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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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Doxorubicin treatment enhances the activity of proteasomes concomitant with a decrease in ubiquitylationA. H1299 cells, ectopically expressing PSMA5-FLAG and His-tagged ubiquitin were treated with doxorubicin or DMSO for 6 hours. Cells were lysed in denaturing conditions. Ubiquitylated proteins were precipitated with Ni-NTA beads, and the ubiquitylated isoforms of PSMA5-FLAG were detected by western blotting with M2 anti-FLAG antibodies. Positions of ubiquitylated isoforms of the PSMA5-FLAG protein are shown.B. 26S proteasomes were purified from K562 cells non-treated (lane 1) or treated with doxorubicin (lane 2). Resulting proteins were separated by SDS-PAGE and visualized by Coomassie staining. Positions of 19S and 20S sub-complexes in the gel are shown.C. Proteasomes extracted from K562 cells non-treated (white bars) or treated with doxorubicin (shaded bars), were compared for their peptidase activities (trypsin-like, chymotrypsin-like and caspase-like) using specific fluorogenic peptides. Peptidase activities of each sample were tested in triplicates. Standard deviations are shown.
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Figure 5: Doxorubicin treatment enhances the activity of proteasomes concomitant with a decrease in ubiquitylationA. H1299 cells, ectopically expressing PSMA5-FLAG and His-tagged ubiquitin were treated with doxorubicin or DMSO for 6 hours. Cells were lysed in denaturing conditions. Ubiquitylated proteins were precipitated with Ni-NTA beads, and the ubiquitylated isoforms of PSMA5-FLAG were detected by western blotting with M2 anti-FLAG antibodies. Positions of ubiquitylated isoforms of the PSMA5-FLAG protein are shown.B. 26S proteasomes were purified from K562 cells non-treated (lane 1) or treated with doxorubicin (lane 2). Resulting proteins were separated by SDS-PAGE and visualized by Coomassie staining. Positions of 19S and 20S sub-complexes in the gel are shown.C. Proteasomes extracted from K562 cells non-treated (white bars) or treated with doxorubicin (shaded bars), were compared for their peptidase activities (trypsin-like, chymotrypsin-like and caspase-like) using specific fluorogenic peptides. Peptidase activities of each sample were tested in triplicates. Standard deviations are shown.

Mentions: Our mass-spec results in K562 cells suggested that DNA damage decreased the overall level of ubiquitylation of the 20S subunits. We wanted to verify the mass-spec data in respect to whether doxorubicin treatment indeed altered the level of proteasome ubiquitylation in vivo using the PSMA5 subunit as an example (Fig. 5A). Thus, we compared the levels of ubiquitylation of ectopically expressed PSMA5 in H1299 cells treated or non-treated with doxorubicin. To enrich the population of ubiquitylated proteins including PSMA5, cells were co-transfected with 6His-ubiquitin followed by the chelate chromatography on Ni-NTA beads. As evidenced from Fig. 5A, the overall level of ubiquitylated PSMA5 decreased significantly in cells treated with doxorubicin compared to control cells. Note, that the cellular levels of PSMA5 in both types of cells were comparable (Fig. 5A). These results suggest that ubiquitylation unlikely targets the PSMA5 protein for degradation but rather plays a regulatory role.


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

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

Doxorubicin treatment enhances the activity of proteasomes concomitant with a decrease in ubiquitylationA. H1299 cells, ectopically expressing PSMA5-FLAG and His-tagged ubiquitin were treated with doxorubicin or DMSO for 6 hours. Cells were lysed in denaturing conditions. Ubiquitylated proteins were precipitated with Ni-NTA beads, and the ubiquitylated isoforms of PSMA5-FLAG were detected by western blotting with M2 anti-FLAG antibodies. Positions of ubiquitylated isoforms of the PSMA5-FLAG protein are shown.B. 26S proteasomes were purified from K562 cells non-treated (lane 1) or treated with doxorubicin (lane 2). Resulting proteins were separated by SDS-PAGE and visualized by Coomassie staining. Positions of 19S and 20S sub-complexes in the gel are shown.C. Proteasomes extracted from K562 cells non-treated (white bars) or treated with doxorubicin (shaded bars), were compared for their peptidase activities (trypsin-like, chymotrypsin-like and caspase-like) using specific fluorogenic peptides. 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
getmorefigures.php?uid=PMC3824523&req=5

Figure 5: Doxorubicin treatment enhances the activity of proteasomes concomitant with a decrease in ubiquitylationA. H1299 cells, ectopically expressing PSMA5-FLAG and His-tagged ubiquitin were treated with doxorubicin or DMSO for 6 hours. Cells were lysed in denaturing conditions. Ubiquitylated proteins were precipitated with Ni-NTA beads, and the ubiquitylated isoforms of PSMA5-FLAG were detected by western blotting with M2 anti-FLAG antibodies. Positions of ubiquitylated isoforms of the PSMA5-FLAG protein are shown.B. 26S proteasomes were purified from K562 cells non-treated (lane 1) or treated with doxorubicin (lane 2). Resulting proteins were separated by SDS-PAGE and visualized by Coomassie staining. Positions of 19S and 20S sub-complexes in the gel are shown.C. Proteasomes extracted from K562 cells non-treated (white bars) or treated with doxorubicin (shaded bars), were compared for their peptidase activities (trypsin-like, chymotrypsin-like and caspase-like) using specific fluorogenic peptides. Peptidase activities of each sample were tested in triplicates. Standard deviations are shown.
Mentions: Our mass-spec results in K562 cells suggested that DNA damage decreased the overall level of ubiquitylation of the 20S subunits. We wanted to verify the mass-spec data in respect to whether doxorubicin treatment indeed altered the level of proteasome ubiquitylation in vivo using the PSMA5 subunit as an example (Fig. 5A). Thus, we compared the levels of ubiquitylation of ectopically expressed PSMA5 in H1299 cells treated or non-treated with doxorubicin. To enrich the population of ubiquitylated proteins including PSMA5, cells were co-transfected with 6His-ubiquitin followed by the chelate chromatography on Ni-NTA beads. As evidenced from Fig. 5A, the overall level of ubiquitylated PSMA5 decreased significantly in cells treated with doxorubicin compared to control cells. Note, that the cellular levels of PSMA5 in both types of cells were comparable (Fig. 5A). These results suggest that ubiquitylation unlikely targets the PSMA5 protein for degradation but rather plays a regulatory role.

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