Protein phosphatase 1 of Leishmania donovani exhibits conserved catalytic residues and pro-inflammatory response.

Protein phosphorylation, governed by kinases and phosphatases, plays a pivotal role in enormous cellular signaling pathways. Although PPP family of serine/threonine phosphatases have been involved in multiplication and growth of trypanosomatid parasites, but comprehensive knowledge is still very limited. In the present study, protein phosphatase 1 from Leishmania donovani (LdPP1) was purified to homogeneity and its structural attributes were explored employing CD and fluorescence spectroscopy as well as bioinformatics methods. The CD analysis revealed an appropriate secondary structure with α-helices content outnumbering the ß-sheets, whereas intrinsic fluorescence study depicted about the buried positioning of tryptophan residues. The three-dimensional structure of LdPP1, determined by homology modeling, displayed all the characteristic features including similar position of metal as well as inhibitor binding site corresponding to the known PP1 structures. Furthermore, ELISA and qRT-PCR results showed that LdPP1 elicit the pro-inflammatory cytokines TNF-α and IL-6 at translated and transcriptional levels in THP1 macrophages. Subsequently, immune effector molecule nitric oxide and transcription factor NF-κB production was also found to be increased upon LdPP1 stimulation. Altogether, this is the first report on PPP phosphatase of trypanosomatid parasite that represents the structural highlights along with protein-mediated immunomodulation in human macrophages.

Structural and functional insights into phosphomannose isomerase: the role of zinc and catalytic residues.

Phosphomannose isomerase (PMI) is a housekeeping enzyme that is found in organisms ranging from bacteria to fungi to mammals and is important for cell-wall synthesis, viability and signalling. PMI is a zinc-dependent enzyme that catalyses the reversible isomerization between mannose 6-phosphate (M6P) and fructose 6-phosphate (F6P), presumably via the formation of a cis-enediol intermediate. The reaction is hypothesized to involve ring opening of M6P, the transfer of a proton from the C2 atom to the C1 atom and between the O1 and O2 atoms of the substrate, followed by ring closure resulting in the product F6P. Several attempts have been made to decipher the role of zinc ions and various residues in the catalytic function of PMI. However, there is no consensus on the catalytic base and the mechanism of the reaction catalyzed by the enzyme. In the present study, based on the structure of PMI from Salmonella typhimurium, site-directed mutagenesis targeting residues close to the bound metal ion and activity studies on the mutants, zinc ions were shown to be crucial for substrate binding. These studies also suggest Lys86 as the most probable catalytic base abstracting the proton in the isomerization reaction. Plausible roles for the highly conserved residues Lys132 and Arg274 could also be discerned based on comparison of the crystal structures of wild-type and mutant PMIs. PMIs from prokaryotes possess a low sequence identity to the human enzyme, ranging between 30% and 40%. Since PMI is important for the virulence of many pathogenic organisms, the identification of catalytically important residues will facilitate its use as a potential antimicrobial drug target.

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

Most of the signaling pathways are regulated by reversible phosphorylation-dephosphorylation which involves enzymes- kinases and phosphatases. 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 and molecular weight determination using Ni-NTA affinity and gel giltration chromatography respectively. 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 ±â€¯0.2 mM-1 and 2.0 ±â€¯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 untreated cells at transcript level too. These observations advocate that LdPP2C generates a pro-inflammatory environment in macrophages and hence plays important role in immunomodulation. Computational modelling showed similar three-dimensional structure and metal binding sites present in other member of PP2C subfamily, while docking studies revealed its interaction with substrate as well as its specific inhibitor. Our study has provided first time reports on enzyme kinetics, structural features and immune response inside the host macrophage of metal-dependent protein phosphatases from a trypanosomatid parasite.

Role of IL-13 Genetic Variants in Signalling of Asthma.

Asthma is a chronic inflammatory disease of the lower airways characterised by intermittent airway narrowing and airflow obstruction. The aim of this study was to examine the association of IL-13 Arg 130 Gln (A/G) and -1112C/T cytokine gene polymorphisms and to know the secretion of IL-13 cytokine levels and the interactions between the IL-13 130A/G and IL-13Rα1/IL-4Rα complex cytokine genes. The study population comprised of atopic and non-atopic asthma patients and healthy controls (HC) (N = 120). Single nucleotide polymorphisms (SNPs) were determined by restriction fragment length polymorphism (RFLP). IL-13 cytokine serum levels were measured by enzyme-linked immunosorbent assay (ELISA), and homology modelling of IL-13 A/G cytokine gene was performed through in silico analysis. In IL-13 130A/G cytokine gene AG, GG genotypes (p < 0.0042, OR = 2.87, CI 1.46-5.65; OR = 1.92, CI 1.06-3.48) were found to be significant in atopic asthma patients vs HC. The mean IL-13 serum cytokine levels were found to be significantly high in atopic (38.48 ± 36.54) and non-atopic (36.05 ± 34.54) asthma patients whereas total serum IgE levels were significantly high at p < 0.0001 in atopic and low in non-atopic asthma patients at p < 0.003 compared to HC. In silico analysis indicated that residue IL-13 130 with charge modifying variants was crucial in ligand-receptor interactions. IL-13 cytokine serum levels were significantly high in atopic and non-atopic asthma patients compared to HC. The GG genotype of IL-13 130A/G cytokine gene might be involved in the induced production of total IgE and IL-13 cytokine serum levels suggesting IL-13 may be important in the signalling of asthma.

Structural and functional analysis of two universal stress proteins YdaA and YnaF from Salmonella typhimurium: possible roles in microbial stress tolerance.

In many organisms "Universal Stress Proteins" (USPs) are induced in response to a variety of environmental stresses. Here we report the structures of two USPs, YnaF and YdaA from Salmonella typhimurium determined at 1.8Å and 2.4Å resolutions, respectively. YnaF consists of a single USP domain and forms a tetrameric organization stabilized by interactions mediated through chloride ions. YdaA is a larger protein consisting of two tandem USP domains. Two protomers of YdaA associate to form a structure similar to the YnaF tetramer. YdaA showed ATPase activity and an ATP binding motif G-2X-G-9X-G(S/T/N) was found in its C-terminal domain. The residues corresponding to this motif were not conserved in YnaF although YnaF could bind ATP. However, unlike YdaA, YnaF did not hydrolyse ATP in vitro. Disruption of interactions mediated through chloride ions by selected mutations converted YnaF into an ATPase. Residues that might be important for ATP hydrolysis could be identified by comparing the active sites of native and mutant structures. Only the C-terminal domain of YdaA appears to be involved in ATP hydrolysis. The structurally similar N-terminal domain was found to bind a zinc ion near the segment equivalent to the phosphate binding loop of the C-terminal domain. Mass spectrometric analysis showed that YdaA might bind a ligand of approximate molecular weight 800daltons. Structural comparisons suggest that the ligand, probably related to an intermediate in lipid A biosynthesis, might bind at a site close to the zinc ion. Therefore, the N-terminal domain of YdaA binds zinc and might play a role in lipid metabolism. Thus, USPs appear to perform several distinct functions such as ATP hydrolysis, altering membrane properties and chloride sensing.

Structures of mannose-6-phosphate isomerase from Salmonella typhimurium bound to metal atoms and substrate: implications for catalytic mechanism.

Mannose-6-phosphate isomerase (MPI) catalyzes the interconversion of mannose 6-phosphate and fructose 6-phosphate. X-ray crystal structures of MPI from Salmonella typhimurium in the apo form (with no metal bound) and in the holo form (with bound Zn2+) and two other structures with yttrium bound at an inhibitory site and complexed with Zn2+ and fructose 6-phosphate (F6P) were determined in order to gain insights into the structure and the isomerization mechanism. Isomerization involves acid/base catalysis with proton transfer between the C1 and C2 atoms of the substrate. His99, Lys132, His131 and Asp270 are close to the substrate and are likely to be the residues involved in proton transfer. The interactions observed at the active site suggest that the ring-opening step is probably catalyzed by His99 and Asp270. An active-site loop consisting of residues 130-133 undergoes conformational changes upon substrate binding. Zn2+ binding induces structural order in the loop consisting of residues 50-54. The metal atom appears to play a role in substrate binding and is probably also important for maintaining the architecture of the active site. Isomerization probably follows the previously suggested cis-enediol mechanism.