Cytosolic phospholipase A2 (cPLA2) has been implicated in cell injury, however, the mechanisms by which cPLA2 acts are unknown. cPLA2 localizes to an intranuclear site in response to serum deprivation resulting in cell death. Two novel nuclear cPLA2-interacting proteins have been isolated. These cPLA2-interactors are homologous to yeast silencing genes, SIR2 and SAS2/SAS3. One of these interacting proteins, PLIP, (for Phospholipase A2-interacting protein), has an amino acid sequence containing a recently described MYST domain. The MYST domain is made up of an atypical C2HC zinc finger and a signature acetyltransferase sequence. The presence of this domain and homologies of PLIP to yeast drosophila and mammalian proteins suggest a potential role in transcriptional regulation for PLIP and thus cPLA2. The expression of PLIP in renal mesangial cells markedly enhances cell injury due to both serum deprivation and TNFalpha. It is likely that an intranuclear complex containing cPLA2 and the two interactor proteins mediates the mesangial cell response to TNFalpha and to serum deprivation.
The first aim of this proposal is to examine mechanisms by which PLIP modulates cPLA2 activity and contributes to mesangial cell susceptibility to injury. The second specific aim is to characterize the second clone 46, to identify its role in cPLA2 regulation, and to determine its role, if any, in cell susceptibility to injury. To achieve these aims, basic techniques in molecular biology and cellular and lipid biochemistry will be used, specifically including site directed mutagenesis and an adenoviral expression system. Transfected constructs encoding a peroxosomal proliferator activated receptor (PPAR) and containing a reporter gene driven by a PPAR response element will be used for a functional readout of intranuclear arachidonic acid release. The completion of this proposal will greatly enhance our understanding of the role and regulation of cPLA2 in cell injury.
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