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In depth proteomic analysis of proteasome inhibitors bortezomib, carfilzomib and MG132 reveals that mortality factor 4-like 1 (MORF4L1) protein ubiquitylation is negatively impacted

Porras-Yakushi, Tanya R. and Reitsma, Justin M. and Sweredoski, Michael J. and Deshaies, Raymond J. and Hess, Sonja (2021) In depth proteomic analysis of proteasome inhibitors bortezomib, carfilzomib and MG132 reveals that mortality factor 4-like 1 (MORF4L1) protein ubiquitylation is negatively impacted. Journal of Proteomics, 241 . Art. No. 104197. ISSN 1874-3919. doi:10.1016/j.jprot.2021.104197. https://resolver.caltech.edu/CaltechAUTHORS:20210414-071610399

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Abstract

Proteasome inhibitors are an important class of chemotherapeutic drugs. In this study, we performed a large-scale ubiquitylome analysis of the three proteasome inhibitors MG132, bortezomib and carfilzomib. Although carfilzomib is currently being used for the treatment of multiple myeloma, it has not yet been subjected to a global ubiquitylome analysis. In this study, we identified more than 14,000 unique sites of ubiquitylation in more than 4400 protein groups. We introduced stringent criteria to determine the correct ubiquitylation site ratios and used five biological replicates to achieve increased statistical power. With the vast amount of data acquired, we made proteome-wide comparisons between the proteasome inhibitors and indicate candidate proteins that will benefit from further study. We find that in addition to the expected increase in ubiquitylation in the majority of proteins, unexpectedly a select few are specifically and significantly decreased in ubiquitylation at specific sites after treatment with proteasome inhibitors. We chose to follow-up on Mortality factor 4-like 1 (MORF4L1), which was significantly decreased in ubiquitylation at lysine 187 and lysine 104 upon proteasome inhibition, but increased in protein abundance by approximately two-fold. We demonstrate that the endogenous protein level of MORF4L1 is highly regulated by the ubiquitin proteasome system. Significance: This study provides a highly curated dataset of more than 14,000 unique sites of ubiquitylation in more than 4400 protein groups. For the proper quantification of ubiquitylation sites, we introduced a higher standard by quantifying only those ubiquitylation sites that are not flanked by neighboring ubiquitylation, thereby avoiding the report of incorrect ratios. The sites identified will serve to identify important targets of the ubiquitin proteasome system and aid to better understand the repertoire of proteins that are affected by inhibiting the proteasome with MG132, bortezomib, and carfilzomib. In addition, we investigated the unusual observation that ubiquitylation of the tumor suppressor Mortality factor 4-like (MORF4L1) protein decreases rather than increases upon proteasome inhibition, which may contribute to an additional anti-tumor effect of bortezomib and carfilzomib.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1016/j.jprot.2021.104197DOIArticle
ORCID:
AuthorORCID
Sweredoski, Michael J.0000-0003-0878-3831
Deshaies, Raymond J.0000-0002-3671-9354
Hess, Sonja0000-0002-5904-9816
Additional Information:© 2021 Published by Elsevier B.V. Received 5 October 2020, Revised 18 March 2021, Accepted 18 March 2021, Available online 10 April 2021. The authors thank the members of the Proteome Exploration Laboratory for their helpful suggestions and critical review of this work. We also thank the members of the Raymond J. Deshaies laboratory for their helpful advice and suggestions. The authors would especially like to thank Thang Van Nguyen for providing us with the immunoprecipitation procedure under re-naturing conditions. This work was supported by the Gordon and Betty Moore Foundation, through Grant GBMF775, the Beckman Institute and the NIH through Grant 1S10RR029594-01A1. J.M.R was supported by F32 grant GM112308 from NIH. Declaration of competing interest: Tanya R. Porras-Yakush, Justin M. Reitsma, Michael J. Sweredoski, and Sonja Hess report no conflict of interest in the publication of this study. Raymond J. Deshaies is SVP, Global Research of Amgen, which commercializes carfilzomib.
Funders:
Funding AgencyGrant Number
Gordon and Betty Moore FoundationGBMF775
Caltech Beckman InstituteUNSPECIFIED
NIH1S10RR029594-01A1
NIHGM112308
Subject Keywords:Ubiquitin; Proteasome inhibitor; GlyGly sites; NEM; Statistics; MORF4L1
DOI:10.1016/j.jprot.2021.104197
Record Number:CaltechAUTHORS:20210414-071610399
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20210414-071610399
Official Citation:Tanya R. Porras-Yakushi, Justin M. Reitsma, Michael J. Sweredoski, Raymond J. Deshaies, Sonja Hess, In-depth proteomic analysis of proteasome inhibitors bortezomib, carfilzomib and MG132 reveals that mortality factor 4-like 1 (MORF4L1) protein ubiquitylation is negatively impacted, Journal of Proteomics, Volume 241, 2021, 104197, ISSN 1874-3919, https://doi.org/10.1016/j.jprot.2021.104197. (https://www.sciencedirect.com/science/article/pii/S1874391921000968)
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:108723
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:17 Apr 2021 00:01
Last Modified:19 Apr 2021 21:13

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