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

Wang, XS; Jiou, J; Cerra, A; Cobbold, SA; Jochem, M; Mak, KHT; Corcilius, L; Silke, J; Payne, RJ; Goddard-Borger, ED; Komander, D; Lechtenberg, BC

Author(s): Wang, XS; Jiou, J; Cerra, A; Cobbold, SA; Jochem, M; Mak, KHT; Corcilius, L; Silke, J; Payne, RJ; Goddard-Borger, ED; Komander, D; Lechtenberg, BC;
Details: Publication Year 2025-06,Volume 8,Issue #6,Page e202503243
Journal Title: Life Science Alliance
Abstract: HOIL-1 is a RING-between-RING-family E3 ubiquitin ligase and a component of the linear ubiquitin chain assembly complex. Although most E3 ubiquitin ligases conjugate ubiquitin to protein lysine sidechains, HOIL-1 has also 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 and 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. We use HOIL-1's in vitro non-proteinaceous ubiquitination activity 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. Finally, we report an engineered, constitutively active HOIL-1 variant that simplifies in vitro generation of ubiquitinated saccharides.
Publisher: Life Sciene Alliance
Keywords: *Ubiquitination; *Ubiquitin-Protein Ligases/metabolism/genetics; Humans; Substrate Specificity; *Ubiquitin/metabolism; *Lysine/metabolism; Catalytic Domain; Histidine/metabolism/chemistry; Serine/metabolism
Research Division(s): Ubiquitin Signalling; Inflammation
PubMed ID: 40169258
Publisher's Version: https://doi.org/10.26508/lsa.202503243
Open Access at Publisher's Site: https://doi.org/10.26508/lsa.202503243
Terms of Use/Rights Notice: Refer to copyright notice on published article.

Creation Date: 2025-04-08 03:06:12

Last Modified: 2025-04-08 03:12:24