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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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The ubiquitylated PSMA5 subunit is distributed among the protein fractions of different molecular weightWhole-cell extracts prepared from H1299 cells expressing endogenous PSMA5 (A) or ectopic PSMA5-FLAG (B) proteins, were subjected to fractionation in the 5-30% glycerol concentration gradient. 20 equal fractions of different densities were collected and analyzed by western-blotting using anti-PSMA5 (endogenous protein) (A) or anti-FLAG (B) (ectopic PSMA5 protein) antibodies.
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Figure 3: The ubiquitylated PSMA5 subunit is distributed among the protein fractions of different molecular weightWhole-cell extracts prepared from H1299 cells expressing endogenous PSMA5 (A) or ectopic PSMA5-FLAG (B) proteins, were subjected to fractionation in the 5-30% glycerol concentration gradient. 20 equal fractions of different densities were collected and analyzed by western-blotting using anti-PSMA5 (endogenous protein) (A) or anti-FLAG (B) (ectopic PSMA5 protein) antibodies.

Mentions: Next, we decided to compare the ubiquitylation patterns of the monomeric PSMA5 subunit versus the one incorporated into the 20S core particle. Thus, H1299 cell extracts containing either endogenous PSMA5 or ectopic PSMA5-FLAG proteins in various proteasomal complexes were separated in glycerol density gradients according to their molecular weights followed by western-blotting using anti-PSMA5 (Fig. 3A) or anti-FLAG antibodies (Fig. 3B), respectively. Both ectopic and endogenous PSMA5 proteins were found in multiple gradient fractions. Specifically, the endogenous PSMA5 protein was observed both in the low molecular weight (LMW) fractions and in the high molecular weight (HMW) fractions. Apparently, the PSMA5 species that belong to the LMW fractions represent a mix of free monomeric subunits, its dimers and possibly a complex with PSMA1 and PSMA3 subunits. The PSMA5 species found in the HMW fraction likely correspond to the full-sized 20S proteasome and its interacting proteins (Fig. 3, A and B). Importantly, endogenous PSMA5 was preferentially di-ubiquitylated in the LMW fraction, whereas PSMA5 in the 20S proteasome HMW fraction was mostly mono-ubiquitylated (Fig. 3A). A similar pattern was observed for the ectopic PSMA5-FLAG protein with the exception that in the LMW fractions it was not only di-ubiquitylated, but also mono- and tri-ubiquitylated (Fig. 3B). Collectively, our results suggest that both endogenous and ectopic alpha-type subunits undergo ubiquitylation in cells. Moreover, the neighbouring subunits of the 20S particle likely restrict the extent of ubiquitylation for PSMA5 to only mono-ubiquitylation. On the contrary, PSMA5 in its free, or heterotrimeric form, is more susceptible to ubiquitylation.


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

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

The ubiquitylated PSMA5 subunit is distributed among the protein fractions of different molecular weightWhole-cell extracts prepared from H1299 cells expressing endogenous PSMA5 (A) or ectopic PSMA5-FLAG (B) proteins, were subjected to fractionation in the 5-30% glycerol concentration gradient. 20 equal fractions of different densities were collected and analyzed by western-blotting using anti-PSMA5 (endogenous protein) (A) or anti-FLAG (B) (ectopic PSMA5 protein) antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: The ubiquitylated PSMA5 subunit is distributed among the protein fractions of different molecular weightWhole-cell extracts prepared from H1299 cells expressing endogenous PSMA5 (A) or ectopic PSMA5-FLAG (B) proteins, were subjected to fractionation in the 5-30% glycerol concentration gradient. 20 equal fractions of different densities were collected and analyzed by western-blotting using anti-PSMA5 (endogenous protein) (A) or anti-FLAG (B) (ectopic PSMA5 protein) antibodies.
Mentions: Next, we decided to compare the ubiquitylation patterns of the monomeric PSMA5 subunit versus the one incorporated into the 20S core particle. Thus, H1299 cell extracts containing either endogenous PSMA5 or ectopic PSMA5-FLAG proteins in various proteasomal complexes were separated in glycerol density gradients according to their molecular weights followed by western-blotting using anti-PSMA5 (Fig. 3A) or anti-FLAG antibodies (Fig. 3B), respectively. Both ectopic and endogenous PSMA5 proteins were found in multiple gradient fractions. Specifically, the endogenous PSMA5 protein was observed both in the low molecular weight (LMW) fractions and in the high molecular weight (HMW) fractions. Apparently, the PSMA5 species that belong to the LMW fractions represent a mix of free monomeric subunits, its dimers and possibly a complex with PSMA1 and PSMA3 subunits. The PSMA5 species found in the HMW fraction likely correspond to the full-sized 20S proteasome and its interacting proteins (Fig. 3, A and B). Importantly, endogenous PSMA5 was preferentially di-ubiquitylated in the LMW fraction, whereas PSMA5 in the 20S proteasome HMW fraction was mostly mono-ubiquitylated (Fig. 3A). A similar pattern was observed for the ectopic PSMA5-FLAG protein with the exception that in the LMW fractions it was not only di-ubiquitylated, but also mono- and tri-ubiquitylated (Fig. 3B). Collectively, our results suggest that both endogenous and ectopic alpha-type subunits undergo ubiquitylation in cells. Moreover, the neighbouring subunits of the 20S particle likely restrict the extent of ubiquitylation for PSMA5 to only mono-ubiquitylation. On the contrary, PSMA5 in its free, or heterotrimeric form, is more susceptible to ubiquitylation.

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