Figure 6.

Inhibition of autophagy decreases cruzipain activity during T. cruzi differentiation. (A) Enzyme assay of total protein extracts from epimastigotes exposed to in vitro metacyclogenesis in the absence or presence of autophagy inhibitors (Baf or Wort), broad-spectrum protease inhibitors (Prot Inh) or K777 drug. Inhibition treatments were made during the complete differentiation cycle, and samples were collected at 72 h to obtain lysates for gel zymography (A) or the reaction with the fluorogenic substrate CBZ-Phe-Arg-MCA (B). In the last case, enzyme activity was assayed at 37°C in a spectrofluorometer (excitation at 370 nm, emission at 460 nm) and the results were converted into μmol of substrate hydrolyzed per min per mg of protein. Arrowhead indicates the position of mature cruzipain. Image (A) and data (B) are representative of three independent experiments