TWO NOVEL CPLA2 BINDING PROTEINS AND CELL DEATH

描述(改编自《调查者摘要》)：胞质 磷脂酶A2(CPLA2)与细胞损伤有关，然而， CPLA2的作用机制尚不清楚。CPLA2本地化到一个 核内部位对血清剥夺导致的细胞的反应 死亡。已分离到两个新的核cPLA2相互作用蛋白。 这些cPLA2相互作用元件与酵母沉默基因sir2同源。 和SAS2/SAS3。其中一种相互作用的蛋白质，plip，(用于 磷脂酶A2相互作用蛋白)，有一个氨基酸序列 包含最近描述的神秘结构域。神秘域是虚构的 一个非典型的C2HC锌指和一个标志性的乙酰转移酶 序列。该结构域的存在及其与酵母的同源性 果蝇和哺乳动物的蛋白质暗示在 Plip和cplA2的转录调控。的表达方式 肾小球系膜细胞中的PLIP显著增强了两者所致的细胞损伤 血清剥夺和肿瘤坏死因子α。很可能是核内的 含有cPLA2和两个相互作用蛋白的复合体介导 系膜细胞对肿瘤坏死因子α和血清剥夺的反应。第一 这项建议的目的是研究plip的调节机制。 CPLA2活性与肾小球系膜细胞易感性 受伤。第二个具体目标是表征第二克隆46， 确定其在cPLA2调节中的作用，并确定其作用，如果 任何情况下，在细胞内对损伤都很敏感。为了实现这些目标，基本 分子生物学、细胞和脂质生物化学技术将 特别是包括定点突变和一种 腺病毒表达系统。编码a基因的转染体 过氧化体增殖物激活受体(PPAR)，含有 由PPAR反应元件驱动的报告基因将用于 核内花生四烯酸释放的功能读数。这个 这项建议的完成将极大地提高我们对 CPLA2在细胞损伤中的作用和调控。

DESCRIPTION (Adapted from Investigator's Abstract): Cytosolic phospholipase A2 (cPLA2) has been implicated in cell injury, however, the mechanisms by which cPLA2 acts are unknown. cPLA2 localizes to an intranuclear site in response to serum deprivation resulting in cell death. Two novel nuclear cPLA2-interacting proteins have been isolated. These cPLA2-interactors are homologous to yeast silencing genes, SIR2 and SAS2/SAS3. One of these interacting proteins, PLIP, (for Phospholipase A2-interacting protein), has an amino acid sequence containing a recently described MYST domain. The MYST domain is made up of an atypical C2HC zinc finger and a signature acetyltransferase sequence. The presence of this domain and homologies of PLIP to yeast drosophila and mammalian proteins suggest a potential role in transcriptional regulation for PLIP and thus cPLA2. The expression of PLIP in renal mesangial cells markedly enhances cell injury due to both serum deprivation and TNFalpha. It is likely that an intranuclear complex containing cPLA2 and the two interactor proteins mediates the mesangial cell response to TNFalpha and to serum deprivation. The first aim of this proposal is to examine mechanisms by which PLIP modulates cPLA2 activity and contributes to mesangial cell susceptibility to injury. The second specific aim is to characterize the second clone 46, to identify its role in cPLA2 regulation, and to determine its role, if any, in cell susceptibility to injury. To achieve these aims, basic techniques in molecular biology and cellular and lipid biochemistry will be used, specifically including site directed mutagenesis and an adenoviral expression system. Transfected constructs encoding a peroxosomal proliferator activated receptor (PPAR) and containing a reporter gene driven by a PPAR response element will be used for a functional readout of intranuclear arachidonic acid release. The completion of this proposal will greatly enhance our understanding of the role and regulation of cPLA2 in cell injury.