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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

High-molecular weight p62 induced by singlet oxygen production.(A) 50 ng GST-p62 was incubated with 0.05% DMSO, 5 µM verteporfin, or 5 µM rose bengal for 1 h at 4°C or 37°C in the dark. (B) Immunoprecipitated p62 from untreated BxPC-3 cells was exposed to 0.1% DMSO, 10 µM verteporfin, or 10 µM rose bengal for 30 min in the dark at 4°C or 37°C or to (C) 0.1% DMSO, 10 µM verteporfin, or 10 µM rose bengal in the dark at 37°C with or without 20 mM histidine. Reactions were immunoblotted for p62. (D) 26 ng purified His-p62 was incubated with 0.05% DMSO, 5 µM verteporfin, or 5 µM rose bengal for 1 h at 4°C or 37°C in the dark or (E) 50 ng purified GST-p62 was incubated with 0.1% DMSO, 10 µM verteporfin, or 10 µM rose bengal for 30 min at 37°C in the dark. Samples were derivatized with DNPH, subjected to SDS-PAGE, and immunoblotted with anti-DNP antibody solution according to the Oxyblot kit manufacturer’s instructions. All images presented are representative of at least 3 independent experiments.
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pone-0114964-g003: High-molecular weight p62 induced by singlet oxygen production.(A) 50 ng GST-p62 was incubated with 0.05% DMSO, 5 µM verteporfin, or 5 µM rose bengal for 1 h at 4°C or 37°C in the dark. (B) Immunoprecipitated p62 from untreated BxPC-3 cells was exposed to 0.1% DMSO, 10 µM verteporfin, or 10 µM rose bengal for 30 min in the dark at 4°C or 37°C or to (C) 0.1% DMSO, 10 µM verteporfin, or 10 µM rose bengal in the dark at 37°C with or without 20 mM histidine. Reactions were immunoblotted for p62. (D) 26 ng purified His-p62 was incubated with 0.05% DMSO, 5 µM verteporfin, or 5 µM rose bengal for 1 h at 4°C or 37°C in the dark or (E) 50 ng purified GST-p62 was incubated with 0.1% DMSO, 10 µM verteporfin, or 10 µM rose bengal for 30 min at 37°C in the dark. Samples were derivatized with DNPH, subjected to SDS-PAGE, and immunoblotted with anti-DNP antibody solution according to the Oxyblot kit manufacturer’s instructions. All images presented are representative of at least 3 independent experiments.

Mentions: To test this hypothesis, we examined the effects of rose bengal, a widely used chemical dye that produces singlet oxygen and is structurally unrelated to verteporfin [41], [43], [48]. GST-p62 was exposed to 5 µM rose bengal or 5 µM verteporfin for 1 h at 4°C or 37°C in the dark (Fig. 3A). A small amount of high-MW GST-p62 was observed in the DMSO control after exposure to heat, again demonstrating that heat alone can generate some high-MW p62 in vitro. Exposure of recombinant p62 to rose bengal at both 4°C and 37°C induced high-MW p62 forms similar to those generated by verteporfin under the same conditions, and combining rose bengal treatment with heat strongly stimulated the formation of high-MW GST-p62 (Fig. 3A). The fact that significant amounts of high-MW GST-p62 forms were generated in reactions containing only recombinant protein, buffered salt, a photosensitizer, and heat indicates that singlet oxygen production causes p62 oxidation and subsequent crosslinking. The in vitro effect of rose bengal was also demonstrated on cellular p62. p62 was immunoprecipitated from untreated cells and then treated with 10 µM rose bengal or 10 µM verteporfin for 30 min at 4°C or 37°C in the dark. Neither had any effect at 4°C, but at 37°C immunoprecipitated p62 was modified by rose bengal to generate a range of high-MW forms as seen with verteporfin treatment (Fig. 3B).


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)

High-molecular weight p62 induced by singlet oxygen production.(A) 50 ng GST-p62 was incubated with 0.05% DMSO, 5 µM verteporfin, or 5 µM rose bengal for 1 h at 4°C or 37°C in the dark. (B) Immunoprecipitated p62 from untreated BxPC-3 cells was exposed to 0.1% DMSO, 10 µM verteporfin, or 10 µM rose bengal for 30 min in the dark at 4°C or 37°C or to (C) 0.1% DMSO, 10 µM verteporfin, or 10 µM rose bengal in the dark at 37°C with or without 20 mM histidine. Reactions were immunoblotted for p62. (D) 26 ng purified His-p62 was incubated with 0.05% DMSO, 5 µM verteporfin, or 5 µM rose bengal for 1 h at 4°C or 37°C in the dark or (E) 50 ng purified GST-p62 was incubated with 0.1% DMSO, 10 µM verteporfin, or 10 µM rose bengal for 30 min at 37°C in the dark. Samples were derivatized with DNPH, subjected to SDS-PAGE, and immunoblotted with anti-DNP antibody solution according to the Oxyblot kit manufacturer’s instructions. All images presented are representative of at least 3 independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4262463&req=5

pone-0114964-g003: High-molecular weight p62 induced by singlet oxygen production.(A) 50 ng GST-p62 was incubated with 0.05% DMSO, 5 µM verteporfin, or 5 µM rose bengal for 1 h at 4°C or 37°C in the dark. (B) Immunoprecipitated p62 from untreated BxPC-3 cells was exposed to 0.1% DMSO, 10 µM verteporfin, or 10 µM rose bengal for 30 min in the dark at 4°C or 37°C or to (C) 0.1% DMSO, 10 µM verteporfin, or 10 µM rose bengal in the dark at 37°C with or without 20 mM histidine. Reactions were immunoblotted for p62. (D) 26 ng purified His-p62 was incubated with 0.05% DMSO, 5 µM verteporfin, or 5 µM rose bengal for 1 h at 4°C or 37°C in the dark or (E) 50 ng purified GST-p62 was incubated with 0.1% DMSO, 10 µM verteporfin, or 10 µM rose bengal for 30 min at 37°C in the dark. Samples were derivatized with DNPH, subjected to SDS-PAGE, and immunoblotted with anti-DNP antibody solution according to the Oxyblot kit manufacturer’s instructions. All images presented are representative of at least 3 independent experiments.
Mentions: To test this hypothesis, we examined the effects of rose bengal, a widely used chemical dye that produces singlet oxygen and is structurally unrelated to verteporfin [41], [43], [48]. GST-p62 was exposed to 5 µM rose bengal or 5 µM verteporfin for 1 h at 4°C or 37°C in the dark (Fig. 3A). A small amount of high-MW GST-p62 was observed in the DMSO control after exposure to heat, again demonstrating that heat alone can generate some high-MW p62 in vitro. Exposure of recombinant p62 to rose bengal at both 4°C and 37°C induced high-MW p62 forms similar to those generated by verteporfin under the same conditions, and combining rose bengal treatment with heat strongly stimulated the formation of high-MW GST-p62 (Fig. 3A). The fact that significant amounts of high-MW GST-p62 forms were generated in reactions containing only recombinant protein, buffered salt, a photosensitizer, and heat indicates that singlet oxygen production causes p62 oxidation and subsequent crosslinking. The in vitro effect of rose bengal was also demonstrated on cellular p62. p62 was immunoprecipitated from untreated cells and then treated with 10 µM rose bengal or 10 µM verteporfin for 30 min at 4°C or 37°C in the dark. Neither had any effect at 4°C, but at 37°C immunoprecipitated p62 was modified by rose bengal to generate a range of high-MW forms as seen with verteporfin treatment (Fig. 3B).

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