Ceramide-1-phosphate transfer protein (CPTP) regulation by phosphoinositides

Abstract

Ceramide-1-phosphate transfer proteins (CPTPs) are members of the glycolipid transfer protein (GLTP) superfamily that shuttle ceramide-1-phosphate (C1P) between membranes. CPTPs regulate cellular sphingolipid homeostasis in ways that impact programmed cell death and inflammation. CPTP downregulation specifically alters C1P levels in the plasma and trans-Golgi membranes, stimulating proinflammatory eicosanoid production and autophagy-dependent inflammasome-mediated cytokine release. However, the mechanisms used by CPTP to target the trans-Golgi and plasma membrane are not well understood. Here, we monitored C1P intervesicular transfer using fluorescence energy transfer (FRET) and showed that certain phosphoinositides (phosphatidylinositol 4,5 bisphosphate (PI-(4,5)P₂) and phosphatidylinositol 4-phosphate (PI-4P)) increased CPTP transfer activity, whereas others (phosphatidylinositol 3-phosphate (PI-3P) and PI) did not. PIPs that stimulated CPTP did not stimulate GLTP, another superfamily member. Short-chain PI-(4,5)P_2, which is soluble and does not remain membrane-embedded, failed to activate CPTP. CPTP stimulation by physiologically relevant PI-(4,5)P₂ levels surpassed that of phosphatidylserine (PS), the only known non-PIP stimulator of CPTP, despite PI-(4,5)P₂ increasing membrane equilibrium binding affinity less effectively than PS. Functional mapping of mutations that led to altered FRET lipid transfer and assessment of CPTP membrane interaction by surface plasmon resonance indicated that di-arginine motifs located in the α-6 helix and the α3-α4 helix regulatory loop of the membrane-interaction region serve as PI-(4,5)P₂ headgroup-specific interaction sites. Haddock modeling revealed specific interactions involving the PI-(4,5)P₂ headgroup that left the acyl chains oriented favorably for membrane embedding. We propose that PI-(4,5)P₂ interaction sites enhance CPTP activity by serving as preferred membrane targeting/docking sites that favorably orient the protein for function.

Keywords: Arabidopsis accelerated cell death (ACD11) protein; GLTP-fold; di-Arg motif; human ceramide-1-phosphate transfer protein (CPTP); human glycolipid transfer protein (GLTP); membrane interaction; phosphatidylinositol-3-phosphate; phosphatidylinositol-4,5-phosphate; phosphatidylinositol-4-phosphate; phosphoinositide regulation.