RESUMO
Oxidative stress induced by arsenite [As(III)] affects protein folding and results in increased levels of misfolded proteins or protein aggregates. Accumulation of misfolded protein aggregates may act as a cue signal for the oligomerization of the autophagic adaptor protein p62, which facilitates recognition of misfolded protein aggregates that are polyubiquitinated with K63 linkages. However, as the autophagic flux is impaired under exposure to As(III), p62 oligomers cannot be cleared by autophagy and accumulate as aggregates with Keap1. This results in the sequestration of Keap1 and the stabilization of Nrf2, which activates the non-canonical Nrf2-Keap1 pathway as an antioxidant response. In this study, we found that polyubiquitination of p62 itself increased upon exposure to As(III) to prevent further oligomerization of p62 and to increase the availability of functional free monomeric p62. We also found that monomeric p62 could also interact with ubiquitinated proteins and that the forced dimerization of p62 was sufficient to increase the interactions with ubiquitinated proteins, probably polyubiquitinated with K63 linkages. Upon exposure to As(III), (1) inability to form oligomeric p62 because of a mutation in the PB1 dimerization domain, or (2) reduced capability to generate monomeric p62 owing to diminished polyubiquitination of p62 itself, resulted in reduced viability of cells. Therefore, upon exposure to As(III), p62 initially needs to form oligomers to activate an antioxidant response pathway. Subsequently, p62 is polyubiquitinated to prevent further oligomerization and ensure the availability of free p62 monomers. We propose that the polyubiquitination of p62 under exposure to As(III) plays an important role in overcoming the impaired autophagic flux by regulating the oligomerization status of p62.
Assuntos
Arsenitos/toxicidade , Autofagia/efeitos dos fármacos , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Fator 2 Relacionado a NF-E2/genética , Estresse Oxidativo/efeitos dos fármacos , Proteína Sequestossoma-1/genética , Animais , Sobrevivência Celular/efeitos dos fármacos , Embrião de Mamíferos , Fibroblastos , Regulação da Expressão Gênica , Células HEK293 , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Camundongos , Fator 2 Relacionado a NF-E2/metabolismo , Cultura Primária de Células , Dobramento de Proteína/efeitos dos fármacos , Multimerização Proteica , Proteína Sequestossoma-1/metabolismo , Transdução de Sinais , UbiquitinaçãoRESUMO
The polyubiquitin gene Ubc is upregulated under oxidative stress induced by arsenite [As(III)]. However, the detailed mechanism of Ubc upregulation and the exact role of ubiquitin (Ub) to protect cells against As(III)-induced toxicity remain unknown. Here, we found that Ubc-/- mouse embryonic fibroblasts (MEFs) exhibited reduced viability under As(III) exposure, although the Nrf2-Keap1 pathway was activated as a cytoprotective response. Intriguingly, due to the reduced polyubiquitination and delayed onset of degradation of Nrf2 in Ubc-/- MEFs, the basal expression levels of Nrf2 target genes were elevated. As(III)-induced accumulation of Ub conjugates occurred in an Nrf2-independent manner, probably due to cellular stress conditions, including reduced proteasomal activity. Increased cellular Ub levels were essential to polyubiquitinate misfolded proteins generated under As(III) exposure and to degrade them by the proteasome. However, when cellular Ub levels decreased, these misfolded proteins were not efficiently polyubiquitinated, but rather accumulated as large protein aggregates inside the cells, causing cytotoxicity. Furthermore, increased activity of the autophagic pathway to clear these aggregates was not observed in Ubc-/- MEFs. Therefore, reduced viability of Ubc-/- MEFs under As(III) exposure may not be due to dysregulation of the Nrf2-Keap1 pathway, but mostly to reduced efficacy to polyubiquitinate and degrade misfolded protein aggregates.
