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Functional dissection of the retrograde Shiga toxin trafficking inhibitor Retro-2

Nature Chemical Biology volume 16, pages 327–336 (2020)Cite this article

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Abstract

The retrograde transport inhibitor Retro-2 has a protective effect on cells and in mice against Shiga-like toxins and ricin. Retro-2 causes toxin accumulation in early endosomes and relocalization of the Golgi SNARE protein syntaxin-5 to the endoplasmic reticulum. The molecular mechanisms by which this is achieved remain unknown. Here, we show that Retro-2 targets the endoplasmic reticulum exit site component Sec16A, affecting anterograde transport of syntaxin-5 from the endoplasmic reticulum to the Golgi. The formation of canonical SNARE complexes involving syntaxin-5 is not affected in Retro-2-treated cells. By contrast, the interaction of syntaxin-5 with a newly discovered binding partner, the retrograde trafficking chaperone GPP130, is abolished, and we show that GPP130 must indeed bind to syntaxin-5 to drive Shiga toxin transport from the endosomes to the Golgi. We therefore identify Sec16A as a druggable target and provide evidence for a non-SNARE function for syntaxin-5 in interaction with GPP130.

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Fig. 1: Retro-2 binds directly to Sec16A.
Fig. 2: Depletion of Sec16A phenocopies Retro-2 effects.
Fig. 3: Retro-2 treatment slows the anterograde transport of Syn5.
Fig. 4: Syn5 SNARE complex formation is not affected by Retro-2.
Fig. 5: Syn5 interacts with GPP130.
Fig. 6: Syn5-GPP130 interaction is required for STxB retrograde trafficking.

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Data and code availability

The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier project accession no. PXD015642. Imaris and Matlab scripts for EEA1 quantification are available on request. All other data supporting the findings of this study are available within the paper and its supplementary information files.

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Acknowledgements

We thank R. Rodriguez (cell imaging of Retro-2.1 and target identification), G. Boncompain (RUSH), C. Viaris De Lesegno (PLA), R. Onclercq-Delic and S. Bombard (intoxication assays), C. Brewee and B. Sancerne (recombinant protein production and cytotoxicity assays), D. Buisson (purification of 15) and E. Chirkin (synthesis of new batches of chemicals) for help with the indicated experiments. We thank V. Sabatet from the Laboratoire de Spectrométrie de Masse Protéomique for myProMS assistance. We acknowledge support from grants from the Agence Nationale pour la Recherche (ANR-11-BSV2-0018 and ANR-14-CE16-0004-03 to L.J., J.B., J.-C.C. and D.G. and ANR-19-CE13-0001-01 to L.J.), the Human Frontier Science Program (RGP0029-2014 to L.J.), the European Research Council (advanced grant no. 340485 to L.J.), the Swedish Research Council (K2015-99X-22877-01-6 to L.J., J.-C.C. and D.G.), the Joint Ministerial Program of R&D against CBRNE Risks (D.G., J.B., J.-C.C. and L.J.), the CEA (D.G., J.B. and J.-C.C.), the Île de France Region DIM Malinf initiative (grant no. 140101 to D.G., J.B. and L.J.), the Région Île-de-France (D.L.) and the Fondation pour la Recherche Médicale (D.L.). The Gillet and Cintrat teams are members of LabEx LERMIT (ANR-10-LABX-33) and the Johannes team is a member of Labex CelTisPhyBio (11-LBX-0038) and Idex Paris Sciences et Lettres (ANR-10-IDEX-0001–02 PSL). We also acknowledge the Cell and Tissue Imaging (PICT-IBiSA) and Nikon Imaging Centre, Institut Curie, a member of the French National Research Infrastructure France-BioImaging (ANR10-INBS-04).

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  1. These authors contributed equally: Alison Forrester, Stefan J. Rathjen, Maria Daniela Garcia-Castillo.

Authors and Affiliations

  1. Institut Curie, PSL Research University, Cellular and Chemical Biology unit, U1143 INSERM, UMR3666 CNRS, Endocytic Trafficking and Intracellular Delivery team, Paris, France

    Alison Forrester, Stefan J. Rathjen, Maria Daniela Garcia-Castillo, Henri-François Renard, César Augusto Valades-Cruz & Ludger Johannes

  2. Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, USA

    Collin Bachert & Adam D. Linstedt

  3. CEA, INRAE, Medicaments et Technologies pour la Sante (MTS), SCBM, Université Paris-Saclay, Gif-sur-Yvette, France

    Audrey Couhert, Mathilde Munier & Jean-Christophe Cintrat

  4. CEA, INRAE, Medicaments et Technologies pour la Sante (MTS), SIMoS, Université Paris-Saclay, Gif-sur-Yvette, France

    Livia Tepshi, Sylvain Pichard, Jennifer Martinez, Raphael Sierocki, Daniel Gillet & Julien Barbier

  5. Institut Curie, Centre de Recherche, PSL Research University, Laboratoire de Spectrométrie de Masse Protéomique, Paris, France

    Florent Dingli & Damarys Loew

  6. Institut Curie, PSL Research University, Cellular and Chemical Biology Unit, U1143 INSERM, UMR3666 CNRS, Membrane Dynamics and Mechanics of Intracellular Signaling team, Paris, France

    Christophe Lamaze

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  1. Alison Forrester
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Contributions

L.J. and D.G. conceived and designed the study. S.J.R., H.-F.R. and M.D.G.-C. performed the click chemistry immunofluorescence experiments and M.D.G.-C. and S.J.R. performed the click chemistry pulldown experiments. S.J.R. performed the in vitro Retro-2 pulldown, the SNARE PLA, the SNARE relocalization, the Syn5-RUSH assay and the GPP130 rescue analysis. S.J.R., M.D.G.-C., J.B. and L.T. performed the intoxication assays. The BLI assay was performed by J.B. and R.S. Purification of the Syn5 and GPP130 variants, the monensin study and the in vitro Syn5 pulldown of the GPP130 variants were performed by C.B. and A.D.L. S.J.R. and M.D.G.-C. performed the Sec16A and syntaxin-5 proteomics analysis and immunofluorescence. A.F. performed the Sec23 kinetic studies, the GFP-Sec16A pulldown and the STxB, GPP130 and Syn5 immunofluorescence with quantification. C.A.V.-C. wrote the scripts and automated the EEA1 colocalization methods. A.C., M.M. and J.-C.C. designed and performed the chemical synthesis of the azide-functionalized Retro-2 derivatives, and J.B. and L.T. characterized their anti-Shiga toxin activity. J.M., S.P. and L.T. prepared and characterized the recombinant Sec16A1266–1678/Sec13 protein complex. F.D. carried out the MS work, and D.L. supervised the MS and proteomic data analysis. S.J.R. and L.J. wrote the paper. A.F., J.-C.C., J.B., D.G., A.D.L. and C.L. critically revised the manuscript and aided in the design and analysis of experiments.

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Correspondence to Daniel Gillet or Ludger Johannes.

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Supplementary information

Supplementary Information

Supplementary Tables 1–2, Figs. 1–7 and Supplementary Note.

Reporting Summary

Supplementary Dataset 1

Proteomics quantification results GFP-Sec16A vehicle versus EGFP.

Supplementary Dataset 2

Proteomics quantification results GFP-Sec16A Retro-2 versus GFP-Sec16A vehicle.

Supplementary Dataset 3

Proteomics quantification results GFP-STX5 versus EGFP.

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Forrester, A., Rathjen, S.J., Daniela Garcia-Castillo, M. et al. Functional dissection of the retrograde Shiga toxin trafficking inhibitor Retro-2. Nat Chem Biol 16, 327–336 (2020). https://doi.org/10.1038/s41589-020-0474-4

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