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.

Ambient Light Promotes Selective Subcellular Proteotoxicity after Endogenous and Exogenous Porphyrinogenic Stress

“…With regard to autophagy, the apoptosis that is induced by photoactivation of the benzoporphyrin derivative, verteporfin, becomes enhanced in Hepa1 cells upon knockdown of the Atg7 (29). Verteporfin causes similar aggregation of the ubiquitin-binding protein p62 in cultured cells, (30), similar to what we previously observed in two porphyria experimental models (11). All isolations and SDS-PAGE analysis were done under ambient light or dark room conditions.…”

Tumor-selective proteotoxicity of verteporfin inhibits colon cancer progression independently of YAP1

“…Structurally, VP belongs to the porphyrin class of drugs. Intracellular porphyrins can cross-link proteins, leading to formation of high-molecular weight oligomers of proteins (33, 37). Therefore, to assess whether the loss of STAT3 signaling by VP was due to oligomerization of STAT3, HCT-116 cells were treated with VP in a dose- and time-dependent manner.…”

“…VP and other related porphyrin molecules induce high molecular oligomers (33, 37). However, hemin, which also inhibits YAP1, could not form protein oligomers (18).…”

YAP/TAZ Are Mechanoregulators of TGF-β-Smad Signaling and Renal Fibrogenesis

“…We show that verteporfin reduces YAP and TAZ mRNA levels, suggesting that decreased synthesis of YAP and TAZ may at least partially explain our findings. However, because recent reports have suggested that verteporfin may also cause protein aggregation through oxidative crosslinking and enhanced degradation, 38,39 other effects of this drug may also play a role.…”

“…Recent reports have also suggested, however, that verteporfin may directly enhance the degradation of certain proteins through oxidative crosslinking and oligomerization. 38,39 Thus, verteporfin may reduce Smad2/3 levels via multiple mechanisms, some of which are likely YAP/TAZ dependent, whereas others may be YAP/TAZ independent.…”