Sec14-like phosphatidylinositol transfer proteins (PITPs) integrate diverse territories of intracellular lipid metabolism with stimulated phosphatidylinositol-4-phosphate production and are discriminating portals for interrogating phosphoinositide signaling. Yet, neither Sec14-like PITPs nor PITPs in general have been exploited as targets for chemical inhibition for such purposes. Herein, we validate what is to our knowledge the first small-molecule inhibitors (SMIs) of the yeast PITP Sec14. These SMIs are nitrophenyl(4-(2-methoxyphenyl)piperazin-1-yl)methanones (NPPMs) and are effective inhibitors in vitro and in vivo. We further establish that Sec14 is the sole essential NPPM target in yeast and that NPPMs exhibit exquisite targeting specificities for Sec14 (relative to related Sec14-like PITPs), propose a mechanism for how NPPMs exert their inhibitory effects and demonstrate that NPPMs exhibit exquisite pathway selectivity in inhibiting phosphoinositide signaling in cells. These data deliver proof of concept that PITP-directed SMIs offer new and generally applicable avenues for intervening with phosphoinositide signaling pathways with selectivities superior to those afforded by contemporary lipid kinase–directed strategies.
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Wymann, M.P. & Schneiter, R. Lipid signalling in disease. Nat. Rev. Mol. Cell Biol. 9, 162–176 (2008).
Janmey, P.A. & Lindberg, U. Cytoskeletal regulation: rich in lipids. Nat. Rev. Mol. Cell Biol. 5, 658–666 (2004).
Henry, S.A., Kohlwein, S.D. & Carman, G.M. Metabolism and regulation of glycerolipids in the yeast Saccharomyces cerevisiae. Genetics 190, 317–349 (2012).
Irvine, R.F. Nuclear lipid signalling. Nat. Rev. Mol. Cell Biol. 4, 349–360 (2003).
Di Paolo, G. & De Camilli, P. Phosphoinositides in cell regulation and membrane dynamics. Nature 443, 651–657 (2006).
Michell, R.H. Inositol derivatives: evolution and functions. Nat. Rev. Mol. Cell Biol. 9, 151–161 (2008).
Strahl, T. & Thorner, J. Synthesis and function of membrane phosphoinositides in budding yeast, Saccharomyces cerevisiae. Biochim. Biophys. Acta 1771, 353–404 (2007).
Varnai, P., Thyagarajan, B., Rohacs, T. & Balla, T. Rapidly inducible changes in phosphatidylinositol 4,5-bisphosphate levels influence multiple regulatory functions of the lipid in intact living cells. J. Cell Biol. 175, 377–382 (2006).
Suh, B.C., Inoue, T., Meyer, T. & Hille, B. Rapid chemically induced changes of PtdIns(4,5)P2 gate KCNQ ion channels. Science 314, 1454–1457 (2006).
Schaaf, G. et al. Functional anatomy of phospholipid binding and regulation of phosphoinositide homeostasis by proteins of the Sec14 superfamily. Mol. Cell 29, 191–206 (2008).
Bankaitis, V.A., Mousley, C.J. & Schaaf, G. The Sec14 superfamily and mechanisms for crosstalk between lipid metabolism and lipid signaling. Trends Biochem. Sci. 35, 150–160 (2010).
Bankaitis, V.A., Malehorn, D.E., Emr, S.D. & Greene, R. The Saccharomyces cerevisiae SEC14 gene encodes a cytosolic factor that is required for transport of secretory proteins from the yeast Golgi complex. J. Cell Biol. 108, 1271–1281 (1989).
Wu, W.I., Routt, S., Bankaitis, V.A. & Voelker, D.R. A new gene involved in the transport-dependent metabolism of phosphatidylserine, PSTB2/PDR17, shares sequence similarity with the gene encoding the phosphatidylinositol/phosphatidylcholine transfer protein, SEC14. J. Biol. Chem. 275, 14446–14456 (2000).
Desfougères, T., Ferreira, T., Bergès, T. & Régnacq, M. SFH2 regulates fatty acid synthase activity in the yeast Saccharomyces cerevisiae and is critical to prevent saturated fatty acid accumulation in response to haem and oleic acid depletion. Biochem. J. 409, 299–309 (2008).
Vincent, P. et al. A Sec14p-nodulin domain phosphatidylinositol transfer protein polarizes membrane growth of Arabidopsis thaliana root hairs. J. Cell Biol. 168, 801–812 (2005).
Lopez, M.C. et al. A phosphatidylinositol/phosphatidylcholine transfer protein is required for differentiation of the dimorphic yeast Yarrowia lipolytica from the yeast to the mycelial form. J. Cell Biol. 125, 113–127 (1994).
Alb, J.G. Jr. et al. Mice lacking phosphatidylinositol transfer protein-α exhibit spinocerebellar degeneration, intestinal and hepatic steatosis, and hypoglycemia. J. Biol. Chem. 278, 33501–33518 (2003).
Nile, A.H., Bankaitis, V.A. & Grabon, A. Mammalian diseases of phosphatidylinositol transfer proteins and their homologs. Clin. Lipidol. 5, 867–897 (2010).
Hoon, S. et al. An integrated platform of genomic assays reveals small-molecule bioactivities. Nat. Chem. Biol. 4, 498–506 (2008); erratum 4, 632 (2008).
Xie, Z. et al. Phospholipase D activity is required for suppression of yeast phosphatidylinositol transfer protein defects. Proc. Natl. Acad. Sci. USA 95, 12346–12351 (1998).
Kennedy, M.A. et al. Srf1 is a novel regulator of phospholipase D activity and is essential to buffer the toxic effects of C16:0 platelet activating factor. PLoS Genet. 7, e1001299 (2011).
Mousley, C.J. et al. Trans-Golgi network and endosome dynamics connect ceramide homeostasis with regulation of the unfolded protein response and TOR signaling in yeast. Mol. Biol. Cell 19, 4785–4803 (2008).
Curwin, A.J., Fairn, G.D. & McMaster, C.R. Phospholipid transfer protein Sec14 is required for trafficking from endosomes and regulates distinct trans-Golgi export pathways. J. Biol. Chem. 284, 7364–7375 (2009).
Salama, S.R., Cleves, A.E., Malehorn, D.E., Whitters, E.A. & Bankaitis, V.A. Cloning and characterization of Kluyveromyces lactis SEC14, a gene whose product stimulates Golgi secretory function in Saccharomyces cerevisiae. J. Bacteriol. 172, 4510–4521 (1990).
Li, X. et al. Identification of a novel family of nonclassic yeast phosphatidylinositol transfer proteins whose function modulates phospholipase D activity and Sec14p-independent cell growth. Mol. Biol. Cell 11, 1989–2005 (2000).
Schaaf, G. et al. Resurrection of a functional phosphatidylinositol transfer protein from a pseudo-Sec14 scaffold by directed evolution. Mol. Biol. Cell 22, 892–905 (2011).
Cleves, A., McGee, T. & Bankaitis, V. Phospholipid transfer proteins: a biological debut. Trends Cell Biol. 1, 30–34 (1991).
Cleves, A.E. et al. Mutations in the CDP-choline pathway for phospholipid biosynthesis bypass the requirement for an essential phospholipid transfer protein. Cell 64, 789–800 (1991).
Li, X. et al. Analysis of oxysterol binding protein homologue Kes1p function in regulation of Sec14p-dependent protein transport from the yeast Golgi complex. J. Cell Biol. 157, 63–77 (2002).
Fang, M. et al. Kes1p shares homology with human oxysterol binding protein and participates in a new regulatory pathway for yeast Golgi-derived transport vesicle biogenesis. EMBO J. 15, 6447–6459 (1996).
Stevens, T., Esmon, B. & Schekman, R. Early stages in the yeast secretory pathway are required for transport of carboxypeptidase Y to the vacuole. Cell 30, 439–448 (1982).
Phillips, S.E. et al. Yeast Sec14p deficient in phosphatidylinositol transfer activity is functional in vivo. Mol. Cell 4, 187–197 (1999).
Rivas, M.P. et al. Pleiotropic alterations in lipid metabolism in yeast sac1 mutants: relationship to “bypass Sec14p” and inositol auxotrophy. Mol. Biol. Cell 10, 2235–2250 (1999).
Hama, H., Schnieders, E.A., Thorner, J., Takemoto, J.Y. & DeWald, D.B. Direct involvement of phosphatidylinositol 4-phosphate in secretion in the yeast Saccharomyces cerevisiae. J. Biol. Chem. 274, 34294–34300 (1999).
Guo, S., Stolz, L.E., Lemrow, S.M. & York, J.D. SAC1-like domains of yeast SAC1, INP52, and INP53 and of human synaptojanin encode polyphosphoinositide phosphatases. J. Biol. Chem. 274, 12990–12995 (1999).
Wood, C.S. et al. PtdIns4P recognition by Vps74/GOLPH3 links PtdIns 4-kinase signaling to retrograde Golgi trafficking. J. Cell Biol. 187, 967–975 (2009).
Roy, A. & Levine, T.P. Multiple pools of phosphatidylinositol 4-phosphate detected using the pleckstrin homology domain of Osh2p. J. Biol. Chem. 279, 44683–44689 (2004).
Baird, D., Stefan, C., Audhya, A., Weys, S. & Emr, S.D. Assembly of the PtdIns 4-kinase Stt4 complex at the plasma membrane requires Ypp1 and Efr3. J. Cell Biol. 183, 1061–1074 (2008).
Sykes, P. A Guidebook to Mechanism in Organic Chemistry 6th edn. (Pearson, 1986).
Lu, Y. et al. C–X...H contacts in biomolecular systems: how they contribute to protein-ligand binding affinity. J. Phys. Chem. B. 113, 12615–12621 (2009).
Auffinger, P., Hays, F.A., Westhof, E. & Ho, P.S. Halogen bonds in biological molecules. Proc. Natl. Acad. Sci. USA 101, 16789–16794 (2004).
Metrangolo, P. & Resnati, G. Halogen bonding: a paradigm in supramolecular chemistry. Chemistry 7, 2511–2519 (2001).
Ryan, M.M., Temple, B.R., Phillips, S.E. & Bankaitis, V.A. Conformational dynamics of the major yeast phosphatidylinositol transfer protein sec14p: insight into the mechanisms of phospholipid exchange and diseases of sec14p-like protein deficiencies. Mol. Biol. Cell 18, 1928–1942 (2007).
Phillips, S.E. et al. The diverse biological functions of phosphatidylinositol transfer proteins in eukaryotes. Crit. Rev. Biochem. Mol. Biol. 41, 21–49 (2006).
Skinner, H.B., Alb, J.G. Jr., Whitters, E.A., Helmkamp, G.M. Jr. & Bankaitis, V.A. Phospholipid transfer activity is relevant to but not sufficient for the essential function of the yeast SEC14 gene product. EMBO J. 12, 4775–4784 (1993).
Sherman, F., Fink, G.R. & Hink, J.B. Methods in Yeast Genetics: A Laboratory Manual (Cold Spring Harbor, 1983).
Ashburner, M. et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat. Genet. 25, 25–29 (2000).
Young, B.P., Craven, R.A., Reid, P.J., Willer, M. & Stirling, C.J. Sec63p and Kar2p are required for the translocation of SRP-dependent precursors into the yeast endoplasmic reticulum in vivo. EMBO J. 20, 262–271 (2001).
Stolz, L.E., Kuo, W.J., Longchamps, J., Sekhon, M.K. & York, J.D. INP51, a yeast inositol polyphosphate 5-phosphatase required for phosphatidylinositol 4,5-bisphosphate homeostasis and whose absence confers a cold-resistant phenotype. J. Biol. Chem. 273, 11852–11861 (1998).
Clarke, N.G. & Dawson, R.M. Alkaline O leads to N-transacylation. A new method for the quantitative deacylation of phospholipids. Biochem. J. 195, 301–306 (1981).
Šali, A. & Blundell, T.L. Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234, 779–815 (1993).
Sha, B., Phillips, S.E., Bankaitis, V.A. & Luo, M. Crystal structure of the Saccharomyces cerevisiae phosphatidylinositol- transfer protein. Nature 391, 506–510 (1998).
Jones, G., Willett, P., Glen, R.C., Leach, A.R. & Taylor, R. Development and validation of a genetic algorithm for flexible docking. J. Mol. Biol. 267, 727–748 (1997).
Friesner, R.A. et al. Extra precision glide: docking and scoring incorporating a model of hydrophobic enclosure for protein-ligand complexes. J. Med. Chem. 49, 6177–6196 (2006).
Cho, A.E., Guallar, V., Berne, B.J. & Friesner, R. Importance of accurate charges in molecular docking: quantum mechanical/molecular mechanical (QM/MM) approach. J. Comput. Chem. 26, 915–931 (2005).
Meng, E.C., Kuntz, I., Abraham, D. & Kellogg, G. Evaluating docked complexes with the HINT exponential function and empirical atomic hydrophobicities. J. Comput. Aided Mol. Des. 8, 299–306 (1994).
Abraham, D.J., Kellogg, G.E., Holt, J.M. & Ackers, G.K. Hydropathic analysis of the non-covalent interactions between molecular subunits of structurally characterized hemoglobins. J. Mol. Biol. 272, 613–632 (1997).
Adamo, J.E. et al. Yeast Cdc42 functions at a late step in exocytosis, specifically during polarized growth of the emerging bud. J. Cell Biol. 155, 581–592 (2001).
Goldstein, A. & Lampen, J.O. β-D-Fructofuranoside fructohydrolase from yeast. Methods Enzymol. 42, 504–511 (1975).
This work was supported by the Robert A. Welch Foundation (V.A.B.) and grant GM44530 (V.A.B.) from the US National Institutes of Health (NIH). R.W.D. was supported by the NIH (HG003317). G. Giaever and C. Nislow were supported by the National Human Genome Research Institute (5RO1-003317-08) and the Canadian Cancer Society (020380). The Texas A&M Laboratory for Molecular Simulation provided software, support and computer time. G.E. Kellogg and eduSoft LC donated HINT software, U. Schlecht (Stanford) assisted with growth analyses, A. Holzenburg and R. Littleton (Texas A&M) assisted with electron microscopy. We are grateful to D. Lew (Duke University) for donating mss4ts alleles.
The authors declare no competing financial interests.
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Nile, A., Tripathi, A., Yuan, P. et al. PITPs as targets for selectively interfering with phosphoinositide signaling in cells. Nat Chem Biol 10, 76–84 (2014). https://doi.org/10.1038/nchembio.1389
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