CHARACTERISATION OF CASPASE-6-DEPENDENT REGULATORY MECHANISMS OF SQSTM1/p62 DROPLET-BASED AUTOPHAGY
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p62, otherwise known as SQSTM1, is the most characterised selective autophagy receptor essential for autophagic cargo recognition, capture and degradation. p62 and its cargo form liquid-liquid phase-separated droplets which operate as assembly platforms for the formation of autophagosomes. It is poorly understood how physiological and pathological conditions modulate p62 droplet-based autophagy. In this study, the effects of cellular toxicity upon the regulation of p62 droplet-based autophagy were investigated. This study discovered that the inflammatory toxicity promotes caspase-6 to cleave p62 at a novel site D256. As the C-terminal cleavage product was undetected via western blotting rendering it unstable, the attention was focused on the N-terminal cleavage product. By utilising a variety of cellular models, p62N was found to dominantly and negatively regulate p62 droplet formation. In vitro phase separation assay confirmed this finding. Caspase-6-generated p62N was also shown to interfere with autophagosome formation and protein aggregate clearance via autophagy. Moreover, caspase-6-generated p62N was found in brain tissue lysates of aged and Huntington’s disease mice. This study has discovered a novel pathway for the regulation of autophagy under certain cytotoxic stimuli. Caspase-6 activity is associated with neurodegenerative diseases such as Alzheimer’s and Huntington’s diseases. As p62 droplet autophagy is critical for protein aggregate clearance, its negative regulation by caspase-6 may play a critical role in disease pathologies.