Non-vesicular trafficking by a ceramide-1-phosphate transfer protein regulates eicosanoids

Abstract

Phosphorylated sphingolipids ceramide-1-phosphate (C1P) and sphingosine-1-phosphate (S1P) have emerged as key regulators of cell growth, survival, migration and inflammation1,2,3,4,5. C1P produced by ceramide kinase is an activator of group IVA cytosolic phospholipase A2α (cPLA2α), the rate-limiting releaser of arachidonic acid used for pro-inflammatory eicosanoid production3,6,7,8,9, which contributes to disease pathogenesis in asthma or airway hyper-responsiveness, cancer, atherosclerosis and thrombosis. To modulate eicosanoid action and avoid the damaging effects of chronic inflammation, cells require efficient targeting, trafficking and presentation of C1P to specific cellular sites. Vesicular trafficking is likely10 but non-vesicular mechanisms for C1P sensing, transfer and presentation remain unexplored11,12. Moreover, the molecular basis for selective recognition and binding among signalling lipids with phosphate headgroups, namely C1P, phosphatidic acid or their lyso-derivatives, remains unclear. Here, a ubiquitously expressed lipid transfer protein, human GLTPD1, named here CPTP, is shown to specifically transfer C1P between membranes. Crystal structures establish C1P binding through a novel surface-localized, phosphate headgroup recognition centre connected to an interior hydrophobic pocket that adaptively expands to ensheath differing-length lipid chains using a cleft-like gating mechanism. The two-layer, α-helically-dominated ‘sandwich’ topology identifies CPTP as the prototype for a new glycolipid transfer protein fold13 subfamily. CPTP resides in the cell cytosol but associates with the trans-Golgi network, nucleus and plasma membrane. RNA interference-induced CPTP depletion elevates C1P steady-state levels and alters Golgi cisternae stack morphology. The resulting C1P decrease in plasma membranes and increase in the Golgi complex stimulates cPLA2α release of arachidonic acid, triggering pro-inflammatory eicosanoid generation.

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Figure 1: CPTP lipid transfer activity and architecture.
Figure 2: CPTP conformation and functional recognition of C1P.
Figure 3: CPTP accommodation and adaptation for C1P species, functional assessment of CPTP and intracellular localization.
Figure 4: CPTP siRNA depletion/rescue effects on cellular C1P levels, arachidonic acid generation and eicosanoid release and model of CPTP cell biological function.

Accession codes

Accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors for human CPTP crystal complexes with various lipids andmouse apo-CPTP have been deposited in the Protein Data Bank. Accession codes are: 2:0-C1P–CPTP (4K80), 8:0-C1P–CPTP (4KF6), 12:0-C1P–CPTP (4K85), 16:0-C1P–CPTP (4K84), 18:1-C1P–CPTP (4K8N), di12:0-PA–CPTP (4KBS) and mouse apo-CPTP (4KBR).

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Acknowledgements

This research was supported by NIH/NCI CA121493 (D.J.P. & R.E.B.), NIH/NIGMS GM45928 (R.E.B.), NIH/NIGMS GM072754 (E.H.H.), NIH/CA154314 (C.E.C.), VA Merit Award (C.E.C.), VA Research Career Scientist Award (C.E.C.), VA Career Devel. Award (D.S.W.), NRS-T32/NIGMS 008695 (D.S.W.), Spanish Ministerio de Ciencia e Innovacion BFU2010-17711 (L.M.), Russian Foundation for Basic Research #12-04-00168 (J.G.M.), Hormel Foundation. (R.E.B.), Abby Rockefeller Mauze Trust (D.J.P.) and Maloris Foundation (D.J.P.). We thank H. Pike for expressing and purifying protein used for transfer activity analyses, K. Karanjeet for preparing cells for confocal and epifluorescence microscopy, and the staff of X-29 beamline at the National Synchrotron Light Source and ID-24-C/E beamlines at the Advanced Photon Source for help.

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D.K.S. carried out all structural analyses and provided evidence for C1P binding by CPTP, generated all CPTP point mutants and wrote text. R.K.K did transfer analyses of wild-type CPTP and CPTP point mutants and wrote text. D.S.W. conducted siRNA CPTP knockdown, rescue, and all lipid analyses and wrote text. Xi.Zo. cloned wild-type CPTP and did PCR analyses of CPTP transcript distribution in human tissues. Xi.Zh. did CPTP transfer rate analyses. S.K.M. prepared CPTP RNAi constructs for microscopy and CPTP overexpression constructs for lipidomics analyses. J.G.M. synthesized all fluorescent lipids. L.M. contributed to structural data interpretation. E.H.H. did fluorescence microscopy of CPTP localization in fixed and living cells and finalized the write-up. C.E.C. directed siRNA knockdown, rescue and related lipidomics analyses and finalized the write-up. D.J.P. directed CPTP structural analyses and finalized the write-up. R.E.B. directed functional analyses after the initial CPTP discovery in his laboratory, finalized the write-up and coordinated and integrated all section write-ups.

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Correspondence to Edward H. Hinchcliffe or Charles E. Chalfant or Rhoderick E. Brown or Dinshaw J. Patel.

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The authors declare no competing financial interests.

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Simanshu, D., Kamlekar, R., Wijesinghe, D. et al. Non-vesicular trafficking by a ceramide-1-phosphate transfer protein regulates eicosanoids. Nature 500, 463–467 (2013). https://doi.org/10.1038/nature12332

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