PROBING THE ACTIVE SITE OF THE MITOCHONDRIAL HOLOCYTOCHROME C SYNTHASE, HCCS

探测线粒体全细胞色素 C 合成酶 (HCCS) 的活性位点

• 批准号: 9279705
• 负责人: Shalon Elizabeth Ledbetter
• 金额: $ 2.9万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-01 至 2016-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9279705
• 关键词: Active Sites; Affect; Amino Acids; Amoeba genus; Animals; Apoptosis; Binding; Binding Sites; Biochemical; Bioenergetics; Biogenesis; Biological Assay; Biological Process; Cell Respiration; Cells; Cellular biology; Cessation of life; Chemicals; Complex; Cysteine; Cytochrome c Group; Cytochromes; Data; Defect; Disease; Electrons; Environment; Escherichia coli; Eukaryota; Eukaryotic Cell; Figs - dietary; Health; Heme; Histidine; Homologous Gene; Human; Human Activities; In Vitro; Knowledge; Label; Length; Life; Mass Spectrum Analysis; Mediating; Metalloproteins; Methods; Mitochondria; Modification; Mutation; Organelles; Pathway interactions; Peptides; Play; Positioning Attribute; Process; Production; Prokaryotic Cells; Property; Prosthesis; Proteins; Protocols documentation; Recombinants; Role; Site; Site-Directed Mutagenesis; Structure; System; Testing; Work; Yeasts; absorption; base; cofactor; crosslink; cytochrome C synthetase; cytochrome c; heme a; human disease; in vivo; insight; microphthalmia-dermal aplasia-sclerocornea syndrome; mutant; thioether

DESCRIPTION (provided by applicant): In eukaryotes, cytochrome c plays a vital role in both aerobic respiration and apoptosis, thus impacting the life and death of a cell. C-type cytochromes are characterized by covalent attachment of a heme co-factor, a modification that is required for its stability and subsequent function. Heme attachment takes place in the mitochondria and is mediated by holocytochrome c synthase, HCCS, the primary component of the eukaryotic cytochrome c biogenesis pathway, also known as System III. Previous studies in animals have shown that defects in HCCS can result in lethality or the human disease microphthalmia with linear skin defects (MLS). Although HCCS was discovered in yeast 25 years ago, the mechanisms underlying HCCS function have yet to be explored, largely due to its poor recombinant expression and instability. Our lab has very recently been successful in overcoming these technical limitations, allowing us to initiate the first comprehensive biochemical analysis of HCCS structure/ function. The catalytic function of HCCS depends on its ability to interact with and coordinate interactions between its substrates, heme and cytochrome c. Therefore, the proposed study seeks to determine which residues and/or domains in HCCS comprise its active site. Aim 1 analyzes the functional consequences of mutations of highly conserved residues in HCCS by examining perturbations in heme binding and cytochrome c recruitment and maturation. The apocytochrome c substrate-binding site of HCCS is directly assessed in Aim 2 by peptide crosslinking, using a UV-crosslinkable cytochrome c peptide containing the heme-attachment site. These studies will provide the first in-depth mechanistic analysis of HCCS, thus advancing our understanding of mitochondrial bioenergetics. Our results will directly impact what is currently known about diseases like MLS that are caused by HCCS abnormalities, as well as contribute to the body of knowledge concerning the many malfunctions observed in mitochondria in other human conditions.

描述（申请人提供）：在真核生物中，细胞色素c在有氧呼吸和细胞凋亡中发挥着至关重要的作用，从而影响细胞的生死。 C 型细胞色素的特点是共价连接血红素辅助因子，这是其稳定性和后续功能所必需的修饰。血红素附着发生在线粒体中，由全细胞色素 c 合酶 HCCS 介导，HCCS 是真核细胞色素 c 生物发生途径（也称为系统 III）的主要组成部分。 此前对动物的研究表明，HCCS 缺陷可导致死亡或伴有线性皮肤缺陷 (MLS) 的人类疾病小眼症。尽管 HCCS 于 25 年前在酵母中被发现，但 HCCS 功能的机制尚未被探索，这主要是由于其重组表达较差且不稳定。我们的实验室最近成功克服了这些技术限制，使我们能够对 HCCS 结构/功能进行首次全面的生化分析。 HCCS 的催化功能取决于其与其底物血红素和细胞色素 c 相互作用并协调它们之间相互作用的能力。因此，拟议的研究旨在确定 HCCS 中的哪些残基和/或结构域构成其活性位点。目标 1 通过检查血红素结合以及细胞色素 c 募集和成熟的扰动来分析 HCCS 中高度保守残基突变的功能后果。在目标 2 中，使用含有血红素附着位点的可 UV 交联的细胞色素 c 肽，通过肽交联直接评估 HCCS 的脱辅基细胞色素 c 底物结合位点。 这些研究将首次对 HCCS 进行深入的机制分析，从而增进我们对线粒体生物能学的理解。我们的结果将直接影响目前对由 HCCS 异常引起的 MLS 等疾病的了解，并有助于了解其他人类疾病中线粒体中观察到的许多功能障碍。