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Cysteine proteases represent a broad class of proteolytic enzymes widely distributed among living organisms. Although well known as typical lysosomal enzymes, cysteine proteases are actually recognized as multi-function enzymes, being involved in antigen processing and presentation, in membrane-bound protein cleavage, as well as in degradation of the cellular matrix and in processes of tissue remodeling. Very recently, it has been shown that the NO(-donor)-mediated chemical modification of the Cys catalytic residue of cysteine proteases, including Coxsackievirus and Rhinovirus cysteine proteases, cruzain, Leishmania infantum cysteine protease, falcipain, papain, as well as mammalian caspases, cathepsins and calpain, blocks the enzyme activity in vitro and in vivo. Here, inhibition of representative cysteine proteases by NO(-donors) is reviewed.

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The hemoflagellate protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease, a progressive fatal cardiomyopathy widespread in South and Central America. Here, we postulate that the preferential colonization of cardiomyocytes by T. cruzi may reflect the role of myoglobin (Mb) as a nitric oxide (NO) scavenger, protecting the parasite from the trypanocidal effects of NO. The proposal of this novel function of Mb is based on knowledge that ferrous oxygenated Mb reacts rapidly and irreversibly with NO yielding nitrate and ferric oxidized Mb, which is reduced back to the physiologically active form by an intracellular reductase. The postulated protective role of Mb on the viability of T. cruzi is reminiscent of that postulated for hemoglobin in protecting intraerythrocytic Plasmodia from the parasiticidal effect of NO. ß

Cysteine proteinases are relevant to several aspects of the parasite life cycle and of parasite±host relationships. Here, a quantitative investigation of the effect of temperature and pH on the total substrate inhibition of cruzipain, the major papain-like cysteine proteinase from Trypanosoma cruzi, is reported. Values of the apparent catalytic and inhibition parameters K m , V max , V max /K m , and K i for the cruzipain-catalysed hydrolysis of N-a-benzyloxycarbonyl-l-phenylalanyl-larginine-(7-amino-4-methylcoumarin) (Z-Phe-Arg-AMC) and azocasein were determined between 10.0 8C and 40.0 8C and between pH 4.5 and 8.5. Values of K m were independent of temperature and pH, whereas values of . As a whole, total substrate inhibition of cruzipain decreased with increasing temperature and pH. These data suggest that both synthetic and protein substrates can bind to the unique active centre of cruzipain either productively or following a binding mode which results in enzyme inhibition. However, allosteric effect(s) cannot be excluded.Keywords: cruzipain; effect of pH; effect of temperature; papain-like cysteine proteinase; total substrate inhibition.The haemoflagellate protozoan parasite Trypanosoma cruzi, the causative agent of the Chagas' disease, afflicts more than 20 million people in Central and South America. A much larger population is considered at risk worldwide [1±3].T. cruzi has a complex life cycle, involving a triatomine bug as haematophagous vector and a mammalian host. The major forms of T. cruzi present in the haematophagous insect vector are a replicative stage, the epimastigote, and a nonreplicative form, the infective metacyclic trypomastigote. In the mammalian host, T. cruzi has an obligate intracellular replicative stage, the amastigote, and a nonreplicative form, the bloodstream trypomastigote [1±3]. The T. cruzi life cycle is affected by changes in temperature and pH, both in the insect vector and in the mammalian host. In the triatomine bug, the optimal temperature range for parasite replication is 23 8C to 27 8C, being higher in mammalian hosts (< 37 8C). Note that the replication rate of T. cruzi increases with temperature. Moreover, T. cruzi replication is facilitated at the pH of the mammalian host cytoplasm (i.e. pH 7.4) [4±8].Cysteine proteinases are relevant to several aspects of the parasite life cycle and of the parasite±host relationship. In particular, cruzipain, the major papain-like cysteine proteinase from T. cruzi, is expressed in all stages of the parasite life cycle, being more abundant in replicating forms and particularly in the insect epimastigote stage. Among others, cruzipain participates in the penetration of T. cruzi trypomastigotes into host cells, in the nutrition of the parasite at the expense of the host, and in the escape mechanisms of the parasite from the immune system of the host [1,2,9]. Moreover, cruzipain is a promising target for antiparasite chemotherapy. Notably, cysteine proteinase inhibitors block replication and differentiation of T. cruzi both in vitro a...

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