Structural analysis of BNIP3 transmembrane interactions

BNIP3跨膜相互作用的结构分析

基本信息

批准号：
7471442
负责人：
Kevin R MacKenzie
金额：
$ 24.19万
依托单位：
RICE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-08-01 至 2010-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7471442
关键词：
Address Adenoviruses Apoptosis Apoptosis Regulator Apoptotic Binding Biochemical Biological Biological Assay C-terminal Caenorhabditis elegans Cell Death Cellular biology Complex Condition Data Detergents Development Dimerization Drug Delivery Systems Exhibits Goals Homo Homodimerization Homologous Gene Hypoxia Ischemia Lateral Length Malignant Neoplasms Mammals Measures Mediating Membrane Membrane Proteins Micelles Mitochondria Mutagenesis Mutation NMR Spectroscopy Neurodegenerative Disorders Organ Orthologous Gene Prevention Principal Investigator Proteins Range Regulation Research Research Personnel Role Role playing therapy Signal Transduction Solutions Specificity Structure Tail Transmembrane Domain Work Yeasts analytical ultracentrifugation base dimer fly genetic regulatory protein in vivo insight man member mutant novel programs protein folding protein function protein protein interaction protein structure research study

项目摘要

DESCRIPTION (provided by applicant): The long range goals of this research are to determine how the transmembrane domain sequences of "BH3-only"apoptotic regulatory proteins result in structural and functional interactions that give rise to programmed cell death. More than a dozen members of the 'BH3-only' subclass of the Bcl-2 superfamily provide cell- and signal-specific inputs to mitochondria-mediated apoptosis in mammals, but the biochemical and structural basis for this control is poorly understood. This project focuses on BNIP3 (Bcl-2/adenovirus E1B 19-kDa protein-interacting protein 3), a "BH3-only" protein that is functionally conserved from C. elegans to man. The transmembrane (TM) domain of BNIP3 is implicated in the pro-apoptotic function of the protein, in homodimerization, and in interactions with the anti-apoptotic protein Bcl-2. This research will determine the sequence specificity and structural basis for these TM domain interactions and will determine how these lateral associations within membranes contribute to the regulation of mitochondria-mediated apoptosis. Elucidating the mechanism of action of these proteins will have implications for our understanding of development, cancer, and neurodegenerative diseases. In the next five years, this proposed research will determine the structural basis for homo- and hetero-oligomerization of the hydrophobic C-terminal transmembrane (TM) domain of the mammalian "BH3-only" protein BNIP3 and its orthologs in C. elegans and D. melanogaster. Saturation mutagenesis experiments will define the sequence requirements for these associations in biological membranes and in detergents, and the structures of BNIP3 homodimers and heteromeric complexes will be determined using solution NMR spectroscopy. Interpretation of the mutagenesis data in the context of structures will elucidate the physical basis for the stability and specificity of protein-protein interactions inside membranes. Measuring the apoptogenic effects of wild type and mutant full-length BNIP3 constructs in vivo will determine the functional role(s) played by TM-TM interactions. The results will reveal fundamental rules of membrane protein folding, stimulate new types of experiments by cell biology researchers, and may identify novel drug targets for prevention of apoptosis-related cell death and organ damage such as hypoxia-induced apoptosis following ischemia.

描述（由申请人提供）：这项研究的远距离目标是确定“仅BH3”的跨膜结构域序列如何导致结构性和功能相互作用，从而导致程序性细胞死亡。 Bcl-2超家族的“仅BH3”亚类的十多个成员为哺乳动物的线粒体介导的细胞凋亡提供了细胞和信号特异性输入，但是该对照的生化和结构基础知之甚少。该项目侧重于BNIP3（Bcl-2/腺病毒E1b 19-kDa蛋白相互作用蛋白3），这是一种“仅BH3”蛋白，它在秀丽隐杆线虫到人的功能上都是功能保守的。 BNIP3的跨膜（TM）结构域与蛋白质的促凋亡功能，均质二聚化以及与抗凋亡蛋白Bcl-2的相互作用有关。这项研究将确定这些TM结构域相互作用的序列特异性和结构基础，并将确定膜内的这些侧向关联如何有助于调节线粒体介导的细胞凋亡。阐明这些蛋白质的作用机理将对我们对发育，癌症和神经退行性疾病的理解产生影响。在接下来的五年中，这项拟议的研究将确定哺乳动物“仅BH3蛋白”蛋白BNIP3及其在exkemans and D. Melanogaster中的疏水性C末端跨膜（TM）结构域的均质和异源性弱化的结构基础。饱和诱变实验将定义生物膜和洗涤剂中这些关联的序列要求，并且将使用溶液NMR光谱法确定BNIP3同型二聚体和杂体配合物的结构。在结构背景下对诱变数据的解释将阐明膜内蛋白质 - 蛋白质相互作用的稳定性和特异性的物理基础。在体内测量野生型和突变体全长BNIP3构建体的凋亡作用将确定TM-TM相互作用所起的功能作用。结果将揭示膜蛋白折叠的基本规则，通过细胞生物学研究人员刺激新型实验，并可以鉴定出预防与凋亡相关的细胞死亡和器官损伤的新型药物靶标，例如缺血后缺氧诱导的凋亡。