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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.Mutations in the p62 PB1 domain that abolish self-oligomerization also abolished crosslinked oligomer formation.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 PB1 mutation on p62 crosslinking by verteporfin.p62−/− MEF cells expressing GFP-p62 wt or GFP-p62 K7A/D69 or p62+/+ MEF cells were exposed to 0.1% DMSO or 10 µM verteporfin for 4 h in complete medium. Cell lysates were immunoblotted for p62 and β-tubulin. The image presented is representative of at least 3 independent experiments.
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pone-0114964-g007: Effect of PB1 mutation on p62 crosslinking by verteporfin.p62−/− MEF cells expressing GFP-p62 wt or GFP-p62 K7A/D69 or p62+/+ MEF cells were exposed to 0.1% DMSO or 10 µM verteporfin for 4 h in complete medium. Cell lysates were immunoblotted for p62 and β-tubulin. The image presented is representative of at least 3 independent experiments.

Mentions: The N-terminal PB1 domain of p62 mediates its interaction with other proteins containing PB1 domains to form heterodimers and homo-oligomers [11], [12], [15]. The PB1 domain of p62 is unique in that it contains both an acidic and a basic interaction surface that enables p62 to self-oligomerize in a front-to-back orientation, thus forming homotypic arrays [11], [14], [15], a property that is critical for targeting p62 to the autophagosome formation site [61]. We tested whether a p62 PB1 mutant (K7A/D69A) that disrupts p62 self-oligomerization can form high-MW p62 in the presence of verteporfin. Using the Tet-On system, GFP-p62 K7A/D69A and GFP-p62 wt were expressed in p62−/− MEF cells and p62 was monitored after 4 h treatment with verteporfin. High-MW GFP-p62 was observed only in the wild-type GFP-p62 expressing cells, and the amount of high-MW GFP-p62 increased significantly after verteporfin treatment (Fig. 7). By contrast, no high-MW p62 was detected in cells expressing the PB1 mutant, even after exposure to verteporfin (Fig. 7). As expected, verteporfin also generated high-MW endogenous p62 in p62+/+ MEF cells (Fig. 7). These results demonstrate that verteporfin-induced production of high-MW p62 crosslink products is dependent on p62 oligomerization through the PB1 domain. Therefore, it is likely that crosslinking amongst p62 monomers occurs via PB1 domain modification by verteporfin.


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 PB1 mutation on p62 crosslinking by verteporfin.p62−/− MEF cells expressing GFP-p62 wt or GFP-p62 K7A/D69 or p62+/+ MEF cells were exposed to 0.1% DMSO or 10 µM verteporfin for 4 h in complete medium. Cell lysates were immunoblotted for p62 and β-tubulin. The image presented is 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-g007: Effect of PB1 mutation on p62 crosslinking by verteporfin.p62−/− MEF cells expressing GFP-p62 wt or GFP-p62 K7A/D69 or p62+/+ MEF cells were exposed to 0.1% DMSO or 10 µM verteporfin for 4 h in complete medium. Cell lysates were immunoblotted for p62 and β-tubulin. The image presented is representative of at least 3 independent experiments.
Mentions: The N-terminal PB1 domain of p62 mediates its interaction with other proteins containing PB1 domains to form heterodimers and homo-oligomers [11], [12], [15]. The PB1 domain of p62 is unique in that it contains both an acidic and a basic interaction surface that enables p62 to self-oligomerize in a front-to-back orientation, thus forming homotypic arrays [11], [14], [15], a property that is critical for targeting p62 to the autophagosome formation site [61]. We tested whether a p62 PB1 mutant (K7A/D69A) that disrupts p62 self-oligomerization can form high-MW p62 in the presence of verteporfin. Using the Tet-On system, GFP-p62 K7A/D69A and GFP-p62 wt were expressed in p62−/− MEF cells and p62 was monitored after 4 h treatment with verteporfin. High-MW GFP-p62 was observed only in the wild-type GFP-p62 expressing cells, and the amount of high-MW GFP-p62 increased significantly after verteporfin treatment (Fig. 7). By contrast, no high-MW p62 was detected in cells expressing the PB1 mutant, even after exposure to verteporfin (Fig. 7). As expected, verteporfin also generated high-MW endogenous p62 in p62+/+ MEF cells (Fig. 7). These results demonstrate that verteporfin-induced production of high-MW p62 crosslink products is dependent on p62 oligomerization through the PB1 domain. Therefore, it is likely that crosslinking amongst p62 monomers occurs via PB1 domain modification by verteporfin.

Bottom Line: Verteporfin was recently found to inhibit autophagosome formation by an unknown mechanism that does not require exposure to light.Mutations in the p62 PB1 domain that abolish self-oligomerization also abolished crosslinked oligomer formation.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