The p160 nuclear receptor co-activator RAC3 exerts an anti-apoptotic role through a cytoplasmatic action.

The p160 nuclear receptor co-activators represent a family of molecules, which are recruited by steroid nuclear receptors as well as other transcription factors that are overexpressed in several tumors. We investigated the role of one member of this family on the sensitivity of cells to apoptosis. We observed that overexpression of the RAC3 (receptor-associated co-activator-3) p160 co-activator inhibits hydrogen peroxide-induced cell death in human embryonic kidney 293 (HEK293) cells. The mechanism involves the activation of anti-apoptotic pathways mediated through enhanced nuclear factor kappa B (NF-kappaB) activity, inhibition of caspase-9 activation, diminished apoptotic-inducing factor (AIF) nuclear localization and a change in the activation pattern of several kinases, including an increase in both AKT and p38 kinase activities, and inhibition of ERK2. Moreover, RAC3 has been found associated with a protein complex containing AIF, Hsp90 and dynein, suggesting a role for the co-activator in the cytoplasmatic nuclear transport of these proteins associated with cytoskeleton. These results demonstrate that there are several molecular pathways that could be affected by their overexpression, including those not restricted to steroid regulation or the nuclear action of co-activators, which results in diminished sensitivity to apoptosis. Furthermore, this could represent one mechanism by which co-activators contribute to tumor development.

Differential subcellular localization of members of the Toxoplasma gondii small heat shock protein family.

The results of this study describe the identification and characterization of the Toxoplasma gondii alpha-crystallin/small heat shock protein (sHsp) family. By database (www.toxodb.org) search, five parasite sHsps (Hsp20, Hsp21, Hsp28, Hsp29, and the previously characterized Hsp30/Bag1) were identified. As expected, they share the homologous alpha-crystallin domain, which is the key characteristic of sHsps. However, the N-terminal segment of each protein contains unique characteristics in size and sequence. Most T. gondii sHsps are constitutively expressed in tachyzoites and fully differentiated bradyzoites, with the exception of Hsp30/Bag1. Interestingly, by subcellular localization we observed that T. gondii sHsps are located in different compartments. Hsp20 is located at the apical end of the cell, Hsp28 is located inside the mitochondrion, Hsp29 showed a membrane-associated labeling, and Hsp21 appeared throughout the cytosol of the parasites. These particular differences in the immunostaining patterns suggest that their targets and functions might be different.

Expression of a cDNA encoding a Toxoplasma gondii protein belonging to the heat-shock 90 family and analysis of its antigenicity.

A cDNA clone (Tgzy85d11.r1) obtained from the Toxoplasma Expressed Sequence Tag project was chosen due to its homology with proteins of the heat shock 90 family. The cDNA encodes 137 amino acids of the C-terminal portion of the Toxoplasma Hsp90 protein (TgHsp90). Serum samples obtained from orally infected BALB/c and C57BL/6 mice showed reactivity against a recombinant TgHsp90 (rTgHsp90) after 8 weeks postinfection. Isotype analysis showed an anti-rTgHsp90 IgG2a/IgG3 response in infected BALB/c and anti-rTgHsp90 IgG1/IgG2a/IgG2b response in infected C57BL/6 mice. Serum samples from individuals chronically and putative acutely infected with T. gondii showed a similar anti-rTgHsp90 IgG response. Our work identifies TgHsp90 as a novel parasite antigen that seems to elicit a higher relation of anti-TgHsp90/anti-T. gondii IgGs during chronic infection in comparison with the acute stage.

Characterisation of a novel interspersed Toxoplasma gondii DNA repeat with potential uses for PCR diagnosis and PCR-RFLP analysis.

A novel Toxoplasma gondii interspersed repeat element (TgIRE), present in most of the tachyzoite chromosomes, was characterised. Two regions on the TgIRE sequence showed high identity to two different T. gondii expressed sequence tag cDNAs of unknown function, which seems to be TgIRE pseudogenes. Two set of primers were designed, 2-2' and 2-3, that amplify products of 1.02 and 0.62 kb, respectively. T. gondii DNA from RH and Me49 strains was amplified with TgIRE 2-2' primers, and the respective 1.02 kb products were digested with several endonucleases. Different fragment patterns by gel electrophoresis were found only with MboI. Sensitivity analysis revealed that the set 2-3 was more sensitive than 2-2', detecting by gel visualisation the amount of DNA equivalent to 1 and 10 parasites, respectively.