The M32 metalloproteinases of Trypanosoma cruzi: a new potential target for chemotherapy.

GABRIELA NIEMIROWICZ, MARÍA SOLÁ, FRANCESC-XAVIER GOMIS-RUTH Y JUAN JOSÉ CAZZULO.

Athens, Georgia.

Simposio; Global Health through Research & 18th Molecular Parasitology/Vector Biology Symposium.; 2008

The Center for Tropical and Emerging Global Diseases.

Trypanosoma cruzi, the protozoan parasite which is the causative agent of the American Trypanosomiasis, Chagas Disease, contains at least three carboxypeptidases: a serine carboxypeptidase (TcSCP) belonging to the S10 family and two metallocarboxypeptidases (TcMCPs) belonging to the M32 family, found up to now only in prokaryotes. The two TcMCPs encoded by the genome of the CL Brener clone have 64 % identity between them. Both genes are present as single copies per haploid genome, and are differentially expressed in the parasite’s life cycle. Whereas TcMCP1 is expressed by the four main stages, TcMCP2 seems to be present only in the stages present in the insect vector. The purified recombinant proteins differed also in specificity; whereas TcMCP1 preferred a C-terminal basic residue, TcMCP2 preferred aromatic and aliphatic non polar residues. TcMCP1 was able to degrade several important hormonal peptides, like bradikinin. Immunofluorescence experiments showed that both TcMCPs are localized in the cytosol. Both enzymes are dimers consisting of identical subunits. The 3-D structure of the 128 kDa TcMCP1 was determined by X-Ray crystallography to a resolution of 2.0 Å. The monomer resembles an elongated cowry shell, with a long, deep, narrow active-site cleft mimicking the aperture. A residue identified as crucial to shape the S1’pocket in TcMCP1, Met 304, was mutated to the respective residue in TcMCP2, an Arg, leading to a TcMCP1 variant with TcMCP2 specificity. If the activity of these enzymes proves to be essential for the parasite, they would be good candidates as possible targets for the development of new drugs against Chagas Disease, since the M32 family is absent from all eukaryotic genomes sequenced so far, with the only exception of trypanosomatids.