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DNA damage-induced ubiquitylation of proteasome controls its proteolytic activity.

Moiseeva TN, Bottrill A, Melino G, Barlev NA - Oncotarget (2013)

Bottom Line: 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.In vivo, doxorubicin increased the activity of proteasomes, paralleling with attenuation of the overall level of proteasome ubiquitylation.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cytology, Russian Academy of Sciences, St-Petersburg, Russia.

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.

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Related in: MedlinePlus

2D electrophoregrams of proteasomal proteins before and after doxorubicin treatmentProteasomes were extracted from control (A) and doxorubicin treated (B) K562 cells and then separated on 2D-gel (isoelectric focusing followed by SDS-PAGE). Separated proteins were visualized by Coomassie staining. Marked spots were excised from the gel and analyzed by mass-spectrometry.
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Figure 1: 2D electrophoregrams of proteasomal proteins before and after doxorubicin treatmentProteasomes were extracted from control (A) and doxorubicin treated (B) K562 cells and then separated on 2D-gel (isoelectric focusing followed by SDS-PAGE). Separated proteins were visualized by Coomassie staining. Marked spots were excised from the gel and analyzed by mass-spectrometry.

Mentions: Several studies suggest that proteasomes play an important role in DNA damage signaling and DNA repair [32]. An important question emerges as whether the activity of proteasomes is regulated upon DNA damage and what is the molecular mechanism behind it. To address this, we first decided to catalogue the posttranslational modifications of 20S proteasome subunits induced by doxorubicin-mediated genotoxic stress by using a proteomic approach. To this end, proteasomal proteins purified from control and DNA damage treated cells were separated by 2D-electrophoresis (Fig. 1) and spots, corresponding to the 20S subunits were excised from the gel and analyzed using LC-MS/MS mass-spectrometry. As shown in Table 1, we were able to identify both novel and previously reported sites for phosphorylation, acetylation and ubiquitylation. The majority of newly identified modification sites are acetylated amino acids including lysines, serines and alanines. In addition, we identified phosphorylation sites on all of the alpha-type subunits, except PSMA7. Finally, two new ubiquitylation sites – Lys15 on PSMB3 and Lys52 on PSMA3 were also detected. Interestingly, several of the newly found acetylation sites were previously reported to be ubiquitylated (Lys45 on PSMA6, Lys165 on PSMA2 and many more [33]).


DNA damage-induced ubiquitylation of proteasome controls its proteolytic activity.

Moiseeva TN, Bottrill A, Melino G, Barlev NA - Oncotarget (2013)

2D electrophoregrams of proteasomal proteins before and after doxorubicin treatmentProteasomes were extracted from control (A) and doxorubicin treated (B) K562 cells and then separated on 2D-gel (isoelectric focusing followed by SDS-PAGE). Separated proteins were visualized by Coomassie staining. Marked spots were excised from the gel and analyzed by mass-spectrometry.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3824523&req=5

Figure 1: 2D electrophoregrams of proteasomal proteins before and after doxorubicin treatmentProteasomes were extracted from control (A) and doxorubicin treated (B) K562 cells and then separated on 2D-gel (isoelectric focusing followed by SDS-PAGE). Separated proteins were visualized by Coomassie staining. Marked spots were excised from the gel and analyzed by mass-spectrometry.
Mentions: Several studies suggest that proteasomes play an important role in DNA damage signaling and DNA repair [32]. An important question emerges as whether the activity of proteasomes is regulated upon DNA damage and what is the molecular mechanism behind it. To address this, we first decided to catalogue the posttranslational modifications of 20S proteasome subunits induced by doxorubicin-mediated genotoxic stress by using a proteomic approach. To this end, proteasomal proteins purified from control and DNA damage treated cells were separated by 2D-electrophoresis (Fig. 1) and spots, corresponding to the 20S subunits were excised from the gel and analyzed using LC-MS/MS mass-spectrometry. As shown in Table 1, we were able to identify both novel and previously reported sites for phosphorylation, acetylation and ubiquitylation. The majority of newly identified modification sites are acetylated amino acids including lysines, serines and alanines. In addition, we identified phosphorylation sites on all of the alpha-type subunits, except PSMA7. Finally, two new ubiquitylation sites – Lys15 on PSMB3 and Lys52 on PSMA3 were also detected. Interestingly, several of the newly found acetylation sites were previously reported to be ubiquitylated (Lys45 on PSMA6, Lys165 on PSMA2 and many more [33]).

Bottom Line: 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.In vivo, doxorubicin increased the activity of proteasomes, paralleling with attenuation of the overall level of proteasome ubiquitylation.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cytology, Russian Academy of Sciences, St-Petersburg, Russia.

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.

Show MeSH
Related in: MedlinePlus