Two novel cPLA2 binding proteins and cell death

描述（由申请人提供）：胞质磷脂酶A2与3种蛋白质相互作用，包括Tip 60（我们称为PLIP的Tip 60剪接变体）和SIRT 2。该基金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。Tip 60和PLIP是增强细胞对凋亡的易感性的乙酰转移酶蛋白。PLIP的促凋亡作用与PGE 2产生的cPLA 2依赖性增加有关。我们最近已经证明，PLIP表达也与pRb水平的显著降低和GO/G1细胞百分比的降低伴随subG的增加有关。这些数据表明PLIP和cPLA 2可能在G2/M检查点起作用，并在DNA损伤后诱导G2/M停滞或凋亡。使用Tip 60特异性抗体，我们已经表明，Tip 60和PLIP定位于肾小球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。SIRT 2与包括SIRT 1在内的其他I类sirtuins共享大面积的同源性。虽然对SIRT 2知之甚少，但SIRT 1与p53活性和细胞凋亡有关。我们已经确定了cPLA 2和SIRT 2之间的结合位点，并表明它包含在一个高度保守的区域。我们的假设是，cPLA 2，通过其与PLIP和/或Tip 6 O和SIRT 2的相互作用，调节DNA损伤后的细胞凋亡。本研究的主要目的是：1）阐明Tip 60、PLIP和SIRT 2的定位及其与cPLA 2相互作用的机制; 2）确定这些蛋白对细胞周期的调控作用，以及这些作用是否与它们与cPLA 2的相互作用以及对cPLA 2依赖的花生四烯酸释放和PGE 2产生的作用有关;（3）探讨Tip 60、PLIP、SIRT 2和cPLA 2在DNA损伤后细胞凋亡调控中的作用。我们将使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。肾小球系膜细胞的细胞周期调控和细胞凋亡在包括糖尿病肾病在内的肾小球肾炎的进展或消退中至关重要。更好地了解这些事件可能使我们能够开发更好地治疗这些失能和昂贵的疾病的疗法，从而限制对肾脏替代治疗的需求。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.