Role of Mammalian PITP Beta in Neurodegenerative Disease

本研究的目的是对一类未被充分研究的蛋白质进行详细分析：哺乳动物磷脂酰肌醇转移蛋白（PITPs）。这些蛋白催化磷脂酰肌醇或磷脂酰胆碱作为单体在体外膜双层之间的运输。然而，哺乳动物细胞中pitp的确切功能及其实现这些功能的机制仍有待阐明。目前的证据表明，pitp在调节磷脂介导的信号转导通路中起核心作用。目前的证据表明，pitp在调节磷脂介导的信号转导途径中发挥核心作用，这些信号转导途径与蛋白质切片、光转导和受体介导的信号传导等多种细胞过程相结合。这些功能界面与人类疾病直接相关，因为已知的两例高等真核生物遗传性PITP不足导致严重的神经退行性疾病，涉及特定神经元群的逐渐丧失。现有的数据表明，PITPs在刺激神经元存活的特定通路中起着基本作用，这种功能与神经元组织中PITPs的富集大致一致。该研究计划代表了一项综合性和多学科的努力，旨在确定特定哺乳动物PITP异构体（PITPbeta）的功能。PITPbeta在物理上与高尔基复合体有关，并在后生动物中一直保存。我们将进行三项调查，以辨别这种蛋白质在哺乳动物，特别是哺乳动物神经系统中的功能。首先，我们采用复杂的光漂白和定量成像技术来比较活细胞中PITPbeta与相关PITPalpha异构体的动态，并确定活细胞中PITPbeta与相关PITPalpha异构体动态的决定因素，并确定这两种pitp的差异定位的决定因素。其次，在定义PITPbeta功能的更直接的方法中，我们将描述通过标准同源基因靶向方法产生的PITPbeta缺陷小鼠，并将产生用于中枢神经系统特异性PITPbeta缺陷的完全发育小鼠。最后，我们将开发PITPbeta缺陷细胞模型，以便在细胞水平上分析PITPbeta功能的机制。所有这些研究都将结合使用新的突变体PITPbeta进行互补实验，旨在解决磷脂结合/转移特性与PITPbeta的任何特定生理功能相关的问题。这些研究将提供新的和基础的信息，这些信息将直接影响哺乳动物中特定的PITP异构体的功能，以及PITP保护哺乳动物神经系统免受神经退行性疾病的分子机制。Bankaitis实验室在从事哺乳动物神经系统退行性疾病研究方面具有独特的优势。Bankaitis实验室在进行这一研究方面处于独特的地位，因为它为这种全面分析开发了必要的遗传和生化系统。

The objective of this research is to undertake a detailed analysis of an under-investigated class of proteins: the mammalian phosphatidylinositol transfer proteins (PITPs). These proteins catalyze the transport of either phosphatidylinositol or phosphatidylcholine, as monomers, between membrane bilayers in vitro. The precise functions of PITPs in mammalian cells, and the mechanisms by which PITPs execute such functions, remain to be elucidated, however. Present evidence suggests that PITPs play central roles in regulating phospholipid-mediated signal transduction pathways that interface. Present evidence suggests that PITPs play central roles in regulating phospholipid-mediated signal transduction pathways that interface with such diverse cellular processes as protein section, photo-transduction, and receptor-mediated signaling. These functional interfaces are of direct relevance to human disease as to two known cases of inherited PITP insufficiency in higher eukaryotes result in dramatic neurodegenerative diseases that involve progressive loss of specific neuronal populations. The available data identify fundamental roles for PITPs in stimulating specific pathways for neuronal survival and this function is broadly consistent with the enrichment of PITPs in neuronal tissues. The research plan represents a comprehensive and multi-disciplinary effort designed to identify the function(s) executed by a specific mammalian PITP isoform (PITPbeta). PITPbeta physically associates with the Golgi complex and is conserved throughout by the metazoans. We will undertake three lines of investigation to discern the function of this protein in mammals and, in particular, the mammalian nervous system. First, we employ sophisticated photobleaching and quantitative imaging techniques to compare the dynamics of PITPbeta in living cells with those of the related PITPalpha isoform, and to identify the determinants that compare the dynamics of PITPbeta in living cells with those of the related PITPalpha isoform, and to identify the determinants that specify the differential localization of these two PITPs. Second, in more directed approaches for defining PITPbeta function, we shall characterize PITPbeta-deficient mice generated by standard homologous gene targeting methods, and will generate fully developed mice induced for central nervous system-specific PITPbeta deficiencies. Finally, we will develop PITPbeta-deficient cell models so that mechanisms of PITPbeta function at the cellular level can be analyzed. All of these studies will be coupled to the use of novel mutant PITPbeta's in complementation experiments designed to address which phospholipid binding/transfer property is relevant to any particular physiological function of PITPbeta. The proposed studies will provide new and fundamental information that will bear directly on the functions of specific PITP isoforms in mammals, and the molecular mechanisms by which PITPs protect the mammalian nervous system from neurodegenerative disease. The Bankaitis laboratory is in a unique position tp undertake the mammalian nervous system from neurodegenerative disease. The Bankaitis laboratory is in a unique position to undertake this line of research as it has developed the requisite genetic and biochemical systems for such comprehensive analyses.