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The cholesterol transfer protein GRAMD1A regulates autophagosome biogenesis

Nature Chemical Biology volume 15, pages 710–720 (2019)Cite this article

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Abstract

Autophagy mediates the degradation of damaged proteins, organelles and pathogens, and plays a key role in health and disease. Thus, the identification of new mechanisms involved in the regulation of autophagy is of major interest. In particular, little is known about the role of lipids and lipid-binding proteins in the early steps of autophagosome biogenesis. Using target-agnostic, high-content, image-based identification of indicative phenotypic changes induced by small molecules, we have identified autogramins as a new class of autophagy inhibitor. Autogramins selectively target the recently discovered cholesterol transfer protein GRAM domain-containing protein 1A (GRAMD1A, which had not previously been implicated in autophagy), and directly compete with cholesterol binding to the GRAMD1A StART domain. GRAMD1A accumulates at sites of autophagosome initiation, affects cholesterol distribution in response to starvation and is required for autophagosome biogenesis. These findings identify a new biological function of GRAMD1A and a new role for cholesterol in autophagy.

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Fig. 1: Autogramins inhibit autophagy.
Fig. 2: Autogramins target GRAMD1A.
Fig. 3: The cholesterol binding and transport activity of the GRAMD1A StART domain is inhibited by autogramins.
Fig. 4: Cholesterol and autogramins target the same binding site within the GRAMD1A StART domain.
Fig. 5: GRAMD1A is required for autophagy initiation.
Fig. 6: GRAMD1A is localized to sites of autophagosome initiation.

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Data availability

The authors declare that the data supporting the findings of this study are available within the paper and its supplementary information files. Additional raw data associated with all figures are available from the corresponding authors upon reasonable request. The atomic structure of the StART domain of GRAMD1C was deposited in the Protein Data Bank under the accession number 6GN5.

References

  1. Mizushima, N., Levine, B., Cuervo, A. M. & Klionsky, D. J. Autophagy fights disease through cellular self-digestion. Nature 451, 1069–1075 (2008).

    Article  CAS  Google Scholar 

  2. Mizushima, N., Yoshimori, T. & Ohsumi, Y. The role of Atg proteins in autophagosome formation. Ann. Rev. Cell. Dev. Biol. 27, 107–132 (2011).

    Article  CAS  Google Scholar 

  3. Rubinsztein, D. C., Codogno, P. & Levine, B. Autophagy modulation as a potential therapeutic target for diverse diseases. Nat. Rev. Drug Discov. 11, 709–730 (2012).

    Article  CAS  Google Scholar 

  4. White, E. Deconvoluting the context-dependent role for autophagy in cancer. Nat. Rev. Cancer 12, 401–410 (2012).

    Article  CAS  Google Scholar 

  5. Laraia, L. et al. Discovery of novel cinchona-alkaloid-inspired oxazatwistane autophagy inhibitors. Angew. Chem. Int. Ed. 56, 2145–2150 (2017).

    Article  CAS  Google Scholar 

  6. Robke, L. et al. Phenotypic identification of a novel autophagy inhibitor chemotype targeting lipid kinase VPS34. Angew. Chem. Int. Ed. 56, 8153–8157 (2017).

    Article  CAS  Google Scholar 

  7. Robke, L. et al. Discovery of the novel autophagy inhibitor aumitin that targets mitochondrial complex I. Chem. Sci. 9, 3014–3022 (2018).

    Article  CAS  Google Scholar 

  8. Dall’Armi, C., Devereaux, K. A. & Di Paolo, G. The role of lipids in the control of autophagy. Curr. Biol. 23, R33–R45 (2013).

    Article  Google Scholar 

  9. Nascimbeni, A. C., Codogno, P. & Morel, E. Phosphatidylinositol‐3‐phosphate in the regulation of autophagy membrane dynamics. FEBS J. 284, 1267–1278 (2017).

    Article  CAS  Google Scholar 

  10. Petiot, A., Ogier-Denis, E., Blommaart, E. F. C., Meijer, A. J. & Codogno, P. Distinct classes of phosphatidylinositol 3′-kinases are involved in signaling pathways that control macroautophagy in HT-29 cells. J. Biol. Chem. 275, 992–998 (2000).

    Article  CAS  Google Scholar 

  11. Kihara, A., Noda, T., Ishihara, N. & Ohsumi, Y. Two distinct Vps34 phosphatidylinositol 3-kinase complexes function in autophagy and carboxypeptidase Y sorting in Saccharomyces cerevisiae. J. Cell Biol. 152, 519–530 (2001).

    Article  CAS  Google Scholar 

  12. Dooley, H. C. et al. WIPI2 links LC3 conjugation with PI3P, autophagosome formation, and pathogen clearance by recruiting Atg12–5-16L1. Mol. Cell 55, 238–252 (2014).

    Article  CAS  Google Scholar 

  13. Konstantinidis, G., Sievers, S. & Wu, Y.-W. in Autophagy in Differentiation and Tissue Maintenance. Methods in Molecular Biology Vol. 1854 (ed. Turksen, K.) 1–9 (Humana Press, 2018).

  14. Balgi, A. D. et al. Screen for chemical modulators of autophagy reveals novel therapeutic inhibitors of mTORC1 signaling. PLoS ONE 4, e7124 (2009).

    Article  Google Scholar 

  15. Peppard, J. V. et al. Identifying small molecules which inhibit autophagy: a phenotypic screen using image-based high-content cell analysis. Curr. Chem. Genom. Trans. Med. 8, 3–15 (2014).

    Article  CAS  Google Scholar 

  16. Liu, J. et al. Beclin1 controls the levels of p53 by regulating the deubiquitination activity of USP10 and USP13. Cell 147, 223–234 (2011).

    Article  CAS  Google Scholar 

  17. Wong, L. H. & Levine, T. P. Lipid transfer proteins do their thing anchored at membrane contact sites… but what is their thing? Biochem. Soc. Trans. 44, 517–527 (2016).

    Article  CAS  Google Scholar 

  18. Khafif, M., Cottret, L., Balagué, C. & Raffaele, S. Identification and phylogenetic analyses of VASt, an uncharacterized protein domain associated with lipid-binding domains in eukaryotes. BMC Bioinformatics 15, 222 (2014).

    Article  Google Scholar 

  19. Doerks, T., Strauss, M., Brendel, M. & Bork, P. GRAM, a novel domain in glucosyltransferases, myotubularins and other putative membrane-associated proteins. Trends Biochem. Sci. 25, 483–485 (2000).

    Article  CAS  Google Scholar 

  20. Begley, M. J. et al. Crystal structure of a phosphoinositide phosphatase, MTMR2: insights into myotubular myopathy and Charcot-Marie-Tooth syndrome. Mol. Cell 12, 1391–1402 (2003).

    Article  CAS  Google Scholar 

  21. Horenkamp, F. A., Valverde, D. P., Nunnari, J. & Reinisch, K. M. Molecular basis for sterol transport by StART‐like lipid transfer domains. EMBO J. 37, e98002 (2018).

    Article  Google Scholar 

  22. Tong, J., Manik, M. K. & Im, Y. J. Structural basis of sterol recognition and nonvesicular transport by lipid transfer proteins anchored at membrane contact sites. Proc. Natl Acad. Sci. USA 115, E856–E865 (2018).

    Article  CAS  Google Scholar 

  23. Sandhu, J. et al. Aster proteins facilitate nonvesicular plasma membrane to ER cholesterol transport in mammalian cells. Cell 175, 514–529 (2018).

    Article  CAS  Google Scholar 

  24. Robers, M. B. et al. Target engagement and drug residence time can be observed in living cells with BRET. Nat. Commun. 6, 10091 (2015).

    Article  CAS  Google Scholar 

  25. Jafari, R. et al. The cellular thermal shift assay for evaluating drug target interactions in cells. Nat. Protoc. 9, 2100–2122 (2014).

    Article  CAS  Google Scholar 

  26. Holm, L. & Rosenström, P. Dali server: conservation mapping in 3D. Nucleic Acids Res. 38, W545–W549 (2010).

    Article  CAS  Google Scholar 

  27. Gatta, A. T. et al. A new family of StART domain proteins at membrane contact sites has a role in ER–PM sterol transport. eLife 4, e07253 (2015).

    Article  Google Scholar 

  28. Kimura, S., Noda, T. & Yoshimori, T. Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3. Autophagy 3, 452–460 (2007).

    Article  CAS  Google Scholar 

  29. Blommaart, E. F. C., Krause, U., Schellens, J. P., Vreeling‐Sindelárová, H. & Meijer, A. J. The phosphatidylinositol 3‐kinase inhibitors wortmannin and LY294002 inhibit autophagy in isolated rat hepatocytes. Eur. J. Biochem. 243, 240–246 (2004).

    Article  Google Scholar 

  30. Ikonen, E. Cellular cholesterol trafficking and compartmentalization. Nat. Rev. Mol. Cell Biol. 9, 125–138 (2008).

    Article  CAS  Google Scholar 

  31. Wijdeven, R. H. et al. Cholesterol and ORP1L-mediated ER contact sites control autophagosome transport and fusion with the endocytic pathway. Nat. Commun. 7, 11808 (2016).

    Article  CAS  Google Scholar 

  32. Sarkar, S. et al. Impaired autophagy in the lipid-storage disorder Niemann-Pick type C1 disease. Cell Rep. 5, 1302–1315 (2013).

    Article  CAS  Google Scholar 

  33. Murley, A. et al. Ltc1 is an ER-localized sterol transporter and a component of ER–mitochondria and ER–vacuole contacts. J. Cell Biol. 209, 539–548 (2015).

    Article  CAS  Google Scholar 

  34. Elbaz-Alon, Y. et al. Lam6 regulates the extent of contacts between organelles. Cell Rep. 12, 7–14 (2015).

    Article  CAS  Google Scholar 

  35. Murley, A. et al. Sterol transporters at membrane contact sites regulate TORC1 and TORC2 signaling. J. Cell Biol. 216, 2679–2689 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Besprozvannaya, M. et al. GRAM domain proteins specialize functionally distinct ER–PM contact sites in human cells. eLife 7, e31019 (2018).

    Article  Google Scholar 

  37. Punnonen, E.-L., Pihakaski, K., Mattila, K., Lounatmaa, K. & Hirsimäki, P. Intramembrane particles and filipin labelling on the membranes of autophagic vacuoles and lysosomes in mouse liver. Cell. Tissue Res. 258, 269–276 (1989).

    Article  CAS  Google Scholar 

  38. Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008).

    Article  CAS  Google Scholar 

  39. Tyanova, S. & Cox, J. in Cancer Systems Biology: Methods and Protocols (ed. von Stechow, L.) 133–148 (Springer, 2018).

  40. Hashimoto, Y., Zhang, S. & Blissard, G. W. Ao38, a new cell line from eggs of the black witch moth, Ascalapha odorata (Lepidoptera: Noctuidae), is permissive for AcMNPV infection and produces high levels of recombinant proteins. BMC Biotechnol. 10, 50 (2010).

    Article  Google Scholar 

  41. Oishi, H., Takaoka, Y., Nishimaki-Mogami, T., Saito, H. & Ueda, M. A novel nuclear receptor ligand, digoxigenin, is a selective antagonist of liver-X-receptors. Chem. Lett. 46, 313–314 (2016).

    Article  Google Scholar 

  42. Kernstock, R. M. & Girotti, A. W. Lipid transfer protein binding of unmodified natural lipids as assessed by surface plasmon resonance methodology. Anal. Biochem. 365, 111–121 (2007).

    Article  CAS  Google Scholar 

  43. Fang, J., Rand, K. D., Beuning, P. J. & Engen, J. R. False EX1 signatures caused by sample carryover during HX MS analyses. Int. J. Mass Spec. 302, 19–25 (2011).

    Article  CAS  Google Scholar 

  44. Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

    Article  CAS  Google Scholar 

  45. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  Google Scholar 

  46. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  48. 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).

    Article  CAS  Google Scholar 

  49. Laskowski, R. A. & Swindells, M. B. LigPlot+: multiple ligand–protein interaction diagrams for drug discovery. J. Chem. Inf. Mod. 51, 2778–2786 (2011).

    Article  CAS  Google Scholar 

  50. Itakura, E. & Mizushima, N. Characterization of autophagosome formation site by a hierarchical analysis of mammalian Atg proteins. Autophagy 6, 764–776 (2010).

    Article  CAS  Google Scholar 

  51. de Chaumont, F. et al. Icy: an open bioimage informatics platform for extended reproducible research. Nat. Methods 9, 690–696 (2012).

    Article  Google Scholar 

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Acknowledgements

This work was supported by the Max Planck Society (H.W.), DFG grant SPP 1623, ERC (ChemBioAP), Vetenskapsrådet (2018-04585) and the Knut and Alice Wallenberg Foundation (Y.-W.W). L.Laraia was supported by a fellowship from the Alexander von Humboldt Stiftung. D.P.C. is supported by a fellowship from the Canadian Institute of Health Research (MFE-152550). We thank S. Sievers and the Compound Management and Screening Center (COMAS), Dortmund, Germany, for compound screening. We thank R. Gasper-Schönenbrücher, K. Estel and the beamline staff for help with data collection at the SLS, Villigen, Switzerland. We thank S. Tooze for the kind gift of EGFP–WIPI2b cells. We acknowledge the Biochemical Imaging Center (BICU) at Umeå University and the National Microscopy Infrastructure, NMI (VR-RFI 2016-00968) for providing assistance in microscopy.

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  1. These authors contributed equally: Luca Laraia, Alexandra Friese.

Authors and Affiliations

  1. Department of Chemical Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany

    Luca Laraia, Alexandra Friese, Walter Hofer, Hacer Karatas, Andreas Brockmeyer, Malte Metz, Beate Schölermann, Ingrid R. Vetter, Slava Ziegler, Petra Janning & Herbert Waldmann

  2. Department of Chemistry, Technical University of Denmark, Lyngby, Denmark

    Luca Laraia

  3. Chemical Genomics Centre of the Max Planck Society, Dortmund, Germany

    Dale P. Corkery, Georgios Konstantinidis, Laura Klewer & Yao-Wen Wu

  4. Department of Chemistry, Umeå University, Umeå, Sweden

    Dale P. Corkery, Georgios Konstantinidis & Yao-Wen Wu

  5. Faculty of Chemistry and Chemical Biology, Technical University Dortmund, Dortmund, Germany

    Nelli Erwin, Mridula Dwivedi, Lei Li, Roland Winter & Herbert Waldmann

  6. Centre for Biological Signalling Studies (BIOSS), University of Freiburg, Freiburg, Germany

    Pablo Rios-Munoz & Maja Köhn

  7. Faculty of Biology, University of Freiburg, Freiburg, Germany

    Pablo Rios-Munoz & Maja Köhn

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Contributions

L.Laraia carried out the analog and probe synthesis, the structure–activity relationship analysis, the initial biological compound validation and the proteomic target identification and initial target validation. A.F. carried out the cloning, expression and purification of all recombinant proteins as well as all fluorescence polarization, DSF, PIP-binding and crystallography experiments. A.F., A.B. and M.M. performed HDX-MS experiments. D.P.C. carried out cell biological characterization of GRAMD1A. G.K. and L.K. carried out autogramin validation experiments. W.H. and H.K. carried out the synthesis of the fluorescent probe. B.S. performed nanoBRET experiments. N.E. and L.Li carried out the cholesterol transfer experiments. M.D. performed AFM experiments. R.W. supervised cholesterol transfer and AFM experiments. P.J. carried out the MS proteomics analysis. I.R.V. analyzed crystallographic data and performed homology modeling and docking experiments. S.Z., P.R.-M. and M.K. provided reagents. All authors analyzed data. L.Laraia, A.F., D.P.C., G.K., Y.-W.W. and H.W. wrote the paper with comments from all other co-authors.

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Correspondence to Yao-Wen Wu or Herbert Waldmann.

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Supplementary information

Supplementary Information

Supplementary Tables 1–3 and Supplementary Figures 1–13

Reporting Summary

Supplementary Note

Supplementary Dataset 1

Mass spectrometry-based proteomics, raw data.

Supplementary Dataset 2

Kinase panel, complete data.

Supplementary Video 1

Live-cell imaging of HeLa cells simultaneously transfected with EGFP–WIPI1 and GRAMD1A–mCherry under starvation conditions.

Supplementary Video 2

Live-cell imaging of HeLa cells simultaneously transfected with EGFP–LC3 and GRAMD1A–mCherry under starvation conditions.

Supplementary Video 3

Additional live-cell imaging of HeLa cells simultaneously transfected with EGFP–LC3 and GRAMD1A–mCherry under starvation conditions.

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Laraia, L., Friese, A., Corkery, D.P. et al. The cholesterol transfer protein GRAMD1A regulates autophagosome biogenesis. Nat Chem Biol 15, 710–720 (2019). https://doi.org/10.1038/s41589-019-0307-5

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