RESUMEN
Serine proteinases in Leishmania (Viannia) braziliensis promastigotes were assessed in this work. This study included the investigation of the enzymatic activity of subcellular fractions obtained from benzamidine affinity chromatography, reverse transcription polymerase chain reactions, and in silico assays of subcellular localization of subtilisin. Promastigote serine proteinases showed gelatinolytic activity with molecular masses of 43 kDa to 170 kDa in the cytosolic fraction and 67 kDa to 170 kDa in the membranous fraction. Serine proteinase activities were detected using N-benzyloxycarbonyl-l-phenylalanyl-l-arginine 7-amino-4-methylcoumarin (Z-FR-AMC) and N-succinyl-l-alanine-l-phenylalanine-l-lysine 7-amino-4-methylcoumarin (Suc-AFK-AMC) as substrates in the cytosolic fraction (Z-FR-AMC = 392 ± 30 µmol.min-1 mg of protein-1 and Suc-AFK-AMC = 252 ± 20 µmol.min-1 mg of protein-1) and in the membranous fraction (Z-FR-AMC = 53 ± 5 µmol.min-1 mg of protein-1 and Suc-AFK-AMC = 63.6 ± 6.5 µmol.min-1 mg of protein-1). Enzyme specificity was shown by inhibition with aprotinin (19% to 80% inhibition) and phenylmethanesulfonyl fluoride (3% to 69%), depending on the subcellular fraction and substrate. The expression of subtilisin (LbrM.13.0860 and LbrM.28.2570) and tryparedoxin peroxidase (LbrM.15.1080) genes was observed by the detection of RNA transcripts 200 bp, 162 bp, and 166 bp long, respectively. Subsequent in silico assays showed LbrM.13.0860 can be located in the cytosol and LbrM.28.2570 in the membrane of the parasite. Data obtained here show the subcellular distribution and expression of serine proteinases, including the subtilisin-like serine proteinases in L. (V.) braziliensis promastigotes.
Asunto(s)
Membrana Celular/metabolismo , Citosol/metabolismo , Leishmania braziliensis/enzimología , Serina Proteasas/genética , Serina Proteasas/metabolismo , Cromatografía de Afinidad , Simulación por Computador , Regulación de la Expresión Génica , Leishmania braziliensis/genética , Peso Molecular , Peroxidasas/genética , Peroxidasas/metabolismo , Transporte de Proteínas , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Sensibilidad y Especificidad , Subtilisina/genética , Subtilisina/metabolismoRESUMEN
Malaria and American Trypanosomiasis constitute major global health problems. The continued emergence and spreading of resistant strains and the limited efficacy and/or safety of currently available therapeutic agents require a constant search for new sources of antiparasitic compounds. In the present study, a fraction enriched in tight-binding protease inhibitors was isolated from the Caribbean coral Plexaura homomalla (Esper, 1792), functionally characterized and tested for their antiparasitic activity against Trypanosoma cruzi and Plasmodium falciparum. The resultant fraction was chromatographically enriched in tight-binding inhibitors active against Papain-like cysteine peptidases (92%) and Pepsin-like aspartyl peptidases (8%). Globally, the inhibitors present in the enriched fraction showed no competition with substrates and apparent Ki values of 1.99 and 4.81nM for Falcipain 2 and Cruzipain, the major cysteine peptidases from P. falciparum and T. cruzi, respectively. The inhibitor-enriched fraction showed promising antiparasitic activity in cultures. It reduced the growth of the chloroquine-resistant P. falciparum strain Dd2 (IC50=0.46µM) and promoted the apparent accumulation of trophozoites, both consistent with a blockade in the hemoglobin degradation pathway. At sub-micromolar concentrations, the inhibitor-enriched fraction reduced the infection of VERO cells by T. cruzi (CL Brener clone) trypomastigotes and interfered with intracellular differentiation and/or replication of the parasites. This study provides new scientific evidence that confirms P. homomalla as an excellent source of tight-biding protease inhibitors for different proteases with biomedical relevance, and suggests that either the individual inhibitors or the enriched fraction itself could be valuable as antiparasitic compounds.