Article | Published:

Covalent targeting of the vacuolar H+-ATPase activates autophagy via mTORC1 inhibition

Abstract

Autophagy is a lysosomal degradation pathway that eliminates aggregated proteins and damaged organelles to maintain cellular homeostasis. A major route for activating autophagy involves inhibition of the mTORC1 kinase, but current mTORC1-targeting compounds do not allow complete and selective mTORC1 blockade. Here, we have coupled screening of a covalent ligand library with activity-based protein profiling to discover EN6, a small-molecule in vivo activator of autophagy that covalently targets cysteine 277 in the ATP6V1A subunit of the lysosomal v-ATPase, which activates mTORC1 via the Rag guanosine triphosphatases. EN6-mediated ATP6V1A modification decouples the v-ATPase from the Rags, leading to inhibition of mTORC1 signaling, increased lysosomal acidification and activation of autophagy. Consistently, EN6 clears TDP-43 aggregates, a causative agent in frontotemporal dementia, in a lysosome-dependent manner. Our results provide insight into how the v-ATPase regulates mTORC1, and reveal a unique approach for enhancing cellular clearance based on covalent inhibition of lysosomal mTORC1 signaling.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Data availability

The data sets generated during and/or analyzed during the current study are available from the corresponding authors on reasonable request.

Ethics declarations

Competing interests

This study was funded by the Novartis Institutes for BioMedical Research and the Novartis-Berkeley Center for Proteomics and Chemistry Technologies. D.K.N. is the director of the Novartis-Berkeley Center for Proteomics and Chemistry Technologies. D.K.N. is a co-founder, share-holder and adviser for Artris Therapeutics and Frontier Medicines. R.Z. is a co-founder, share-holder and adviser for Frontier Medicines.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  1. 1.

    Khaminets, A., Behl, C. & Dikic, I. Ubiquitin-dependent and independent signals in selective autophagy. Trends Cell Biol. 26, 6–16 (2016).

  2. 2.

    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).

  3. 3.

    Galluzzi, L., Bravo-San Pedro, J. M., Levine, B., Green, D. R. & Kroemer, G. Pharmacological modulation of autophagy: therapeutic potential and persisting obstacles. Nat. Rev. Drug Discov. 16, 487–511 (2017).

  4. 4.

    Dikic, I. & Elazar, Z. Mechanism and medical implications of mammalian autophagy. Nat. Rev. Mol. Cell Biol. 19, 349–364 (2018).

  5. 5.

    Perera, R. M. & Zoncu, R. The lysosome as a regulatory hub. Annu. Rev. Cell Dev. Biol. 32, 223–253 (2016).

  6. 6.

    Saxton, R. A. & Sabatini, D. M. mTOR signaling in growth, metabolism, and disease. Cell 169, 361–371 (2017).

  7. 7.

    Settembre, C. et al. A lysosome-to-nucleus signalling mechanism senses and regulates the lysosome via mTOR and TFEB. EMBO J. 31, 1095–1108 (2012).

  8. 8.

    Menzies, F. M., Fleming, A. & Rubinsztein, D. C. Compromised autophagy and neurodegenerative diseases. Nat. Rev. Neurosci. 16, 345–357 (2015).

  9. 9.

    Barmada, S. J. et al. Autophagy induction enhances TDP43 turnover and survival in neuronal ALS models. Nat. Chem. Biol. 10, 677–685 (2014).

  10. 10.

    Tsvetkov, A. S. et al. A small-molecule scaffold induces autophagy in primary neurons and protects against toxicity in a Huntington disease model. Proc. Natl Acad. Sci. USA 107, 16982–16987 (2010).

  11. 11.

    Fu, Y. et al. A toxic mutant huntingtin species is resistant to selective autophagy. Nat. Chem. Biol. 13, 1152–1154 (2017).

  12. 12.

    Fox, J. H. et al. The mTOR kinase inhibitor Everolimus decreases S6 kinase phosphorylation but fails to reduce mutant huntingtin levels in brain and is not neuroprotective in the R6/2 mouse model of Huntington’s disease. Mol. Neurodegener. 5, 26 (2010).

  13. 13.

    Duarte-Silva, S. et al. Combined therapy with m-TOR-dependent and -independent autophagy inducers causes neurotoxicity in a mouse model of Machado–Joseph disease. Neuroscience 313, 162–173 (2016).

  14. 14.

    Ghosh, A. & Greenberg, M. E. Distinct roles for bFGF and NT-3 in the regulation of cortical neurogenesis. Neuron 15, 89–103 (1995).

  15. 15.

    Kuruvilla, R., Ye, H. & Ginty, D. D. Spatially and functionally distinct roles of the PI3-K effector pathway during NGF signaling in sympathetic neurons. Neuron 27, 499–512 (2000).

  16. 16.

    Brunet, A. et al. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 96, 857–868 (1999).

  17. 17.

    Sancak, Y. et al. Ragulator-Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids. Cell 141, 290–303 (2010).

  18. 18.

    Zoncu, R. et al. mTORC1 senses lysosomal amino acids through an inside-out mechanism that requires the vacuolar H+-ATPase. Science 334, 678–683 (2011).

  19. 19.

    Castellano, B. M. et al. Lysosomal cholesterol activates mTORC1 via an SLC38A9-Niemann-Pick C1 signaling complex. Science 355, 1306–1311 (2017).

  20. 20.

    Sancak, Y. et al. The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1. Science 320, 1496–1501 (2008).

  21. 21.

    Kim, E., Goraksha-Hicks, P., Li, L., Neufeld, T. P. & Guan, K.-L. Regulation of TORC1 by Rag GTPases in nutrient response. Nat. Cell Biol. 10, 935–945 (2008).

  22. 22.

    Forgac, M. Vacuolar ATPases: rotary proton pumps in physiology and pathophysiology. Nat. Rev. Mol. Cell Biol. 8, 917–929 (2007).

  23. 23.

    Zhao, J., Benlekbir, S. & Rubinstein, J. L. Electron cryomicroscopy observation of rotational states in a eukaryotic V-ATPase. Nature 521, 241–245 (2015).

  24. 24.

    Jewell, J. L. et al. Metabolism. Differential regulation of mTORC1 by leucine and glutamine. Science 347, 194–198 (2015).

  25. 25.

    Dechant, R., Saad, S., Ibáñez, A. J. & Peter, M. Cytosolic pH regulates cell growth through distinct GTPases, Arf1 and Gtr1, to promote Ras/PKA and TORC1 activity. Mol. Cell 55, 409–421 (2014).

  26. 26.

    Bar-Peled, L., Schweitzer, L. D., Zoncu, R. & Sabatini, D. M. Ragulator is a GEF for the rag GTPases that signal amino acid levels to mTORC1. Cell 150, 1196–1208 (2012).

  27. 27.

    Kaizuka, T. et al. An autophagic flux probe that releases an internal control. Mol. Cell 64, 835–849 (2016).

  28. 28.

    Spradlin, J. N. et al. Harnessing the anti-cancer natural product nimbolide for targeted protein degradation. bioRxiv Preprint at https://doi.org/10.1101/436998 (2018).

  29. 29.

    Anderson, K. E., To, M., Olzmann, J. A. & Nomura, D. K. Chemoproteomics-enabled covalent ligand screening reveals a thioredoxin-caspase 3 interaction disruptor that impairs breast cancer pathogenicity. ACS Chem. Biol. 12, 2522–2528 (2017).

  30. 30.

    Grossman, E. A. et al. Covalent ligand discovery against druggable hotspots targeted by anti-cancer natural products. Cell Chem. Biol. 24, 1368–1376.e4 (2017).

  31. 31.

    Bateman, L. A. et al. Chemoproteomics-enabled covalent ligand screen reveals a cysteine hotspot in reticulon 4 that impairs ER morphology and cancer pathogenicity. Chem. Commun. 53, 7234–7237 (2017).

  32. 32.

    Weerapana, E. et al. Quantitative reactivity profiling predicts functional cysteines in proteomes. Nature 468, 790–795 (2010).

  33. 33.

    Backus, K. M. et al. Proteome-wide covalent ligand discovery in native biological systems. Nature 534, 570–574 (2016).

  34. 34.

    Chen, Y.-C. et al. Covalent modulators of the vacuolar ATPase. J. Am. Chem. Soc. 139, 639–642 (2017).

  35. 35.

    Steinberg, B. E. et al. A cation counterflux supports lysosomal acidification. J. Cell Biol. 189, 1171–1186 (2010).

  36. 36.

    Bar-Peled, L. et al. A tumor suppressor complex with GAP activity for the Rag GTPases that signal amino acid sufficiency to mTORC1. Science 340, 1100–1106 (2013).

  37. 37.

    Roczniak-Ferguson, A. et al. The transcription factor TFEB links mTORC1 signaling to transcriptional control of lysosome homeostasis. Sci. Signal. 5, ra42 (2012).

  38. 38.

    Martina, J. A., Chen, Y., Gucek, M. & Puertollano, R. MTORC1 functions as a transcriptional regulator of autophagy by preventing nuclear transport of TFEB. Autophagy 8, 903–914 (2012).

  39. 39.

    Sreedharan, J. et al. TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis. Science 319, 1668–1672 (2008).

  40. 40.

    Joseph, G.A. et al. Inhibition of mTORC1 signaling in aged rats counteracts the decline in muscle mass and reverses multiple parameters of muscle signaling associated with sarcopenia. bioRxiv Preprint at https://www.biorxiv.org/content/10.1101/591891v1 (2019)

  41. 41.

    Ramos, F. J. et al. Rapamycin reverses elevated mtorc1 signaling in lamin A/C-deficient mice, rescues cardiac and skeletal muscle function, and extends survival. Sci. Transl. Med. 4, 144ra103 (2012).

  42. 42.

    Masiero, E. et al. Autophagy is required to maintain muscle mass. Cell Metab. 10, 507–515 (2009).

  43. 43.

    Yano, T. et al. Clinical impact of myocardial mTORC1 activation in nonischemic dilated cardiomyopathy. J. Mol. Cell. Cardiol. 91, 6–9 (2016).

  44. 44.

    Thoreen, C. C. et al. An ATP-competitive mammalian target of rapamycin inhibitor reveals rapamycin-resistant functions of mTORC1. J. Biol. Chem. 284, 8023–8032 (2009).

  45. 45.

    Rodrik-Outmezguine, V. S. et al. Overcoming mTOR resistance mutations with a new-generation mTOR inhibitor. Nature 534, 272–276 (2016).

  46. 46.

    García-Martínez, J. M. et al. Ku-0063794 is a specific inhibitor of the mammalian target of rapamycin (mTOR). Biochem. J. 421, 29–42 (2009).

  47. 47.

    Sarbassov, D. D. et al. Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB. Mol. Cell 22, 159–168 (2006).

  48. 48.

    Lamming, D. W. et al. Rapamycin-induced insulin resistance is mediated by mTORC2 loss and uncoupled from longevity. Science 335, 1638–1643 (2012).

  49. 49.

    Arai, S. et al. Rotation mechanism of Enterococcus hirae V1-ATPase based on asymmetric crystal structures. Nature 493, 703–707 (2013).

  50. 50.

    Mazhab-Jafari, M. T. et al. Atomic model for the membrane-embedded VO motor of a eukaryotic V-ATPase. Nature 539, 118–122 (2016).

  51. 51.

    Smith, P. K. et al. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76–85 (1985).

  52. 52.

    Xu, T. et al. ProLuCID: an improved SEQUEST-like algorithm with enhanced sensitivity and specificity. J. Proteomics 129, 16–24 (2015).

  53. 53.

    Benjamin, D. I. et al. Ether lipid generating enzyme AGPS alters the balance of structural and signaling lipids to fuel cancer pathogenicity. Proc. Natl Acad. Sci. USA 110, 14912–14917 (2013).

  54. 54.

    Counihan, J. L., Wiggenhorn, A. L., Anderson, K. E. & Nomura, D. K. Chemoproteomics-enabled covalent ligand screening reveals aldh3a1 as a lung cancer therapy target. ACS Chem. Biol. 13, 1970–1977 (2018).

  55. 55.

    Louie, S. M. et al. GSTP1 is a driver of triple-negative breast cancer cell metabolism and pathogenicity. Cell Chem. Biol. 23, 567–578 (2016).

  56. 56.

    Medina-Cleghorn, D. et al. Mapping proteome-wide targets of environmental chemicals using reactivity-based chemoproteomic platforms. Chem. Biol. 22, 1394–1405 (2015).

  57. 57.

    Roberts, A. M. et al. Chemoproteomic screening of covalent ligands reveals UBA5 as a novel pancreatic cancer target. ACS Chem. Biol. 12, 899–904 (2017).

  58. 58.

    Ward, C. C. et al. Covalent ligand screening uncovers a RNF4 E3 ligase recruiter for targeted protein degradation applications. ACS Chem. Biol. https://doi.org/10.1021/acschembio.8b01083 (2018).

Download references

Acknowledgements

We thank the members of the Nomura Research Group, the Zoncu laboratory and Novartis Institutes for BioMedical Research for critical reading of the manuscript. This work was supported by Novartis Institutes for BioMedical Research and the Novartis-Berkeley Center for Proteomics and Chemistry Technologies (NB-CPACT) for C.Y.S.C., C.A.B., B.F., C.C.W. and D.K.N., National Institutes of Health (grant no. NIEHS R01ES028096 for D.K.N. and C.Y.S.C., no. NCI F31CA225173 for C.C.W., no. NCI DP2CA195761 for R.Z. and no. NIGMS R01GM112948 for J.A.O.), the Shurl & Kay Curci Foundation Faculty Scholars grant (R.Z.) and the National Research Foundation funded by the South Korean government for H.R.S. (grant no. 2017R1C1B2007409). This study was also supported by the Mark Foundation for Cancer Research ASPIRE award (D.K.N.). Confocal imaging experiments were conducted at the CRL Molecular Imaging Center, supported by the Helen Wills Neuroscience Institute and Gordon and Betty Moore Foundation (UC Berkeley). We would like to thank H. Aaron and F. Ives for their microscopy training and assistance. We also thank R. Zalpuri at the University of California Berkeley Electron Microscope Laboratory for advice and assistance in electron microscopy sample preparation and data collection.

Author information

C.Y.S.C., H.R.S., R.Z. and D.K.N. conceived the project and wrote the paper. C.Y.S.C., H.R.S., C.A.B., B.F., R.Z. and D.K.N. designed and performed the experiments. C.Y.S.C., H.R.S., C.A.B., C.C.W., R.Z. and D.K.N. analyzed the data. J.A.O. provided reagents.

Competing interests

This study was funded by the Novartis Institutes for BioMedical Research and the Novartis-Berkeley Center for Proteomics and Chemistry Technologies. D.K.N. is the director of the Novartis-Berkeley Center for Proteomics and Chemistry Technologies. D.K.N. is a co-founder, share-holder and adviser for Artris Therapeutics and Frontier Medicines. R.Z. is a co-founder, share-holder and adviser for Frontier Medicines.

Correspondence to Roberto Zoncu or Daniel K. Nomura.

Supplementary information

Supplementary Information

Supplementary Tables 1–2, Supplementary Figs. 1–16

Reporting Summary

Synthetic Procedures

Synthetic Procedures

Supplementary Dataset 1

Autophagy activation screening data in MEF and HEK293A cells.

Supplementary Dataset 2

isoTOP-ABPP analysis of EN6 in situ treatment in MEF cells.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark
Fig. 1: Covalent ligand screen for autophagy activators.
Fig. 2: Target identification and validation of EN6.
Fig. 3: EN6 inhibits mTORC1 recruitment to the lysosome.
Fig. 4: EN6 effects on TFEB, lysosomal acidification and clearance of TDP-43 aggregates.
Fig. 5: EN6 inhibits mTORC1 signaling in mice.
Fig. 6: Scheme of v-ATPase-mTORC1 regulation of autophagy and mechanism of EN6 action.