Hypothesis

Aging increases macromolecular crowding and alters chaperone availability, which selectively diminishes low‑affinity, transient interactions within highly connected PPI hubs while stabilizing high‑affinity complexes. This interaction‑loss rewires hub topology from a dynamic signaling scaffold to a rigid, aggregation‑prone network, driving functional decline and proteostasis failure.

Predictions

1. In old tissues, hub proteins will show a significant decrease in the number of transient (≤1 s) interactors detected by time‑resolved crosslinking MS, whereas the number of stable (>10 s) interactors will increase or remain unchanged.
2. The loss of transient contacts will correlate with increased local viscosity measured by fluorescence recovery after photobleaching (FRAP) and with elevated levels of inflammatory signaling (e.g., IFN‑stimulated genes) that further promote crowding.
3. Chronic rapamycin treatment in aged mice will restore the youthful balance of transient/stable hub interactions and reduce hub‑associated aggregation, an effect that will be attenuated in autophagy‑deficient (Atg5‑KO) mice.
4. Hub proteins that lose the greatest proportion of transient interactions will be enriched for those whose RNAi knockdown extends C. elegans lifespan, linking interaction‑loss specificity to longevity regulation.

Experimental Approach

• Sample preparation: isolate nuclear and cytoplasmic fractions from young (3 mo) and old (24 mo) male and female mice liver and muscle; include rapamycin‑treated and vehicle controls.
• Interaction mapping: apply fast‑fixing crosslinking (e.g., DSSO with quenching at 0.5 s, 2 s, 5 s) followed by AP‑MS and quantitative MS to capture interaction lifetimes.
• Crowding measurement: perform FRAP on GFP‑tagged hub proteins in situ to infer intracellular viscosity.
• Validation: use proximity‑labeling (TurboID) to map steady‑state interactome and compare with crosslinking data; assess aggregate formation by filter‑trap assay and immunoblot for insoluble hub fractions.
• Genetic tests: repeat in muscle‑specific Atg5 knockout mice to test autophagy dependence.

Potential Implications

If confirmed, this hypothesis would reposition PPI hub dynamics—not just static connectivity—as a central effector of aging. It would explain why hub proteins are both highly connected and sensitive to longevity interventions: their transient interaction capacity is a rheostat that crowds, inflammation, and autophagy can tune. Therapeutic strategies aimed at modulating macromolecular crowding (e.g., osmolytes, crowd‑modulating peptides) or boosting chaperone‑mediated remodeling could preserve hub plasticity and extend healthspan.