Dataset Information

A GCN2-Like eIF2? Kinase (LdeK1) of Leishmania donovani and Its Possible Role in Stress Response.

ABSTRACT: Translation regulation in Leishmania parasites assumes significance particularly because they encounter myriad of stresses during their life cycle. The eukaryotic initiation factor 2? (eIF2?) kinases, the well-known regulators of translation initiation in higher eukaryotes have now been found to control various processes in these protozoan parasites as well. Here, we report on cloning and characterization of a GCN2-like eIF2? kinase from L. donovani and also on its modulation during nutrient starvation. We cloned a GCN2-like kinase from L. donovani, which we named as LdeK1 and validated it to be a functional eIF2? kinase by in vitro kinase assay. LdeK1 was found to be localized in the cytoplasm of the promastigotes with a five-fold higher expression in this stage of the parasite as compared to the axenic amastigotes. Phosphorylation of eIF2? and a G1-arrest was observed in response to nutrient starvation in the wild-type parasites. In contrast, phosphorylation was significantly impaired in a dominant-negative mutant of LdeK1 during this stress with a subsequent failure to bring about a G1-arrest during cell cycle. Thus, LdeK1 is a functional GCN2-like kinase of L. donovani which responds to nutrient starvation by phosphorylating its substrate, eIF2? and a G1-arrest in the cell cycle. Nutrient starvation is encountered by the parasites inside the vector which triggers metacyclogenesis. We therefore propose that global translational regulation by activation of LdeK1 followed by eIF2? phosphorylation and G1-arrest during nutrient starvation in the gut of sandfly vector could be one of the mechanisms to retool the cellular machinery required for metacyclogenesis of Leishmania promastigotes.

SUBMITTER: Rao SJ

PROVIDER: S-EPMC4889150 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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A GCN2-Like eIF2α Kinase (LdeK1) of Leishmania donovani and Its Possible Role in Stress Response.

Rao Shilpa J SJ Meleppattu Shimi S Pal Jayanta K JK

PloS one 20160601 6

Translation regulation in Leishmania parasites assumes significance particularly because they encounter myriad of stresses during their life cycle. The eukaryotic initiation factor 2α (eIF2α) kinases, the well-known regulators of translation initiation in higher eukaryotes have now been found to control various processes in these protozoan parasites as well. Here, we report on cloning and characterization of a GCN2-like eIF2α kinase from L. donovani and also on its modulation during nutrient sta ...[more]

PMID: 27248816

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Project description:GCN2 (general control nonrepressed 2) is a serine/threonine-protein kinase that regulates translation in response to stressors such as amino acid and purin deprivation, cold shock, wounding, cadmium, and UV-C exposure. Activated GCN2 phosphorylates the α-subunit of the eukaryotic initiation factor 2 (eIF2) leading to a drastic inhibition of protein synthesis and shifting translation to specific mRNAs. To investigate the role of GCN2 in responses to UV-B radiation its activity was analyzed through eIF2α phosphorylation assays in mutants of the specific UV-B and stress signaling pathways of Arabidopsis thaliana. EIF2α phosphorylation was detectable 30 min after UV-B exposure, independent of the UV-B photoreceptor UV RESISTANCE LOCUS8 and its downstream signaling components. GCN2 dependent phosphorylation of eIF2α was also detectable in mutants of the stress related MAP kinases, MPK3 and MPK6 and their negative regulator map kinase phosphatase1 (MKP1). Transcription of downstream components of the UV-B signaling pathway, the Chalcone synthase (CHS) was constitutively higher in gcn2-1 compared to wildtype and further increased upon UV-B while GLUTATHIONE PEROXIDASE7 (GPX7) behaved similarly to wildtype. The UVR8 independent FAD-LINKED OXIDOREDUCTASE (FADox) had a lower basal expression in gcn2-1 which was increased upon UV-B. Since high fluence rates of UV-B induce DNA damage the expression of the RAS ASSOCIATED WITH DIABETES PROTEIN51 (RAD51) was quantified before and after UV-B. While the basal expression was similar to wildtype it was significantly less induced upon UV-B in the gcn2-1 mutant. This expression pattern correlates with the finding that gcn2 mutants develop less cyclobutane pyrimidine dimers after UV-B exposure. Quantification of translation with the puromycination assay revealed that gcn2 mutants have an increased rate of translation which was also higher upon UV-B. Growth of gcn2 mutants to chronic UV-B exposure supports GCN2's role as a negative regulator of UV-B responses. The elevated resistance of gcn2 mutants towards repeated UV-B exposure points to a critical role of GCN2 in the regulation of translation upon UV-B.

Project description:Leishmania secretes over 151 proteins during in vitro cultivation. Cellular functions of one such novel protein: mevalonate kinase is discussed here; signifying its importance in Leishmania infection. Visceral Leishmaniasis is a persistent infection, caused by Leishmania donovani in Indian subcontinent. This persistence is partly due to phagocytosis and evasion of host immune response. The underlying mechanism involves secretory proteins of Leishmania parasite; however, related studies are meagre. We have identified a novel secretory Leishmania donovani glycoprotein, Mevalonate kinase (MVK), and shown its importance in parasite internalization and immuno-modulation. In our studies, MVK was found to be secreted maximum after 1 h temperature stress at 37°C. Its secretion was increased by 6.5-fold in phagolysosome-like condition (pH ~5.5, 37°C) than at pH ~7.4 and 25°C. Treatment with MVK modulated host immune system by inducing interleukin-10 and interleukin-4 secretion, suppressing host's ability to kill the parasite. Peripheral blood mononuclear cell (PBMC)-derived macrophages infected with mevalonate kinase-overexpressing parasites showed an increase in intracellular parasite burden in comparison to infection with vector control parasites. Mechanism behind the increase in phagocytosis and immunosuppression was found to be phosphorylation of mitogen-activated protein (MAP) kinase pathway protein, Extracellular signal-regulated kinases-1/2, and actin scaffold protein, cortactin. Thus, we conclude that Leishmania donovani Mevalonate kinase aids in parasite engulfment and subvert the immune system by interfering with signal transduction pathways in host cells, which causes suppression of the protective response and facilitates their persistence in the host. Our work elucidates the involvement of Leishmania in the process of phagocytosis which is thought to be dependent largely on macrophages and contributes towards better understanding of host pathogen interactions.

Dataset Information

A GCN2-Like eIF2? Kinase (LdeK1) of Leishmania donovani and Its Possible Role in Stress Response.

Publications

A GCN2-Like eIF2α Kinase (LdeK1) of Leishmania donovani and Its Possible Role in Stress Response.

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