Hypothesis

The selectivity of autophagy is governed not only by ubiquitination but by a kinetic hierarchy in which cargo receptors compete for limited LC3‑binding sites based on avidity and phosphorylation state. When this hierarchy is flattened—by uniform receptor overexpression or by blunting phosphorylation gradients—high‑avidity cargos (e.g., damaged mitochondria) lose priority, leading to premature degradation of low‑avidity substrates and a mismatch between supply and demand that drives age‑related proteostatic failure.

Mechanistic Basis

Selective autophagy receptors such as p62/SQSTM1, NBR1, OPTN, and NDP52 contain LC3‑interacting regions (LIR) and ubiquitin‑binding domains. Their effective avidity arises from oligomerization, post‑translational modifications (especially TBK1‑mediated phosphorylation of the ubiquitin‑associated domain), and local ubiquitin chain density. ’s synthesis shows that under starvation, cargos with higher avidity recruit the Atg1 complex more efficiently, effectively “winning” the limited autophagosome‑nucleation slots [https://pmc.ncbi.nlm.nih.gov/articles/PMC4871809/]. If receptor avidity is artificially equalized—by expressing a non‑phosphorylatable p62 mutant that still oligomerizes, or by saturating the cytosol with a soluble LIR peptide—competition is disrupted. Low‑avidity cargos then gain access to autophagosomes, depleting the machinery before high‑avidity substrates can be engaged. The resulting selective deficit manifests as accumulation of damaged mitochondria and protein aggregates, which are hallmarks of cellular aging.

Testable Predictions

1. Cells expressing a phosphorylation‑deficient p62 (S403A) will show increased autophagic flux toward cytosolic proteins (measured by long‑lived protein degradation) but decreased mitophagy (mt‑Keima assay) compared with wild‑type p62 [https://doi.org/10.1101/2025.05.06.650963].
2. In aged mouse muscle, overexpressing a soluble LIR peptide will accelerate the onset of sarcopenia, as evidenced by reduced grip strength and increased fibrosis, whereas the same peptide in young mice will have minimal effect [https://pmc.ncbi.nlm.nih.gov/articles/PMC5602610/].
3. Restoring the avidity gradient—by introducing a phosphomimetic p62 (S403E) that enhances LC3 binding—will rescue mitophagy in p62‑deficient fibroblasts and extend replicative lifespan in human diploid fibroblasts [https://pubmed.ncbi.nlm.nih.gov/33730405/].

Experimental Approach

• Generate CRISPR‑edited C2C12 myoblasts expressing p62‑S403A or p62‑S403E under a doxycycline‑inducible promoter. Measure LC3‑II turnover with bafilomycin A1, ubiquitin‑positive puncta, and mitochondrial membrane potential (TMRM) over a 48‑h starvation period.
• Perform longitudinal studies in 24‑month‑old mice treated with AAV9‑delivered soluble LIR peptide versus control AAV9‑GFP. Assess muscle mass, histology, and autophagy flux (LC3‑II/I ratio, p62 accumulation) at 3‑month intervals.
• Use proximity‑labeling (BioID) to quantify receptor competition for LC3 under varying avidity conditions, confirming that low‑avidity cargos increase LC3 association when high‑avidity receptors are blocked.

If the hierarchy is indeed a determinant of autophagic efficiency, flattening it should uncouple bulk flux from selective quality control, producing a selective loss that predicts functional decline. Conversely, reinforcing the gradient should preserve selectivity and delay age‑related phenotypes.