Ceramide-1-phosphate transfer protein (CPTP) regulation by phosphoinositides
2021; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1016/j.jbc.2021.100600
ISSN1083-351X
AutoresYong‐Guang Gao, Xiuhong Zhai, Ivan Boldyrev, Julian G. Molotkovsky, Dinshaw J. Patel, Lucy Malinina, Rhoderick E. Brown,
Tópico(s)Lipid Membrane Structure and Behavior
ResumoCeramide-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. 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. Lipid intracellular transport by vesicular and nonvesicular mechanisms helps maintain distinct lipid compositions associated with various cell organelles. Vesicular lipid transport involves budding and fission of vesicles from source membranes followed by trafficking and fusion with destination membranes. Nonvesicular lipid transport occurs via lipid transfer proteins (LTPs) that acquire and release their specific lipid cargoes during transient interaction with source and destination membranes (1de Saint-Jean M. Delfosse V. Douguet D. Chicanne G. Payrastre B. Bourguet W. Antonny B. Drin G. Osh4p exchanges sterols for phosphatidylinositol 4-phosphate between lipid bilayers.J. Cell Biol. 2011; 195: 965-978Crossref PubMed Scopus (272) Google Scholar, 2Grabon A. Bankaitis V.A. McDermott M.I. The interface between phosphatidylinositol transfer protein function and phosphoinositide signaling in higher eukaryotes.J. Lipid Res. 2019; 60: 242-268Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 3Holthuis J.C. Menon A.K. Lipid landscapes and pipelines in membrane homeostasis.Nature. 2014; 510: 48-57Crossref PubMed Scopus (536) Google Scholar, 4Lipp N.F. Ikhlef S. Milanini J. Drin G. Lipid exchangers: Cellular functions and mechanistic links with phosphoinositide metabolism.Front. Cell. Dev. Biol. 2020; 8: 663Crossref PubMed Scopus (15) Google Scholar, 5Luo J. Jiang L.Y. Yang H. Song B.L. Intracellular cholesterol transport by sterol transfer proteins at membrane contact sites.Trends Biochem. Sci. 2019; 44: 273-292Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 6Malinina L. Patel D.J. Brown R.E. How alpha-helical motifs form functionally diverse lipid-binding compartments.Annu. Rev. Biochem. 2017; 86: 609-636Crossref PubMed Scopus (16) Google Scholar, 7Malinina L. Simanshu D.K. Zhai X. Samygina V.R. Kamlekar R. Kenoth R. Ochoa-Lizarralde B. Malakhova M.L. Molotkovsky J.G. Patel D.J. Brown R.E. Sphingolipid transfer proteins defined by the GLTP-fold.Q. Rev. Biophys. 2015; 48: 281-322Crossref PubMed Scopus (23) Google Scholar, 8Mishra S.K. Gao Y.G. Zou X. Stephenson D.J. Malinina L. Hinchcliffe E.H. Chalfant C.E. Brown R.E. Emerging roles for human glycolipid transfer protein superfamily members in the regulation of autophagy, inflammation, and cell death.Prog. Lipid Res. 2020; 78: 101031Crossref PubMed Scopus (11) Google Scholar, 9Olkkonen V.M. Li S. Oxysterol-binding proteins: Sterol and phosphoinositide sensors coordinating transport, signaling and metabolism.Prog. Lipid Res. 2013; 52: 529-538Crossref PubMed Scopus (122) Google Scholar, 10Prinz W.A. Bridging the gap: Membrane contact sites in signaling, metabolism, and organelle dynamics.J. Cell Biol. 2014; 205: 759-769Crossref PubMed Scopus (276) Google Scholar, 11Wong L.H. Copic A. Levine T.P. Advances on the transfer of lipids by lipid transfer proteins.Trends Biochem. Sci. 2017; 42: 516-530Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 12Wong L.H. Gatta A.T. Levine T.P. Lipid transfer proteins: The lipid commute via shuttles, bridges and tubes.Nat. Rev. Mol. Cell Biol. 2019; 20: 85-101Crossref PubMed Scopus (173) Google Scholar). Variations in LTP transfer mechanisms include: i) shuttling of the amphitropic LTP back and forth through the aqueous milieu between membranes or ii) pendulum-like swinging of membrane-associated protein containing an LTP domain between closely apposed membranes. LTPs involved in the nonvesicular trafficking of sphingolipids (SLs) between membranes include ceramide transfer protein (CERT) (13Kumagai K. Hanada K. Structure, functions and regulation of CERT, a lipid-transfer protein for the delivery of ceramide at the ER-Golgi membrane contact sites.FEBS Lett. 2019; 593: 2366-2377Crossref PubMed Scopus (38) Google Scholar, 14Yamaji T. Hanada K. Sphingolipid metabolism and interorganellar transport: Localization of sphingolipid enzymes and lipid transfer proteins.Traffic. 2015; 16: 101-122Crossref PubMed Scopus (107) Google Scholar), certain SL activator proteins (15Leon L. Tatituri R.V. Grenha R. Sun Y. Barral D.C. Minnaard A.J. Bhowruth V. Veerapen N. Besra G.S. Kasmar A. Peng W. Moody D.B. Grabowski G.A. Brenner M.B. Saposins utilize two strategies for lipid transfer and CD1 antigen presentation.Proc. Natl. Acad. Sci. U. S. A. 2012; 109: 4357-4364Crossref PubMed Scopus (37) Google Scholar, 16Ran Y. Fanucci G.E. Ligand extraction properties of the GM2 activator protein and its interactions with lipid vesicles.Biophys. J. 2009; 97: 257-266Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 17Schulze H. Sandhoff K. Sphingolipids and lysosomal pathologies.Biochim. Biophys. Acta. 2014; 1841: 799-810Crossref PubMed Scopus (69) Google Scholar, 18Teyton L. Role of lipid transfer proteins in loading CD1 antigen-presenting molecules.J. Lipid Res. 2018; 59: 1367-1373Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar), and members of the glycolipid transfer protein (GLTP) superfamily (6Malinina L. Patel D.J. Brown R.E. How alpha-helical motifs form functionally diverse lipid-binding compartments.Annu. Rev. Biochem. 2017; 86: 609-636Crossref PubMed Scopus (16) Google Scholar, 7Malinina L. Simanshu D.K. Zhai X. Samygina V.R. Kamlekar R. Kenoth R. Ochoa-Lizarralde B. Malakhova M.L. Molotkovsky J.G. Patel D.J. Brown R.E. Sphingolipid transfer proteins defined by the GLTP-fold.Q. Rev. Biophys. 2015; 48: 281-322Crossref PubMed Scopus (23) Google Scholar, 8Mishra S.K. Gao Y.G. Zou X. Stephenson D.J. Malinina L. Hinchcliffe E.H. Chalfant C.E. Brown R.E. Emerging roles for human glycolipid transfer protein superfamily members in the regulation of autophagy, inflammation, and cell death.Prog. Lipid Res. 2020; 78: 101031Crossref PubMed Scopus (11) Google Scholar, 19Brown R.E. Mattjus P. Glycolipid transfer proteins.Biochim. Biophys. Acta. 2007; 1771: 746-760Crossref PubMed Scopus (70) Google Scholar, 20Mattjus P. Glycolipid transfer proteins and membrane interaction.Biochim. Biophys. Acta. 2009; 1788: 267-272Crossref PubMed Scopus (56) Google Scholar, 21Mattjus P. Specificity of the mammalian glycolipid transfer proteins.Chem. Phys. Lipids. 2016; 194: 72-78Crossref PubMed Scopus (13) Google Scholar). In the GLTP superfamily, evolutionary modifications have led to the two-layer, all-α-helical GLTP-fold becoming adapted for binding SLs containing either phosphate or sugar headgroups, thus distinguishing two GLTP families. Examples of the phosphate headgroup-specific family include human ceramide-1-phosphate transfer protein (CPTP) and the plant CPTP ortholog, accelerated cell death-11 protein (ACD11) (22Simanshu D.K. Kamlekar R.K. Wijesinghe D.S. Zou X. Zhai X. Mishra S.K. Molotkovsky J.G. Malinina L. Hinchcliffe E.H. Chalfant C.E. Brown R.E. Patel D.J. Non-vesicular trafficking by a ceramide-1-phosphate transfer protein regulates eicosanoids.Nature. 2013; 500: 463-467Crossref PubMed Scopus (134) Google Scholar, 23Simanshu D.K. Zhai X. Munch D. Hofius D. Markham J.E. Bielawski J. Bielawska A. Malinina L. Molotkovsky J.G. Mundy J.W. Patel D.J. Brown R.E. Arabidopsis accelerated cell death 11, ACD11, is a ceramide-1-phosphate transfer protein and intermediary regulator of phytoceramide levels.Cell Rep. 2014; 6: 388-399Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), whereas glycolipid-specific members include human GLTP and phosphatidylinositol-4-phosphate adapter protein-2 (FAPP2) (19Brown R.E. Mattjus P. Glycolipid transfer proteins.Biochim. Biophys. Acta. 2007; 1771: 746-760Crossref PubMed Scopus (70) Google Scholar, 24Airenne T.T. Kidron H. Nymalm Y. Nylund M. West G. Mattjus P. Salminen T.A. Structural evidence for adaptive ligand binding of glycolipid transfer protein.J. Mol. Biol. 2006; 355: 224-236Crossref PubMed Scopus (45) Google Scholar, 25D'Angelo G. Polishchuk E. Di Tullio G. Santoro M. Di Campli A. Godi A. West G. Bielawski J. Chuang C.C. van der Spoel A.C. Platt F.M. Hannun Y.A. Polishchuk R. Mattjus P. De Matteis M.A. Glycosphingolipid synthesis requires FAPP2 transfer of glucosylceramide.Nature. 2007; 449: 62-67Crossref PubMed Scopus (328) Google Scholar, 26Kamlekar R.K. Simanshu D.K. Gao Y.G. Kenoth R. Pike H.M. Prendergast F.G. Malinina L. Molotkovsky J.G. Venyaminov S.Y. Patel D.J. Brown R.E. The glycolipid transfer protein (GLTP) domain of phosphoinositol 4-phosphate adaptor protein-2 (FAPP2): Structure drives preference for simple neutral glycosphingolipids.Biochim. Biophys. Acta. 2013; 1831: 417-427Crossref PubMed Scopus (22) Google Scholar, 27Ochoa-Lizarralde B. Gao Y.G. Popov A.N. Samygina V.R. Zhai X. Mishra S.K. Boldyrev I.A. Molotkovsky J.G. Simanshu D.K. Patel D.J. Brown R.E. Malinina L. Structural analyses of 4-phosphate adaptor protein 2 yield mechanistic insights into sphingolipid recognition by the glycolipid transfer protein family.J. Biol. Chem. 2018; 293: 16709-16723Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar, 28Malinina L. Malakhova M.L. Kanack A.T. Lu M. Abagyan R. Brown R.E. Patel D.J. The liganding of glycolipid transfer protein is controlled by glycolipid acyl structure.PLoS Biol. 2006; 4e362Crossref PubMed Scopus (50) Google Scholar, 29Malinina L. Malakhova M.L. Teplov A. Brown R.E. Patel D.J. Structural basis for glycosphingolipid transfer specificity.Nature. 2004; 430: 1048-1053Crossref PubMed Scopus (100) Google Scholar, 30Samygina V.R. Popov A.N. Cabo-Bilbao A. Ochoa-Lizarralde B. Goni-de-Cerio F. Zhai X. Molotkovsky J.G. Patel D.J. Brown R.E. Malinina L. Enhanced selectivity for sulfatide by engineered human glycolipid transfer protein.Structure. 2011; 19: 1644-1654Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). In all cases, the two-layer α-helical GLTP-fold binds the SL in "sandwich-like" fashion such that the initial phosphate or sugar residue of the SL headgroup is bound to the protein surface and the hydrocarbon chains are enveloped within a hydrophobic pocket. Emerging information indicates that GLTP superfamily members can function in vivo as molecular sensors and/or presentation devices involved in lipid metabolic regulation and signaling processes. GLTP, FAPP2, CPTP, and ACD11 have been implicated in the in vivo regulation of SL homeostatic levels and intracellular distributions (22Simanshu D.K. Kamlekar R.K. Wijesinghe D.S. Zou X. Zhai X. Mishra S.K. Molotkovsky J.G. Malinina L. Hinchcliffe E.H. Chalfant C.E. Brown R.E. Patel D.J. Non-vesicular trafficking by a ceramide-1-phosphate transfer protein regulates eicosanoids.Nature. 2013; 500: 463-467Crossref PubMed Scopus (134) Google Scholar, 23Simanshu D.K. Zhai X. Munch D. Hofius D. Markham J.E. Bielawski J. Bielawska A. Malinina L. Molotkovsky J.G. Mundy J.W. Patel D.J. Brown R.E. Arabidopsis accelerated cell death 11, ACD11, is a ceramide-1-phosphate transfer protein and intermediary regulator of phytoceramide levels.Cell Rep. 2014; 6: 388-399Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 25D'Angelo G. Polishchuk E. Di Tullio G. Santoro M. Di Campli A. Godi A. West G. Bielawski J. Chuang C.C. van der Spoel A.C. Platt F.M. Hannun Y.A. Polishchuk R. Mattjus P. De Matteis M.A. Glycosphingolipid synthesis requires FAPP2 transfer of glucosylceramide.Nature. 2007; 449: 62-67Crossref PubMed Scopus (328) Google Scholar, 31Halter D. Neumann S. van Dijk S.M. Wolthoorn J. de Maziere A.M. Vieira O.V. Mattjus P. Klumperman J. van Meer G. Sprong H. Pre- and post-Golgi translocation of glucosylceramide in glycosphingolipid synthesis.J. Cell Biol. 2007; 179: 101-115Crossref PubMed Scopus (231) Google Scholar, 32Kjellberg M.A. Backman A.P. Ohvo-Rekila H. Mattjus P. Alternation in the glycolipid transfer protein expression causes changes in the cellular lipidome.PLoS One. 2014; 9e97263Crossref PubMed Scopus (18) Google Scholar, 33Mishra S.K. Gao Y.G. Deng Y. Chalfant C.E. Hinchcliffe E.H. Brown R.E. CPTP: A sphingolipid transfer protein that regulates autophagy and inflammasome activation.Autophagy. 2018; 14: 862-879Crossref PubMed Scopus (32) Google Scholar, 34Mishra S.K. Stephenson D.J. Chalfant C.E. Brown R.E. Upregulation of human glycolipid transfer protein (GLTP) induces necroptosis in colon carcinoma cells.Biochim. Biophys. Acta Mol. Cell Biol. Lipids. 2019; 1864: 158-167Crossref PubMed Scopus (13) Google Scholar). In the case of human CPTP, siRNA-induced downregulation not only stimulates proinflammatory eicosanoid production but also triggers autophagy-dependent, inflammasome-mediated release of interleukin-1β and -18 and pyroptosis by macrophage-like surveillance cells (22Simanshu D.K. Kamlekar R.K. Wijesinghe D.S. Zou X. Zhai X. Mishra S.K. Molotkovsky J.G. Malinina L. Hinchcliffe E.H. Chalfant C.E. Brown R.E. Patel D.J. Non-vesicular trafficking by a ceramide-1-phosphate transfer protein regulates eicosanoids.Nature. 2013; 500: 463-467Crossref PubMed Scopus (134) Google Scholar, 33Mishra S.K. Gao Y.G. Deng Y. Chalfant C.E. Hinchcliffe E.H. Brown R.E. CPTP: A sphingolipid transfer protein that regulates autophagy and inflammasome activation.Autophagy. 2018; 14: 862-879Crossref PubMed Scopus (32) Google Scholar). CPTP intracellular docking sites include the trans-Golgi, a C1P production site by ceramide kinase, as well as the plasma and nuclear membranes (22Simanshu D.K. Kamlekar R.K. Wijesinghe D.S. Zou X. Zhai X. Mishra S.K. Molotkovsky J.G. Malinina L. Hinchcliffe E.H. Chalfant C.E. Brown R.E. Patel D.J. Non-vesicular trafficking by a ceramide-1-phosphate transfer protein regulates eicosanoids.Nature. 2013; 500: 463-467Crossref PubMed Scopus (134) Google Scholar). CPTP depletion leads to approximately fourfold in vivo elevations of intracellular C1P (mostly 16:0-C1P species) that accumulate in trans-Golgi-enriched membrane fractions and decrease in plasma-membrane-enriched fractions. The elevated C1P levels in the TGN stimulate arachidonic acid release and drive downstream production of proinflammatory eicosanoids (22Simanshu D.K. Kamlekar R.K. Wijesinghe D.S. Zou X. Zhai X. Mishra S.K. Molotkovsky J.G. Malinina L. Hinchcliffe E.H. Chalfant C.E. Brown R.E. Patel D.J. Non-vesicular trafficking by a ceramide-1-phosphate transfer protein regulates eicosanoids.Nature. 2013; 500: 463-467Crossref PubMed Scopus (134) Google Scholar), presumably reflecting activation of cytoplasmic phospholipase A2α via C1P binding to its C2 domain (35Stahelin R.V. Subramanian P. Vora M. Cho W. Chalfant C.E. Ceramide-1-phosphate binds group IVA cytosolic phospholipase a2 via a novel site in the C2 domain.J. Biol. Chem. 2007; 282: 20467-20474Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 36Ward K.E. Bhardwaj N. Vora M. Chalfant C.E. Lu H. Stahelin R.V. The molecular basis of ceramide-1-phosphate recognition by C2 domains.J. Lipid Res. 2013; 54: 636-648Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). GLTP superfamily members such as CPTP, ACD11, and GLTP lack known lipid-binding domains (LBDs) (e.g., PH, PZ, C1, C2) that target various proteins to select phosphoglycerides in intracellular membranes (37Cho W. Stahelin R.V. Membrane-protein interactions in cell signaling and membrane trafficking.Annu. Rev. Biophys. Biomol. Struct. 2005; 34: 119-151Crossref PubMed Scopus (479) Google Scholar, 38Corbalan-Garcia S. Gomez-Fernandez J.C. Signaling through C2 domains: More than one lipid target.Biochim. Biophys. Acta. 2014; 1838: 1536-1547Crossref PubMed Scopus (140) Google Scholar, 39Hirano Y. Gao Y.G. Stephenson D.J. Vu N.T. Malinina L. Simanshu D.K. Chalfant C.E. Patel D.J. Brown R.E. Structural basis of phosphatidylcholine recognition by the C2-domain of cytosolic phospholipase A2alpha.Elife. 2019; 8e44760Crossref PubMed Scopus (19) Google Scholar, 40Lemmon M.A. Membrane recognition by phospholipid-binding domains.Nat. Rev. Mol. Cell Biol. 2008; 9: 99-111Crossref PubMed Scopus (1114) Google Scholar, 41Moravcevic K. Oxley C.L. Lemmon M.A. Conditional peripheral membrane proteins: Facing up to limited specificity.Structure. 2012; 20: 15-27Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 42Stahelin R.V. Lipid binding domains: More than simple lipid effectors.J. Lipid Res. 2009; 50 Suppl: S299-S304Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 43Stahelin R.V. Scott J.L. Frick C.T. Cellular and molecular interactions of phosphoinositides and peripheral proteins.Chem. Phys. Lipids. 2014; 182: 3-18Crossref PubMed Scopus (80) Google Scholar). To determine whether CPTP and related GLTP homologs contain targeting motifs for specific phosphoglycerides embedded in membranes, we investigated the regulatory effects exerted by various phosphoinositides (PIPs) on SL transfer by CPTP, ACD11, or GLTP and their membrane partitioning. We focused on PIPs present in the trans-Golgi (e.g., phospatidylinositol-4-phosphate; PI-4P) and plasma membrane (phospatidylinositol-4,5-bisphosphate; PI-(4,5)P2) due to earlier findings of CPTP enrichment at these intracellular sites (22Simanshu D.K. Kamlekar R.K. Wijesinghe D.S. Zou X. Zhai X. Mishra S.K. Molotkovsky J.G. Malinina L. Hinchcliffe E.H. Chalfant C.E. Brown R.E. Patel D.J. Non-vesicular trafficking by a ceramide-1-phosphate transfer protein regulates eicosanoids.Nature. 2013; 500: 463-467Crossref PubMed Scopus (134) Google Scholar). The data are consistent with PIP-specific headgroup interaction sites existing on CPTP but not GLTP that serve a dual role of enhancing SL transfer activity while also acting as preferred targeting/docking sites in specific membranes in vivo. Mapping of the PIP-selective motifs in C1P-specific GLTP-folds within membrane interaction regions reveals a role for the recently discovered ID-loop (α3-α4 helices connecting loop) (27Ochoa-Lizarralde B. Gao Y.G. Popov A.N. Samygina V.R. Zhai X. Mishra S.K. Boldyrev I.A. Molotkovsky J.G. Simanshu D.K. Patel D.J. Brown R.E. Malinina L. Structural analyses of 4-phosphate adaptor protein 2 yield mechanistic insights into sphingolipid recognition by the glycolipid transfer protein family.J. Biol. Chem. 2018; 293: 16709-16723Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). To assess whether certain PIPs can activate the SL transfer activities of various GLTP superfamily members (human CPTP, plant CPTP-ACD11, human GLTP), we used an established fluorescence resonance energy transfer (FRET) approach that monitors the real-time kinetics of the complete SL transfer reaction, i.e., SL uptake by protein from "SL-source" membrane vesicles and SL delivery by protein to "destination" membrane vesicles (44Kenoth R. Brown R.E. Kamlekar R.K. In vitro measurement of sphingolipid intermembrane transport illustrated by GLTP superfamily members.Methods Mol. Biol. 2019; 1949: 237-256Crossref PubMed Scopus (3) Google Scholar). A more complete description of the FRET assay is provided as Supporting information and illustrated in Fig. S1. Inclusion of various long-chain PIPs (PI-(4,5)P2, PI-4P, PI-3P) or PI in SL-source POPC vesicles was found to exert different effects on the SL intermembrane transfer rates catalyzed by CPTP and GLTP (Fig. 1) and by ACD11 (Fig. S2) at physiologic ionic strength. The PIP concentrations in the model membranes were kept low to mimic the physiological situation (45Schink K.O. Tan K.W. Stenmark H. Phosphoinositides in control of membrane dynamics.Annu. Rev. Cell Dev. Biol. 2016; 32: 143-171Crossref PubMed Scopus (164) Google Scholar) and the buffer contained EDTA to block potential effects by polyvalent cations such as calcium (46Wen Y. Vogt V.M. Feigenson G.W. Multivalent cation-bridged PI(4,5)P2 clusters form at very low concentrations.Biophys. J. 2018; 114: 2630-2639Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Notably, significant stimulation of C1P transfer rates occurred when 2, 4, or 6 mol% of PI-(4,5)P2 or PI-4P was present in the C1P source (donor) vesicles (Fig. 1, A–E). In contrast, PI-3P and PI failed to stimulate and inhibited, respectively, the C1P transfer activity of CPTP and exerted minimal effects on ACD11 (Fig. S2). Notably, GalCer transfer rates by GLTP were unaffected by PI and PI-3P as well as by 2 and 4 mol% PI-4P but were moderately decreased by 4 and 6 mol% PI-(4,5)P2 (Fig. 1F). To directly assess the ability of CPTP and ACD11 to interact with PI-(4,5)P2 and PI-4P, protein–lipid overlay assays were performed (47Dowler S. Kular G. Alessi D.R. Protein lipid overlay assay.Sci. STKE. 2002; 2002pl6Crossref PubMed Scopus (204) Google Scholar). Fig. S3 shows that both CPTP and ACD11 exhibit relatively strong binding interactions with PI-(4,5)P2 and PI-4P compared with various other anionic and zwitterionic phosphoglycerides, neutral lipids, and sulfatide. The observation of CPTP and ACD11 binding to phosphatidylserine (PS) and phosphatidic acid (PA) in the protein–lipid overlay assay, albeit moderate in intensity, is consistent with earlier findings for these two phosphoglycerides (48Zhai X. Gao Y.G. Mishra S.K. Simanshu D.K. Boldyrev I.A. Benson L.M. Bergen 3rd, H.R. Malinina L. Mundy J. Molotkovsky J.G. Patel D.J. Brown R.E. Phosphatidylserine stimulates ceramide 1-phosphate (C1P) intermembrane transfer by C1P transfer proteins.J. Biol. Chem. 2017; 292: 2531-2541Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). In this previous study, SL transfer activity by CPTP and ACD11, but not GLTP, was found to be stimulated by membrane-embedded PS, but not by PA although no specific interaction site was identified. We therefore hypothesized that the CPTP membrane interaction region contains a specific binding site for targeting the PI-(4,5)P2 and/or PI-4P headgroups. These PIP headgroups presumably would act as a tethering/activation site to help favorably orient CPTP for C1P uptake during membrane interaction while the PIP acyl chains remain embedded in the membrane interior. To test this idea, we assessed whether C1P transfer by CPTP is stimulated by "soluble" PI-(4,5)P2 with short acyl chains (di-octanoyl- PI-(4,5)P2). We expected little or no activation by "soluble" PI-(4,5)P2 due to its much weaker anchoring in the POPC bilayer vesicle and its high aqueous solubility (cmc > 4 mM; (49Collins M.D. Gordon S.E. Short-chain phosphoinositide partitioning into plasma membrane models.Biophys. J. 2013; 105: 2485-2494Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar)) compared with di-oleoyl PI-(4,5)P2. Indeed, replacing di-18:1 PI-(4,5)P2 with di-8:0-PI-(4,5)P2 in the SL-source vesicles resulted in no significant stimulation in C1P transfer rates by CPTP (Fig. S4). To test whether long-chain PI-(4,5)P2 is a better stimulator of CPTP than long-chain PS, we compared in side-by-side fashion. The data in Figure 2 show that PI-(4,5)P2 is a better stimulator of CPTP activity than PS at physiologically relevant PI-(4,5)P2 membrane concentrations (≤6 mol% in POPC). To quantifiably assess and compare the extent to which the long-chain PIPs, PS, and other anionic phosphoglycerides impact the membrane-binding affinity of CPTP, we relied on FRET involving CPTP Tyr/Trp (energy donor) and POPC membrane vesicles containing dansyl-phosphatidylethanolamine (PE) (energy acceptor). Figure 3, A and B show the FRET responses produced by CPTP adsorption to POPC vesicles as a function of their concentration when containing equal amounts of PI-(4,5)P2, PI-4P, PI-3P, or PI (Fig. 3A) as well as PS, PA, or PG (Fig. 3B). Analyses of the binding isotherms resulted in the relative equilibrium binding affinity constants (Kd) shown in Table 1. The Kd values for POPC vesicles containing PI-(4,5)P2, PI-4P, and PI-3P are six- to seven-fold lower than that for pure POPC vesicles (Table 1), whereas the Kd for POPC vesicles containing PI is only 2.3-fold lower. Interestingly, including PS and PA yielded relative Kd values even lower than the PIP values, which are comparable to that elicited by the presence of PG. The binding response for POPC vesicles containing PS and PA (relative to pure PC vesicles) agrees with earlier qualitative data showing that both PS and PA enhance membrane binding by plant CPTP, i.e., ACD11, but only PS stimulates transfer activity of ACD11 and CPTP, but not GLTP (48Zhai X. Gao Y.G. Mishra S.K. Simanshu D.K. Boldyrev I.A. Benson L.M. Bergen 3rd, H.R. Malinina L. Mundy J. Molotkovsky J.G. Patel D.J. Brown R.E. Phosphatidylserine stimulates ceramide 1-phosphate (C1P) intermembrane transfer by C1P transfer proteins.J. Biol. Chem. 2017; 292: 2531-2541Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar).Table 1Relative equilibrium binding affinity of CPTP for POPC vesicles containing different anionic phosphoglyceridesLipidKd (μM)Relative to POPCPI-(4,5)P20.26 ± 0.046.8PI-4P0.26 ± 0.036.8PI-3P0.29 ± 0.046.1PI0.75 ± 0.092.3PS0.07 ± 0.0225.1PA0.11 ± 0.0216.0PG0.24 ± 0.047.3PC1.76 ± 0.201.0Anionic phosphoglycerides = 10 mol%. Open table in a new tab Anionic phosphoglycerides = 10 mol%. To determine the impact of long-chain PI-(4,5)P2 on CPTP association and dissociation to/from PC membranes, surface plasmon resonance (SPR) analyses were performed. SPR provides real-time insights into protein adsorption and desorption to/from the membrane associated with SL uptake or release, i.e., transfer "half-reactions." The insights help understand the protein-mediated lipid transfer process, which involves: i) LTP association with the membrane; ii) lipid uptake by membrane-associated LTP; iii) LTP/cargo-lipid desorption from the membrane; iv) LTP/cargo-lipid association with acceptor membrane; v) LTP release of lipid cargo into the membrane; vi) lipid-free LTP desorption from acceptor membrane (19Brown R.E. Mattjus P. Glycolipid transfer proteins.Biochim. Biophys. Acta. 2007; 1771: 746-760Crossref PubMed Scopus (70) Google Scholar, 50Rao C.S. Chung T. Pike H.M. Brown R.E. Glycoli
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