Hypothesis

Core idea: Age‑dependent lysosomal alkalinization triggers v‑ATPase dysfunction and ATP shortage, causing lysosomal membrane permeabilization (LMP) that releases cathepsins B and D into the cytosol [1]. As cystatin B and C decline with age [2], the proteolytic activity of these cathepsins rises unchecked. We propose that cytosolic cathepsins directly cleave the shelterin complex—specifically TRF2 and POT1—at conserved hinge regions, generating telomere deprotection that mimics replicative telomere loss. This cleavage converts lysosomal proteolytic entropy into telomeric informational entropy, making telomere length a quantitative readout of cumulative cathepsin activity rather than a pure cell‑division counter.

Mechanistic steps

1. LMP initiation – Mitochondrial ROS and ATP deficit impair v‑ATPase, raising lysosomal pH and weakening the membrane [1];
2. Cathepsin release – Lysosomal alkalinization reduces cathepsin activity inside the lysosome but permits leakage; cytosolic cathepsins encounter a reducing environment that favors their proteolytic activity [2];
3. Shelterin cleavage – Cathepsin B (preferring Arg‑Lys sites) and cathepsin D (preferring hydrophobic residues) cleave TRF2 at amino acids 425‑438 and POT1 at 210‑225, disrupting telomere‑binding duplexes; this generates uncapped telomeres that activate ATM/ATR signaling and produce telomere‑associated DNA damage foci (TAFs);
4. Informational entropy accumulation – Each cleavage event adds irreversible proteolytic damage to the telomere‑protective scaffold, increasing the entropy of the telomere‑information system; telomere shortening observed by Q‑FISH or TRF reflects loss of shelterin‑mediated protection rather than solely nucleolytic erosion;
5. Feedback loops – Uncapped telomeres exacerbate mitochondrial ROS via p53‑mediated metabolic reprogramming, further impairing v‑ATPase and sustaining LMP [6]; inflammasome activation by cathepsin‑processed NLRP3 amplifies IL‑1β, driving senescence [4] and reinforcing cystatin downregulation.

Testable predictions

• Genetic: Overexpressing cystatin B in aged mice will reduce cytosolic cathepsin activity, preserve TRF2/POT1 intact (verified by western blot of cytosolic fractions), and attenuate telomere shortening measured by Q‑FISH without altering telomerase expression.
• Pharmacological: Treating senescent human fibroblasts with a selective cathepsin B inhibitor (e.g., Ca‑074Me) or cathepsin D inhibitor (pepstatin A analog) will decrease shelterin cleavage products (detected by neo‑epitope antibodies) and slow the rate of telomere attrition over 30 population doublings.
• Mutational: Introducing non‑cleavable TRF2 (R425A/K428A) and POT1 (L212A/L215A) knock‑in alleles will confer resistance to LMP‑induced telomere dysfunction, evidenced by reduced TAFs and lower senescence‑associated secretory phenotype (SASP) despite high cathepsin levels.
• Biomarker: Serum cathepsin B/cystatin C ratio will correlate with peripheral blood leukocyte telomere length (LTL) across human cohorts, independent of age and leukocyte turnover rates.

Falsifiability If inhibition of cathepsins or overexpression of cystatin B fails to preserve shelterin integrity or telomere length in aged cells, or if shelterin‑cleavage mutants do not protect telomeres despite high cathepsin activity, the hypothesis would be refuted, indicating that telomere shortening is driven primarily by nucleolytic erosion rather than proteolytic entropy.

Broader implication Linking lysosomal proteolytic entropy to telomeric informational entropy reframes aging as a thermodynamic cost of maintaining protein‑nucleic acid homeostasis under declining proteostatic capacity, suggesting that interventions targeting the lysosomal‑cathepsin‑shelterin axis could decouple metabolic decline from genomic instability.