P62/SQSTM1 upregulates NQO1 transcription via Nrf2/Keap1a signaling pathway to resist microcystins-induced oxidative stress in freshwater mussel Cristaria plicata.

Microcystins (MCs) are the most frequent and widely distributed type of cyanotoxin in aquatic systems, and they cause an imbalance of the body's oxidative system. In a previous experiment, we demonstrated that the mollusk Cristaria plicata can protect against MC-induced oxidative damage through the nuclear factor erythroid 2-related factor 2(Nrf2)/Kelch-like epichlorohydrin-related protein-1 (Keap1) pathway. Here, we evaluated whether selective autophagy affects the Nrf2/Keap1a anti-oxidative stress pathway in C. plicata. Full-length cDNA sequences of p62/SQSTM1 from C. plicata (Cpp62) were divided into 2484 bp fragments. From N-terminal to C-terminal, the amino acid sequence of Cpp62 contained PB1 (Phox and Bem1p domain), ZNF (zinc finger domain) chain, LIR (LC3 interacting region) and UBA (ubiquitin-associated domain) domains, but not the KIR (Keap1 interacting region) domain. We confirmed that Cpp62 did not bind to CpKeap1a in vitro, and the relative level of Cpp62 was the highest in the hepatopancreas. Moreover, MCs significantly upregulated the mRNA and protein levels of Cpp62 in the hepatopancreas after CpKeap1a knockdown, whereas Nrf2 upregulated the transcription levels of Cpp62, suggesting that MCs increased Cpp62 expression via the Nrf2/Keap1a signaling pathway. Moreover, Cpp62 and CpNrf2 proteins have a strong affinity for the NQO1 promoter, but MCs inhibited the ability of CpNrf2 and Cpp62 to upregulate luciferase activity. The results show that Nrf2 and the p62 protein induced p62 expression by binding to ARE (antioxidant response element) sequences in the p62 promoter of C. plicata, thereby promoting p62 to resist MC-induced oxidative stress. Therefore, we speculate that MCs induce p62-dependent autophagy in C. plicata, resulting in the inhibition of Nrf2 transcription and Cpp62 promoter activity. These findings help to reveal the mechanism by which the p62-Nrf2/Keap1 pathway mitigates MC-induced oxidative damage in mussels.