DNA damage-induced ubiquitylation of proteasome controls its proteolytic activity

Tatiana N Moiseeva,Gerry Melino,Andrew Bottrill,Nickolai A Barlev

doi:10.18632/oncotarget.1060

Abstract

Stability of proteins is largely controlled by post-translational covalent modifications. Among those, ubiquitylation plays a central role as it marks the proteins for proteasome-dependent degradation. Proteolytic activities of proteasomes are critical for execution of various cellular processes, including DNA damage signaling and repair. However, very little is known about the regulation of proteasomal activity in cells during genotoxic stress. Here we investigated post-translational modifications of the 20S proteasomal subunits upon DNA damage induced by doxorubicin. Using mass-spectrometry, we found novel sites of phosphorylation and ubiquitylation in multiple proteasome subunits upon doxorubicin treatment. Ectopic co-expression of proteasome subunits and tagged ubiquitin confirmed the presence of ubiquitylated forms of PSMA5, PSMA1, PSMA3 and PSMB5 in cells. Moreover, we demonstrated that ubiquitylation in vitro inhibited chymotrypsin-like and caspase-like activities of proteasomes. In vivo, doxorubicin increased the activity of proteasomes, paralleling with attenuation of the overall level of proteasome ubiquitylation. Collectively, our results suggest a novel mechanism whereby the proteolytic activities of proteasomes are dynamically regulated by ubiquitylation upon DNA damage.

Highlights

Proteasomes are multi-protein complexes, widely known to participate in protein degradation by ubiquitindependent and ubiquitin-independent proteolysis
We first decided to catalogue the posttranslational modifications of 20S proteasome subunits induced by doxorubicin-mediated genotoxic stress by using a proteomic approach
Proteasomes were extracted from control (A) and doxorubicin treated (B) K562 cells and separated on 2D-gel

Summary

Introduction

Proteasomes are multi-protein complexes, widely known to participate in protein degradation by ubiquitindependent and ubiquitin-independent proteolysis. Beta-subunits are responsible for proteolysis, while the main function of alpha-rings is to regulate an access of a substrate to the proteolytic chamber [1,2]. A second proteasome inhibitor, arfilzomib, was approved for treatment of the same disease [6]. This preliminary success makes proteasomes an appealing potential therapeutic target and highlights the importance of studying the regulatory mechanisms that control proteasome activities. Inhibition of interactions between specific ubiquitin ligases and their protein targets results in abrogation of their ubiquitin-dependent proteasomal degradation, and is considered a promising therapeutic strategy [7, 8, 9]

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Conclusion