The gene repertoire of the main cysteine protease of Trypanosoma cruzi, cruzipain, reveals four sub-types with distinct active sites

Abstract

Cruzipains are the main papain-like cysteine proteases of Trypanosoma cruzi, the protozoan parasite that causes Chagas disease. Encoded by a multigenic family, previous studies have estimated the presence of dozens of copies spread over multiple chromosomes in different parasite strains. Here, we describe the complete gene repertoire of cruzipain in three parasite strains, their genomic organization, and expression pattern throughout the parasite life cycle. Furthermore, we have analyzed primary sequence variations among distinct family members as well as structural differences between the main groups of cruzipains. Based on phylogenetic inferences and residue positions crucial for enzyme function and specificity, we propose the classification of cruzipains into two families (I and II), whose genes are distributed in two or three separate clusters in the parasite genome, according with the strain. Family I comprises nearly identical copies to the previously characterized cruzipain 1/cruzain, whereas Family II encompasses three structurally distinct sub-types, named cruzipain 2, cruzipain 3, and cruzipain 4. RNA-seq data derived from the CL Brener strain indicates that Family I genes are mainly expressed by epimastigotes, whereas trypomastigotes mainly express Family II genes. Significant differences in the active sites among the enzyme sub-types were also identified, which may play a role in their substrate selectivity and impact their inhibition by small molecules.

Structure-Based Optimization of Quinazolines as Cruzain and TbrCATL Inhibitors

“…In this regard, it is noteworthy that multiple genes encode cruzipains, and while cruzain is a truncated version of cruzipain 1, other isoforms have been described. , Compared to cruzipain 1, cruzipain 2 differs in active site residues and specificity for substrates and inhibitors . Other members of this family also differ in residues in the S3–S1′ pockets, including substitutions in residues that frequently interact with cruzain ligands (e.g., Glu208 and Leu67), likely resulting in different specificity and affinity profiles …”