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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

The alpha type subunits of the 20S proteasome undergo ubiquitylation in vivoH1299 cells transiently co-expressing one of the indicated FLAG-tagged proteasome subunits and His-tagged ubiquitin were lysed in denaturing conditions. Ubiquitylated proteins were precipitated with Ni-NTA beads, and the ubiquitylated isoforms of PSMA5-FLAG, PSMB5-FLAG (A) and PSMA1-FLAG, PSMA3-FLAG (B) subunits were separated by SDS-PAGE followed by western-blotting with anti-FLAG antibodies. Positions of ubiquitylated subunits are indicated.
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Figure 2: The alpha type subunits of the 20S proteasome undergo ubiquitylation in vivoH1299 cells transiently co-expressing one of the indicated FLAG-tagged proteasome subunits and His-tagged ubiquitin were lysed in denaturing conditions. Ubiquitylated proteins were precipitated with Ni-NTA beads, and the ubiquitylated isoforms of PSMA5-FLAG, PSMB5-FLAG (A) and PSMA1-FLAG, PSMA3-FLAG (B) subunits were separated by SDS-PAGE followed by western-blotting with anti-FLAG antibodies. Positions of ubiquitylated subunits are indicated.

Mentions: To validate the results of mass-spectrometry on ubiquitylated proteasome subunits, we individually examined the ubiquitylation status of ectopically expressed PSMA5, PSMA1, PSMA3 and PSMB5 subunits in the presence of tagged ubiquitin. To this end, whole-cell extracts were prepared from H1299 cells co-transfected with respective FLAG-tagged proteasome subunits and a vector, expressing His-tagged ubiquitin. 6His-ubiquitylated proteins were then affinity purified on the Ni-NTA resin followed by the western blot analysis using FLAG antibodies to detect tagged proteasomal subunits. The results of experiments shown in Fig. 2 suggest that all four subunits investigated were present in various ubiquitylated forms. The PSMB5 subunit was represented mostly as a mono-ubiquitylated species and this modification largely affected the proteolytically non-processed form of PSMB5 (Fig. 2A, compare lanes 4 and 7). The PSMA5 ubiquitylation pattern was more heterogeneous, presumably due to additional non-ubiquitin covalent modifications, such as acetylation, phosphorylation and/or O-glycosylation. These modifications are known to affect the protein mobility in SDS-PAGE. However, the majority of PSMA5 proteins in the presence of ubiquitin was distributed between two zones, which likely correspond to mono- and di-ubiquitylated forms (Fig. 2a, compare lanes 3 and 6). It should also be noted that most of the PSMA5 species retained on Ni2+ beads were ubiquitylated, because no PSMA5 protein was detected on beads in the absence of 6His-ubiquitin (Fig. 2A, compare lanes 2 and 3). In the same experiment, PSMA1 and PSMA3 subunits were preferentially mono-ubiquitylated, although di- and tri-ubiquitylated isoforms were also detected but at a lesser extent (Fig. 2B).


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

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

The alpha type subunits of the 20S proteasome undergo ubiquitylation in vivoH1299 cells transiently co-expressing one of the indicated FLAG-tagged proteasome subunits and His-tagged ubiquitin were lysed in denaturing conditions. Ubiquitylated proteins were precipitated with Ni-NTA beads, and the ubiquitylated isoforms of PSMA5-FLAG, PSMB5-FLAG (A) and PSMA1-FLAG, PSMA3-FLAG (B) subunits were separated by SDS-PAGE followed by western-blotting with anti-FLAG antibodies. Positions of ubiquitylated subunits are indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: The alpha type subunits of the 20S proteasome undergo ubiquitylation in vivoH1299 cells transiently co-expressing one of the indicated FLAG-tagged proteasome subunits and His-tagged ubiquitin were lysed in denaturing conditions. Ubiquitylated proteins were precipitated with Ni-NTA beads, and the ubiquitylated isoforms of PSMA5-FLAG, PSMB5-FLAG (A) and PSMA1-FLAG, PSMA3-FLAG (B) subunits were separated by SDS-PAGE followed by western-blotting with anti-FLAG antibodies. Positions of ubiquitylated subunits are indicated.
Mentions: To validate the results of mass-spectrometry on ubiquitylated proteasome subunits, we individually examined the ubiquitylation status of ectopically expressed PSMA5, PSMA1, PSMA3 and PSMB5 subunits in the presence of tagged ubiquitin. To this end, whole-cell extracts were prepared from H1299 cells co-transfected with respective FLAG-tagged proteasome subunits and a vector, expressing His-tagged ubiquitin. 6His-ubiquitylated proteins were then affinity purified on the Ni-NTA resin followed by the western blot analysis using FLAG antibodies to detect tagged proteasomal subunits. The results of experiments shown in Fig. 2 suggest that all four subunits investigated were present in various ubiquitylated forms. The PSMB5 subunit was represented mostly as a mono-ubiquitylated species and this modification largely affected the proteolytically non-processed form of PSMB5 (Fig. 2A, compare lanes 4 and 7). The PSMA5 ubiquitylation pattern was more heterogeneous, presumably due to additional non-ubiquitin covalent modifications, such as acetylation, phosphorylation and/or O-glycosylation. These modifications are known to affect the protein mobility in SDS-PAGE. However, the majority of PSMA5 proteins in the presence of ubiquitin was distributed between two zones, which likely correspond to mono- and di-ubiquitylated forms (Fig. 2a, compare lanes 3 and 6). It should also be noted that most of the PSMA5 species retained on Ni2+ beads were ubiquitylated, because no PSMA5 protein was detected on beads in the absence of 6His-ubiquitin (Fig. 2A, compare lanes 2 and 3). In the same experiment, PSMA1 and PSMA3 subunits were preferentially mono-ubiquitylated, although di- and tri-ubiquitylated isoforms were also detected but at a lesser extent (Fig. 2B).

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