Site-specific ubiquitylation and SUMOylation using genetic-code expansion and sortase

Abstract

Post-translational modification of proteins with ubiquitin and ubiquitin-like proteins (Ubls) is central to the regulation of eukaryotic cellular processes. Our ability to study the effects of ubiquitylation, however, is limited by the difficulty to prepare homogenously modified proteins in vitro and by the impossibility to selectively trigger specific ubiquitylation events in living cells. Here we combine genetic-code expansion, bioorthogonal Staudinger reduction and sortase-mediated transpeptidation to develop a general tool to ubiquitylate proteins in an inducible fashion. The generated ubiquitin conjugates display a native isopeptide bond and bear two point mutations in the ubiquitin C terminus that confer resistance toward deubiquitinases. Nevertheless, physiological integrity of sortase-generated diubiquitins in decoding cellular functions via recognition by ubiquitin-binding domains is retained. Our approach allows the site-specific attachment of Ubls to nonrefoldable, multidomain proteins and enables inducible and ubiquitin-ligase-independent ubiquitylation of proteins in mammalian cells, providing a powerful tool to dissect the biological functions of ubiquitylation with temporal control.

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Fig. 1: General scheme showing sortase-mediated ubiquitylation (sortylation).
Fig. 2: Site-specific incorporation of AzGGK into proteins in E. coli.
Fig. 3: Srt5M-mediated ubiquitylation of GGK-bearing proteins.
Fig. 4: Srt2A-mediated ubiquitylation of GGK-bearing proteins.
Fig. 5: Site-specific SUMOylation of GGK-bearing proteins.
Fig. 6: Incorporation of AzGGK into proteins in mammalian cells and sortase-mediated ubiquitylation and SUMOylation of proteins in living HEK293T cells.

Data availability

The authors declare that the data supporting the findings of this study are available in the paper and its supplementary information files. Raw data and other materials are available upon reasonable request.

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Acknowledgements

This work was supported by the Excellence Initiative CIPSM and the DFG through the following programmes: GRK1721, SFB1309 and SPP1623 (to K.L.) as well as SFB1035 project B12 to V.R.I.K. and B10 to K.L. K.L. is a Mössbauer Professor at TUM-IAS. We thank C. Biertümpfel, MPI Martinsried for PCNA plasmids. Natively ubiquitylated PCNA was a generous gift from C. Biertümpfel. We thank M. Vermeulen, Radboud Insitute for Molecular Life sciences for GST-TAB2-NZF plasmid.

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K.L. conceived the research plan and experimental strategy. M.F. synthesized UAAs, performed all experiments in bacteria, including cloning, expression, purification of proteins and ubiquitylation/SUMOylation assays, as well as enzymatic assays and pull-down assays. A.-D.B. created PylRS libraries and evolved AzGGKRS. D.H.-G. performed initial mammalian cell experiments, including site-specific incorporation of AzGGK into HEK293T cells, and V.B. and A.B. performed ubiquitylation and SUMOylation assays in live HEK293T cells. A.J. and V.R.I.K. performed MD simulations. All authors analyzed data, and K.L. wrote the paper with input from the other authors.

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Correspondence to Kathrin Lang.

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We have filed a patent concerning the sortylation approach.

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Supplementary Tables 1–9, Supplementary Figures 1–23, Supplementary Notes 1 and 2

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Fottner, M., Brunner, A., Bittl, V. et al. Site-specific ubiquitylation and SUMOylation using genetic-code expansion and sortase. Nat Chem Biol 15, 276–284 (2019). https://doi.org/10.1038/s41589-019-0227-4

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