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.
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