Impact of losing hRpn13 Pru or UCHL5 on proteasome clearance of ubiquitinated proteins and RA190 cytotoxicity

Author:

Osei Amponsa,Vasty

Sridharan, Vinidhra

Tandon,Mayank

Evans,Christine

Klarmann,Kim

Cheng,Oscar

Lack,Justin

Chari,Raj

Walters,Kylie [ORCID]
Author Address

Protein Processing Section, Structural Biophysics Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA., Collaborative Bioinformatics Resource, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA., Advanced Biomedical Computational Science, Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA., Genome Modification Core, Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA., CCR-Frederick Flow Cytometry Core, Basic Science Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21701, USA., Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21702, USA., NIAID Collaborative Bioinformatics Resource (NCBR), National Institute of Allergy and Infectious Diseases, National Institutes of Health, Frederick MD 21702., Protein Processing Section, Structural Biophysics Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA kylie.walters@nih.gov.,

Abstract:

hRpn13/ADRM1 links substrate recruitment with deubiquitination at the proteasome through its proteasome- and ubiquitin-binding Pru domain and DEUBAD domain, which binds and activates deubiquitinating enzyme (DUB) UCHL5/Uch37. Here, we edit the HCT116 colorectal cancer cell line to delete part of the hRpn13 Pru, producing cells that express truncated hRpn13 (trRpn13), which is competent for UCHL5 binding, but defective for proteasome interaction. trRpn13 cells demonstrates reduced levels of proteasome-bound ubiquitinated proteins, indicating that loss of hRpn13 function at proteasomes cannot be fully compensated for by the two other dedicated substrate receptors (hRpn1 and hRpn10). Previous studies indicate that loss of full-length hRpn13 causes corresponding reduction of UCHL5. We find UCHL5 levels unaltered in trRpn13 cells, but that hRpn11 is elevated in ?hRpn13 and trRpn13, perhaps from cell stress. Despite the ~90 DUBs in human cells, including two others in addition to UCHL5 at the proteasome, we found deletion of UCHL5 from HCT116 cells to cause increased levels of ubiquitinated proteins in whole cell extract and at proteasomes, suggesting that UCHL5 activity cannot be fully assumed by other DUBs. We also report anti-cancer molecule RA190, which binds covalently to hRpn13 and UCHL5, to require hRpn13 Pru and not UCHL5 for cytotoxicity.

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