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Ubiquitylation beyond protein targets

Sophie Ye , Minglei Zhao

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Vita > Cutting Edge > DOI: 10.15302/vita.2026.06.0043
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Ubiquitylation beyond protein targets

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Ubiquitylation is increasingly recognized to extend beyond canonical protein substrates to include chemically diverse non-protein targets. In a recent Nature paper, Jochem et al. establish NoPro-clipping as a discovery and quantification workflow for non-proteinaceous ubiquitylation, revealing dynamic glycogen ubiquitylation in cells and tissues, as well as previously unrecognized ubiquitylation of the metabolites glycerol and spermine.

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Ubiquitylation has long been established in the biological canon as a process by which cells regulate proteostasis. Canonically, ubiquitylation involves the formation of an iso-peptide bond between a ubiquitin subunit via its C-terminal glycine and the ε-amino group of a lysine residue on the target protein. Within the realm of protein ubiquitylation, there are also non-canonical paths that can be taken to form thiol- or oxi-ester linkages to cysteine or serine/threonine residues. However, increasingly within the field, new non-proteinaceous substrates of ubiquitylation are being identified, including lipids1, polysaccharides2,3, nucleic acids4-7, and exogenous small molecules8,9 (Fig. 1a).
Regardless of substrate identity, ubiquitylation is carried out through a conserved three-step enzymatic cascade consisting of ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and finally a substrate-specific ubiquitin ligase (E3). It is the E3 ubiquitin ligase that confers the greatest substrate specificity to each given ubiquitylation process. Despite growing evidence that ubiquitin can modify non-protein substrates, the field has lacked a general method to systematically discover and quantify new non-canonical ubiquitylation events. In their recent publication, Jochem et al. leveraged the glycogen-modifying activity of HOIL-1L to establish a new non-proteinaceous ubiquitylation identification workflow and quantified glycogen ubiquitylation both in vitro and in vivo10.
This workflow, termed non-protein Ub-clipping (NoPro-clipping), involves a series of steps that isolate ubiquitylated non-proteinaceous substrates and convert them into enriched and quantifiable species for high-sensitivity mass spectrometry (MS). Given a sample containing non-proteinaceous ubiquitylated species, bacterial ubiquitin-clippases are first introduced to cleave ubiquitin internally, leaving only the C-terminal Gly-Gly (GG) motif attached to the substrate. This effectively converts the ubiquitome into a “GG-ome”. Proteinaceous species are then removed by molecular-weight filtration, and sortase A, a bacterial transpeptidase found natively in Staphylococcus aureus, is used to selectively peptide-label the resulting GG-modified non-protein substrates. The peptide label designed by Jochem et al., coined ClipTag, is a biotinylated LPXTG motif-containing peptide with an ester (depsi) bond between threonine and glycine, which drives the sortase reaction to completion. The sortase treatment enables enrichment and peptide-centric LC-MS acquisition, thereby allowing sensitive identification and quantification of the peptide-labelled species (Fig. 1b). In this study, the authors applied this workflow to quantify glycogen ubiquitylation; since glycogen is a complex, branched polysaccharide of variable mass, an extra step involving cleavage of ubiquitylated glycogen (Ub-glycogen) by α-amylase prior to clippase treatment was introduced.
The identification and fluorescent labelling of Ub-glycogen for in cellulo visualization, coupled with the NoPro-Clipping workflow for quantification, allowed the authors to gain new insights into the potential biological functions and relevance of Ub-glycogen. In particular, they observed a colocalization of Ub-glycogen, HOIL-1L, and the lysosomal marker LAMP1, which could be decoupled by introducing an E1-inhibitor or in cell lines possessing only a non-functional HOIL-1L mutant, suggesting that Ub-glycogen may promote lysosomal trafficking. The authors also reported a striking increase (~300-fold after normalization) in Ub-glycogen when GBE1, an enzyme that mediates 1,6-glucoside linkage formation and thus creates the branched architecture of glycogen, was knocked out. Loss of GBE1 is associated with the formation of polyglucosan bodies which are linked to several human diseases, including Anderson’s disease, adult polyglucosan body disease (APBD), and glycogen storage disease type IV (GSD IV)10. It is yet unclear what the functional consequence of this marked increase in Ub-glycogen upon GBE1 malfunction could be, but this correlation may provide an important entry point for further elucidating disease-associated mechanisms. The authors found particularly high levels of Ub-glycogen in liver tissue and showed that glycogen levels in mouse liver dropped after 6 h of fasting as expected, whereas Ub-glycogen levels increased 8-fold. Given the liver’s central role in glycogen metabolism, this discovery may reveal a previously unrecognized mechanism for regulating glycogen homeostasis.
Beyond their exploration of Ub-glycogen, Jochem et al. also tested the capabilities of their NoPro-Clipping workflow in discovery of previously unrecognized ubiquitylation substrates. After a meticulous analysis including multiple steps of filtering, they identified two hits of interest, which they matched through metabolite database searching to glycerol and spermine, respectively. Having identified these new species of interest, they then sought to quantify their endogenous ubiquitylation levels in Huh7 human liver cancer cells; they found ubiquitylated glycerol levels at 35 fmol/mg of total protein, and ubiquitylated spermine at 16 fmol/mg of total protein. Both species were also detected endogenously in mouse liver lysates. These novel findings demonstrate that ubiquitylation can occur on metabolites that had not previously been recognized as ubiquitin substrates. The biological significance of these modifications remains to be further elucidated.
There are still potential limitations to the NoPro-Clipping method, many of which reflect areas require broader validation. For example, not all macromolecules may be equally susceptible to Ub-clipping, which could influence detection efficiency and skew results especially during quantification. The workflow may also require substrate-specific optimization, which poses limits on the ease of discovery of new targets. The most pertinent example here would be glycogen, which requires an additional α-amylase treatment step to standardize and make feasible its measurement. Nonetheless, NoPro-Clipping represents a powerful new approach for discovering and quantifying non-proteinaceous ubiquitylation. In this study, its application has revealed observations of substantial biological interest, including evidence consistent with ubiquitin-dependent glycophagy and the possibility that ubiquitin is a previously unrecognized regulator of glycogen metabolism. Both ideas require further exploration and validation. More broadly, as NoPro-Clipping and related approaches continue to improve in sensitivity, substrate compatibility, and analytical workflows, many additional forms of non-canonical ubiquitylation are likely to be uncovered across diverse biomolecular classes and cellular contexts. The key implication is that ubiquitylation is no longer confined to the protein world. Together with other recent reports, this work broadens the scope of ubiquitin biology and sets the stage for the field to shift from discovery toward mechanistic studies that define the writers, erasers, and readers of these new modifications, and ultimately toward establishing their physiological and disease relevance.

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The Author(s) 2026. Published by Higher Education Press. This is an Open Access article distributed under the terms of the CC BY license (https://creativecommons.org/licenses/by/4.0/).

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Ye, S., Zhao, M.  Ubiquitylation beyond protein targets  Vita https://doi.org/10.15302/vita.2026.06.0043 ()
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