Leishmania donovani PP2C: kinetics, structural attributes and in vitro immune response

Publication date: Available online 28 June 2018Source: Molecular and Biochemical ParasitologyAuthor(s): Jakkula Pranay, Rahila Qureshi, Atif Iqbal, S.R. Sagurthi, Insaf A. QureshiAbstractMost of the signaling pathways are regulated by the reversible phosphorylation-dephosphorylation which involves enzymes- kinases and phosphatases. The current knowledge about the protein phosphatases in parasites like Trypanosoma and Leishmania is very minimal despite their enormousity. In present study, full length ORF of Leishmania donovani PP2C was cloned into expression vector followed by purification using Ni-NTA affinity chromatography and molecular weight determination using gel filtration chromatography. Purified LdPP2C was found to be enzymatically active, while inhibition study suggested that sanguinarine acts as a non-competitive inhibitor. CD and fluorescence spectroscopy results indicated towards an adequate protein conformation from pH 3.5 to 8.5. The quenching constant (Ksv) and free energy (ΔG) of LdPP2C was found to be 11.1 ± 0.2 mM-1 and 2.0 ± 1.1 kcal mol-1 in presence of acrylamide and urea respectively. The protein was found to elicit the innate immune functions through upregulation of pro-inflammatory cytokines (TNF-α and IL-6) as well as nitric oxide generation. Simultaneously, these cytokines were found to be fairly higher in protein treated cells as compared to the untreated cells at transcript level too. These observations advocate that LdPP2C generat...
Source: Molecular and Biochemical Parasitology - Category: Parasitology Source Type: research