P-281 - Missing GPx8 activity in ER impacts on lipid composition

Abstract

Cys-containing GPx8 is an intrinsic ER enzyme, whose silencing leads to H2O2 leakage. While studying GPx8 in HeLa cells, we observed that enzyme silencing deeply impacts on ER membrane lipid composition. ESI-Q-TOF-MS, integrated with principal component analysis, revealed that missing activity leads to an enrichment of neutral glycosphingolipids (GSL) and sphingomyelins (SM), but not glycerophospholipid (PL). Moreover, in all PL but phosphatidylethanonolamine, PUFA decreased. The deepest depletion of arachidonic acid was observed in phosphatidylinositol where the 18:0/18:1 replaced for up to 50% of the 18:0/20:4. Real time PCR analysis showed 30 to 50% increase of the enzymes for the ceramide (CER) biosynthesis. While expression of the CER transfer protein, involved in translocating CER from the ER to the Golgi for sphingomyelin biosynthesis, was also increased, sphingomyelin synthase 1 was not modified. This suggests that the increase of SM results from increased substrate availability due to activation of the upstream biosynthetic pathway. This evidence links the increased H2O2 efflux from ER due GPx8 silencing, and thus a more oxidizing environment, to a metabolic adaptation encompassing a modulation of specific lipid biosynthetic pathways.