Preprint / Version 1

Cellular synthesis of ubiquitin-based probes for spatially resolved deubiquitinase profiling

This article is a preprint and has not been certified by peer review.

Authors

    Jinghao Chen,   Xinyu Zhang,   Yang Huang,   Gang Li,   Yu-Hsuan Tsai
    Yu-Hsuan Tsai
Categories
Chemical Biology
Keywords
Activity-based protein profiling; Deubiquitinase (DUB); Proximity-induced chemistry; Sortase A (SrtA); Ubiquitination

Abstract

Deubiquitinases (DUBs) orchestrate ubiquitin homeostasis, yet their compartment-specific activities remain underexplored. We developed a genetically encoded strategy to assembly activity-based ubiquitin probes in cellulo via sortase A (SrtA)-mediated transpeptidation. Co-targeting SrtA and its ubiquitin substrate to the outer mitochondrial membrane reduced off-target transpeptidation and enabled spatially resolved profiling in live cells. Quantitative proteomics identified distinct sets of cytosolic, nuclear, and cell line specific DUBs in HEK293T and MCF7 cells. This localization-tunable, delivery-free strategy expands the chemical biology toolkit for mapping DUB activity within native cellular environments.

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DOI:

https://doi.org/10.65215/9am1sc06

Submission ID:

36

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Posted

2025-11-30

How to Cite

Chen, J., Zhang, X., Huang, Y., Li, G., & Tsai, Y.-H. (2025). Cellular synthesis of ubiquitin-based probes for spatially resolved deubiquitinase profiling. LangTaoSha Preprint Server. https://doi.org/10.65215/9am1sc06

Declaration of Competing Interests

The authors declare no competing interests to disclose.

Copyright

The copyright holder for this preprint is the author/funder.

Creative Commons License

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.