Phosphatidylinositol Signaling and Human Disease

描述（由申请人提供）：该基金的主要目标是确定导致人类疾病的磷脂酰肌醇信号通路缺陷。肌醇（1，3，4）-P3 5/6-激酶在抑制TNF诱导的细胞凋亡中的作用将被研究。这种作用是由于肌醇磷酸产物还是由于凋亡途径中蛋白质的磷酸化？将产生缺乏肌醇或蛋白激酶活性的重组激酶的突变形式，并测试其阻断TNF凋亡的能力。导致肌醇六磷酸的酶将在293细胞中过表达或被RNAi抑制，以阐明更高肌醇磷酸在细胞凋亡、mRNA的核输出和细胞内囊泡运输中的功能。B的作用。将通过确定假鼻疽蛋白BobB是否是磷酸肌醇磷酸酶来研究其在该生物体的毒力中的作用。BopB的两种人类同源物的底物特异性将通过克隆编码这些蛋白质的cDNA并在Sf 9细胞中产生重组蛋白来确定。将通过研究肌微管蛋白相关蛋白6和7之间的复合物来进行肌微管蛋白基因家族（其突变导致几种不同的人类疾病）的继续研究，所述肌微管蛋白相关蛋白6和7与无活性的肌微管蛋白相关蛋白9具有酶活性。复合物形成在劳氏综合征发病机制中的作用将通过确定在该疾病中突变的肌醇多磷酸5-磷酸酶（OCRL）的功能来研究。将研究OCRL在高尔基体囊泡运输中的作用，并研究突变的OCRL对肾近端小管的NBC 1碳酸氢盐转运蛋白的加工和/或活性的影响。人类NBC 1突变产生Lowe综合征的表型，这一发现表明这种转运蛋白功能障碍

DESCRIPTION (provided by applicant): The major goal of this grant is to define defects in the phosphatidylinositol signaling pathway that result in human diseases. The role of inositol (1,3,4)-P3 5/6-kinase in inhibition of TNF-induced apoptosis will be investigated. Is the effect due to some inositol phosphate product or to phosphorylation of a protein of the apoptotic pathway? Mutant forms of recombinant kinase that lack either inositol or protein kinase activity will be created and tested for their ability to block TNF apoptosis. The enzymes leading to inositol hexaphosphate will be overexpressed in 293 cells or inhibited by RNAi to elucidate functions for the higher inositol phosphates in apoptosis, nuclear export of mRNA, and intracellular vesicle trafficking. The role of the B. pseudomallei protein BobB in virulence of this organism will be studied by determining whether it is an inositol phosphate phosphatase. The substrate specificity of two human homologues of BopB will be determined by cloning cDNA's encoding these proteins and producing recombinant protein in Sf9 cells. Continued studies of the myotubularin gene family (mutations of which cause several different human diseases) will be performed by investigating complexes between myotubularin related proteins 6 and 7 that are enzymatically active with the inactive myotubularin related protein 9. The role of complex formation in the pathogenesis of Lowe syndrome will be studied by determining the function of the inositol polyphosphate 5-phosphatase (OCRL) that is mutated in that disease. The role of OCRL in Golgi vesicle trafficking will be studied and the effect of mutated OCRL on the processing and/or activity of the NBC1 bicarbonate transporter of renal proximal tubules will be investigated. Malfunction of this transporter is suggested by the finding that mutations in NBC1 in humans produce a phenocopy of Lowe syndrome