Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

PITPs as targets for selectively interfering with phosphoinositide signaling in cells

Nature Chemical Biology volume 10, pages 76–84 (2014)Cite this article

Subjects

Abstract

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: NPPMs specifically inactivate Sec14.
Figure 2: NPPM SARs.
Figure 3: Sec14 is the essential cellular target of bioactive NPPMs.
Figure 4: Sec14-active NPPMs exhibit compartment-specific inhibition of PtdIns-(4)-phosphate signaling.
Figure 5: NPPMs discriminate between Sec14- and Sfh4-mediated PtdIns-4-P signaling.
Figure 6: Model for NPPM-mediated inhibition of Sec14.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

  1. Wymann, M.P. & Schneiter, R. Lipid signalling in disease. Nat. Rev. Mol. Cell Biol. 9, 162–176 (2008).

    CAS  PubMed  Google Scholar 

  2. Janmey, P.A. & Lindberg, U. Cytoskeletal regulation: rich in lipids. Nat. Rev. Mol. Cell Biol. 5, 658–666 (2004).

    CAS  PubMed  Google Scholar 

  3. Henry, S.A., Kohlwein, S.D. & Carman, G.M. Metabolism and regulation of glycerolipids in the yeast Saccharomyces cerevisiae. Genetics 190, 317–349 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Irvine, R.F. Nuclear lipid signalling. Nat. Rev. Mol. Cell Biol. 4, 349–360 (2003).

    CAS  PubMed  Google Scholar 

  5. Di Paolo, G. & De Camilli, P. Phosphoinositides in cell regulation and membrane dynamics. Nature 443, 651–657 (2006).

    CAS  PubMed  Google Scholar 

  6. Michell, R.H. Inositol derivatives: evolution and functions. Nat. Rev. Mol. Cell Biol. 9, 151–161 (2008).

    CAS  PubMed  Google Scholar 

  7. Strahl, T. & Thorner, J. Synthesis and function of membrane phosphoinositides in budding yeast, Saccharomyces cerevisiae. Biochim. Biophys. Acta 1771, 353–404 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 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).

    CAS  PubMed  Google Scholar 

  12. 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).

    CAS  PubMed  Google Scholar 

  13. 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).

    CAS  PubMed  Google Scholar 

  14. 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).

    PubMed  Google Scholar 

  15. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 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).

    CAS  PubMed  Google Scholar 

  17. 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).

    CAS  PubMed  Google Scholar 

  18. Nile, A.H., Bankaitis, V.A. & Grabon, A. Mammalian diseases of phosphatidylinositol transfer proteins and their homologs. Clin. Lipidol. 5, 867–897 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 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).

    CAS  PubMed  Google Scholar 

  20. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Cleves, A., McGee, T. & Bankaitis, V. Phospholipid transfer proteins: a biological debut. Trends Cell Biol. 1, 30–34 (1991).

    CAS  PubMed  Google Scholar 

  28. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 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).

    CAS  PubMed  Google Scholar 

  32. Phillips, S.E. et al. Yeast Sec14p deficient in phosphatidylinositol transfer activity is functional in vivo. Mol. Cell 4, 187–197 (1999).

    CAS  PubMed  Google Scholar 

  33. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 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).

    CAS  PubMed  Google Scholar 

  35. 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).

    CAS  PubMed  Google Scholar 

  36. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 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).

    CAS  PubMed  Google Scholar 

  38. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Sykes, P. A Guidebook to Mechanism in Organic Chemistry 6th edn. (Pearson, 1986).

  40. 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).

    CAS  PubMed  Google Scholar 

  41. Auffinger, P., Hays, F.A., Westhof, E. & Ho, P.S. Halogen bonds in biological molecules. Proc. Natl. Acad. Sci. USA 101, 16789–16794 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Metrangolo, P. & Resnati, G. Halogen bonding: a paradigm in supramolecular chemistry. Chemistry 7, 2511–2519 (2001).

    CAS  PubMed  Google Scholar 

  43. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Phillips, S.E. et al. The diverse biological functions of phosphatidylinositol transfer proteins in eukaryotes. Crit. Rev. Biochem. Mol. Biol. 41, 21–49 (2006).

    CAS  PubMed  Google Scholar 

  45. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Sherman, F., Fink, G.R. & Hink, J.B. Methods in Yeast Genetics: A Laboratory Manual (Cold Spring Harbor, 1983).

  47. Ashburner, M. et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat. Genet. 25, 25–29 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. 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).

    CAS  PubMed  Google Scholar 

  50. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Šali, A. & Blundell, T.L. Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234, 779–815 (1993).

    PubMed  Google Scholar 

  52. Sha, B., Phillips, S.E., Bankaitis, V.A. & Luo, M. Crystal structure of the Saccharomyces cerevisiae phosphatidylinositol- transfer protein. Nature 391, 506–510 (1998).

    CAS  PubMed  Google Scholar 

  53. 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).

    CAS  PubMed  Google Scholar 

  54. 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).

    CAS  PubMed  Google Scholar 

  55. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. 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).

    CAS  PubMed  Google Scholar 

  57. 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).

    CAS  PubMed  Google Scholar 

  58. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. Goldstein, A. & Lampen, J.O. β-D-Fructofuranoside fructohydrolase from yeast. Methods Enzymol. 42, 504–511 (1975).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

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.

Author information

Authors and Affiliations

  1. Department of Molecular and Cellular Medicine, Texas A&M University, College Station, Texas, USA

    Aaron H Nile, Ashutosh Tripathi, Peihua Yuan, Carl J Mousley & Vytas A Bankaitis

  2. Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, USA

    Aaron H Nile, Ashutosh Tripathi, Peihua Yuan, Carl J Mousley & Vytas A Bankaitis

  3. Department of Chemistry, Texas A&M University, College Station, Texas, USA

    Aaron H Nile, Ashutosh Tripathi, Peihua Yuan, Carl J Mousley & Vytas A Bankaitis

  4. Department of Cell and Developmental Biology, Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA

    Aaron H Nile, Sweety D Shah & Vytas A Bankaitis

  5. Laboratory for Molecular Modeling, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

    Ashutosh Tripathi, Denise Teotico Pohlhaus & Alexander Tropsha

  6. Department of Biochemistry, Stanford Genome Technology Center, Stanford University, Palo Alto, California, USA

    Sundari Suresh, Ronald W Davis & Robert P St. Onge

  7. Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada

    Iain M Wallace

  8. R.L. Juliano Structural Bioinformatics Core University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

    Brenda Temple

  9. Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, British Columbia, Canada

    Corey Nislow & Guri Giaever

Authors
  1. Aaron H Nile
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ashutosh Tripathi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Peihua Yuan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Carl J Mousley
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sundari Suresh
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Iain M Wallace
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Sweety D Shah
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Denise Teotico Pohlhaus
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Brenda Temple
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Corey Nislow
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Guri Giaever
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Alexander Tropsha
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Ronald W Davis
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Robert P St. Onge
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Vytas A Bankaitis
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.H.N. was involved in all experimentation, design, data analysis, manuscript preparation and figure production. A. Tripathi, with assistance from B.T., D.T.P. and A. Tropsha, was involved in designing computational experiments, data analysis, manuscript preparation and figure production. P.Y. and C.J.M. performed electron microscopy and invertase and CPY experiments, and S.D.S. generated site-directed mutants and purified proteins. I.M.W., C.N., S.S. and R.P.S. selected initial SAR compounds, performed and designed chemogenomic profiling. R.W.D. and G.G. advised chemogenomic studies. V.A.B. was involved in all aspects of experimental design, data analysis and manuscript preparation.

Corresponding authors

Correspondence to Ashutosh Tripathi or Vytas A Bankaitis.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1–3 and Supplementary Figures 1–25. (PDF 20340 kb)

Supplementary Note 1

Significant GIs observed (−2 ≤ S ≥ 2). (PDF 5387 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.1389

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing