Oxidative locking of p62 LC3 interaction creates a kinetic trap that reprograms the autophagy substrate hierarchy during aging
Hypothesis
Age‑related decline in selective autophagy stems from a kinetic trap in which oxidized p62 LC3‑interacting region (LIR) forms stable, non‑degradative condensates that sequester LC3 and prevent TBK1‑dependent phosphorylation of competing receptors, thereby freezing the substrate hierarchy in favor of aggrephagy and starving mitophagy/ER‑phagy.
Mechanistic Basis
- p62/SQSTM1 normally drives aggrephagy via its high‑affinity LIR motif and PB1‑mediated liquid‑liquid phase separation, concentrating LC3 into condensates that outcompete diffuse substrates under limiting flux (febs.onlinelibrary.wiley.com/doi/10.1111/febs.15824).
- With age, cumulative oxidative stress promotes disulfide‑linked crosslinking of p62 LIR cysteines, increasing its avidity for LC3 and rendering the condensate resistant to disassembly (febs.onlinelibrary.wiley.com/doi/10.1111/febs.15824).
- Concurrently, declining TBK1 kinase activity reduces phosphorylation of OPTN, NDP52 and TAX1BP1 LIR‑proximal serines, weakening their ability to compete for LC3 (febs.onlinelibrary.wiley.com/doi/10.1111/febs.15824).
- The result is a self‑reinforcing loop: oxidized p62 condensates sequester LC3, TBK1 cannot modify competing receptors, and the hierarchy remains locked on aggrephagy while mitophagy and ER‑phagy fail.
Testable Predictions
- Oxidation state correlation – In aged human fibroblasts, p62 LIR will show higher disulfide‑bond formation and reduced LC3 turnover compared with young cells.
- Condensate stability – Fluorescence recovery after photobleaching (FRAP) of p62‑GFP will reveal slower mobility in aged cells, indicating a more solid‑like phase.
- Rescue by LIR reduction – Expressing a cysteine‑to‑serine p62 LIR mutant (C→S) in aged cells will decrease LC3 sequestration, restore TBK1‑dependent phosphorylation of OPTN/NDP52, and increase mitophagy flux.
- TBK1 bypass – Introducing phosphomimetic OPTN (S→D) or NDP52 mutants in aged cells will outcompete oxidized p62 for LC3, re‑establishing selective mitochondrial clearance even when p62 remains oxidized.
- Antioxidant intervention – Treatment with mito‑targeted antioxidants (e.g., MitoQ) will lower p62 LIR oxidation, increase LC3 availability to mitophagy receptors, and improve mitochondrial health in aged tissues.
Experimental Approach
- Biochemical: Immunoprecipitate p62 from young vs. aged fibroblasts, assay LIR oxidation via maleimide‑shift gels and measure LC3 binding affinity by surface plasmon resonance.
- Imaging: Use mCherry‑GFP‑LC3 tandem reporter and mt‑Keima to quantify aggrephagy vs. mitophagy flux; perform FRAP on p62‑GFP condensates.
- Genetic: CRISPR‑knock‑in of p62 LIR C→S; lentiviral expression of phosphomimetic OPTN/NDP52; TBK1 overexpression or kinase‑dead controls.
- Pharmacological: Acute MitoQ or NAC treatment; assess rescue of LC3 receptor competition and downstream ROS/DNA damage markers.
Expected Outcomes
If the hypothesis is correct, reducing p62 LIR oxidation or enhancing competing receptor phosphorylation will selectively increase mitophagy/ER‑phagy without globally upregulating autophagy flux, thereby improving cellular homeostasis in aged models. Conversely, preventing TBK1 activity or forcing p62 oxidation will phenocopy the hierarchical collapse seen in aging, even in young cells.
Implications
This reframes aging‑associated autophagy decline not as a simple loss of machinery but as a malleable, redox‑regulated competition for LC3. Therapeutic strategies that selectively modulate p62 LIR redox state or boost TBK1‑dependent receptor phosphorylation could restore selective autophagy specificity and mitigate age‑related pathology.