Two novel cPLA2 binding proteins and cell death

Brigham And Women'S Hospital, Boston MA

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Abstract

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.