Cytochrome C biogenesis

细胞色素C生物合成

基本信息

批准号：
9983433
负责人：
Robert G. Kranz
金额：
$ 23.56万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1994
资助国家：
美国
起止时间：
1994-09-01 至 2021-08-31
项目状态：
已结题

项目摘要

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.

项目摘要（从当前奖励期 - GM47909，20 - 23年）细胞色素是大多数生物的有氧和厌氧生长必不可少的血红素蛋白，包括人类病原体。最近，很明显，专用的装配因素至关重要用于细胞色素生物发生。 C型细胞色素的生物发生由三种途径之一发生，系统I，II或III。系统I有八个（CCMABCDEFGH），系统II有两个（CCSBA）专用装配因子（膜蛋白），而线粒体系统III使用一种称为全染色物C合酶（HCCS）。因为只有原核生物使用系统I和II，并且它们起作用像青霉素的靶标一样，在细胞质膜之外，这些途径代表潜力新抗菌化合物的靶标。而且，人类HCC的变体负责遗传病MLS和线粒体病理在癌症，肌病中实施神经系统和其他疾病。 C型细胞色素具有血红素，该血红素在两个处共价附着在两个半胱氨酸（在CXXCH基序上），每种系统的合成酶进行的反应。这项研究检查系统I，II和III中的蛋白质如何将血红素连接到启示色C上，包括 CXXCH基序在合成酶（CCMF/H，CCSBA，HCCS）上相互作用，以及这些合成酶如何功能。 CXXCH结合位点在所有合成酶上代表我们的主要空隙对途径的了解以及合成酶功能的体外重构是巨大的挑战领域。该提案在净化系统I，II和III的所有蛋白质方面都利用了最近的成功来自重组大肠杆菌。对于大多数这些纯化成分，内源性血红素是共同的纯化的，支持血红素转运，红氧控制和固定机制的分析。三提出了目标，分析系统I（AIM 1），系统II（AIM 2）和System III（AIM 3）。系统我是用两个步骤描述。步骤1是CCMABCD介导的合成和周质的释放 Holoccme（即血红素）。 AIM 1A分析此步骤，在CCMC中建立残差与血红素相互作用，以进行运输和检验我们关于Holoccme形成机制的假设。在步骤2，Holoccme伴侣血红素到CCMF/H合成酶，以附着在启示色素上。三种合成酶中的每一个都描述了共同的目标：建立CXXCH结合位点 CCMF/H（AIM 1B），CCSBA（AIM 2）和HCC（AIM 3）。这将使用体内和体外交联方法与纯化的合成酶。对于每个纯化的合成酶（AIM 1B， CCMF/h），CCSBA（AIM 2），HCCS（AIM 3），将开发体外附件测定法（即附件血红素到启示色素c）。此外，AIM 3将测试并进一步阐明我们假设的四个 HCC的生物发生模型。这里的结果将揭示所有C型细胞色素生物发生的分子机制。

项目成果

期刊论文数量（43）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Heme ligand identification and redox properties of the cytochrome c synthetase, CcmF.

DOI：
10.1021/bi201508t
发表时间：
2011-12-20
期刊：
BIOCHEMISTRY
影响因子：
2.9
作者：
Francisco, Brian San;Bretsnyder, Eric C.;Rodgers, Kenton R.;Kranz, Robert G.
通讯作者：
Kranz, Robert G.

ABC transporters associated with cytochrome c biogenesis.

DOI：
10.1016/s0923-2508(01)01203-7
发表时间：
2001-04
期刊：
Research in microbiology
影响因子：
2.6
作者：
Barry S. Goldman;R. Kranz
通讯作者：
Barry S. Goldman;R. Kranz

Essential histidine pairs indicate conserved haem binding in epsilonproteobacterial cytochrome c haem lyases.

DOI：
10.1099/mic.0.042838-0
发表时间：
2010-12
期刊：
Microbiology (Reading, England)
影响因子：
0
作者：
Kern M;Scheithauer J;Kranz RG;Simon J
通讯作者：
Simon J

In vitro reconstitution reveals major differences between human and bacterial cytochrome c synthases.

DOI：
10.7554/elife.64891
发表时间：
2021-05-11
期刊：
eLife
影响因子：
7.7
作者：
Sutherland MC;Mendez DL;Babbitt SE;Tillman DE;Melnikov O;Tran NL;Prizant NT;Collier AL;Kranz RG
通讯作者：
Kranz RG

Conserved residues of the human mitochondrial holocytochrome c synthase mediate interactions with heme.

DOI：
10.1021/bi500704p
发表时间：
2014-08-19
期刊：
BIOCHEMISTRY
影响因子：
2.9
作者：
Babbitt, Shalon E.;San Francisco, Brian;Bretsnyder, Eric C.;Kranz, Robert G.
通讯作者：
Kranz, Robert G.