Two novel cPLA2 binding proteins and cell death

CPLA2与三种蛋白质相互作用，包括TipGO，我们称之为PLIP的TipGO剪接变体，以及SIRT2。这项应用的目的是研究这些蛋白在系膜细胞凋亡中的分子基础和作用。Tip60和PLIP是乙酰基转移酶蛋白，可增强细胞对凋亡的敏感性。我们最近证明，PLIP的表达与GO/G1期细胞的显著减少有关，与视网膜母细胞瘤蛋白的减少有关。系膜细胞内源性PLIP增加 提示PLIP可能在DNA损伤后的G2/M检查点或之后诱导细胞凋亡。SIRT2最近被确定为参与有丝分裂退出的调节蛋白。我们发现SIRT2在基因毒性应激后系膜细胞中表达增加，其表达与TipGO的增加有关。我们发现这些蛋白在MRL/Faslpr小鼠的肾脏中减少，这表明在狼疮肾小球肾炎的发展中可能起到了作用。我们的假设是，PLIP、Tip60和SIRT2在系膜细胞DMA损伤后调节关键的细胞周期事件；它们的功能是通过它们与cPLA2的相互作用而介导的，这些蛋白的丢失伴随并介导了肾小球肾炎的系膜改变。本研究的目的有三个：1)确定Tip60、PLIP和SIRT在系膜细胞正常细胞周期中的表达及其依赖于cPLA2的功能；2)确定TipGO和SIRT2在DNA损伤诱导的系膜细胞凋亡中的作用，并确定pRb降解在PLIP相关的凋亡中的作用；3)确定TipGO在细胞内转运的不同基础 和检测TipGO中潜在的核定位和核输出信号(NES)，以及SIRT2和TipGO剪接变异体在MRL/Faslpr肾脏中的表达，以提示这些蛋白在肾小球肾炎发生发展中的潜在作用。我们将用分子生物学和细胞生物化学的基本方法来回答这些问题。肾小球系膜细胞细胞周期事件和细胞凋亡的调控在肾小球肾炎的进展或消退中起着至关重要的作用。对这些事件的更好理解可能会让我们开发出更好地治疗这些使人丧失能力和代价高昂的疾病的疗法。

cPLA2 interacts with three proteins, including TipGO, a TipGO splice variant which we have called PLIP, and SIRT2. The goals of this application have been to study the molecular basis for and roles of these proteins in mesangial cell apoptosis. Tip60 and PLIP are acetyltransferase proteins that enhance cell susceptibility to apoptosis. We have recently demonstrated that PLIP expression is associated with a marked decrease in GO/G1 phase cells associated with a decrease in retinoblastoma protein. Endogenous PLIP is increased in mesangial cells following genotoxic stress suggesting that PLIP may induce apoptosis at or after the G2/M checkpoint following DMA damage. SIRT2 has recently been identified as a regulatory protein involved in mitotic exit. We show that SIRT2 is increased in mesangial cells following genotoxic stress and its expression is associated with an increase in TipGO. We show that these proteins are decreased in kidneys of MRL/Faslpr mice suggesting a potential role in the development of lupus glomerulonephritis. Our hypothesis is that PLIP, Tip60 and SIRT2 modulate critical cell cycle events following DMA damage in mesangial cells; that their functions are mediated via their interaction with cPLA2 and that loss of these proteins accompanies and mediates mesangial changes in glomerulonephritis. This proposal includes three specific Aims: 1) To determine the expression and cPLA2-dependent functions of Tip60, PLIP and SIRT during the normal cell cycle of mesangial cells; 2) To determine the roles of TipGO and SIRT2 in DNA-damage induced apoptosis in mesangial cells and to determine the role of pRb degradation in PLIP-associated apoptosis; 3) To determine the basis for differences in intracellular trafficking of TipGO and PLIP and examine potential nuclear localization and nuclear export signal (NES) in TipGO, and to characterize the expression of SIRT2 and TipGO splice variants in MRL/Faslpr kidneys in order to suggest a potential role for these proteins in the development of glomerulonephritis. We will use basic methods of molecular biology and cellular biochemistry to answer these questions. The regulation of cell cycle events and apoptosis in the mesangial cell is critical in the progression or resolution of glomerulonephritides. A better understanding of these events may allow us to develop therapies to better treat these incapacitating and costly diseases.