Role of Mammalian PITP Beta in Neurodegenerative Disease

本研究的目的是进行详细分析的一类研究不足的蛋白质：哺乳动物磷脂酰肌醇转移蛋白（PITP）。这些蛋白质在体外催化磷脂酰肌醇或磷脂酰胆碱作为单体在膜双层之间的转运。然而，PITP在哺乳动物细胞中的确切功能以及PITP执行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途径中发挥着重要作用。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部分、光转导和受体介导的信号转导等多种细胞过程相互作用。这些功能界面与人类疾病直接相关，因为高等真核生物中遗传性PITP不足的两个已知病例导致涉及特定神经元群体进行性丧失的显著神经退行性疾病。现有的数据确定了PITP在刺激神经元存活的特定途径中的基本作用，并且这种功能与PITP在神经元组织中的富集大致一致。该研究计划代表了一个全面的和多学科的努力，旨在确定由特定的哺乳动物PITP亚型（PITPbeta）执行的功能。PITPbeta与高尔基复合体物理上相关，并且在后生动物中始终保守。我们将进行三条线的调查，以辨别这种蛋白质在哺乳动物，特别是哺乳动物的神经系统中的功能。首先，我们采用先进的光漂白和定量成像技术，比较动态的PITPbeta在活细胞与相关的PITPalpha亚型，并确定的决定因素，比较动态的PITPbeta在活细胞与相关的PITPalpha亚型，并确定的决定因素，指定这两个PITPs的差异定位。第二，在更直接的方法来定义PITPbeta功能，我们将表征PITPbeta缺陷小鼠产生的标准同源基因靶向方法，并将产生完全发育的小鼠诱导中枢神经系统特异性PITPbeta缺陷。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。所有这些研究都将与新的突变PITPbeta在互补实验中的应用相结合，该互补实验旨在解决哪种磷脂结合/转移特性与PITPbeta的任何特定生理功能相关。拟议的研究将提供新的和基本的信息，将直接承担特定的PITP亚型在哺乳动物中的功能，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实验室处于一个独特的位置，承担哺乳动物神经系统的神经退行性疾病。Bankaitis实验室在进行这一研究方面处于独特的地位，因为它已经开发了进行这种全面分析所需的遗传和生物化学系统。

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.