Parkin-mediated ubiquitination of hif-α modulates hypoxia signaling in Megalobrama amblycephala

Megalobrama amblycephala is an economically important fish species in Chinese aquaculture, but its high sensitivity to hypoxia poses a major challenge for farming. This study aims to investigate the role of the E3 ubiquitin ligase parkin in regulating hypoxia signaling in fish. Under hypoxic stress, parkin expression was significantly upregulated in oxygen-sensitive tissues (brain and gills) and embryos of M. amblycephala, consistent with the expression of hypoxia-responsive genes. Ma-parkin was found to exhibit high conservation in its key functional domains. Mechanistically, it directly interacts with Ma-hif-α, promoting their ubiquitination and proteasomal degradation via E3 ligase activity, thereby suppressing hif-mediated transcriptional activation. Critical residues T74 and C333 were identified as essential for this activity; mutations at these sites impaired Ma-hif-α degradation and ubiquitination, as well as HRE transactivation. Furthermore, we found that Ma-hif-α can regulate the transcription of parkin, forming a negative feedback loop to fine-tune the hypoxic response. Our study demonstrates that parkin serves as a key negative regulator of the hypoxia signaling pathway in M. amblycephala, operating through a finely tuned feedback mechanism. The E3 ubiquitin ligase activity of parkin, dependent on critical residues T74 and C333, directly mediates hif-α ubiquitination and degradation. The reciprocal regulation between hif-α and parkin forms a regulatory circuit that modulates the hypoxic response. These findings not only reveal a conserved adaptive mechanism to hypoxia in fish but also identify specific molecular targets for genetic improvement of hypoxia tolerance in aquaculture species.