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SQSTM1/p62 droplets play crucial roles in droplets-based macroautophagy/autophagy including selective autophagy and bulk autophagy. We observed that under several stress milieus, SQSTM1 droplets entirely colocalize with P-body markers, and these stress-induced SQSTM1 droplets contain mRNAs. We thus determined that under certain stress conditions, autophagic SQSTM1 droplets are converted to a type of enlarged P-bodies, designated SQSTM1/p62-dependent P-bodies (pd-PBs). Stress-enhanced SQSTM1 droplet formation drives the nucleation of pd-PBs through the interaction between SQSTM1 and the RNA-binding protein DDX6. Furthermore, pd-PBs sequester PYCARD, facilitating the assembly of NLRP3 inflammasomes, and in turn induce inflammation-related cytotoxicity. Our study suggests that under stress settings, autophagic SQSTM1 droplets are transformed to pd-PBs, underlining a critical role of SQSTM1 in P-body condensation.

Autophagy and ribonucleoprotein granules, such as P-bodies (PBs) and stress granules, represent vital stress responses to maintain cellular homeostasis. SQSTM1/p62 phase-separated droplets are known to play critical roles in selective autophagy; however, it is unknown whether p62 can exist as another form in addition to its autophagic droplets. Here, we found that, under stress conditions, including proteotoxicity, endotoxicity, and oxidation, autophagic p62 droplets are transformed to a type of enlarged PBs, termed p62-dependent P-bodies (pd-PBs). p62 phase separation is essential for the nucleation of pd-PBs. Mechanistically, pd-PBs are triggered by enhanced p62 droplet formation upon stress stimulation through the interactions between p62 and DDX6, a DEAD-box ATPase. Functionally, pd-PBs recruit the NLRP3 inflammasome adaptor ASC to assemble the NLRP3 inflammasome and induce inflammation-associated cytotoxicity. Our study shows that p62 droplet-to-PB transformation acts as a stress response to activate the NLRP3 inflammasome process, suggesting that persistent pd-PBs lead to NLRP3-dependent inflammation toxicity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

SQSTM1/p62, as a major autophagy receptor, forms droplets that are critical for cargo recognition, nucleation, and clearance. p62 droplets also function as liquid assembly platforms to allow the formation of autophagosomes at their surfaces. It is unknown how p62-droplet formation is regulated under physiological or pathological conditions. Here, we report that p62-droplet formation is selectively blocked by inflammatory toxicity, which induces cleavage of p62 by caspase-6 at a novel cleavage site D256, a conserved site across human, mouse, rat, and zebrafish. The N-terminal cleavage product is relatively stable, whereas the C-terminal product appears undetectable. Using a variety of cellular models, we show that the p62 N-terminal caspase-6 cleavage product (p62-N) plays a dominant-negative role to block p62-droplet formation. In vitro p62 phase separation assays confirm this observation. Dominant-negative regulation of p62-droplet formation by caspase-6 cleavage attenuates p62 droplets dependent autophagosome formation. Our study suggests a novel pathway to modulate autophagy through the caspase-6-p62 axis under certain stress stimuli., (© 2021. The Author(s).)

SQSTM1/p62 is an autophagy receptor, forming droplets to sequester intracellular polyubiquitinated cargo and mediate its delivery for autophagic clearance. SQSTM1 droplets can function as platforms to allow the formation of autophagosomes at their surfaces. It would be interesting to understand how SQSTM1-droplet formation is regulated. We have shown that inflammatory toxicity induces SQSTM1 cleavage by CASP6 at a novel cleavage site, D256. The C-terminal cleavage product is unlikely to be functional, because it is hardly detectable, possibly due to its rapid turnover. The SQSTM1 N-terminal cleavage product (SQSTM1-N) exerts a dominant-negative effect on SQSTM1-droplet production, in turn attenuating SQSTM1 droplets-based autophagosome formation. Our study suggests that the CASP6-SQSTM1 axis negatively regulates SQSTM1 droplets-based autophagy under certain stress conditions.