PIRT Family Modulation of TRPM8

 描述（由申请人提供）：本提案的长期目标是了解TRPM 8如何通过PIRT进行调节。该提议的动机是与调节TRPM 8信号级联相关的显著健康相关性。TRPM 8是一种离子通道，在人体中充当主要的冷传感器。该通道最初被发现是一种致癌基因，在许多类型的癌症中上调，目前正在评估作为抗癌治疗的靶点。最近，TRPM 8已被确定为肥胖和慢性疼痛的药理学干预的有吸引力的靶标。最近的研究表明，PIRT是一种具有两个跨膜螺旋的小膜蛋白，它直接与TRPM 8相互作用，并改变对各种刺激的反应。调制的机制目前还不清楚，也没有关于这个复杂的结构信息。在这个建议中，我们将使用核磁共振光谱（NMR），电生理学，生物化学和计算结构生物学，以产生一个实验限制的TRPM 8-PIRT膜蛋白复合物的综合结构模型。目标1将分离和表征的区域的蛋白质，所需的复合物的形成，使用电生理学，核磁共振结合，和生物化学研究的混合。这一目标也将首次探测TRPM 8-PIRT复合物的化学计量，并确定磷酸肌醇的PIRT脂质结合位点。目的2将产生人膜蛋白PIRT的第一个结构，比较TRPM 8模型，以及TRPM 8-PIRT复合物的实验限制的整合结构模型。目标1和目标2在本质上是独立的，但又是合作的：目标1的结果可用于指导目标2中的综合结构生物学，目标2的输出可用于生成假设并指导复杂功能决定簇的分离。我们已经产生了重要的初步电生理学，核磁共振和计算数据，表明这些目标是可行的，在这个建议的时间范围内。对于目标1，NMR和电生理学数据表明TRPM 8电压敏感结构域是PIRT调节的关键，并且我们已经分离出TRPM 8中对功能调节至关重要的特定残基。目标2的初步数据表明，PIRT的NMR结构研究将导致这种蛋白质的第一个结构。对TRPM 8的PIRT调节的结构和功能分析将增加我们对如何调节该通道的理解。此外，了解PIRT的结构和功能可以提供一种替代的治疗途径来调节TRP通道功能，具有潜在的更少的脱靶效应。

 DESCRIPTION (provided by applicant): The long-term objective of this proposal is to understand how TRPM8 is modulated by PIRT. The motivation for this proposal is the significant health relevance associated with regulating TRPM8 signaling cascades. TRPM8 is an ion channel that functions as the primary cold sensor in humans. This channel was initially found to be an oncogene that is upregulated in a number of types of cancer and is currently under evaluation as a target of anti-cancer therapy. More recently, TRPM8 has been identified as an attractive target for pharmacological intervention of obesity and chronic pain. It was recently shown that PIRT, a small membrane protein with two transmembrane helices, directly interacts withTRPM8 and alters the response to diverse stimuli. The mechanism of modulation is not currently understood and there is no structural information regarding this complex. In this proposal we will use nuclear magnetic resonance spectroscopy (NMR), electrophysiology, biochemistry, and computational structural biology to generate an experimentally restrained integrative structural model of the TRPM8-PIRT membrane protein complex. Aim 1 will isolate and characterize the regions of the proteins that are required for complex formation using a mix of electrophysiology, NMR binding, and biochemistry studies. This aim will also, for the first time, probe the TRPM8-PIRT complex stoichiometry and identify the PIRT lipid binding site for phosphoinositides. Aim 2 will produce the first structure of the human membrane protein PIRT, a comparative TRPM8 model, and an experimentally restrained integrative structural model of the TRPM8-PIRT complex. Aims 1 and 2 are independent yet cooperative in nature: the results from Aim 1 can be used to guide the integrative structural biology in Aim 2 and the output from Aim 2 can be used to generate hypotheses and instruct the isolation of the complex functional determinants. We have generated significant preliminary electrophysiology, NMR, and computational data that suggest these aims are feasible during the timeframe of this proposal. For Aim 1, both NMR and electrophysiology data that suggest the TRPM8 voltage-sensing domain is key to PIRT modulation and we have isolated specific residues in TRPM8 that are key to functional modulation. Preliminary data for Aim 2 suggest that NMR structural studies of PIRT will result in the first structure of this protein. The proposed structural and functional analysisof PIRT modulation of TRPM8 will increase our understanding of how to regulate this channel. Moreover, understanding the structure and function of PIRT may provide an alternative therapeutic pathway to modulate TRP channel function with potentially fewer off target effects.