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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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Ubiquitylation of proteasomes in vitro inhibits their proteolytic activitiesA. Proteasomes extracted from untreated K562 cells were ubiquitylated in vitro using biotinylated ubiquitin and different E2 enzymes: UbcH1, UbcH2, UbcH3, UbcH5a, UbcH5b, UbcH5c, UbcH6, UbcH7, UbcH8, UbcH10, and UbcH13. Samples were then subjected to SDS-PAGE followed by transfer to nitrocellulose membrane. Ubiquitylated proteins were detected using the streptavidin-biotin system. The two right lanes represent control reactions without ubiquitin or without proteasomes.B and C. Proteasomes were ubiquitylated as described above in the presence of either UbcH1, UbcH2, UbcH3, UbcH5a, UbcH5b, UbcH6, UbcH7 or UbcH13 ubiquitin-conjugating enzymes followed by the measurement of chymotrypsin-like (B) and caspase-like (C) activities using fluorogenic peptides as specific substrates. In each case, activity of the non-ubiquitylated proteasome was arbitrary set as 1. Peptidase activities of each sample were tested in triplicates. Standard deviations are shown.
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Figure 4: Ubiquitylation of proteasomes in vitro inhibits their proteolytic activitiesA. Proteasomes extracted from untreated K562 cells were ubiquitylated in vitro using biotinylated ubiquitin and different E2 enzymes: UbcH1, UbcH2, UbcH3, UbcH5a, UbcH5b, UbcH5c, UbcH6, UbcH7, UbcH8, UbcH10, and UbcH13. Samples were then subjected to SDS-PAGE followed by transfer to nitrocellulose membrane. Ubiquitylated proteins were detected using the streptavidin-biotin system. The two right lanes represent control reactions without ubiquitin or without proteasomes.B and C. Proteasomes were ubiquitylated as described above in the presence of either UbcH1, UbcH2, UbcH3, UbcH5a, UbcH5b, UbcH6, UbcH7 or UbcH13 ubiquitin-conjugating enzymes followed by the measurement of chymotrypsin-like (B) and caspase-like (C) activities using fluorogenic peptides as specific substrates. In each case, activity of the non-ubiquitylated proteasome was arbitrary set as 1. Peptidase activities of each sample were tested in triplicates. Standard deviations are shown.

Mentions: To assess the effect of ubiquitylation on the proteolytic activities of the 20S complex, we performed in vitro ubiquitylation assay followed by the peptidase activity assay (Fig 4). A collection of purified E2 ubiquitin conjugating enzymes (Enzo Life Sciences) was tested for their ability to ubiquitylate 20S proteasomes isolated from K562 cells (Fig. 4A). From eleven E2 ligases tested eight (UbcH1, UbcH2, UbcH3, UbcH5a, UbcH5b, UbcH6, UbcH7, and UbcH13) exhibited ubiquitylation activity towards the 20S proteasomal proteins (Fig. 4A). Those eight ligases were subsequently used for ubiquitylation of the 20S complex followed by the measurement of caspase-like and chymotrypsin-like activities of proteasomes (Fig. 4B, C). The results of experiment showed that ubiquitylation by all E2 enzymes inhibited to a various extent both caspase-like and chymotrypsin-like activities of proteasomes (Fig. 4B and 4C, respectively). Interestingly, there was no strict correlation between the number of ubiquitylated subunits and the efficiency of repression. This suggests that mono-ubiquitylation of even one subunit of the 20S complex may result in strong inhibitory effect.


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

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

Ubiquitylation of proteasomes in vitro inhibits their proteolytic activitiesA. Proteasomes extracted from untreated K562 cells were ubiquitylated in vitro using biotinylated ubiquitin and different E2 enzymes: UbcH1, UbcH2, UbcH3, UbcH5a, UbcH5b, UbcH5c, UbcH6, UbcH7, UbcH8, UbcH10, and UbcH13. Samples were then subjected to SDS-PAGE followed by transfer to nitrocellulose membrane. Ubiquitylated proteins were detected using the streptavidin-biotin system. The two right lanes represent control reactions without ubiquitin or without proteasomes.B and C. Proteasomes were ubiquitylated as described above in the presence of either UbcH1, UbcH2, UbcH3, UbcH5a, UbcH5b, UbcH6, UbcH7 or UbcH13 ubiquitin-conjugating enzymes followed by the measurement of chymotrypsin-like (B) and caspase-like (C) activities using fluorogenic peptides as specific substrates. In each case, activity of the non-ubiquitylated proteasome was arbitrary set as 1. Peptidase activities of each sample were tested in triplicates. Standard deviations are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 4: Ubiquitylation of proteasomes in vitro inhibits their proteolytic activitiesA. Proteasomes extracted from untreated K562 cells were ubiquitylated in vitro using biotinylated ubiquitin and different E2 enzymes: UbcH1, UbcH2, UbcH3, UbcH5a, UbcH5b, UbcH5c, UbcH6, UbcH7, UbcH8, UbcH10, and UbcH13. Samples were then subjected to SDS-PAGE followed by transfer to nitrocellulose membrane. Ubiquitylated proteins were detected using the streptavidin-biotin system. The two right lanes represent control reactions without ubiquitin or without proteasomes.B and C. Proteasomes were ubiquitylated as described above in the presence of either UbcH1, UbcH2, UbcH3, UbcH5a, UbcH5b, UbcH6, UbcH7 or UbcH13 ubiquitin-conjugating enzymes followed by the measurement of chymotrypsin-like (B) and caspase-like (C) activities using fluorogenic peptides as specific substrates. In each case, activity of the non-ubiquitylated proteasome was arbitrary set as 1. Peptidase activities of each sample were tested in triplicates. Standard deviations are shown.
Mentions: To assess the effect of ubiquitylation on the proteolytic activities of the 20S complex, we performed in vitro ubiquitylation assay followed by the peptidase activity assay (Fig 4). A collection of purified E2 ubiquitin conjugating enzymes (Enzo Life Sciences) was tested for their ability to ubiquitylate 20S proteasomes isolated from K562 cells (Fig. 4A). From eleven E2 ligases tested eight (UbcH1, UbcH2, UbcH3, UbcH5a, UbcH5b, UbcH6, UbcH7, and UbcH13) exhibited ubiquitylation activity towards the 20S proteasomal proteins (Fig. 4A). Those eight ligases were subsequently used for ubiquitylation of the 20S complex followed by the measurement of caspase-like and chymotrypsin-like activities of proteasomes (Fig. 4B, C). The results of experiment showed that ubiquitylation by all E2 enzymes inhibited to a various extent both caspase-like and chymotrypsin-like activities of proteasomes (Fig. 4B and 4C, respectively). Interestingly, there was no strict correlation between the number of ubiquitylated subunits and the efficiency of repression. This suggests that mono-ubiquitylation of even one subunit of the 20S complex may result in strong inhibitory effect.

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