞色素c的附着)。此外，目标3将测试并进一步阐明我们假设的HCCS生物发生的四步模型。结果将揭开所有C型细胞色素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.