神经酰胺-1-磷酸转移蛋白(CPTP)是糖脂转移蛋白(GLTP)超家族的成员,可在膜之间穿梭神经酰胺-1-磷酸(C1P)。CPTP以影响程序性细胞死亡和炎症的方式调节细胞鞘脂稳态。CPTP下调特异性改变血浆和跨高尔基体膜中的C1P水平,刺激促炎性类二十烷酸的产生和自噬依赖性炎性体介导的细胞因子释放。然而,CPTP用于靶向跨高尔基体和质膜的机制尚不清楚。这里,我们使用荧光能量转移(FRET)监测C1P囊泡间转移,并显示某些磷酸肌醇(磷脂酰肌醇4,5双磷酸(PI-(4,5)P2)和磷脂酰肌醇4-磷酸(PI-4P))增加CPTP转移活性,而其他(磷脂酰肌醇3-磷酸(PI-3P)和PI)则没有。刺激CPTP的PIP不刺激GLTP,另一个超家族成员。短链PI-(4,5)P2是可溶性的,不会保持膜包埋,无法激活CPTP。生理相关的PI-(4,5)P2水平的CPTP刺激超过了磷脂酰丝氨酸(PS),唯一已知的CPTP非PIP刺激器,尽管PI-(4,5)P2增加膜平衡结合亲和力的有效性低于PS。导致FRET脂质转移改变的突变的功能作图以及通过表面等离子体共振评估CPTP膜相互作用表明,位于膜相互作用区域的α-6螺旋和α3-α4螺旋调节环中的双精氨酸基序充当PI-(4,5)P2头基特异性相互作用位点。Haddock建模揭示了涉及PI-(4,5)P2头基的特定相互作用,使酰基链有利于膜嵌入。我们建议PI-(4,5)P2相互作用位点通过充当优选的膜靶向/对接位点来增强CPTP活性,从而有利地定向蛋白质的功能。
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)P2, 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.