Structural analysis of BNIP3 transmembrane interactions

描述（由申请人提供）：本研究的长期目标是确定“仅BH 3“凋亡调节蛋白的跨膜结构域序列如何导致结构和功能相互作用，从而引起程序性细胞死亡。Bcl-2超家族的“仅BH 3”亚类的十几个成员为哺乳动物中的细胞凋亡介导的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知之甚少。该项目的重点是BNIP 3（Bcl-2/腺病毒E1 B 19-kDa蛋白相互作用蛋白3），一种功能上保守于C. BNIP 3的跨膜（TM）结构域涉及蛋白质的促凋亡功能、同源二聚化以及与抗凋亡蛋白Bcl-2的相互作用。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关联如何有助于调节细胞凋亡。阐明这些蛋白质的作用机制将对我们理解发育、癌症和神经退行性疾病产生影响。 在接下来的五年里，这项拟议的研究将确定哺乳动物“BH 3-only”蛋白BNIP 3及其在C中的直系同源物的疏水C末端跨膜（TM）结构域的同源和异源寡聚化的结构基础。elegans和D.黑腹菌饱和诱变实验将定义生物膜和洗涤剂中这些缔合的序列要求，并且将使用溶液NMR光谱法确定BNIP 3同二聚体和异二聚体复合物的结构。在结构的背景下的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全长BNIP 3构建体的致突变作用将确定TM-TM相互作用所起的功能作用。这些结果将揭示膜蛋白折叠的基本规则，刺激细胞生物学研究人员进行新类型的实验，并可能确定新的药物靶点，用于预防缺血相关的细胞死亡和器官损伤，如缺血后缺氧诱导的细胞凋亡。

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.