The RBR E3 ubiquitin ligase HOIL-1 can ubiquitinate diverse non-proteinaceous substrates in vitro

Wang, X.; Jiou, J.; Cerra, A.; Cobbold, S. A.; Jochem, M.; Mak, K. H. T.; Corcilius, L.; Payne, R. J.; Goddard-Borger, E. D.; Komander, D.; Lechtenberg, B. C.

doi:10.1101/2024.11.29.626046

bioRxiv · 10.1101/2024.11.29.626046

The RBR E3 ubiquitin ligase HOIL-1 can ubiquitinate diverse non-proteinaceous substrates in vitro

Wang, X.·Jiou, J.·Cerra, A.·Cobbold, S. A.·Jochem, M.·Mak, K. H. T.·Corcilius, L.·Payne, R. J.·Goddard-Borger, E. D.·Komander, D.·Lechtenberg, B. C.

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Abstract

HOIL-1 is a RING-between-RING (RBR)-family E3 ubiquitin ligase and component of the linear ubiquitin chain assembly complex (LUBAC). While most E3 ubiquitin ligases conjugate ubiquitin to protein lysine sidechains, HOIL-1 has been reported to ubiquitinate hydroxyl groups in protein serine and threonine sidechains and glucosaccharides, such as glycogen and its building block maltose, in vitro. However, HOIL-1 substrate specificity is currently poorly defined. Here we show that HOIL-1 is unable to ubiquitinate lysine but can efficiently ubiquitinate serine as well as a variety of model and physiologically relevant di- and monosaccharides in vitro. We identify a critical catalytic histidine residue, His510, in the flexible catalytic site of HOIL-1 that enables this O-linked ubiquitination and prohibits ubiquitin discharge onto lysine sidechains. Finally, we utilise HOIL-1s in vitro non-proteinaceous ubiquitination activity and an engineered, constitutively active HOIL-1 variant to produce preparative amounts of different ubiquitinated saccharides that can be used as tool compounds and standards in the rapidly emerging field of non-proteinaceous ubiquitination.

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Wang, X., Jiou, J., Cerra, A., Cobbold, S. A., Jochem, M., Mak, K. H. T., Corcilius, L., Payne, R. J., Goddard-Borger, E. D., Komander, D., Lechtenberg, B. C.. 2024-11-30. The RBR E3 ubiquitin ligase HOIL-1 can ubiquitinate diverse non-proteinaceous substrates in vitro. https://doi.org/10.1101/2024.11.29.626046

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