Hypothesis

The selectivity hierarchy of autophagy is not merely a passive reflection of cargo receptor abundance but is actively tuned by a redox‑sensitive switch on the ubiquitin‑binding protein p62/SQSTM1. Organelle‑specific reactive oxygen species (ROS) fluxes generate distinct cysteine oxidation patterns on p62 that modulate its affinity for ubiquitinated substrates, thereby determining whether the isolation membrane preferentially engulfs ER, mitochondria, or protein aggregates. In young cells, fasting‑induced ROS bursts from mitochondria favor oxidation of p62 Cys105, enhancing its binding to ER‑associated ubiquitin conjugates and promoting early ERophagy. With age, mitochondrial ROS buffering declines, shifting the redox environment toward a more oxidized state that constitutively modifies p62 Cys258, reducing ER affinity and increasing preference for cytosolic protein aggregates. This rewiring of the p62 redox switch disrupts the canonical autophagy triage, causing premature clearance of soluble aggregates while sparing damaged ER, ultimately triggering ER stress‑driven senescence.

Mechanistic Basis

• p62 contains multiple redox‑reactive cysteines (Cys105, Cys258, Cys269) that can form sulfenic acid or disulfide modifications in response to H₂O₂ • [8]
• Oxidation of Cys105 increases the ubiquitin‑associated (UBA) domain’s binding kinetics for K63‑linked chains enriched on ER‑resident misfolded proteins • (inferred from structural studies of UBA domains)
• Persistent oxidation of Cys258 sterically hinders the Phox and Bem1p (PB1) oligomerization domain, favoring p62 monomers that preferentially bind p97/VCP‑associated aggregates • [6]
• During fasting, mitochondrial ROS spikes transiently oxidize Cys105, ticking the hierarchy toward ERophagy; as fasting prolongs, cytosolic ROS from NADPH oxidases dominate, shifting the signal to Cys258 and promoting aggrephagy.

Predictions & Experimental Design

1. ROS‑specific p62 mutants – Generate knock‑in cells expressing C105S (cannot be oxidized) or C258S (constitutively reduced). Monitor organelle turnover using mt‑Keima, ER‑Keima, and aggregate‑Keima reporters during 12‑, 24‑, and 36‑h fasts. Prediction: C105S cells will show delayed ERophagy and excess aggregate accumulation; C258S cells will exhibit premature aggrephagy and ER stress markers (BiP, CHOP).
2. Redox biosensor – Express a roGFP2‑Orp1 targeted to the cytosol and mitochondrial matrix to map ROS fluxes alongside p62 oxidation state (detected via biotin‑switch assay). Expect a temporal correlation: early mitochondrial ROS peak ↔ Cys105 oxidation ↔ ER‑Keima flux; late cytosolic ROS peak ↔ Cys258 oxidation ↔ aggregate‑Keima flux.
3. In vivo aging test – Treat young and old mice with the ROS scavenger MitoQ or NADPH oxidase inhibitor GKT137831 during intermittent fasting. Measure p62 cysteine oxidation (Western blot with anti‑sulfenic antibody) and tissue‑specific autophagy hierarchy. Prediction: In old mice, MitoQ will restore youthful Cys105 oxidation pattern, rescuing ERophagy and improving glucose tolerance.

Implications

If the p62 redox switch governs autophagy hierarchy, then age‑related loss of substrate selectivity—not merely reduced autophagic flux—drives proteostatic collapse. Therapeutically, targeting specific p62 cysteines with small‑molecule redox modulators could reinstate the proper triage order, extending healthspan without globally up‑regulating autophagy, which risks deleterious self‑consumption.

References [1] https://pmc.ncbi.nlm.nih.gov/articles/PMC4871809/ [2] https://hms.harvard.edu/news/self-eating-decisions [3] https://pmc.ncbi.nlm.nih.gov/articles/PMC8388447/ [4] https://int.livhospital.com/hours-of-fasting-for-cell-repair/ [5] https://doi.org/10.1080/15548627.2019.1586258 [6] https://doi.org/10.1074/jbc.m113.457408 [7] https://doi.org/10.1083/jcb.201610113 [8] https://doi.org/10.1101/2025.05.27.656309 [9] https://doi.org/10.1038/s41467-020-15119-w