Lysosomal Acidification Gates Age‑Dependent Rewiring of Intrinsically Disordered Protein Interactomes

Hypothesis

Age‑dependent lysosomal alkalinization selectively disrupts transient interactions of intrinsically disordered regions (IDRs) that rely on acidic pH‑mediated electrostatic switches, leading to organ‑specific interactome rewiring that drives proteostasis collapse.

Mechanistic Rationale

Lysosomal lumen pH rises from ~4.5 in young cells to >6.0 in aged tissues, weakening protonation of histidine and aspartate residues in IDRs. Many signaling hubs (e.g., FUS, TDP‑43, RBPs) contain low‑complexity domains that bind partners via charge‑dependent motifs that are strongest at acidic pH. When the lumen alkalinizes, these motifs lose positive charge, decreasing affinity for acidic patches on partner proteins. The result is a bias loss of transient, low‑affinity PPIs while stable complexes remain largely intact. Because lysosomal pH varies by cell type and organ, the pattern of interaction loss mirrors the organ‑specific epigenetic aging signatures seen in the mouse ATAC‑seq atlas.

This mechanism links two otherwise separate observations: (1) the accumulation of non‑ubiquitinated proteins due to slowed turnover, and (2) the organ‑specific synchrony of chromatin accessibility changes. Loss of IDR‑mediated contacts impairs the recruitment of ubiquitin ligases and autophagy receptors to substrates, further slowing degradation and creating a feedback loop that exacerbates lysosomal dysfunction.

Testable Predictions

1. In young versus old mouse liver, brain, and muscle, BioID‑based proximity labeling of an IDR‑bait (e.g., FUS‑RRM) will show a significant reduction in labeling of known acidic‑patch partners only in aged samples.
2. Restoring lysosomal acidity by overexpressing TFEB or treating with vacuolar‑ATPase activators will rescue the lost IDR interactions without altering overall protein expression levels.
3. The magnitude of interaction loss will correlate with local lysosomal pH measured by ratiometric LysoSensor dyes and with downstream ubiquitination rates of the bait’s substrates.
4. Sex‑specific differences in lysosomal pH will produce sex‑biased interactome rewiring, matching observed differences in lifespan and disease susceptibility.

Experimental Design

• Model: C57BL/6 mice, male and female, 3 mo (young) vs 24 mo (old).
• Bait: TurboID fused to the low‑complexity domain of FUS (or an analogous IDR). Express via AAV under a ubiquitous promoter; validate similar expression across ages and sexes by western blot.
• Labeling: 10 min biotin pulse, streptavidin pull‑down, LC‑MS/MS.
• Readouts: (a) quantitative changes in prey abundance (label‑free quantification), (b) lysosomal pH per tissue using LysoSensor Yellow/Blue ratiometric imaging, (c) ubiquitination of FUS‑dependent substrates (K‑ε‑GG enrichment).
• Intervention: Parallel cohorts receive AAV‑TFEB or chloroquine‑low dose to alkalinize lysosomes as control.
• Analysis: Compare interaction networks; test prediction that loss is greatest in tissues with highest pH increase (brain > liver > muscle). Use linear regression to link pH shift to interaction score change.

Potential Pitfalls and Alternatives

If lysosomal pH does not differ with age, the hypothesis is falsified; alternative mechanisms (e.g., oxidative modification of IDRs) would then be explored. If TFEB overexpression fails to restore interactions despite correcting pH, the defect may lie downstream in chaperone availability.

By directly measuring how organelle acidity shapes the transient interactome, this work bridges the gap between proteostasis turnover data and the missing age‑resolved PPI maps.