Biology subjects

Levantovsky, S.

Publications and source records attributed to Levantovsky, S..

3 recordsLinked to original sources

bioRxiv2026

Ubiquitin ligase RNF126 exerts functions in lysosomal damage response and lysosomal homeostasis maintenance

The response to lysosomal membrane permeabilization involves extensive ubiquitylation at the lysosome; however, the ubiquitin ligases identified so far cannot account for the complexity of the response. Using extensive spatial proteomics, we identified RNF126 as a new ligase that is robustly recruited to lysosomes in response to a range of different damage triggers across distinct cell types. Recruitment is mediated independent of BAG6 via an arginine-rich stretch in the low-complexity region of RNF126 to mostly unacidified lysosomes in the perinuclear region. Ubiquitin remnant mass spectrometry identified several damage-induced RNF126 substrates, among them known lysosomal damage response components and potential new factors that might drive regeneration from lysosomal damage. Consistently, RNF126 deletion resulted in persistent lysosomal swelling. In addition, we uncovered a constitutive role of RNF126 in lysosomal homeostasis maintenance. Hence, our findings identified the ubiquitin ligase RNF126 as novel lysosomal health regulating factor with functions in homeostasis and stress conditions.

cell biology↗

bioRxiv2025

ATXN3 regulates lysosome regeneration after damage by targeting K48-K63-branched ubiquitin chains

The cellular response to lysosomal damage involves fine-tuned mechanisms of membrane repair, lysosome regeneration and lysophagy, but how these different processes are coordinated is not fully understood. Here we show in human cells that the deubiquitinating enzyme ATXN3 helps restore integrity of the lysosomal system after damage by targeting K48-K63-linked branched ubiquitin chains on regenerating lysosomes. We find that ATXN3 translocates to lysosomes after different types of damage and there colocalizes with its partner, VCP/p97. ATXN3 recruitment occurs late after the initial repair of a subset of lysosomes and after phagophore formation on terminally damaged lysosomes. Of note, inactivation of ATXN3 by induced degradation, depletion or knock-out impairs clearance of damaged lysosomes and full restoration of lysosomal capacity. Mechanistically, ATXN3, along with VCP/p97, turns over K48-K63-branched ubiquitin conjugates on LAMP1-positive, phosphatidylinositol-(4,5)-bisphosphate-decorated compartments that are not yet fully re-acidified indicating involvement in lysosome regeneration. Our findings identify a key role of ATXN3 in restoring lysosomal function after lysosomal membrane damage and uncover K48-K63-branched ubiquitin chain-regulated regeneration as a critical element of the lysosomal damage stress response.

cell biology↗

bioRxiv2023

A ubiquitin-specific, proximity-based labeling approach for the identification of ubiquitin ligase substrates

Ubiquitination of proteins is central to protein homeostasis and other cellular processes including DNA repair, vesicular transport, cell-division etc. The process of ubiquitination is conserved from yeast to humans and is carried out by the sequential action of three enzymes: E1, E2 and E3. There are an estimated >600 E3 ligases in humans that execute ubiquitination of specific target proteins in a spatio-temporal manner to elicit desired signaling effects. Here, we developed a ubiquitin-specific proximity-based labeling method to selectively biotinylate substrates of a given ubiquitin ligase. Our method exploits the proximity and the relative orientation of the E3-ligase catalytic domain with respect to ubiquitin observed in the enzymatic intermediate-state structures of E3-E2[~]Ub. By fusing the biotin ligase BirA and an Avi-tag variant to the candidate E3 ligase and ubiquitin, respectively, we were able to specifically enrich bona fide substrates and potential new substrates of a ligase using a one-step streptavidin pulldown under denaturing conditions. As proof-of-principle, we applied our method, which we named Ub-POD, to the RING E3 ligase RAD18. RAD18 ubiquitinates DNA-sliding clamp PCNA upon UV-induced DNA damage. We identified PCNA and several other critical players in the DNA damage repair pathway in a single RAD18 Ub-POD experiment. We went on to validate DNA replicase POLE as a possible new substrate of RAD18. Through RAD18 Ub-POD, we were also able to pin down the cellular localization of RAD18-mediated ubiquitination to the damaged DNA nuclear puncta using streptavidin immunofluorescence. Furthermore, we applied Ub-POD to TRAF6, another RING ubiquitin ligase involved in NF-{kappa}B signaling and successfully identified known and potentially new TRAF6 substrates. Finally, we adapted our method to the U-box-type E3 ubiquitin ligase CHIP to demonstrate that we can identify substrates of two major classes of mammalian ubiquitin ligases. We anticipate that our method and principle could be widely adapted to all classes of ubiquitin ligases to identify substrates and localize the cellular site(s) of ubiquitination.

biochemistry↗