Two novel cPLA2 binding proteins and cell death

描述(申请人提供)：胞浆磷脂酶A2与3种蛋白质相互作用，包括Tip60，我们称之为PLIP的Tip60剪接变异体，以及SIRT2。这项资助的目标是研究这些蛋白质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。PLIP的促凋亡作用与依赖cPLA2的PGE2生成增加有关。我们最近发现，plip的表达还与pRb水平的显著降低以及处于GO/G1期的细胞比例的降低伴随着亚G的增加有关。提示plip和cPLA2可能在G2/M检查点发挥作用，在DNA损伤后诱导G2/M期停滞或细胞凋亡。使用Tip60特异性抗体，我们已经证明Tip60和PLIP定位于系膜细胞的不同细胞室，这表明它们可能不具有相同的功能。SIRT2与包括SIRT1在内的其他I类sirtuins有很大的同源性。尽管对SIRT2知之甚少，但SIRT1与P53活性和细胞凋亡有关。我们已经确定了cPLA2和SIRT2之间的结合位点，并表明它包含在一个高度保守的区域内。我们的假设是，cPLA2通过与PLIP和/或Tip60以及与SIRT2的相互作用来调控DNA损伤后的细胞凋亡。本研究的目的包括3个方面：1)阐明Tip60、PLIP和SIRT2的定位及其与cPLA2相互作用的机制；2)确定这些蛋白在细胞周期调控中的作用，以及这些作用是否与它们与cPLA2的相互作用以及对依赖cPLA2的花生四烯酸的释放和PGE2的产生有关；以及3)确定和阐明Tip60、plIP、SIRT2和cPLA2在DNA损伤后的细胞凋亡调控中的作用。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节在包括糖尿病肾病在内的肾小球肾炎的进展或消退中起着至关重要的作用。更好地了解这些事件可能会让我们开发治疗方法，更好地治疗这些丧失能力和昂贵的疾病，从而限制对肾脏替代疗法的需求。鉴于每年有大量患者开始接受透析，这解决了一个关键的公共卫生问题。

DESCRIPTION (provided by applicant): Cytosolic phospholipase A2 interacts with 3 proteins, including Tip60, a Tip60 splice variant which we have called PLIP, and SIRT2. The goals of this grant 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. PLIP's proapoptotic effect is associated with a cPLA2-dependent increase in PGE2 generation. We have recently demonstrated that PLIP expression is also associated with a striking decrease in pRb levels and a decrease in the percentage of cells in GO/G1 accompanied by an increase in subG. This data suggests that PLIP and cPLA2 may function at the G2/M checkpoint and induce G2/M arrest or apoptosis following DNA damage. Using a Tip60-specific antibody, we have shown that Tip60 and PLIP localize to different cellular compartments of mesangial cells, suggesting that they may not share identical functions. SIRT2 shares large areas of homology with other class I sirtuins, including SIRT1. Although little is known about SIRT2, SIRT1 has been associated with p53 activity and with apoptosis. We have identified the binding site between cPLA2 and SIRT2 and show that it is contained within a highly conserved region. Our hypothesis is that cPLA2, via its interaction with PLIP and/or Tip6O and with SIRT2 modulates apoptosis following DNA damage. This proposal includes 3 specific aims: 1) To elucidate the mechanisms underlying the localization of Tip60, PLIP and SIRT2 and their interaction to cPLA2; 2) To determine the effects of these proteins on cell cycle regulation and whether these effects are functionally related to their interaction with cPLA2 and on cPLA2-dependent arachidonic acid release and PGE2 generation; and 3) To determine and elucidate the role of Tip60, PLIP, SIRT2 and cPLA2 in regulation of apoptosis following DNA damage. 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, including diabetic nephropathy. A better understanding of these events may allow us to develop therapies to better treat these incapacitating and costly diseases and thus limit the need for renal replacement therapy. Given the large number of patients that are initiated on dialysis each year, this addresses a critical public health problem.