Leishmania infantum NTPDase1 and NTPDase2 play an important role in infection and nitric oxide production in macrophages.

Leishmania infantum, the causative agent of American Visceral Leishmaniasis (VL), is known for its ability to modulate the host immune response to its own favor. Ecto-nucleoside triphosphate diphosphohydrolase (ENTPDase) represents a family of enzymes that hydrolyze nucleotides and are involved in nucleotide-dependent biological processes. L. infantum has two ENTPDases, namely LiNTPDase1 and LiNTPDase2. Here, we used genetic tools to overexpress or abolish the expression of LiNTPDase1 and -2 to assess their role in parasite growth in culture and macrophage infection. While LiNTPDase1 or 2-overexpressing clones showed no morphological or growth changes in promastigotes, LiNTPDase2 overexpression increased macrophage adhesion and infection by 50% and 30%, respectively. The individual LiNTPDase1 and 2 knockout mutants showed lag in growth profile, which was reversed by the addition of adenine and guanine to the culture media. Moreover, the morphology of the knockout mutants even in supplemented media was changed to an amastigote-like form. The double knockout of both genes was lethal and a mechanism of compensation of deletion of one isoform was detected in these mutants. Correspondingly, the absence of LiNTPDase1 or LiNTPDase2 led to a dramatic reduction in in vitro infection (∼90%). Interestingly, nitric oxide production was decreased in both knockout mutants during infection, which suggests that both LiNTPDases can inhibit macrophage responses against the parasite. Overall, our results show important roles of LiNTPDase1 and -2 concerning in vitro macrophage infection and reinforce their use as potential targets to control Leishmania infections.

ENTPDases from Pathogenic Trypanosomatids and Purinergic Signaling: Shedding Light towards Biotechnological Applications.

ENTPDases are enzymes known for hydrolyzing extracellular nucleotides and playing an essential role in controlling the nucleotide signaling via nucleotide/purinergic receptors P2. Moreover, ENTPDases, together with Ecto-5´-nucleotidase activity, affect the adenosine signaling via P1 receptors. These signals control many biological processes, including the immune system. In this context, ATP is considered as a trigger to inflammatory signaling, while adenosine (Ado) induces anti-inflammatory response. The trypanosomatids Leishmania and Trypanosoma cruzi, pathogenic agents of Leishmaniasis and Chagas Disease, respectively, have their own ENTPDases named "TpENTPDases," which can affect the nucleotide signaling, adhesion and infection, in order to favor the parasite. Besides, TpENTPDases are essential for the parasite nutrition, since the Purine De Novo synthesis pathway is absent in them, which makes these pathogens dependent on the intake of purines and nucleopurines for the Salvage Pathway, in which TpENTPDases also take place. Here, we review information regarding TpNTPDases, including their known biological roles and their effect on the purinergic signaling. We also highlight the roles of these enzymes in parasite infection and their biotechnological applications, while pointing to future developments.

Combined SRPK and AKT pharmacological inhibition is synergistic in T-cell acute lymphoblastic leukemia cells.

The serine/arginine protein kinases respond to the EGFR-PI3K-AKT signaling module in the context of pre-mRNA alternative splicing regulation. These enzymes (notably SRPK1 and SRPK2) have been found dysregulated in a variety of cancers, which suggests them as promising drug targets in oncology. SRPK2 has been related to leukemia cells proliferation and found preferentially overexpressed in T-cell acute lymphoblastic leukemia (T-ALL). Previously, synergistic combination between vincristine and SRPK inhibitors has been observed in leukemia cells in vitro. Herein we sought to evaluate the in vitro combinatory effects of inhibiting SRPK and multiple other kinase targets from the EGFR pathway in T-ALL, a hematological malignancy with a still poor prognosis. We found that the combined SRPK and AKT pharmacological inhibition is synergistic in Jurkat, CCRF-CEM, and TALL-1 (all T-ALL) but not in HL60, an acute myelogenous leukemia cell lineage. Combined treatments also impaired SR proteins phosphorylation in accordance with an improved suppression of SRPK activity. Furthermore, the synergism of treatments seemed associated with apoptosis triggering, as revealed by flow cytometry analyses. Taken together, these results suggest the therapeutic potential of the combined SRPK and AKT pharmacological inhibition against T-ALL.