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，专门的组装途径和因子对细胞色素生物发生至关重要，这一点已经变得明显。C型细胞色素的组装通过三个途径之一进行，称为系统I、II和III。系统I和II分别有9个和4个组装因子(膜蛋白)，而系统III使用一种称为细胞色素C血红素裂解酶的单一酶。系统III中的缺陷会导致某些人类遗传病。因为只有原核生物、植物和原生动物使用系统I或II，所以这些途径是抗菌剂的潜在靶标。C型细胞色素具有两个半胱氨酸共价连接到凋亡素的血红素，必须还原才能附着在血红素上。由于c型细胞色素的组装发生在内膜的外表面，细胞必须将在细胞内合成的还原的血红素输送到分泌的未折叠的凋亡素。在这一应用中，我们利用我们之前在重组大肠杆菌中重组所有三个系统的成功，研究了对每个系统最优的血红素在体内的浓度和特征。将建立血红素传递途径。此外，每个系统的最终细胞色素c合成酶(连接酶)活性所需的蛋白质(S)将被提纯，并对血红素和细胞色素c的连接特性、结合和机制进行表征。基于我们的发现，某些金属卟啉是系统I和II途径的特异性抑制剂，其他卟啉类似物将被测试它们与细胞色素c连接的能力(或抑制生物发生)，进一步描述对连接至关重要的血红素的性质。综上所述，这项研究将解决哪些蛋白质(S)构成细胞色素c合成酶，血红素是如何输送的，每个系统需要什么浓度和特性的血红素。结果将有助于揭开为什么自然界中出现了三种截然不同的途径以及针对它们的机制。

DESCRIPTION (provided by applicant): Cytochromes are heme proteins essential for the aerobic and anaerobic growth of most organisms, including human pathogens. Relatively recently it has become clear that dedicated assembly pathways and factors are crucial for cytochrome biogenesis. The assembly of c-type cytochromes occurs by one of three pathways, called systems I, II, and III. Systems I and II have nine and four assembly factors (membrane proteins) respectively, while system III uses a single enzyme called cytochrome c heme lyase. Defects in system III result in certain human genetic diseases. Because only prokaryotes, plants, and protozoa use systems I or II, these pathways represent potential targets for antimicrobial agents. The c-type cytochromes possess heme that is covalently ligated to the apocytochrome at two cysteines, which must be reduced for attachment to the heme. Since assembly of c-type cytochromes occurs at the outer surface of the inner membrane, cells must deliver reduced heme, synthesized inside the cell, to the secreted unfolded apocytochrome. In this application we take advantage of our previous success for reconstituting all three systems in recombinant Escherichia coli to study the in vivo concentrations and features of heme that are optimal for each system. The heme delivery pathways will be established. Furthermore, the protein(s) required for the final cytochrome c synthetase (ligase) activity of each system will be purified and characterized for heme and apocytochrome c ligation properties, binding, and mechanisms. Based on our findings that certain metal porphyrins are specific inhibitors of the systems I and II pathways, other porphyrin analogs will be tested for their ability to be ligated to apocytochrome c (or to inhibit biogenesis), further delineating the properties of heme that are critical for ligation. In summary, this study will address which protein(s) constitute the cytochrome c synthetases, how heme is delivered, what concentrations and features of heme are required for each system. Results will help unravel why three very different pathways have emerged in nature and mechanisms to target them.