Hypothesis

SIRT6 does not merely increase the overall flux of autophagy; it reprograms the selective hierarchy by deacetylating the autophagy receptor p62/SQSTM1, thereby shifting substrate preference from bulk cytosolic proteins toward damaged mitochondria and protein aggregates. This deacetylation‑driven reordering explains why NAD⁺ repletion improves healthspan only when SIRT6 is present, and why autophagy upregulation without SIRT6 fails to extend lifespan.

Mechanistic Basis

• SIRT6 targets p62 lysine residues: Structural modeling predicts that SIRT6 can deacetylate p62 at K420 and K440, residues located within its ubiquitin‑associated (UBA) domain. Deacetylation increases the domain’s affinity for K63‑linked ubiquitin chains on mitochondrial outer‑membrane proteins (e.g., MFN2) and on p62‑binding aggregates, while decreasing affinity for monoubiquitinated cytosolic proteins.
• Altered oligomerization: Deacetylated p62 forms more stable oligomers, enhancing its ability to nucleate phagophores around selected cargo. This shifts the cargo‑first selective autophagy pathway upstream of the ULK1‑ATG9A‑PI3Kc1 cascade, making substrate choice a primary determinant of autophagosome formation.
• NAD⁺‑SIRT6 axis: Elevated NAD⁺ activates SIRT6, leading to p62 deacetylation and a autophagy hierarchy that prioritizes mitochondrially derived ROS‑producing substrates and aggregation‑prone proteins. In SIRT6‑deficient cells, despite high NAD⁺, p62 remains acetylated, autophagy proceeds in a bulk‑like mode, and deleterious substrates accumulate.

Predictions & Experimental Design

1. Acetylation status of p62 correlates with NAD⁺ levels and SIRT6 activity
   • Measure p62‑K420/K440 acetylation by immunoblot in wild‑type, SIRT6 KO, and SIRT6‑overexpressing mouse tissues under basal, NAD⁺‑boosted (NMN), and NAD⁺‑depleted (FK866) conditions.
   • Falsification: If p62 acetylation does not change with NAD⁺/SIRT6 manipulation, the mechanism is untenable.
2. Deacetylated p62 shows altered ubiquitin‑chain preference
   • Perform pull‑down assays using recombinant acetylated vs. deacetylated p62 UBA domain incubated with ubiquitinated MFN2, aggregated tau, and cytosolic GST‑Ub. Quantify binding by ELISA.
   • Falsification: Equal binding across substrates would refute selectivity shift.
3. Cargo selectivity determines longevity outcomes
   • Generate knock‑in mice expressing acetylation‑mimic (K420Q/K440Q) and acetylation‑dead (K420R/K440R) p62. Assess mitochondrial protein turnover (using mito‑QC reporter), aggregate load (filter‑trap assay), and lifespan under NMN supplementation.
   • Falsification: If acetylation‑dead p62 does not improve mitochondrial clearance or extend lifespan despite elevated NAD⁺, the hypothesis fails.
4. Pharmacological modulation of p62 acetylation recapitulates SIRT6 effects
   • Treat cells with a small‑molecule inhibitor of p62 acetyltransferase (e.g., p300/CBP activator) and measure autophagic flux toward mitochondria (mt‑Keima) versus bulk (LC3‑II turnover).
   • Falsification: Lack of preferential mitochondrial clearance would indicate p62 acetylation is not the key node.

Potential Outcomes

• Support: Demonstrating that SIRT6‑mediated p62 deacetylation redirects autophagy to degrade damaged mitochondria and toxic aggregates would position the acetylation status of p62 as a molecular switch linking NAD⁺ metabolism to selective autophagy and aging.
• Refute: Finding that p62 acetylation does not alter substrate preference, or that manipulating p62 acetylation fails to affect longevity despite changes in bulk autophagy, would require revising the model to consider other SIRT6 targets (e.g., ULK1, ATG proteins) or alternative mechanisms of hierarchy control.

This framework provides a clear, falsifiable path to test whether the quality—not just the quantity—of autophagic cargo determines the anti‑aging effects of NAD⁺‑SIRT6 signaling.