STRUCTURE/FUNCTION OF NERVE GROWTH FACTOR/NEUROTROPHINS

描述(改编自申请者的摘要)：神经营养因子发挥作用 在许多类型神经元的发育和维持中起主要作用。 神经生长因子相关神经营养因子家族(BDNF、NT-3、NT-4/5)相互作用 与低亲和力神经营养素受体(LANR)和 Trk原癌基因受体酪氨酸激酶(Trk)建立信号转导机制 在有反应的外周神经元(感觉、交感)和中枢神经元内 神经元(胆碱能、多巴胺能)。所解决的问题是 识别、特异性和信号转导的性质 细胞内部的受体，即构象如何变化 都是通过膜传递的。这些项目将成为 涉及神经营养因子的蛋白质-蛋白质相互作用的动力学- 受体(S)结合采用化学、免疫化学、重组、 诱变和光谱技术与动力学和热力学 纯化的神经营养因子和纯化的受体成分分析。这个 该项目的具体目标是：(1)重组，部分加工 NGF(1-120)在某些生物检测中的活性降低，这表明 NGF活性可能受细胞外蛋白分解过程的调节。 NGF(1-120)的生理意义，包含C- 末端二肽，将通过进一步鉴定其特性来确定 特异性，通过在C末端产生突变，并通过测量 C-末端未加工神经生长因子在神经中的数量和分布 组织。(2)倒转的功能和结构作用 NGF在LANR和Trk结合中的环将由 诱变。神经营养素受体之间的相互作用将是 用热力学循环分析神经生长因子的多重突变 联动。(3)位置上芳香氨基酸的贡献 52-54和NGF的N末端对功能、稳定性和结构的影响 将由点突变和缺失突变决定。构象 突变体的稳定性(在前三个目标中)将是 以生物物理化学技术为特征的；它们的特异性将 通过与重组Trk家族受体结合和通过 PC12细胞、背根和结节感觉神经元的生物测定， 交感神经元和多巴胺能神经元及(4)电位 神经营养因子与神经营养因子相互作用时的构象变化 LANR受体胞外区(LAN-RED)和Trk受体 胞外区(TRK-RED)家族将通过时间- 分辨荧光和圆二色谱研究。这些项目 这里概述的是对理解分子基础很重要的 神经营养因子对中枢神经系统和三叉神经节中特定神经元的影响 受衰老或阿尔茨海默氏症等神经疾病的影响 疾病、帕金森氏病和神经病。

DESCRIPTION (adapted from the applicant's abstract): Neurotrophins play a major role in development and maintenance of many types of neurons. The family of NGF related neurotrophins (BDNF, NT-3, NT-4/5) interact with a low affinity neurotrophin receptor (LANR) and with the family of trk proto-oncogene receptor tyrosine kinases (Trk) to establish signaling within responsive peripheral neurons (sensory, sympathetic) and central neurons (cholinergic, dopaminergic). The question addressed is the nature of the recognition, specificity, and signal transduction through the receptor to the inside of the cell, i.e., how conformational changes are communicated across the membrane. These projects will characterize the dynamics of protein-protein interactions involved in neurotrophin- receptor(s) binding by using chemical, immunochemical, recombinant, mutagenic, and spectroscopic techniques with kinetic and thermodynamic analyses on purified neurotrophin and purified receptor components. The Specific Aims of this project are: (1) Recombinant, partially processed NGF (1-120) has diminished activity in certain bioassays suggesting that NGF activity may be regulated by extracellular proteolytic processing. The physiological importance of the NGF (1-120), containing the C- terminal dipeptide, will be determined by further characterizing its specificity, by making mutants at the C-terminus, and by measuring the amounts and distribution of C-terminal unprocessed NGF in nervous tissue. (2) The functional and structural role of the reverse turn loops of NGF in binding both LANR and Trk will be determined by mutagenesis. Interactions between neurotrophin receptors will be analyzed using multiple mutations of NGF with thermodynamic cycle linkage. (3) The contribution of the aromatic amino acids at positions 52-54 and the N-terminus of NGF to function, stability and structure will be determined by point and deletion mutagenesis. The conformation and stability of the mutants (in these first three aims) will be characterized by biophysical chemical techniques; their specificity will be determined by binding to recombinant Trk family receptors and by bioassays with PC12 cells, dorsal root and nodose sensory neurons, sympathetic neurons and dopaminergic neurons and (4) Potential conformational changes upon interaction of the neurotrophins with the LANR receptor extracellular domain (LAN-RED) and with the Trk receptor extracellular domain (TRK-RED) family will be determined using time- resolved fluorescence and circular dichroism studies. The projects outlined here are important in understanding the molecular basis of neurotrophin effects on specific neurons in the CNS and PNS that may be affected in aging or in neurological disorders such as Alzheimer's disease, Parkinsons disease, and neuropathies.