A Peptidase A1 domain protein from Stropharia rugosoannulata: ACE inhibition and multi-pathway vascular modulation

A Peptidase A1 domain-containing protein (SRP, 43.1 kDa, >95% purity) was isolated from the edible mushroom Stropharia rugosoannulata and characterized for its structural properties, and ACE inhibitory and vascular modulatory activities. This represents the first report of an intact Peptidase A1 family protein exhibiting bioactivities beyond its canonical proteolytic role. SRP inhibited ACE (IC₅₀ = 108.84 ± 3.42 μg mL^-1) through strong intermolecular interactions, with Glu264 contributing 45% (-20.76 kcal mol^-1) of the total binding energy and additional contributions from conserved active-site residues. Isothermal titration calorimetry confirmed high-affinity binding to ACE (K_D = 1.12 × 10^-8 M), driven by both enthalpic and entropic contributions with entropy as the dominant thermodynamic force, suggesting a complex binding mode involving both primary and secondary interaction sites. In a zebrafish hypertension model, SRP at 62.5 μg mL^-1 decreased cardiac output by 20-24% and increased vessel diameter by 17-18%, with effects comparable to those of captopril at the tested concentration. Integrative transcriptomic and proteomic analyses revealed that SRP reversed the downregulation of core genes and key proteins, modulating multiple vascular homeostasis pathways including calcium signaling, vascular smooth muscle contraction, Toll-like receptor signaling, and neuroactive ligand-receptor interaction. These findings expand the known functional repertoire of Peptidase A1 family proteins and highlight the multi-pathway modulatory potential of food-derived intact proteins.