Cezar Martins de Sá

On the chromatin structure of Trypanosoma cruzi.

The chromatin structure of Trypanosoma cruzi was investigated. It was found that, as in other eukaryotes, the chromatin is organized in repeating units, the nucleosomes containing about 200 base pairs of DNA associated with histones. While there is no difference in the DNA size in nucleosomes from T. cruzi and from rat liver nuclei, the histone population of T. cruzi differs in various aspects. Of particular interest is the presence of two basic proteins, possibly histone H1 subcomponents, with very high electrophoretic mobilities.

Heterologous production of Aspergillus fumigatus keratinase in Pichia pastoris

Aspergillus fumigatus Fresenius was previously shown to grow in mineral medium containing chicken feather flour as carbon and nitrogen source. Substantial proteolytic keratin-degrading activity was present in the culture supernatant after 24–72 h of growth at 42 °C. The keratinase was successfully purified by a single ion exchange chromatographic procedure and had a molecular mass of 31 kDa as determined by SDS–PAGE. The keratinase cDNA was expressed in Pichia pastoris cells and the recombinant clones were shown to be able to produce substantial caseinolytic, azo-keratinolytic and keratinolytic activities. SDS–PAGE and Western-blotting analysis using antibody against keratinase of A. fumigatus showed the presence of a single protein in the culture supernatants of several recombinant P. pastoris cells. This protein had a molecular mass corresponding to that of the A. fumigatus keratinase. The enzyme production profile showed that theP. pastoris recombinant cells produced an increasing amount of proteolytic and azo-keratinolytic activities over a 72 h growth period. Dry weight determination analysis indicated that 10% of the keratin flour was hydrolysed over a 24 h incubation period with 510 U (caseinolytic activity) of the recombinant keratinase.

Effects of an anticarcinogenic Bowman-Birk protease inhibitor on purified 20S proteasome and MCF-7 breast cancer cells.

Proteasome inhibitors have been described as an important target for cancer therapy due to their potential to regulate the ubiquitin-proteasome system in the degradation pathway of cellular proteins. Here, we reported the effects of a Bowman-Birk-type protease inhibitor, the Black-eyed pea Trypsin/Chymotrypsin Inhibitor (BTCI), on proteasome 20S in MCF-7 breast cancer cells and on catalytic activity of the purified 20S proteasome from horse erythrocytes, as well as the structural analysis of the BTCI-20S proteasome complex. In vitro experiments and confocal microscopy showed that BTCI readily crosses the membrane of the breast cancer cells and co-localizes with the proteasome in cytoplasm and mainly in nucleus. Indeed, as indicated by dynamic light scattering, BTCI and 20S proteasome form a stable complex at temperatures up to 55°C and at neutral and alkaline pHs. In complexed form, BTCI strongly inhibits the proteolytic chymotrypsin-, trypsin- and caspase-like activities of 20S proteasome, indicated by inhibition constants of 10−7 M magnitude order. Besides other mechanisms, this feature can be associated with previously reported cytostatic and cytotoxic effects of BTCI in MCF-7 breast cancer cells by means of apoptosis.

Adenosine-rich elements present in the 5′-untranslated region of PABP mRNA can selectively reduce the abundance and translation of CAT mRNAs in vivo

The poly(A)‐binding protein (PABP) is a highly conserved eukaryotic protein whose synthesis is regulated at the post‐transcriptional level. The binding of PABP to the poly(A)‐rich element found in the 5′‐untranslated region (5′UTR) of PABP mRNA specifically inhibits its own translation. In this report, we show that similar adenosine‐rich elements in the 5′UTR of the chloramphenicol acetyl‐transferase (CAT) gene can significantly reduce the reporter mRNA abundance and translation in human 293 cells. The reduction in mRNA level, but not CAT expression, is dependent on the size of the 5′UTR poly(A) element. Furthermore, one 5′UTR‐tethered PABP molecule is enough to inhibit CAT expression without affecting its mRNA level. We propose that the control of PABP synthesis may involve mRNA decay and the repression of translation.

Trypanosoma cruzi : Effect of the infection on the 20S proteasome in non-immune cells

Human infection with the protozoan Trypanosoma cruzi leads to Chagas disease. After 10-20 years of the normal acute phase, this disease develops to a chronic phase characterized mainly by dilated congestive cardiomyopathy. The mechanisms involved in the chronic phase are poorly understood, and it has been suggested that the parasite evades immune surveillance by down regulating the MHC class I antigen processing pathway. Here we analyzed whether composition or expression of the 20S proteasome, the major proteinase responsible for the generation of MHC class I ligands, were altered upon infection of HeLa cells by T. cruzi. Two-dimensional gel electrophoresis and RT-PCR experiments comparing non-infected and infected cells did not show differences between the composition of 20S proteasome or expression of its subunits. However, the proteasomes trypsin- and chymotrypsin-like activities were 2.5 and 3.6 times higher in infected cells than in non-infected cells. Our results suggest that in vitro T. cruzi infection of human or rat cells do not alter the expression of 20S proteasomal subunits or particle composition, and fails to induce the formation of immunoproteasome. However, a significant increase in the trypsin- and chymotrypsin-like activities of the host proteasome was observed.

Trypanosoma cruzi Infection Down-Modulates the Immunoproteasome Biosynthesis and the MHC Class I Cell Surface Expression in HeLa Cells

Generally, Trypanosoma cruzi infection in human is persistent and tends to chronicity, suggesting that the parasite evade the immune surveillance by down regulating the intracellular antigen processing routes. Within the MHC class I pathway, the majority of antigenic peptides are generated by the proteasome. However, upon IFN-γ stimulation, the catalytic constitutive subunits of the proteasome are replaced by the subunits β1i/LMP2, β2i/MECL-1 and β5i/LMP7 to form the immunoproteasome. In this scenario, we analyzed whether the expression and activity of the constitutive and the immunoproteasome as well as the expression of other components of the MHC class I pathway are altered during the infection of HeLa cells with T. cruzi. By RT-PCR and two-dimensional gel electrophoresis analysis, we showed that the expression and composition of the constitutive proteasome is not affected by the parasite. In contrast, the biosynthesis of the β1i, β2i, β5i immunosubunits, PA28β, TAP1 and the MHC class I molecule as well as the proteasomal proteolytic activities were down-regulated in infected-IFN-γ-treated cell cultures. Taken together, our results provide evidence that the protozoan T. cruzi specifically modulates its infection through an unknown posttranscriptional mechanism that inhibits the expression of the MHC class I pathway components.

Optimization of a protein synthesizing lysate system from Trypanosoma cruzi

A protein-synthesizing lysate system from Trypanosoma cruzi epimastigotes analogous to the rabbit reticulocyte lysate system was established. The system was optimized by the ‘classical’ method where one of the factors is varied while the others are kept constant. With this the following optima were found: [Mg2+]: 1.0 mM, [K+]: 60 mM, T: 25°C, pH: 7.5. This method was compared with the ‘sequential simplex’ method [Long, D.E. (1969) Anal. Chim. Acta 46, 93–100], a method designed to optimize rationally interdependent factors in biological systems. The optima as determined with this method were: [Mg2+]: 1.02 mM, [K+]: 63 mM, T: 25.5°C, pH: 7.25. At these values the system incorporated 43% more amino acids into proteins than a system optimized with the ‘classical’ method. Fluorographic analysis of the proteins synthesized by the system shows that while proteins in the molecular weight range between 14 000 and 45 000 are synthesized in amounts comparable to the in vivo situation, the higher molecular weight proteins (>45 000) are synthesized in lesser quantities.

Leukemogenesis in Down Syndrome

Constitutional trisomy 21 or Down syndrome (DS) is the most common human genetic aneuploidy caused by the presence of all or part of an extra 21 chromosome. The incidence of DS is estimated at 1 per 700 births (Malinge et al., 2009) and is the most common genetic factor predisposing to childhood leukemia. People with DS present several clinical phenotypes, including cognitive impairment, craniofacial dysmorphy, gastrointestinal tract abnormalities, congenital heart defects, endocrine abnormalities, neuropathology leading to dementia and immunological defects. Concerning the hematopoietic system, children with DS frequently show abnormalities in platelet counts, macrocytosis and an increased prevalence of leukemia (Lange, 2000; Roizen & Amarose, 1993).