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Induction of Covalently Crosslinked p62 Oligomers with Reduced Binding to Polyubiquitinated Proteins by the Autophagy Inhibitor Verteporfin.

Donohue E, Balgi AD, Komatsu M, Roberge M - PLoS ONE (2014)

Bottom Line: Verteporfin was recently found to inhibit autophagosome formation by an unknown mechanism that does not require exposure to light.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada.

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.

No MeSH data available.


Related in: MedlinePlus

Effect of verteporfin on p62 in cells.(A) MCF-7 EGFP-LC3 cells were exposed to 0.1% DMSO, 100 nM bafilomycin A1, or 10 µM verteporfin for 8 h in the presence or absence of serum and cell lysates were immunoblotted for p62. β-tubulin was monitored as a loading control. (B) MCF-7 EGFP-LC3 cells were exposed to 0.1% DMSO or 10 µM verteporfin for 4 h. Indicated amounts of each lysate were immunoprecipitated with anti-p62 antibody and analyzed by western blot. (C) MCF-7 EGFP-LC3 cells were exposed to 0.1% DMSO, 10 µM verteporfin, or 100 nM bafilomycin A1 for 4 h in complete medium. The cells were fixed and stained with p62 antibody, and images were acquired by confocal microscopy. Scale bar, 10 µm. (D) MCF-7 EGFP-LC3 cells were exposed to 0.1% DMSO, 100 nM bafilomycin A1, or 10 µM verteporfin for 4 h in complete medium. Cell lysates were collected, quantified, and normalized in the presence or absence of overhead laboratory light as indicated. 0.5 µg of lysate was used to examine p62 levels by western blotting. All images presented are representative of at least 3 independent experiments.
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pone-0114964-g001: Effect of verteporfin on p62 in cells.(A) MCF-7 EGFP-LC3 cells were exposed to 0.1% DMSO, 100 nM bafilomycin A1, or 10 µM verteporfin for 8 h in the presence or absence of serum and cell lysates were immunoblotted for p62. β-tubulin was monitored as a loading control. (B) MCF-7 EGFP-LC3 cells were exposed to 0.1% DMSO or 10 µM verteporfin for 4 h. Indicated amounts of each lysate were immunoprecipitated with anti-p62 antibody and analyzed by western blot. (C) MCF-7 EGFP-LC3 cells were exposed to 0.1% DMSO, 10 µM verteporfin, or 100 nM bafilomycin A1 for 4 h in complete medium. The cells were fixed and stained with p62 antibody, and images were acquired by confocal microscopy. Scale bar, 10 µm. (D) MCF-7 EGFP-LC3 cells were exposed to 0.1% DMSO, 100 nM bafilomycin A1, or 10 µM verteporfin for 4 h in complete medium. Cell lysates were collected, quantified, and normalized in the presence or absence of overhead laboratory light as indicated. 0.5 µg of lysate was used to examine p62 levels by western blotting. All images presented are representative of at least 3 independent experiments.

Mentions: p62 is a multifunctional protein commonly used to monitor autophagic flux [21]. As an adapter protein, p62 tethers polyubiquitinated proteins destined for degradation to the membrane of nascent autophagosomes via LC3 and it is degraded along with its cargo [22], [23]. In a recent study on the therapeutic effect of verteporfin in a pancreatic cancer xenograft model, we observed that verteporfin caused the appearance of an altered form of p62 that showed reduced electrophoretic mobility in denaturing SDS-PAGE conditions, which we denoted as high-MW p62 [7]. To characterize this unusual form of p62 and to investigate the mechanism of action of verteporfin, we first compared its effect on p62 levels to those of bafilomycin A1, a v-ATPase inhibitor that prevents autophagosomal degradation. When MCF-7 cells, which display low basal autophagy in complete cell culture medium containing serum [5], were exposed to 100 nM bafilomycin A1 for 8 h, there was no visible increase in cellular p62 levels compared to untreated cells (Fig. 1A), confirming low basal autophagy. Exposing cells to serum-free medium to stimulate autophagic flux caused a noticeable decrease in p62 levels that was prevented by co-incubation with 100 nM bafilomycin A1 (Fig. 1A), demonstrating degradation of p62 by autophagy under these conditions.


Induction of Covalently Crosslinked p62 Oligomers with Reduced Binding to Polyubiquitinated Proteins by the Autophagy Inhibitor Verteporfin.

Donohue E, Balgi AD, Komatsu M, Roberge M - PLoS ONE (2014)

Effect of verteporfin on p62 in cells.(A) MCF-7 EGFP-LC3 cells were exposed to 0.1% DMSO, 100 nM bafilomycin A1, or 10 µM verteporfin for 8 h in the presence or absence of serum and cell lysates were immunoblotted for p62. β-tubulin was monitored as a loading control. (B) MCF-7 EGFP-LC3 cells were exposed to 0.1% DMSO or 10 µM verteporfin for 4 h. Indicated amounts of each lysate were immunoprecipitated with anti-p62 antibody and analyzed by western blot. (C) MCF-7 EGFP-LC3 cells were exposed to 0.1% DMSO, 10 µM verteporfin, or 100 nM bafilomycin A1 for 4 h in complete medium. The cells were fixed and stained with p62 antibody, and images were acquired by confocal microscopy. Scale bar, 10 µm. (D) MCF-7 EGFP-LC3 cells were exposed to 0.1% DMSO, 100 nM bafilomycin A1, or 10 µM verteporfin for 4 h in complete medium. Cell lysates were collected, quantified, and normalized in the presence or absence of overhead laboratory light as indicated. 0.5 µg of lysate was used to examine p62 levels by western blotting. All images presented are representative of at least 3 independent experiments.
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Related In: Results  -  Collection

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pone-0114964-g001: Effect of verteporfin on p62 in cells.(A) MCF-7 EGFP-LC3 cells were exposed to 0.1% DMSO, 100 nM bafilomycin A1, or 10 µM verteporfin for 8 h in the presence or absence of serum and cell lysates were immunoblotted for p62. β-tubulin was monitored as a loading control. (B) MCF-7 EGFP-LC3 cells were exposed to 0.1% DMSO or 10 µM verteporfin for 4 h. Indicated amounts of each lysate were immunoprecipitated with anti-p62 antibody and analyzed by western blot. (C) MCF-7 EGFP-LC3 cells were exposed to 0.1% DMSO, 10 µM verteporfin, or 100 nM bafilomycin A1 for 4 h in complete medium. The cells were fixed and stained with p62 antibody, and images were acquired by confocal microscopy. Scale bar, 10 µm. (D) MCF-7 EGFP-LC3 cells were exposed to 0.1% DMSO, 100 nM bafilomycin A1, or 10 µM verteporfin for 4 h in complete medium. Cell lysates were collected, quantified, and normalized in the presence or absence of overhead laboratory light as indicated. 0.5 µg of lysate was used to examine p62 levels by western blotting. All images presented are representative of at least 3 independent experiments.
Mentions: p62 is a multifunctional protein commonly used to monitor autophagic flux [21]. As an adapter protein, p62 tethers polyubiquitinated proteins destined for degradation to the membrane of nascent autophagosomes via LC3 and it is degraded along with its cargo [22], [23]. In a recent study on the therapeutic effect of verteporfin in a pancreatic cancer xenograft model, we observed that verteporfin caused the appearance of an altered form of p62 that showed reduced electrophoretic mobility in denaturing SDS-PAGE conditions, which we denoted as high-MW p62 [7]. To characterize this unusual form of p62 and to investigate the mechanism of action of verteporfin, we first compared its effect on p62 levels to those of bafilomycin A1, a v-ATPase inhibitor that prevents autophagosomal degradation. When MCF-7 cells, which display low basal autophagy in complete cell culture medium containing serum [5], were exposed to 100 nM bafilomycin A1 for 8 h, there was no visible increase in cellular p62 levels compared to untreated cells (Fig. 1A), confirming low basal autophagy. Exposing cells to serum-free medium to stimulate autophagic flux caused a noticeable decrease in p62 levels that was prevented by co-incubation with 100 nM bafilomycin A1 (Fig. 1A), demonstrating degradation of p62 by autophagy under these conditions.

Bottom Line: Verteporfin was recently found to inhibit autophagosome formation by an unknown mechanism that does not require exposure to light.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada.

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.

No MeSH data available.


Related in: MedlinePlus