title: Induction of Covalently Crosslinked p62 Oligomers with Reduced Binding to Polyubiquitinated Proteins by the Autophagy Inhibitor Verteporfin.
abstract: Autophagy is a cellular catabolic process responsible for the degradation of cytoplasmic constituents, including organelles and long-lived proteins, that helps maintain cellular homeostasis and protect against various cellular stresses. Verteporfin is a benzoporphyrin derivative used clinically in photodynamic therapy to treat macular degeneration. Verteporfin was recently found to inhibit autophagosome formation by an unknown mechanism that does not require exposure to light. We report that verteporfin directly targets and modifies p62, a scaffold and adaptor protein that binds both polyubiquitinated proteins destined for degradation and LC3 on autophagosomal membranes. Western blotting experiments revealed that exposure of cells or purified p62 to verteporfin causes the formation of covalently crosslinked p62 oligomers by a mechanism involving low-level singlet oxygen production. Rose bengal, a singlet oxygen producer structurally unrelated to verteporfin, also produced crosslinked p62 oligomers and inhibited autophagosome formation. Co-immunoprecipitation experiments demonstrated that crosslinked p62 oligomers retain their ability to bind to LC3 but show defective binding to polyubiquitinated proteins. Mutations in the p62 PB1 domain that abolish self-oligomerization also abolished crosslinked oligomer formation. Interestingly, small amounts of crosslinked p62 oligomers were detected in untreated cells, and other groups noted the accumulation of p62 forms with reduced SDS-PAGE mobility in cellular and animal models of oxidative stress and aging. These data indicate that p62 is particularly susceptible to oxidative crosslinking and lead us to propose a model whereby oxidized crosslinked p62 oligomers generated rapidly by drugs like verteporfin or over time during the aging process interfere with autophagy.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Donohue
authors.affiliation: Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Aruna D
authors.initials: AD
authors.lastname: Balgi
authors.affiliation: Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan.
authors.forename: Masaaki
authors.initials: M
authors.lastname: Komatsu
authors.affiliation: Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Michel
authors.initials: M
authors.lastname: Roberge
mesh_terms: D000071182:Autophagosomes / Q000378:metabolism; D001343:Autophagy / Q000187:drug effects*; D006801:Humans; D061986:MCF-7 Cells; D008856:Microscopy, Fluorescence; D010588:Phagosomes / Q000187:drug effects; D011166:Porphyrins / Q000494:pharmacology*; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012395:Rose Bengal / Q000494:pharmacology; D054875:Ubiquitination; D000077362:Verteporfin
chemical_list: D011166:Porphyrins; D016271:Proto-Oncogene Proteins c-myc; D011994:Recombinant Proteins; D000077362:Verteporfin; D012395:Rose Bengal