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: 8717157
• 负责人: Shalon Elizabeth Ledbetter
• 金额: $ 5.15万
• 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 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8717157
• 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; 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; public health relevance; 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（由申请人提供）：在真核生物中，细胞色素c在有氧呼吸和细胞凋亡中起着至关重要的作用，从而影响细胞的生命和死亡。C型细胞色素的特征在于血红素辅因子的共价连接，这是其稳定性和后续功能所需的修饰。血红素附着发生在线粒体中，并由全细胞色素c合成酶（HCCS）介导，HCCS是真核细胞色素c生物合成途径（也称为系统III）的主要组分。 先前的动物研究表明，HCCS的缺陷可导致致命性或人类疾病小眼症伴线性皮肤缺损（MLS）。虽然HCCS是在25年前在酵母中发现的，但HCCS功能的潜在机制尚未被探索，主要是由于其重组表达差和不稳定性。我们的实验室最近成功地克服了这些技术限制，使我们能够启动第一个全面的HCCS结构/功能的生化分析。 HCCS的催化功能取决于其与底物血红素和细胞色素c相互作用和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中哪些残基和/或结构域包含其活性位点。目的1通过研究血红素结合和细胞色素c募集和成熟的扰动，分析HCCS中高度保守残基突变的功能后果。HCCS的脱辅基细胞色素c底物结合位点在Aim 2中通过肽交联直接评估，使用含有血红素附着位点的UV可交联细胞色素c肽。 这些研究将提供第一个深入的机制分析HCCS，从而推进我们的线粒体生物能量学的理解。我们的研究结果将直接影响目前已知的由HCCS异常引起的MLS等疾病，并有助于了解在其他人类条件下在线粒体中观察到的许多故障。