title: Dynamics of the degradation of ubiquitinated proteins by proteasomes and autophagy: association with sequestosome 1/p62.
abstract: Proteotoxicity resulting from accumulation of damaged/unwanted proteins contributes prominently to cellular aging and neurodegeneration. Proteasomal removal of these proteins upon covalent polyubiquitination is highly regulated. Recent reports proposed a role for autophagy in clearance of diffuse ubiquitinated proteins delivered by p62/SQSTM1. Here, we compared the turnover dynamics of endogenous ubiquitinated proteins by proteasomes and autophagy by assessing the effect of their inhibitors. Autophagy inhibitors bafilomycin A1, ammonium chloride, and 3-methyladenine failed to increase ubiquitinated protein levels. The proteasome inhibitor epoxomicin raised ubiquitinated protein levels at least 3-fold higher than the lysosomotropic agent chloroquine. These trends were observed in SK-N-SH cells under serum or serum-free conditions and in WT or Atg5(-/-) mouse embryonic fibroblasts (MEFs). Notably, chloroquine considerably inhibited proteasomes in SK-N-SH cells and MEFs. In these cells, elevation of p62/SQSTM1 was greater upon proteasome inhibition than with all autophagy inhibitors tested and was reduced in Atg5(-/-) MEFs. With epoxomicin, soluble p62/SQSTM1 associated with proteasomes and p62/SQSTM1 aggregates contained inactive proteasomes, ubiquitinated proteins, and autophagosomes. Prolonged autophagy inhibition (96 h) failed to elevate ubiquitinated proteins in rat cortical neurons, although epoxomicin did. Moreover, prolonged autophagy inhibition in cortical neurons markedly increased p62/SQSTM1, supporting its degradation mainly by autophagy and not by proteasomes. In conclusion, we clearly demonstrate that pharmacologic or genetic inhibition of autophagy fails to elevate ubiquitinated proteins unless the proteasome is affected. We also provide strong evidence that p62/SQSTM1 associates with proteasomes and that autophagy degrades p62/SQSTM1. Overall, the function of p62/SQSTM1 in the proteasomal pathway and autophagy requires further elucidation.
authors.affiliation: Department of Biological Sciences, Hunter College of City University of New York, New York, New York 10065, USA.
authors.forename: Natura
authors.initials: N
authors.lastname: Myeku
authors.forename: Maria E
authors.initials: ME
authors.lastname: Figueiredo-Pereira
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001343:Autophagy / Q000187:drug effects; D000071187:Autophagy-Related Protein 5; D002457:Cell Extracts; D045744:Cell Line, Tumor; D002540:Cerebral Cortex / Q000166:cytology; D003374:Coumarins / Q000378:metabolism; D005260:Female; D006801:Humans; D006868:Hydrolysis / Q000187:drug effects; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000172:deficiency; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D011247:Pregnancy; D011480:Protease Inhibitors / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D061988:Proteasome Inhibitors; D055503:Protein Multimerization / Q000187:drug effects; D020836:Protein Structure, Quaternary; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D000071456:Sequestosome-1 Protein; D013997:Time Factors; D057149:Ubiquitinated Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C495773:Atg5 protein, mouse; D000071187:Autophagy-Related Protein 5; D002457:Cell Extracts; D003374:Coumarins; D008869:Microtubule-Associated Proteins; C114703:N-succinyl-leucyl-leucyl-valyl-tyrosyl-7-amino-4-methylcoumarin; D009842:Oligopeptides; D011480:Protease Inhibitors; D061988:Proteasome Inhibitors; C099718:SQSTM1 protein, human; D000071456:Sequestosome-1 Protein; D057149:Ubiquitinated Proteins; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease