Protein Phosphatase PP2C Identification in Entamoeba spp.

Entamoeba histolytica is the causative agent of amoebiasis, and Entamoeba dispar is its noninvasive morphological twin. Entamoeba invadens is a reptilian parasite. In the present study, Western blot, phosphatase activity, immunofluorescence, and bioinformatic analyses were used to identify PP2C phosphatases of E. histolytica, E. dispar, and E. invadens. PP2C was identified in trophozoites of all Entamoeba species and cysts of E. invadens. Immunoblotting using a Leishmania mexicana anti-PP2C antibody recognized a 45.2 kDa PP2C in all species. In E. histolytica and E. invadens, a high molecular weight element PP2C at 75 kDa was recognized, mainly in cysts of E. invadens. Immunofluorescence demonstrated the presence of PP2C in membrane and vesicular structures in the cytosol of all species analyzed. The ~75 kDa PP2C of Entamoeba spp. shows the conserved domain characteristic of phosphatase enzymes (according to in silico analysis). Possible PP2C participation in the encystation process was discussed.

Towards DNA-damage induced autophagy: A Boolean model of p53-induced cell fate mechanisms.

How a cell determines a given phenotype upon damaged DNA is an open problem. Cell fate decisions happen at cell cycle checkpoints and it is becoming clearer that the p53 pathway is a major regulator of cell fate decisions involving apoptosis or senescence upon DNA damage, especially at G1/S. However, recent results suggest that this pathway is also involved in autophagy induction upon DNA damage. To our knowledge, in this work we propose the first model of the DNA damage-induced G1/S checkpoint contemplating the decision between three phenotypes: apoptosis, senescence, and autophagy. The Boolean model is proposed based on experiments with U87 glioblastoma cells using the transfection of miR-16 that can induce a DNA damage response. The wild-type case of the model shows that DNA damage induces the checkpoint and the coexistence of the three phenotypes (tristable dynamics), each with a different probability. We also predict that the positive feedback involving ATM, miR-16, and Wip1 has an influence on the tristable state. The model predictions were compared to experiments of gain and loss of function in other three different cell lines (MCF-7, A549, and U2OS) presenting agreement. For p53-deficient cell lines such as HeLa, H1299, and PC-3, our model contemplates the experimental observation that the alternative AMPK pathway can compensate this deficiency. We conclude that at the G1/S checkpoint the p53 pathway (or, in its absence, the AMPK pathway) can regulate the induction of different phenotypes in a stochastic manner in the U87 cell line and others.

MicroRNA-16 feedback loop with p53 and Wip1 can regulate cell fate determination between apoptosis and senescence in DNA damage response.

Cell fate regulation is an open problem whose comprehension impacts several areas of the biosciences. DNA damage induces cell cycle checkpoints that activate the p53 pathway to regulate cell fate mechanisms such as apoptosis or senescence. Experiments with different cell types show that the p53 pathway regulates cell fate through a switch behavior in its dynamics. For low DNA damage the pathway presents an oscillatory pattern associated with intense DNA damage repair while for high damage there are no oscillations and either p53 concentration increases inducing apoptosis or the cell enters a senescence state. Apoptosis and senescence phenotypes seem to have compensatory functions in tissues and the microRNA 16-1 (miR-16) is involved in the regulation of the fate between both phenotypes in cancer cells. To investigate the regulation of cell fate we developed a logical model of the G1/S checkpoint in DNA damage response that takes into account different levels of damage and contemplates the influence of miR-16 through its positive feedback loop formed with p53 and Wip1. The model reproduces the observed cellular phenotypes in experiments: oscillatory (for low DNA damage) regulated by negative feedback loops involving mainly p53 and Mdm2 and apoptotic or senescent (for high DNA damage) regulated by the positive p53/Wip1/miR-16 feedback loop. We find good agreement between the level of DNA damage and the probability of the phenotype produced according to experiments. We also find that this positive feedback makes senescent and apoptotic phenotypes to be determined stochastically (bistable), however controlling the expression level of miR-16 allows the control of fate determination as observed experimentally.

Identification of novel biomarkers associated with poor patient outcomes in invasive breast carcinoma.

Breast carcinoma (BC) corresponds to 23 % of all cancers in women, with 1.38 million new cases and 460,000 deaths worldwide annually. Despite the significant advances in the identification of molecular markers and different modalities of treatment for primary BC, the ability to predict its metastatic behavior is still limited. The purpose of this study was to identify novel molecular markers associated with distinct clinical outcomes in a Brazilian cohort of BC patients. We generated global gene expression profiles using tumor samples from 24 patients with invasive ductal BC who were followed for at least 5 years, including a group of 15 patients with favorable outcomes and another with nine patients who developed metastasis. We identified a set of 58 differentially expressed genes (p ≤ 0.01) between the two groups. The prognostic value of this metastasis signature was corroborated by its ability to stratify independent BC patient datasets according to disease-free survival and overall survival. The upregulation of B3GNT7, PPM1D, TNKS2, PHB, and GTSE1 in patients with poor outcomes was confirmed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) in an independent sample of patients with BC (47 with good outcomes and eight that presented metastasis). The expression of BCL2-associated agonist of cell death (BAD) protein was determined in 1276 BC tissue samples by immunohistochemistry and was consistent with the reduced BAD mRNA expression levels in metastatic cases, as observed in the oligoarray data. These findings point to novel prognostic markers that can distinguish breast carcinomas with metastatic potential from those with favorable outcomes.

Leishmania mexicana: promastigotes and amastigotes secrete protein phosphatases and this correlates with the production of inflammatory cytokines in macrophages.

Phosphatase activity of Leishmania spp. has been shown to deregulate the signalling pathways of the host cell. We here show that Leishmania mexicana promastigotes and amastigotes secrete proteins with phosphatase activity to the culture medium, which was higher in the Promastigote Secretion Medium (PSM) as compared with the Amastigote Secretion Medium (ASM) and was not due to cell lysis, since parasite viability was not affected by the secretion process. The biochemical characterization showed that the phosphatase activity present in PSM was higher in dephosphorylating the peptide END (pY) INASL as compared with the peptide RRA (pT)VA. In contrast, the phosphatase activity in ASM showed little dephosphorylating capacity for both peptides. Inhibition assays demonstrated that the phosphatase activity of both PSM and ASM was sensible only to protein tyrosine phosphatases inhibitors. An antibody against a protein phosphatase 2C (PP2C) of Leishmania major cross-reacted with a 44·9 kDa molecule in different cellular fractions of L. mexicana promastigotes and amastigotes, however, in PSM and ASM, the antibody recognized a protein about 70 kDa. By electron microscopy, the PP2C was localized in the flagellar pocket of amastigotes. PSM and ASM induced the production of tumor necrosis factor alpha, IL-1ß, IL-12p70 and IL-10 in human macrophages.