Phosphoproteomic studies of alamandine signaling in CHO-MrgD and human pancreatic carcinoma cells: An antiproliferative effect is unveiled.

Alamandine is a heptapeptide from the renin-angiotensin system (RAS) with similar structure/function to angiotensin-(1-7) [ang-(1-7)], but they act via different receptors. It remains elusive whether alamandine is an antiproliferative agent like ang-(1-7). The goal of this study was to evaluate the potential antiproliferative activity of alamandine and the underlying cellular signaling. We evaluated alamandine effect in the tumoral cell lines Mia PaCa-2 and A549, and in the nontumoral cell lines HaCaT, CHO and CHO transfected with the alamandine receptor MrgD (CHO-MrgD). Alamandine was able to reduce the proliferation of the tumoral cell lines in a MrgD-dependent fashion. We did not observe any effect in the nontumoral cell lines tested. We also performed proteomics and phosphoproteomics to study the alamandine signaling in Mia PaCa-2 and CHO-MrgD. Data suggest that alamandine induces a shift from anaerobic to aerobic metabolism in the tumoral cells, induces a negative regulation of PI3K/AKT/mTOR pathway and activates the transcriptional factor FoxO1; events that could explain, at least partially, the observed antiproliferative effect of alamandine. This study provides for the first time a comprehensive investigation of the alamandine signaling in tumoral (Mia PaCa-2) and nontumoral (CHO-MrgD) cells, highlighting the antiproliferative activity of alamandine/MrgD and its possible antitumoral effect.

Characterization of the Trypanosoma cruzi Rad51 gene and its role in recombination events associated with the parasite resistance to ionizing radiation.

The Rad51 gene encodes a highly conserved enzyme involved in DNA double-strand break (DSB) repair and recombination processes. We cloned and characterized the Rad51 gene from Trypanosoma cruzi, the protozoan parasite that causes Chagas disease. This gene is expressed in all three forms of the parasite life cycle, with mRNA levels that are two-fold more abundant in the intracellular amastigote form. The recombinase activity of the TcRad51 gene product was verified by an increase in recombination events observed in transfected mammalian cells expressing TcRad51 and containing two inactive copies of the neomycin-resistant gene. As a component of the DSB repair machinery, we investigated the role of TcRad51 in the resistance to ionizing radiation and zeocin treatment presented by T. cruzi. When exposed to gamma irradiation, different strains of the parasite survive to dosages as high as 1 kGy. A role for TcRad51 in this process was evidenced by the increased expression of its mRNA after irradiation. Furthermore, transfected parasites over-expressing TcRad51 have a faster kinetics of recovery of the normal pattern of chromosomal bands after irradiation as well as a higher resistance to zeocin treatment than do wild-type cultures.

Angiotensins as therapeutic targets beyond heart disease.

The renin-angiotensin system (RAS) plays a pivotal role in cardiovascular and hydro-electrolyte homeostasis. Blockade of the RAS as a therapeutic strategy for treating hypertension and related cardiovascular diseases is well established. However, actions of the RAS go far beyond the targets initially described. In this regard, the recent identification of novel components of the RAS, including angiotensin-(1-7) [Ang-(1-7)], Ang-(1-9), and alamandine, have opened new possibilities for interfering with the development and manifestations of cardiovascular and non-cardiovascular diseases. In this article, we briefly review novel targets for angiotensins and its therapeutic implications in diverse areas, including cancer, inflammation, and glaucoma.

Unveiling benznidazole's mechanism of action through overexpression of DNA repair proteins in Trypanosoma cruzi.

Benznidazole (BZ) is the most commonly used drug for the treatment of Chagas disease. Although BZ is known to induce the formation of free radicals and electrophilic metabolites within the parasite Trypanosoma cruzi, its precise mechanisms of action are still elusive. Here, we analyzed the survival of T. cruzi exposed to BZ using genetically modified parasites overexpressing different DNA repair proteins. Our results indicate that BZ induces oxidation mainly in the nucleotide pool, as heterologous expression of the nucleotide pyrophosphohydrolase MutT (but not overexpression of the glycosylase TcOgg1) increased drug resistance in the parasite. In addition, electron microscopy indicated that BZ catalyzes the formation of double-stranded breaks in the parasite, as its genomic DNA undergoes extensive heterochromatin unpacking following exposure to the drug. Furthermore, the overexpression of proteins involved in the recombination-mediated DNA repair increased resistance to BZ, reinforcing the idea that the drug causes double-stranded breaks. Our results also show that the overexpression of mitochondrial DNA repair proteins increase parasite survival upon BZ exposure, indicating that the drug induces lesions in the mitochondrial DNA as well. These findings suggest that BZ preferentially oxidizes the nucleotide pool, and the extensive incorporation of oxidized nucleotides during DNA replication leads to potentially lethal double-stranded DNA breaks in T. cruzi DNA.

Trypanosoma cruzi gene expression in response to gamma radiation.

Trypanosoma cruzi is an organism highly resistant to ionizing radiation. Following a dose of 500 Gy of gamma radiation, the fragmented genomic DNA is gradually reconstructed and the pattern of chromosomal bands is restored in less than 48 hours. Cell growth arrests after irradiation but, while DNA is completely fragmented, RNA maintains its integrity. In this work we compared the transcriptional profiles of irradiated and non-irradiated epimastigotes at different time points after irradiation using microarray. In total, 273 genes were differentially expressed; from these, 160 were up-regulated and 113 down-regulated. We found that genes with predicted functions are the most prevalent in the down-regulated gene category. Translation and protein metabolic processes, as well as generation of precursor of metabolites and energy pathways were affected. In contrast, the up-regulated category was mainly composed of obsolete sequences (which included some genes of the kinetoplast DNA), genes coding for hypothetical proteins, and Retrotransposon Hot Spot genes. Finally, the tyrosyl-DNA phosphodiesterase 1, a gene involved in double-strand DNA break repair process, was up-regulated. Our study demonstrated the peculiar response to ionizing radiation, raising questions about how this organism changes its gene expression to manage such a harmful stress.

Trypanosoma cruzi MSH2: Functional analyses on different parasite strains provide evidences for a role on the oxidative stress response.

Components of the DNA mismatch repair (MMR) pathway are major players in processes known to generate genetic diversity, such as mutagenesis and DNA recombination. Trypanosoma cruzi, the protozoan parasite that causes Chagas disease has a highly heterogeneous population, composed of a pool of strains with distinct characteristics. Studies with a number of molecular markers identified up to six groups in the T. cruzi population, which showed distinct levels of genetic variability. To investigate the molecular basis for such differences, we analyzed the T. cruzi MSH2 gene, which encodes a key component of MMR, and showed the existence of distinct isoforms of this protein. Here we compared cell survival rates after exposure to genotoxic agents and levels of oxidative stress-induced DNA in different parasite strains. Analyses of msh2 mutants in both T. cruzi and T. brucei were also used to investigate the role of Tcmsh2 in the response to various DNA damaging agents. The results suggest that the distinct MSH2 isoforms have differences in their activity. More importantly, they also indicate that, in addition to its role in MMR, TcMSH2 acts in the parasite response to oxidative stress through a novel mitochondrial function that may be conserved in T. brucei.

Overview of DNA Repair in Trypanosoma cruzi, Trypanosoma brucei, and Leishmania major.

A wide variety of DNA lesions arise due to environmental agents, normal cellular metabolism, or intrinsic weaknesses in the chemical bonds of DNA. Diverse cellular mechanisms have evolved to maintain genome stability, including mechanisms to repair damaged DNA, to avoid the incorporation of modified nucleotides, and to tolerate lesions (translesion synthesis). Studies of the mechanisms related to DNA metabolism in trypanosomatids have been very limited. Together with recent experimental studies, the genome sequencing of Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major, three related pathogens with different life cycles and disease pathology, has revealed interesting features of the DNA repair mechanism in these protozoan parasites, which will be reviewed here.

Cloning and characterization of DNA polymerase eta from Trypanosoma cruzi: roles for translesion bypass of oxidative damage.

We report the cloning and characterization of the DNA polymerase eta gene from Trypanosoma cruzi (TcPoleta), the causative agent of Chagas disease. This protein, which can bypass cyclobutane pyrimidine dimers, contains motifs that are conserved between Y family polymerases. In vitro assays showed that the recombinant protein is capable of synthesizing DNA in undamaged primer-templates. Intriguingly, T. cruzi overexpressing TcPoleta does not increase its resistance to UV-light (with or without caffeine) or cisplatin, despite the ability of the protein to enhance UV resistance in a RAD30 mutant of Saccharomyces cerevisiae. Parasites overexpressing TcPoleta are also unable to restore growth after treatment with zeocin or gamma irradiation. T. cruzi overexpressing TcPoleta are more resistant to treatment with hydrogen peroxide (H(2)O(2)) compared to nontransfected cells. The observed H(2)O(2) resistance could be associated with its ability to bypass 8-oxoguanine lesions in vitro. The results presented here suggest that TcPoleta is able to bypass UV and oxidative lesions. However the overexpression of the gene only interferes in response to oxidative lesions, possibly due to the presence of these lesions during the S phase.

Schistosoma mansoni: the IMP4 gene is involved in DNA repair/tolerance after treatment with alkylating agent methyl methane sulfonate.

Using a functional complementation strategy, we have isolated a Schistosoma mansoni cDNA that complemented Escherichia coli mutant strains which are defective in the DNA base excision repair pathway. This cDNA partially complemented the MMS-sensitive phenotype of these strains. The sequence of the isolated cDNA was homologous to genes involved in the RNA metabolism pathway, especially ScIMP4 of Saccharomyces cerevisiae. To establish whether the S. mansoni cDNA clone could complement yeast ScIMP4-defective mutants, we constructed a yeast haploid strain that coded for a truncated Imp4p protein. This mutant strain was treated with different DNA damaging agents, but showed only MMS sensitivity. The functional homology between the ScIMP4 gene and the cDNA from S. mansoni was verified by partial complementation of the mutant yeast with the worm's gene. This gene appears to be involved in DNA repair and RNA metabolism in both S. mansoni and S. cerevisiae.