Sex‑biased extracellular vesicle signaling remodels the aged interactome via protease‑dependent cleavage of hub proteins

Hypothesis

Aging‑associated, sex‑dependent epigenetic remodeling increases the secretion of extracellular vesicles (EVs) enriched with specific proteases (e.g., cathepsin‑L homologs). These EVs travel systemically and cleave exposed interaction interfaces on highly connected hub proteins, thereby rewiring the organism‑wide protein‑protein interaction (PPI) network in a sex‑specific manner.

Mechanistic Reasoning

Single‑cell ATAC‑seq across 21 mouse organs revealed ~300,000 aging‑altered genomic regions, ~40% of which are sex‑dependent [1][2]. Such coordinated chromatin shifts likely alter transcriptional programs governing EV biogenesis and cargo sorting. Spatial proteomics in mouse brain showed that healthy aging versus Alzheimer’s diverges in glycosylation and extracellular‑matrix protein patterns [3], hinting at altered secreted vesicle composition. The human longevity interactome identified hub proteins with age‑expression changes [4] and validated mTOR interactors in C. elegans using static yeast two‑hybrid data [5], but no study has measured how these hubs lose or gain partners in vivo over time.

We propose that sex‑biased epigenetic changes upregulate genes encoding EV‑associated proteases (e.g., cpl‑1 cathepsin‑L in worms, Ctss in mammals). Packaged into EVs, these proteases are released into the pseudocoelom or hemolymph, diffuse, and transiently expose cleavage sites on hub proteins that are otherwise protected in stable complexes. Cleavage reduces binding affinity for specific partners, leading to a measurable decline in PPI frequency without altering hub protein abundance. Because EV secretion and protease content differ between sexes, the resulting interactome remodeling is sex‑specific, providing a mechanistic link between the observed epigenomic synchrony and network‑level aging phenotypes.

Experimental Design

1. Generate tissue‑specific TurboID hubs – Endogenously tag a conserved hub (e.g., let‑7‑regulated RAF ortholog) with TurboID in hermaphrodite and male C. elegans (and analogous hub in Drosophila).
2. Proximity labeling across the lifespan – Feed biotin at young (day 3), mid (day 8), and old (day 15) adulthood, capture biotinylated interactors via streptavidin pulldown, and quantify by label‑free mass spectrometry.
3. Perturb EV and protease pathways – Knock down rsb‑1/cop‑1 (EV biogenesis) or cpl‑1 (cathepsin‑L) using sex‑specific RNAi or CRISPRi; repeat proximity labeling.
4. Readouts – Compare (a) hub‑partner enrichment scores, (b) hub protein levels (Western blot), and (c) organismal lifespan/healthspan under each condition.

Predictions and Falsifiability

• Prediction 1: In wild‑type animals, old hermaphrodites will show a significant loss (≥30 %) of specific hub‑partner interactions compared with young hermaphrodites, while males will exhibit a distinct pattern of loss/gain (sex‑specific rewiring).
• Prediction 2: Reducing EV secretion (rsb‑1 RNAi) will attenuate the age‑dependent interaction loss in both sexes, bringing the old‑young interaction profile closer to that of young animals.
• Prediction 3: Protease inhibition (cpl‑1 RNAi or cathepsin‑L inhibitor) will rescue the lost interactions without altering hub protein abundance, indicating a cleavage‑dependent mechanism.

Falsification: If EV or protease perturbation fails to modify the age‑dependent PPI changes, or if interaction remodeling occurs identically in both sexes despite sex‑specific knockdowns, the hypothesis would be refuted. Likewise, if hub protein levels rather than interaction frequencies drive the observed network shifts, the protease‑centric mechanism would be unsupported.

This framework directly tests whether sex‑biased EV‑mediated proteolysis underlies organism‑wide, age‑dependent interactome remodeling, bridging mammalian epigenomic atlases with functional, dynamic PPI interrogation in invertebrate aging models.