biostudies-literatureUnknown8(6)Wang XSHOIL-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 prohibitsLife science alliancee202503243https://www.ebi.ac.uk/biostudies/studies/S-EPMC11962058biostudies-literatureThe RBR E3 ubiquitin ligase HOIL-1 can ubiquitinate diverse non-protein substrates in vitro.PMC11962058Lechtenberg BCCerra AGoddard-Borger EDCobbold SAMak KHTPayne RJWang XSCorcilius LJochem MSilke JJiou JKomander DfalseThe RBR E3 ubiquitin ligase HOIL-1 can ubiquitinate diverse non-protein substrates in vitro.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 prohibits2025-01-01T00:00:00Z2025 Jun2026-06-02T06:51:30.724Z2025-07-01T03:05:35.733ZS-EPMC119620584016925810.26508/lsa.202503243