Structural analysis of BNIP3 transmembrane interactions

描述(申请人提供)：这项研究的长期目标是确定“只有BH3”的凋亡调控蛋白的跨膜域序列如何导致结构和功能相互作用，从而导致细胞程序性死亡。在Bcl2超家族中，有十多个BH3亚类成员为线粒体介导的哺乳动物细胞凋亡提供细胞和信号特异性输入，但这种控制的生化和结构基础尚不清楚。本项目的重点是BNIP3(Bcl2/腺病毒E1B19-kDa蛋白相互作用蛋白3)，它是一种在线虫和人类之间功能保守的“BH3-Only”蛋白。BNIP3的跨膜(TM)结构域与该蛋白的促凋亡功能、同源二聚化以及与抗凋亡蛋白Bc l-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，并将确定这些膜内的横向联系如何有助于调节线粒体介导的细胞凋亡。阐明这些蛋白的作用机制将对我们理解发育、癌症和神经退行性疾病具有重要意义。 在接下来的五年里，这项拟议的研究将确定哺乳动物“仅BH3”蛋白BNIP3及其同源蛋白BNIP3的疏水C末端跨膜(TM)结构域的同源和异源寡聚的结构基础。饱和诱变实验将确定生物膜和洗涤剂中这些结合的序列要求，并将使用溶液核磁共振光谱确定BNIP3同源二聚体和异构体复合体的结构。在结构的背景下解释突变数据将阐明膜内蛋白质-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型全长BNIP3结构的促凋亡作用将确定TM-TM相互作用所起的功能作用(S)。这些结果将揭示膜蛋白折叠的基本规律，激发细胞生物学研究人员进行新型实验，并可能为预防缺血后与细胞凋亡相关的细胞死亡和器官损伤(如低氧诱导的细胞凋亡)寻找新的药物靶点。

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.