Phenotype-Targeted Inference of Flux-Enzyme Correlations in Adipocyte Metabolism

脂肪细胞代谢中通量-酶相关性的表型靶向推断

• 批准号: 8036855
• 负责人: KYONGBUM LEE
• 金额: $ 25.95万
• 依托单位: TUFTS UNIVERSITY MEDFORD
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8036855
• 关键词: ATP-Binding Cassette Transporters; Adipocytes; Anabolism; Binding; Biochemical; Biogenesis; Biological; Biological Assay; Chloride Channels; Complement; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Data; Disease; Enzymes; Epithelial; Eukaryota; Event; Family; Functional disorder; Glycine; Goals; Homologous Gene; Human; Hydrolysis; In Vitro; Length; Mediating; Membrane; Metabolism; Methods; Minor; Modeling; Mutate; Mutation; Nucleotides; Phenotype; Play; Prokaryotic Cells; Property; Protein Binding Domain; Protein Biosynthesis; Protein Family; Proteins; Regulation; Research; Roentgen Rays; Role; Sodium Chloride; Structure; Tissues; Transmembrane Domain; Virulence; Walkers; Water; dimer; functional loss; human disease; innovation; insight; member; novel; pathogen; protein function; research study; solute

Cystic fibrosis, a disease of altered water and salt secretion across epithelial tissues, is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is a member of the ABC-transporter family of proteins and functions as a chloride channel; loss of the functional chloride channel activity is causative of CF. The ATP-Binding Cassette (ABC-) transporter superfamily of proteins is highly conserved across prokaryotes and eukaryotes, facilitating solute transport across biological membranes. ABC-transporter proteins are minimally composed of a dimer of highly conserved, cytosolic nucleotide-binding domains (NBDs), which provides the energy for solute transport through a dimer of transmembrane domains (TMDs). ATP binding and hydrolysis within the NBDs is regulated by the canonical Walker A and B nucleotide-binding sequences, as well as a sequence unique to the ABC-transporter family of proteins ¿ the signature sequence, or LSGGQRxR. While the Walker A and B sequences are well characterized, the structural and functional properties of the LSGGQRxR sequence are not known. Preliminary data suggest that the LSGGQRxR sequence critically contributes to the biosynthesis and function of CFTR and other ABC-transporter proteins. Functional regulation of NBD-NBD association events is critically altered by substitution of the glycine residues, resulting in either hyperactive or inactive channels. Alterations to the RxR sequence alter channel biosynthesis with only minor effects on channel activity. The major goal of this project is to elucidate the structural and functional roles of the LSGGQRxR sequence in regulating CFTR-channel and ABC-transporter biosynthesis and function. To accomplish this, we have developed methods for the expression, purification and biophysical characterization of the isolated NBD proteins from CFTR and two homologues (human ABCC6 and the bacterial Mj0796). Using a combination of X-ray crystallographic, nucleotide binding and hydrolysis, and biochemical approaches, we will evaluate the specific structural and functional roles of the LSGGQRxR signature sequence. These in vitro data will complement experiments evaluating the biosynthesis and function of full-length protein to provide a detailed model for the regulation of ABC-transporter biogenesis and mechanochemistry by the signature sequence. The specific aims of this application are: (1) Characterize the nucleotide-binding and hydrolysis properties of CFTR NBD1 and NBD2, (2) Elucidate the role of the signature sequence di-glycine residues in ATP-mediated NBD association and function, and (3) Characterize the role of the RxR sequence on local NBD structure and CFTR biosynthesis. The research proposed in this application will provide novel insight into the previously undefined structural and functional roles of this highly conserved LSGGQRxR sequence. The innovative use of in vitro biochemical, structural and enzymatic assays to complement studies of full-length protein biosynthesis and function will refine models of the biogenesis and mechanochemistry of these medically important proteins. Project

囊性纤维化是一种跨上皮组织的水和盐分泌改变的疾病，由 囊性纤维化跨膜电导调节因子(CFTR)。CFTR是ABC-Transporter的成员 蛋白家族，起氯离子通道的作用；功能氯离子通道活性的丧失是 致病因素。三磷酸腺苷结合盒(ABC-)转运蛋白超家族高度保守 跨原核生物和真核生物，促进溶质跨生物膜的运输。ABC-传送器 蛋白质最小限度地由高度保守的胞质核苷酸结合结构域(NBD)的二聚体组成， 它为溶质通过跨膜结构域二聚体(TMD)的运输提供能量。ATP NBDS内的结合和水解由典型的Walker A和B核苷酸结合调节 序列，以及ABC-转运蛋白家族独有的序列，即签名序列， 或LSGGQRxR。虽然Walker A和B序列被很好地表征，但结构和功能 LSGGQRxR序列的属性是未知的。初步数据显示，LSGGQRxR 序列对CFTR和其他ABC转运蛋白的生物合成和功能起着至关重要的作用。 NBD-NBD结合事件的功能调节通过甘氨酸残基的取代而严重改变， 从而导致过度活跃或不活跃的通道。RXR序列的改变改变了通道的生物合成 对通道活动只有很小的影响。这个项目的主要目标是阐明结构和 LSGGQRxR序列在调控CFTR通道和ABC转运蛋白生物合成中的作用 和功能。为了实现这一点，我们开发了表达、纯化和生物物理的方法 从CFTR及其两个同源物(人ABCC6和 细菌Mj0796)。使用X射线结晶学、核苷酸结合和水解的组合，以及 生化方法，我们将评估LSGGQRxR的特定结构和功能作用 签名序列。这些体外数据将补充评估生物合成和功能的实验 为ABC转运蛋白的生物发生和调控提供详细的模型 机械力化学由签名序列决定。本申请的具体目的是：(1)描述 Cftr nbd1和nbd2的核苷酸结合和水解性，(2)阐明了cftr的作用 特征序列二甘氨酸残基在ATP介导的NBD结合和功能中的作用，以及(3) 表征RXR序列在局部NBD结构和CFTR生物合成中的作用。这项研究 本申请中提出的建议将为以前未定义的结构和功能提供新的见解 这个高度保守的LSGGQRxR序列的作用。体外生化、结构和生物化学的创新应用 补充全长蛋白质生物合成和功能研究的酶分析将完善 这些医学上重要的蛋白质的生物发生和机械化学。 项目