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)P 2 ) 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)P 2 levels surpassed that of phosphatidylserine (PS), the only known non-PIP stimulator of CPTP, despite PI-(4,5)P 2 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)P 2 headgroup-specific interaction sites. Haddock modeling revealed specific interactions involving the PI-(4,5)P 2 headgroup that left the acyl chains oriented favorably for membrane embedding. We propose that PI-(4,5)P 2 interaction sites enhance CPTP activity by serving as preferred membrane targeting/docking sites that favorably orient the protein for function.

Ceramide-1-phosphate transfer protein promotes sphingolipid reorientation needed for binding during membrane interaction

“…3 E ,blue trace). This finding is consistent with a recent study showing CPTP transfer activity stimulation via a PI-(4,5)P 2 headgroup interaction site containing a di-Arg motif in the α-3/α-4 helices connecting loop ( 21 ).…”

“…Figure 3 B, C illustrate differing conformations that the α-3/α-4 helices connecting loop can assume when CPTP-containing bound C1P interacts with the POPC bilayer, as revealed by TTClust, relative to the X-ray crystallographic structure of the holo-protein determined in the absence of the POPC bilayer. The dynamic flexibility of the α-3/α-4 helices connecting loop enables close approach to the phosphoglyceride headgroup region of the bilayer ( supplemental Video S1 ), consistent with recent findings indicating the presence of a PI-(4,5)P 2 docking site within the α-3/α-4 helices connecting loop ( 21 ).…”

“…The α-3/α-4 helices connecting loop in the GLTP-fold has previously been implicated in the regulation of the differing glycolipid specificity in FAPP2-GLTPH domain and GLTP ( 12 ), and more recently has been found to harbor a PI-(4,5)P 2 docking site in CPTP ( 21 ). Therefore, we analyzed the dynamics associated with the α-3/α-4 helices connecting loop in more detail.…”

“…So, although the issue of what guides FAPP2 to specific membranes is reasonably well established, only recently have insights begun to emerge for small, single-domain GLTP superfamily members. CPTP has been shown to harbor-specific sequence motifs within its membrane interaction region that interact with select cytoplasm-facing anionic phosphoglycerides (e.g., phosphatidylserine and specific phosphoinositides) to enhance operating efficiency by optimizing protein orientation during membrane docking ( 20 , 21 ).…”

The phosphoinositide code is read by a plethora of protein domains

“…The ceramide-1-phosphate transfer protein (CPTP) does not contain such modules yet localizes to Golgi and plasma membranes. Its lipid transport activity may be stimulated by direct binding of PI4P and PI(4,5)P 2 to an exposed basic-binding surface on its all-helical GLTP domain fold [159], potentially explaining its localization to sites where ceramide is produced and delivered.…”