Hypothesis

Aging‑associated interactome rewiring begins in the kidney and spleen before detectable changes in bulk protein abundance, and this early rewiring is mediated by sex‑dependent destabilization of chaperone‑rich protein‑protein interaction (PPI) hubs. Loss of these hubs triggers a cascade that overwhelms proteostasis networks, leading to tissue‑specific aging signatures.

Rationale

• Proteomic profiling shows kidney and spleen exhibit the earliest organ‑specific aging changes [1].
• In cognitively healthy centenarians, proteins such as FHL1, EZR, CRK, CAP2, and PHACTR1—many of which act as signaling or cytoskeletal hubs—are preserved, suggesting hub stability correlates with resilience [2].
• Evolutionary conservation of longevity‑associated PPI networks links invertebrate longevity genes to human muscle expression patterns [3], indicating that certain hubs are functionally important across species.
• Proteostasis decline is a recognized hallmark of aging [4]; however, the temporal order between interactome perturbation and proteostatic failure remains unclear.

We propose that specific PPI hubs enriched for cochaperones (e.g., HSP90AA1, HSP70, and their co‑chaperones) are disproportionately vulnerable in kidney and spleen. Their disruption occurs early, before substantial changes in the steady‑state levels of the hub proteins themselves, and exhibits marked sex differences due to hormonal regulation of chaperone expression and activity.

Predictions

1. Early PPI loss: In young adult mice (3 months), affinity purification coupled with mass spectrometry (AP‑MS) will reveal kidney‑ and spleen‑specific reductions in the interaction degree of chaperone hub proteins, while total protein abundance of those hubs remains unchanged relative to 12‑month‑old controls.
2. Sex divergence: Female mice will show a slower decline in hub connectivity compared with males, correlating with higher basal expression of estrogen‑responsive chaperone co‑factors.
3. Proteostasis lag: Markers of proteostatic stress (e.g., ubiquitinated protein load, Hsp70 induction) will appear significantly later (≥6 months after the PPI deficit) in the same tissues.
4. Causal link: Targeted preservation of a chosen hub (e.g., CRISPR‑mediated knock‑in of a stabilization mutation in HSP90AA1) will delay both the PPI rewiring and the onset of proteostatic stress, extending healthspan markers in kidney and spleen.

Experimental Design

• Cohorts: Male and female C57BL/6J mice at 3, 6, 9, and 12 months (n = 5 per sex/time point).
• AP‑MS: Perform kidney and spleen lysates using antibodies against HSP90AA1 and EZR (a known centenarian‑preserved hub). Quantify interactome size, edge weight, and network clustering coefficients.
• Proteomics: Parallel label‑free quantitative proteomics to monitor total hub protein levels and global abundance shifts.
• Proteostasis read‑outs: Western blot for K48‑linked ubiquitin, Hsp70, and LC3‑II; fluorescence‑based autophagy flux assays.
• Intervention: AAV9‑mediated delivery of a stabilization‑mutant HSP90AA1 (K→R at the acetylation site) to 3‑month‑old mice; assess interactome and proteostasis at 9 months.
• Statistical tests: Two‑way ANOVA (sex × age) for network metrics; post‑hoc Tukey for pairwise comparisons. Significance set at p < 0.05.

Potential Outcomes

• Support: Observed early, sex‑biased reduction in hub interactions preceding proteostatic stress would validate the hypothesis. Rescue of hub connectivity delaying proteostasis failure would further establish causality.
• Refute: If hub PPI changes occur only after or concurrently with protein abundance alterations, or if sex differences are absent, the hypothesis would need revision, pointing to alternative drivers such as transcriptional dysregulation or post‑translational modifications unrelated to chaperone hubs.

Implications

Confirming this hypothesis would shift the focus of aging interactome research from static abundance maps to dynamic, tissue‑specific, and sex‑aware network topology. It would highlight early‑acting chaperone hubs as viable targets for interventions aimed at preserving proteostasis and extending healthspan.