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The cholesterol transfer protein GRAMD1A regulates autophagosome biogenesis

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.

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.

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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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Authors

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.

Corresponding authors

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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