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，从而影响细胞的生死。c型细胞色素的特点是血红素辅助因子的共价附着，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合成酶（HCCS）介导，全细胞色素c合成酶是真核细胞色素c生物生成途径的主要组成部分，也被称为系统III。