Importance of aspartate 4 in the Mg dependent regulation of Leishmania major PAS domain-containing phosphoglycerate kinase.

Magnesium is an important divalent cation for the regulation of catalytic activity. Recently, we have described that the Mg binding through the PAS domain inhibits the phosphoglycerate kinase (PGK) activity in PAS domain-containing PGK from Leishmania major (LmPAS-PGK) at neutral pH 7.5, but PGK activity is derepressed at acidic pH 5.

The ChaC family of γ-glutamyl cyclotransferases is required for Leishmania to switch to a slow growth state and for long-term survival of the parasite.

The ChaC family of γ-glutamyl cyclotransferases is conserved throughout all Kingdoms and catalyzes the degradation of GSH. So far, the ChaC family proteins in trypanosomal parasites are missing in the literature. Here, we report two members of the ChaC family of γ-glutamyl cyclotransferases (LmChaC and LmChaC) in the unicellular pathogen Leishmania.

Glyceraldehyde-3-phosphate dehydrogenase present in extracellular vesicles from Leishmania major suppresses host TNF-alpha expression.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) fulfills various physiological roles that are unrelated to its glycolytic function. However, to date, the nonglycolytic function of GAPDH in trypanosomal parasites is absent from the literature. Exosomes secreted from Leishmania, like entire parasites, were found to have a significant impact on macrophage cell signaling and function, indicating cross talk with the host immune system.

Regulation of Leishmania major PAS domain-containing phosphoglycerate kinase by cofactor Mg ion at neutral pH.

Recently, we described the PAS domain-containing phosphoglycerate kinase (PGK) from Leishmania major (LmPAS-PGK) that shows acidic pH (5.5)-dependent optimum catalytic activity. The PAS domain of LmPAS-PGK is expected to regulate PGK activity during catalysis, but the mechanism of regulation by PAS domain at the molecular level is uncharacterized.

PAS domain-containing phosphoglycerate kinase deficiency in results in increased autophagosome formation and cell death.

Per-Arnt-Sim (PAS) domains are structurally conserved and present in numerous proteins throughout all branches of the phylogenetic tree. Although PAS domain-containing proteins are major players for the adaptation to environmental stimuli in both prokaryotic and eukaryotic organisms, these types of proteins are still uncharacterized in the trypanosomatid parasites, and In addition, PAS-containing phosphoglycerate kinase (PGK) protein is uncharacterized in the literature. Here, we report a PAS domain-containing PGK (LmPAS-PGK) in the unicellular pathogen The modeled structure of N-terminal of this protein exhibits four antiparallel β sheets centrally flanked by α helices, which is similar to the characteristic signature of PAS domain.

Loss of virulence in NAD(P)H cytochrome b5 oxidoreductase deficient Leishmania major.

Leishmania promastigotes have the ability to synthesize essential polyunsaturated fatty acids de novo and can grow in lipid free media. Recently, we have shown that NAD(P)H cytochrome b5 oxidoreductase (Ncb5or) enzyme in Leishmania acts as the redox partner for Δ12 fatty acid desaturase, which catalyses the conversion of oleate to linoleate. So far, the exact role of Leishmania derived linoleate synthesis is still incomplete in the literature.

Control of catalysis in globin coupled adenylate cyclase by a globin-B domain.

The globin coupled heme containing adenylate cyclase from Leishmania major (HemAC-Lm) has two globin domains (globin-A and globin-B). Globin-B domain (210-360 amino acids) may guide the interaction between globin-A and adenylate cyclase domains for the regulation of catalysis. We investigated the role of globin-B domain in HemAC-Lm by constructing a series of mutants namely Δ209 (209 amino acids deleted), Δ360 (360 amino acids deleted), H161A, H311A and H311A-Δ209.

Endoplasmic reticulum stress responses in Leishmania.

Perturbation of endoplasmic reticulum (ER) homeostasis can lead to an accumulation of misfolded proteins within the ER lumen causing initiation of ER stress. To reestablish homeostasis and mitigate the stress, a series of adaptive intracellular signaling pathways termed the unfolded protein response (UPR) are activated. ER stress is of considerable interest to parasitologists because it takes place in parasites subjected to adverse environmental conditions.

The ferrous-dioxy complex of Leishmania major globin coupled heme containing adenylate cyclase: the role of proximal histidine on its stability.

Recently we have described the globin-coupled heme containing adenylate cyclase from Leishmania major (HemAC-Lm) that shows an O2 dependent cAMP signaling (Sen Santara, et. al. Proc.

Identification of proximal and distal axial ligands in Leishmania major pseudoperoxidase.

Previous optical and electron paramagnetic resonance (EPR) spectroscopic studies of the newly discovered peroxynitrite scavenging pseudoperoxidase from Leishmania major (LmPP) suggested that ferric LmPP contained a six-coordinate low-spin (6cLS) heme with a thiolate ligand, presumably a cysteine, bound to its heme iron. To identify the axial ligands of LmPP, we exploit a systematic mutational analysis of potential heme ligands. On the basis of UV-visible and EPR spectroscopy, we report that the substitution of the proximal His206 with alanine in LmPP alters the 6cLS to a five-coordinate high spin (5cHS) form at pH 4.

Globin-coupled heme containing oxygen sensor soluble adenylate cyclase in Leishmania prevents cell death during hypoxia.

Globin and adenylate cyclase play individually numerous crucial roles in eukaryotic organisms. Comparison of the amino acid sequences of globins and adenylate cyclase from prokaryotic to eukaryotic organisms suggests that they share an early common ancestor, even though these proteins execute different functions in these two kingdoms. The latest studies of biological signaling molecules in both prokaryotic and eukaryotic organisms have discovered a new class of heme-containing proteins that act as sensors.

Effect of distal His mutation on the peroxynitrite reactivity of Leishmania major peroxidase.

The conserved distal histidine in peroxidases has been considered to play a major role as a general acid-base catalyst for heterolytic cleavage of an OO bond in H2O2. However, heme peroxidases react with peroxynitrite to form transient intermediates but the role of the distal histidine in this reaction is still unknown. In order to investigate catalytic roles of the histidine at the distal cavity, two Leishmania major peroxidase (LmP) mutants (H68E, H68V) were prepared.

Mutation of Val90 to His in the pseudoperoxidase from Leishmania major enhances peroxidase activity.

Pseudoperoxidase from Leishmania major (LmPP) catalyzes the breakdown of peroxynitrite though it can hardly react with H(2)O(2). Our modeling structure predicts that a conserved His to Val switch near the distal heme pocket of LmPP may determine the profile of its H(2)O(2) activity. To test this hypothesis, we have generated complementary mutations in the LmPP (V90H) and studied the formation of Compounds I and II.

Protection against peroxynitrite by pseudoperoxidase from Leishmania major.

Heme proteins share the ability to detoxify reactive nitrogen intermediates (NO and peroxynitrite). But, to date, no heme-containing enzymatic defense against toxic reactive nitrogen intermediates has been discovered in Leishmania species. We have cloned, expressed, and characterized a pseudoperoxidase from Leishmania major (LmPP) that is capable of detoxifying peroxynitrite (ONOO(-)).

NAD(P)H cytochrome b5 oxidoreductase deficiency in Leishmania major results in impaired linoleate synthesis followed by increased oxidative stress and cell death.

NAD(P)H cytochrome b(5) oxidoreductase (Ncb5or), comprising cytochrome b(5) and cytochrome b(5) reductase domains, is widely distributed in eukaryotic organisms. Although Ncb5or plays a crucial role in lipid metabolism of mice, so far no Ncb5or gene has been reported in the unicellular parasitic protozoa Leishmania species. We have cloned, expressed, and characterized Ncb5or gene from Leishmania major.

Ascorbate peroxidase acts as a novel determiner of redox homeostasis in Leishmania.

Significance: Reactive oxygen species (ROS) are produced as natural byproducts of metabolism and respiration. While physiological levels of ROS are required for vital cellular functions (e.g.

Role of K(+) binding residues in stabilization of heme spin state of Leishmania major peroxidase.

The endogenous cation in peroxidases may contribute to the type of heme coordination. Here a series of ferric and ferrous derivatives of wild-type Leishmania major peroxidase (LmP) and of engineered K(+) site mutants of LmP, lacking potassium cation binding site, has been examined by electronic absorption spectroscopy at 25°C. Using UV-visible spectrophotometry, we show that the removal of K(+) binding site causes substantial changes in spin states of both the ferric and ferrous forms.

Role of proximal methionine residues in Leishmania major peroxidase.

The active site architecture of Leishmania major peroxidase (LmP) is very similar with both cytochrome c peroxidase and ascorbate peroxidase. We utilized point mutagenesis to investigate if the conserved proximal methionine residues (Met248 and Met249) in LmP help in controlling catalysis. Steady-state kinetics of methionine mutants shows that ferrocytochrome c oxidation is <2% of wild type levels without affecting the second order rate constant of first phase of Compound I formation, while the activity toward a small molecule substrate, guaiacol or iodide, increases.

Endoplasmic reticulum stress-induced apoptosis in Leishmania through Ca2+-dependent and caspase-independent mechanism.

Numerous reports have shown that mitochondrial dysfunctions play a major role in apoptosis of Leishmania parasites, but the endoplasmic reticulum (ER) stress-induced apoptosis in Leishmania remains largely unknown. In this study, we investigate ER stress-induced apoptotic pathways in Leishmania major using tunicamycin as an ER stress inducer. ER stress activates the expression of ER-localized chaperone protein BIP/GRP78 (binding protein/identical to the 78-kDa glucose-regulated protein) with concomitant generation of intracellular reactive oxygen species.

Ascorbate peroxidase from Leishmania major controls the virulence of infective stage of promastigotes by regulating oxidative stress.

Background: Peroxidase represents a heterogeneous group of distinct enzyme family that plays extremely diverse biological functions. Ascorbate peroxidase from Leishmania major (LmAPX) has been shown to be central to the redox defense system of Leishmania. To investigate further its exact physiological role in Leishmania, we attempted to create LmAPX -knockout mutants by gene replacement in L.

Role of C-terminal acidic cluster in stabilization of heme spin state of ascorbate peroxidase from Leishmania major.

Architecture of hemoprotein is solely responsible for different nature of heme coordination. Here we report that substitution of the acidic surface residue Glu226 to Ala in ascorbate peroxidase from Leishmania major alters the 5 coordinate high spin (5cHS) to a 6 coordinate low spin (6cLS) form at pH 7.5.

Overexpression of mitochondrial Leishmania major ascorbate peroxidase enhances tolerance to oxidative stress-induced programmed cell death and protein damage.

Ascorbate peroxidase from Leishmania major (LmAPX) is one of the key enzymes for scavenging of reactive oxygen species generated from the mitochondrial respiratory chain. We have investigated whether mitochondrial LmAPX has any role in oxidative stress-induced apoptosis. The measurement of reduced glutathione (GSH) and protein carbonyl contents in cellular homogenates indicates that overexpression of LmAPX protects Leishmania cells against depletion of GSH and oxidative damage of proteins by H(2)O(2) or camptothecin (CPT) treatment.

Leishmania major ascorbate peroxidase overexpression protects cells against reactive oxygen species-mediated cardiolipin oxidation.

Heme peroxidases are a class of multifunctional redox-active proteins found in all organisms. We recently cloned, expressed, and characterized an ascorbate peroxidase from Leishmania major (LmAPX) that was capable of detoxifying hydrogen peroxide. Localization studies using green fluorescent protein fusions revealed that LmAPX was localized within the mitochondria by its N-terminal signal sequence.

Chenopodium album seed extract-induced sperm cell death: exploration of a plausible pathway.

Background: This study was conducted for to explore the plausible pathway of Chenopodium album seed extract (CAE)-mediated sperm cell death.

Study Design: The role of CAE for its spermicidal action was assessed by (a) measuring lipid peroxidation, protein carbonyl content and intracellular glutathione content in CAE exposed sperm cells; (b) assaying antioxidant enzymes like catalase and superoxide dismutase (SOD); (c) analyzing protein expressions by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis; (d) fluorimetric measurement of intracellular H(2)O(2) level and generation of reactive oxygen species (ROS) in CAE-treated sperm cells; and (e) DNA ladder formation study.

Results: CAE-induced sperm death is due to (a) lipid peroxidation of the sperm cell membrane, oxidation of some critical cellular proteins and depletion of intracellular reduced gluthathione, indicating production of ROS; (b) activation of Mn-SOD and inactivation of catalase favoring endogenous accumulation of H(2)O(2); (c) generation of O(2)(*-) at an enhanced rate during oxidative stress as evidenced by increased Mn-SOD activity and protein expression; (d) accumulation of ROS in spermatozoa reflected in the fluorimetric experiments; and (e) increased production of O(2)(*-) and H(2)O(2) induced apoptosis-like death in sperm cells as observed by DNA ladder formation.

Role of tryptophan-208 residue in cytochrome c oxidation by ascorbate peroxidase from Leishmania major-kinetic studies on Trp208Phe mutant and wild type enzyme.

Ascorbate peroxidase from L. Major (LmAPX) is a functional hybrid between cytochrome c peroxidase (CCP) and ascorbate peroxidase (APX). We utilized point mutagenesis to investigate if a conserved proximal tryptophan residue (Trp208) among Class I peroxidase helps in controlling catalysis.