Age‑induced hub proteinphase separation drives interactome erosion preceding proteostatic decline

Hypothesis

We propose that aging promotes stress‑dependent phase separation of highly connected hub proteins, which sequesters interaction partners and erodes the PPI network before measurable declines in protein abundance or turnover occur. This interactome erosion initiates proteostatic failure rather than being a downstream consequence.

Mechanistic Basis

Hub proteins identified in longevity networks tend to be disordered, multifunctional, and enriched in low‑complexity domains 2. With age, cumulative oxidative stress and altered post‑translational modifications (e.g., lysine acetylation) reduce net charge and increase hydrophobicity, lowering the saturation concentration for phase separation. Consequently, hub proteins transition from soluble, dynamic interactors to stable condensates that capture binding sites but impede exchange. The resulting loss of transient PPIs disrupts signaling cascades and chaperone recruitment, amplifying misfolded protein load. Early proteostasis decline, nucleolar disorganization and mitochondrial dysfunction observed in hierarchical hallmark maps 3 can thus be traced to this upstream interactome collapse.

Testable Predictions

1. In aged C. elegans and mouse tissues, endogenous hub proteins (e.g., HSF‑1, AKT‑1) will show increased co‑localization with Phase‑Separation markers (e.g., DDX‑4, FUS) compared with young controls.
2. Fluorescence recovery after photobleaching (FRAP) of hub‑protein condensates will reveal markedly reduced mobility in old animals, indicating a shift from liquid‑like to solid‑like states.
3. Genetic or pharmacological reduction of hub‑protein phase propensity (e.g., overexpression of the small HSP HSP‑16.2 or treatment with 1,6‑hexanediol) will restore PPI density measured by affinity‑purification mass spectrometry and delay the onset of proteostatic markers such as ubiquitin‑positive aggregates.
4. Rescue of interactome integrity will correlate with improved tissue‑specific functional readouts (motility, neuronal firing) and extended lifespan, independent of global protein synthesis rates.

Experimental Approach

• Sample preparation: Synchronized young (day 1) and old (day 10) C. elegans; young (3 mo) and old (24 mo) mouse brain and liver.
• Condensate detection: Endogenous tagging of hub proteins with GFP; immunofluorescence for established phase‑separation markers; super‑resolution imaging to quantify co‑occurrence.
• Dynamic assays: FRAP on GFP‑hub proteins in live tissues to extract mobile fraction and half‑time of recovery.
• Interactome mapping: TurboID‑based proximity biotinylation followed by LC‑MS/MS in young vs. old samples, with and without 1,6‑hexanediol treatment.
• Functional readouts: Motility assays, pharyngeal pumping, electrophysiological recordings; lifespan tracking under intervention conditions.
• Statistical analysis: Compare condensate frequency, FRAP recovery, PPI edge counts, and survival curves using mixed‑effects models; falsify hypothesis if no age‑dependent increase in hub condensates or if condensate reduction fails to modify PPI network or longevity.

Potential Implications

If validated, this hypothesis repositions phase‑driven interactome erosion as a proximate driver of aging, offering a concrete target for interventions that preserve protein‑protein dynamics. It also explains why proteasome activity and turnover assays alone miss early network decay, guiding future efforts toward mapping dynamic PPI states across the lifespan.