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.

Moiseeva, T., Bottrill, A., Melino, G., Barlev, N. (2013). DNA damage-induced ubiquitylation of proteasome controls its proteolytic activity. ONCOTARGET, 4(9), 1338-1348.

DNA damage-induced ubiquitylation of proteasome controls its proteolytic activity

MELINO, GENNARO;
2013-09-01

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.
set-2013
Pubblicato
Rilevanza internazionale
Articolo
Esperti anonimi
Settore BIO/10 - BIOCHIMICA
Settore BIO/11 - BIOLOGIA MOLECOLARE
English
Phosphorylation; DNA Damage; Humans; Protein Processing, Post-Translational; Proteasome Endopeptidase Complex; Ubiquitination; Cell Line, Tumor; K562 Cells; Antibiotics, Antineoplastic; Doxorubicin
Moiseeva, T., Bottrill, A., Melino, G., Barlev, N. (2013). DNA damage-induced ubiquitylation of proteasome controls its proteolytic activity. ONCOTARGET, 4(9), 1338-1348.
Moiseeva, T; Bottrill, A; Melino, G; Barlev, N
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2108/102687
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