
==== Front
bioRxiv
BIORXIV
bioRxiv
2692-8205
Cold Spring Harbor Laboratory

10.1101/2024.09.02.610852
preprint
1
Article
Design of linked-domain protein inhibitors of UBE2D as tools to study cellular ubiquitination
Bukhari Zara
Gu Li http://orcid.org/0000-0001-7044-6444

Nederstigt Anneroos E. http://orcid.org/0000-0002-3695-3677

Cope Logan J.
Bolhuis Derek L. http://orcid.org/0000-0002-9062-9739

Harvey Kim
Allen Tristan
Hill Spencer
Yang Yujie
Lawson Guy
Lu Cai
Tran Tommy
Pineda Leah
Low Leanne
Chiang Andrew
Song Jason
Fong Michelle V.
Rangel Vanessa M.
Chan William K.
Kleiger Gary http://orcid.org/0000-0003-3924-1680

Goldfarb Dennis http://orcid.org/0000-0002-1584-5423

Vierra Craig A.
Brown Nicholas G. http://orcid.org/0000-0002-6141-0164

Harrison Joseph S. http://orcid.org/0000-0002-2118-6524

02 9 2024
2024.09.02.610852https://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License, which allows reusers to distribute, remix, adapt, and build upon the material in any medium or format for noncommercial purposes only, and only so long as attribution is given to the creator.
http://biorxiv.org/lookup/doi/10.1101/2024.09.02.610852
nihpp-2024.09.02.610852.pdf
ABSTRACT

Ubiquitin (Ub) is a post-translational modification that largely controls proteostasis through mechanisms spanning transcription, translation, and notably, protein degradation. Ub conjugation occurs through a hierarchical cascade of three enzyme classes (E1, E2, and E3s) involving >1000 proteins that regulate the ubiquitination of proteins. The E2 Ub-conjugating enzymes are the midpoint, yet their cellular roles remain under-characterized, partly due to a lack of inhibitors. For example, the cellular roles of the promiscuous E2 UBE2D/UBCH5 are not well described. Here, we develop a highly selective, multivalent, engineered protein inhibitor for the UBE2D family that simultaneously targets the RING- and backside-binding sites. In HeLa cells, these inhibitors phenocopy knockdown of UBE2D by reducing the IC 50 to cisplatin and whole-cell proteomics reveal an increased abundance of ∼20% of the identified proteins, consistent with reduced Ub degradation and proteotoxic stress. These precision tools will enable new studies probing UBE2D’s central role in proteome management.
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