Cysteine and Aspartyl Proteases Contribute to Protein Digestion in the Gut of Freshwater Planaria
Fig 8
SmedCB shows a transient spike of protease activity following digestion and amputation, while chemical inhibition of both cathepsin B and L leads to a significant decrease in regeneration.
(A) Whole worm lysate from S. mediterranea was labeled with the activity-based probe DCG-04 and resolved using SDS-PAGE. Western blot against the biotin tag of DCG-04 was used to analyze activity levels. S. mediterranea lysate showed an increase in cathepsin B labeling following digestion (day 4–6) before returning to base levels by day 14. B) When S. mediterranea worms were amputated following feeding, the increase in DCG-04 signal extended to day 8 before returning to baseline. The large upper band was quantified as SmedCB activity; five micrograms of total protein was used in each reaction with 10μM DCG-04. (C) Worms treated with SmedCB dsRNA did not show any significant defects in regeneration compared to untreated controls, but treatment with K11777 (D) showed a highly significant decrease in growth compared to vehicle worms. This effect was dose dependent. Relative changes in area are reported to account for any differences in initial size of worms selected. A negative value indicates that fragments shrank over time (heads), while positive values show relative growth. K11777 treated worms, on average, grew less (pharynxes, tails) and shrank more (heads), than untreated worms. These results were highly significant at 40 and 50μM treatment in all fragments, but not significant at lower concentrations.