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Substrate biasing in UCHL5 proteoforms
Biorxiv : the Preprint Server for Biology
|July 29, 2026
Summary
UCHL5 proteoforms regulate substrate selectivity. N-terminal ubiquitination activates deubiquitinating enzymes for monoubiquitin, while Rpn13/Adrm1 binding restores debranching activity, revealing molecular mechanisms for enzyme regulation.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Proteolytic deubiquitinating enzymes (DUBs) play crucial roles in cellular processes by removing ubiquitin tags.
- DUBs exhibit complex regulatory mechanisms, often involving proteoforms that dictate substrate specificity and cleavage activity.
- Understanding these regulatory mechanisms is key to deciphering cellular signaling pathways and disease states.
Purpose of the Study:
- To elucidate the substrate-biased regulation of the deubiquitinating enzyme UCHL5 by its proteoforms.
- To investigate the role of N-terminal ubiquitination in modulating UCHL5 activity towards different ubiquitin substrates.
- To determine how interactions with regulatory partners like Rpn13/Adrm1 influence UCHL5's deubiquitinating function.
Main Methods:
- Utilized crystallographic and spectroscopic techniques to analyze UCHL5 proteoform structures and interactions.
- Investigated the impact of N-terminal ubiquitination on UCHL5's substrate cleavage capabilities.
- Examined the effect of Rpn13/Adrm1 binding on UCHL5 inhibition and debranching activity.
Main Results:
- Demonstrated that N-terminal ubiquitination activates UCHL5 towards monoubiquitin substrates, a conserved feature across homologs.
- Revealed that N-terminal ubiquitin binds to an allosteric site, inhibiting branched chain substrate cleavage.
- Showed that association with Rpn13/Adrm1 relieves this inhibition, restoring debranching activity and potentially controlling proteasome-associated functions.
Conclusions:
- Established the molecular basis for substrate selectivity in UCHL5, highlighting the importance of proteoform regulation.
- Uncovered a novel regulatory mechanism where N-terminal ubiquitination acts as an allosteric switch for DUB activity.
- Provided insights into how interactions with regulatory proteins fine-tune DUB function, impacting ubiquitin homeostasis and cellular signaling.
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