Indirect Ubiquitination: Noncovalent Ubiquitin Tethering Independent of Endogenous Ubiquitination Machinery for Targeted Protein Degradation

For Bioorganic 2025.2: A new strategy termed indirect ubiquitination is presented, designing ubiquitin conjugates targeting nuclear factor κB (NF-κB) and Bcl-2. They demonstrate that monoubiquitin noncovalently linked to target proteins is sufficient to trigger proteasomal degradation, expanding the design possibilities for targeted protein degradation beyond traditional PROTACs. Major Comments: While UbAT-NFdecoy shows promising in vitro degradation, the strategy is less effective in living cells (Fig. 3C). This is attributed to rapid deubiquitination and limited uptake, but more discussion on how intracellular stability can be systematically improved would be beneficial. Further research on stability of monoubiquitinated proteins might also be advantageous. How specific is indirect ubiquitination for the intended targets? The Fig. S3C shows that UbAT-SCRM does not degrade NF-κB, but does indirect tethering risk nonspecific degradation of other proteins binding the ligand with lower affinity? Additional experiments showing global proteome changes could support the claim of target-specific degradation. The authors stated that disordered regions in p65 promote degradation (Fig. 2C). It would strengthen the paper to discuss whether this method would be limited to proteins with accessible/disordered regions or if structured targets could also be degraded effectively. Minor Comments: Minor typos and grammatical inconsistencies appear occasionally. Fig. 2A: Modify “UbAT-NFdecov” labels to “UbAT-NFdecoy”. Figs. S6–S8: Be more specific on figure legends in SI if MG132, Bortezomib are present or absent. Figs. 3A, S10, S11: Add the number of replicates and statistical parameters to validate flow cytometry-based conclusions on uptake efficiency. The rationale behind choosing Bcl-2 and NF-κB needs to be articulated more directly. New strategy for protein degradation independent of endogenous ubiquitination machinery is introduced, giving new opportunities for targeting undruggable proteins. However, cellular limitations and practical implementation challenges should be discussed more. Addressing the above points will improve the manuscript’s impact and clarity.

From Bioorganic 2025: The paper showed a new strategy of ubiquited protein degradation independent of E3 ligase. Different from previously existing PROTAC strategies using heterobivalent chimeric molecules to introduce E3 ubiquitin ligases, the authors linked ubiquitin to a target-binding ligand covalently, and the resultant complex was applied to tether targeted proteins. Designed and synthesized chimeric molecules combining ubiquitin with target-specific ligands such as NFdecoy for NF-kB or peptides for Bcl-2. Ubiquitin-DNA conjugates induced degradation of p50 in vitro. The efficiency was comparable to non-covalently ubiquitinated substrates. The conjugates also caused proteolysis of endogenous NF-κB subunits in cytoplasmic extracts. UbAT–NFdecoy showed stronger degradation of p65 than Ub–NFdecoy. Major Comments: Authors used only one ubiquitin tail for degradation. Please add more discussion/verification that monoubiquitin is enough, as a polyubiquitin tail is generally required for protein degradation. Fig. S12 and Fig. 3C both showed limited or no significant target degradation in living cells, despite success in cell extracts. Susceptibility of the ubiquitin-ligand linkage to deubiquitylation in the complex intracellular environment, which can cleave the ubiquitin moiety before it can mediate degradation. The UbAT mutation, conferring some in vitro resistance, was insufficient in cells. Delivery of the chimeras into cells, particularly the Ub-DNA conjugates, was less efficient than the ligand alone, potentially limiting the intracellular concentration needed for effective target binding and degradation. Delivering these biomacromolecules effectively and specifically to target cells in vivo remains a challenge, though compatibility with existing nucleic acid/protein carriers is noted. Improve chimera stability or maintain sufficient intracellular concentration (e.g., continuous expression from plasmids) for intracellular degradation. Minor Comments: Can include more figures in the introduction part for better understanding. Spelling errors in SI. Impact and Future Direction: Nucleic acid-based expression opens an opportunity to combine this technology with mRNA therapeutics or viral delivery.