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. The mutant W208F enzyme had no effect on both apparent dissociation constant of the enzyme-cytochrome c complex and K(m) value for cytochrome c indicating that cytochrome c binding affinity to the enzyme did not alter after mutation. Surprisingly, the mutant was 1000 times less active than the wild type in cytochrome c oxidation without affecting the second order rate constant of compound I formation. Our diode array stopped-flow spectral studies showed that the substrate unbound wild type enzyme reacts with H(2)O(2) to form compound I (compound II type spectrum), which was quite different from that of compound I in W208F mutant as well as horseradish peroxidase (HRP). The spectrum of the compound I in wild type LmAPX showed a red shift from 409 nm to 420 nm with equal intensity, which was broadly similar to those of known Trp radical. In case of compound I for W208F mutant, the peak in the Soret region was decreased in heme intensity at 409 nm and was not shifted to 420 nm suggesting this type of spectrum was similar to that of the known porphyrin pi-cation radical. In case of an enzyme-H(2)O(2)-ascorbate system, the kinetic for formation and decay of compound I and II of a mutant enzyme was almost identical to that of a wild type enzyme. Thus, the results of cytochrome c binding, compound I formation rate and activity assay suggested that Trp208 in LmAPX was essential for electron transfer from cytochrome c to heme ferryl but was not indispensable for ascorbate or guaiacol oxidation.     

Modulation of physiological responses with TiO<Subscript>2</Subscript> nano-particle in <Emphasis Type="Italic">Azolla pinnata</Emphasis> R.Br. under 2,4-D toxicity

The present work is emphasised with the herbicidal tolerance of Azolla pinnata R.Br. and its modulation with TiO₂ nano-particle. Both carbohydrate and nitrogen metabolism were effected with 2,4-D as herbicide and in few cases TiO₂-NP had recovered few detrimental effects. From the nutrient status in Azolla it recorded the recovery of nitrogen as well as potassium by TiO₂-NP but not in case of phosphorus. However, a conversion of nitrate to ammonium was more induced by TiO₂-NP under herbicidal toxicity. Similar results were obtained for inter-conversion of amino acid–nitrate pool, but no changes with glutamine synthase activity with TiO₂-NP. Initially, the effects of 2,4-D was monitored with changes of chlorophyll content but had not been recovered with nanoparticle. Photosynthetic reserves expressed as both total and reducing sugar were insensitive to TiO₂-NP interference but activity of soluble and wall bound invertase was in reverse trend as compared to control. The 2,4-D mediated changes of redox and its oxidative stress was ameliorated in plants with over expressed ADH activity. As a whole the Azolla bio system with TiO₂ supplementation may be useful in sustenance against 2,4-D toxicity through recovery of nitrogen metabolism. Thus, Azolla-TiO₂-NP bio system would be realised to monitor the herbicidal toxicity in soil and its possible bioremediation.     

Reduced LINE-1 methylation is associated with arsenic-induced genotoxic stress in children

Early life exposure to arsenic has profound effect towards development of arsenic induced toxic outcomes. Some districts in the state of West Bengal, India are highly affected by arsenic, mainly through ground water. In children, not much of the toxic outcomes like dermatological lesions are observed but it is thought that the exposure leads to transient alteration in their biological processes that leads to various deleterious health effects later on. We evaluated the global methylation status by analyzing the LINE-1 methylation profile in children from arsenic exposed region between the age group 5–15 years along with the cytogenetic stress induced by arsenic as measured by lymphocyte micronucleus (MN) frequency. A total of 52 arsenic exposed and 32 unexposed children were analyzed. Whole blood DNA was used to measure the LINE-1 methylation by qRT-MSP. We found a significant association of MN-frequency in exposed individuals with highly depleted LINE-1 methylation compared to the exposed individuals with near baseline (which was comparable to unexposed control) methylation index as well as with those with the hypermethylated LINE-1 promoters. From our results, we interpret that LINE-1 methylation index may serve as a potent global epigenetic mark to detect the degree of arsenic genotoxicity at a very early age. We propose that this may be utilized to determine the extent of toxic influence exerted by arsenic, from a very early age.     

Effect of thiocyanate on the peroxidase and pseudocatalase activities of Leishmania major ascorbate peroxidase

We report here that the Leishmania major ascorbate peroxidase (LmAPX), having similarity with plant ascorbate peroxidase, catalyzes the oxidation of suboptimal concentration of ascorbate to monodehydroascorbate (MDA) at physiological pH in the presence of added H(2)O(2) with concurrent evolution of O(2). This pseudocatalatic degradation of H(2)O(2) to O(2) is solely dependent on ascorbate and is blocked by a spin trap, alpha-phenyl-n-tert-butyl nitrone (PBN), indicating the involvement of free radical species in the reaction process. LmAPX thus appears to catalyze ascorbate oxidation by its peroxidase activity, first generating MDA and H(2)O with subsequent regeneration of ascorbate by the reduction of MDA with H(2)O(2) evolving O(2) through the intermediate formation of O(2)(-). Interestingly, both peroxidase and ascorbate-dependent pseudocatalatic activity of LmAPX are reversibly inhibited by SCN(-) in a concentration dependent manner. Spectral studies indicate that ascorbate cannot reduce LmAPX compound II to the native enzyme in presence of SCN(-). Further kinetic studies indicate that SCN(-) itself is not oxidized by LmAPX but inhibits both ascorbate and guaiacol oxidation, which suggests that SCN(-) blocks initial peroxidase activity with ascorbate rather than subsequent nonenzymatic pseudocatalatic degradation of H(2)O(2) to O(2). Binding studies by optical difference spectroscopy indicate that SCN(-) binds LmAPX (Kd = 100 +/- 10 mM) near the heme edge. Thus, unlike mammalian peroxidases, SCN(-) acts as an inhibitor for Leishmania peroxidase to block ascorbate oxidation and subsequent pseudocatalase activity.     

Mutational analysis of the tetrahydrobiopterin-binding site in inducible nitric-oxide synthase.

Inducible nitric-oxide synthase (iNOS) is a hemeprotein that requires tetrahydrobiopterin (H4B) for activity. The influence of H4B on iNOS structure-function is complex, and its exact role in nitric oxide (NO) synthesis is unknown. Crystal structures of the mouse iNOS oxygenase domain (iNOSox) revealed a unique H4B-binding site with a high degree of aromatic character located in the dimer interface and near the heme. Four conserved residues (Arg-375, Trp-455, Trp-457, and Phe-470) engage in hydrogen bonding or aromatic stacking interactions with the H4B ring. We utilized point mutagenesis to investigate how each residue modulates H4B function. All mutants contained heme ligated to Cys-194 indicating no deleterious effect on general protein structure. Ala mutants were monomers except for W457A and did not form a homodimer with excess H4B and Arg. However, they did form heterodimers when paired with a full-length iNOS subunit, and these were either fully or partially active regarding NO synthesis, indicating that preserving residue identities or aromatic character is not essential for H4B binding or activity. Aromatic substitution at Trp-455 or Trp-457 generated monomers that could dimerize with H4B and Arg. These mutants bound Arg and H4B with near normal affinity, but Arg could not displace heme-bound imidazole, and they had NO synthesis activities lower than wild-type in both homodimeric and heterodimeric settings. Aromatic substitution at Phe-470 had no significant effects. Together, our work shows how hydrogen bonding and aromatic stacking interactions of Arg-375, Trp-457, Trp-455, and Phe-470 influence iNOSox dimeric structure, heme environment, and NO synthesis and thus help modulate the multiple effects of H4B.     

Tryptophan 409 controls the activity of neuronal nitric-oxide synthase by regulating nitric oxide feedback inhibition.

The heme of neuronal nitric-oxide synthase participates in oxygen activation but also binds self-generated NO during catalysis resulting in reversible feedback inhibition. We utilized point mutagenesis to investigate if a conserved tryptophan residue (Trp-409), which engages in pi-stacking with the heme and hydrogen bonds to its axial cysteine ligand, helps control catalysis and regulation by NO. Surprisingly, mutants W409F and W409Y were hyperactive compared with the wild type regarding NO synthesis without affecting cytochrome c reduction, reductase-independent N-hydroxyarginine oxidation, or Arg and tetrahydrobiopterin binding. In the absence of Arg, NADPH oxidation measurements showed that electron flux through the heme was actually slower in the Trp-409 mutants than in wild-type nNOS. However, little or no NO complex accumulated during NO synthesis by the mutants, as opposed to the wild type. This difference was potentially related to mutants forming unstable 6-coordinate ferrous-NO complexes under anaerobic conditions even in the presence of Arg and tetrahydrobiopterin. Thus, Trp-409 mutations minimize NO feedback inhibition by preventing buildup of an inactive ferrous-NO complex during the steady state. This overcomes the negative effect of the mutation on electron flux and results in hyperactivity. Conservation of Trp-409 among different NOS suggests that the ability of this residue to regulate heme reduction and NO complex formation is important for enzyme physiologic function.     

Co-solvent effects on structure and function properties of savinase: solvent-induced thermal stabilization

The industrial utilization of savinase is mainly constrained by its stability limitations. In the present study, the irreversible thermoinactivation of savinase has been evaluated at 70 degrees C, and various possible mechanisms for irreversible thermoinactivation of savinase were examined. The main process seemed to be autodigestion of savinase at higher temperatures. To improve the thermal stability of the enzyme, the effect of two co-solvents (sorbitol and trehalose) on the enzyme's activity and stability was investigated. Both osmolytes prevented the autolysis of savinase at 70 degrees C without inactivating the enzyme; furthermore, the structural and kinetic stabilities of the enzyme increased in the presence of additives.     

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 H₂O₂ or camptothecin (CPT) treatment. Confocal microscopy and fluorescence spectroscopy data have revealed that the intracellular elevation of Ca²⁺ attained by the LmAPX-overexpressing cells was always below that attained in control cells. Flow cytometry assay data and confocal microscopy observation strongly suggest that LmAPX overexpression protects cells from H₂O₂-induced mitochondrial membrane depolarization as well as ATP decrease. Western blot data suggest that overexpression of LmAPX shields against H₂O₂- or CPT-induced cytochrome c and endonuclease G release from mitochondria and subsequently their accumulation in the cytoplasm. Caspase activity assay by flow cytometry shows a lower level of caspase-like protease activity in LmAPX-overexpressing cells under apoptotic stimuli. The data on phosphatidylserine exposed on the cell surface and DNA fragmentation results show that overexpression of LmAPX renders the Leishmania cells more resistant to apoptosis provoked by H₂O₂ or CPT treatment. Taken together, these results indicate that constitutive overexpression of LmAPX in the mitochondria of L. major prevents cells from the deleterious effects of oxidative stress, that is, mitochondrial dysfunction and cellular death.In multicellular organisms, mitochondria are the major physiological source of reactive oxygen species (ROS) within cells and also are important checkpoints for the control of programmed cell death (27). There are increasing numbers of reports that describe apoptosis- or programmed cell death-like processes in unicellular organisms also, such as trypanosomatids (4, 60), bacteria (20, 25), yeasts (34), and Plasmodium (3). Among the kinetoplastid parasites, Trypanosoma and Leishmania are the most carefully studied genera where apoptotic features are well established (49). Several reports have shown that mitochondrial dysfunction or an imbalance of antioxidant homeostasis causes an increase in mitochondrion-generated ROS, which include H₂O₂, superoxide radical anions, singlet oxygen, and hydroxyl radicals. These species have all been implicated in apoptosis (16, 26, 28, 41). Increasing evidence has been presented to support that ROS homeostasis regulates two major types of important physiological processes and exerts diverse functions within cells. One type of function includes damage or oxidation of cellular macromolecules (DNA, proteins, and lipids), which can lead to necrotic cell death or protein modification (7). The second type of function includes the activation of cellular signaling cascades that regulate proliferation, detoxification, DNA repair, or apoptosis (11). The detoxification of toxic mitochondrial ROS in cells occurs through a variety of cellular antioxidant enzymes, such as superoxide dismutase, which detoxifies cells from superoxide released into the mitochondrial matrix, and several other antioxidant proteins, such as catalase, glutathione (GSH) peroxidase, and peroxiredoxins, which are known to catalyze further degradation of H₂O₂ (44). During its life cycle, the Leishmania sp. encounters a pool of ROS that is generated either by its own physiological processes or as a result of host immune reaction and drug metabolism. However, unlike most eukaryotes, Leishmania lacks catalase- and selenium-containing GSH peroxidases, enzymes that play a front-line role in detoxifying ROS. Hence, the mechanism by which it resists the toxic effects of H₂O₂ remains poorly understood.Recently, we cloned, expressed and characterized the unusual heme-containing ascorbate peroxidase from Leishmania major (LmAPX) and observed that the expression of LmAPX is increased when Leishmania cells are treated with exogenous H₂O₂ (1, 18). This enzyme is a functional hybrid between cytochrome c peroxidase and APX, owing to its ability to use both ascorbate and cytochrome c as reducing electron donors (58). Colocalization studies by confocal microscopy, submitochondrial fractionation analysis of the isolated mitochondria, and subsequent Western blot analysis with anti-LmAPX antibody have confirmed that the mature enzyme is present in intermembrane space side of the inner membrane. It has also been shown that overexpression of LmAPX causes a decrease in the mitochondrial ROS burden, an increase in tolerance to H₂O₂, and protection against cardiolipin oxidation under oxidative stress (18). Although previous studies have shown that Leishmania species use superoxide dismutase (23), peroxiredoxins (8), intracellular thiols (14), lipophosphoglycan (13), trypanothione (5), HSP 70 (a heat shock protein) (36), tryparedoxin peroxidase (29), and APX (18) for detoxification of ROS, it is still unclear how the antioxidants protect against oxidative stress-induced apoptotic events in the unicellular organism Leishmania.Since the LmAPX protein is localized in the mitochondria, we hypothesized that it would be a key protein for the maintenance of mitochondrial functions due to its antioxidant properties via its ROS-scavenging function (18). To test this hypothesis, we overexpressed LmAPX in Leishmania major cells and investigated whether overexpression of LmAPX can confer resistance to oxidant-mediated mitochondrial damage as well as oxidative stress-induced cell death. In this study, we provide evidence that the overexpression of LmAPX in Leishmania cells can indeed protect against camptothecin (CPT) or H₂O₂-mediated mitochondrial damage as measured by various parameters, including disruption of mitochondrial membrane potential (Δψ_m), decrease of ATP production, and cytochrome c and endonuclease G release from mitochondria. Cells overexpressing LmAPX were also protected against oxidative stress-induced protein carbonylation, DNA fragmentation, and apoptosis. To the best of our knowledge, this is the first report of a mitochondrial hemeperoxidase that controls the ROS-induced mitochondrial death pathway.     

The membrane-proximal intracellular domain of the epidermal growth factor receptor underlies negative cooperativity in ligand binding

The binding of EGF induces dimerization of its receptor, leading to the stimulation of its intracellular tyrosine kinase activity. Kinase activation occurs within the context of an asymmetric dimer in which one kinase domain serves as the activator for the other kinase domain but is not itself activated. How ligand binding is related to the formation and dynamics of this asymmetric dimer is not known. The binding of EGF to its receptor is negatively cooperative--that is, EGF binds with lower affinity to the second site on the dimer than to the first site on the dimer. In this study, we analyzed the binding of (125)I-EGF to a series of EGF receptor mutants in the intracellular juxtamembrane domain and demonstrate that the most membrane-proximal portion of this region plays a significant role in the genesis of negative cooperativity in the EGF receptor. The data are consistent with a model in which the binding of EGF to the first site on the dimer induces the formation of one asymmetric kinase dimer. The binding of EGF to the second site is required to disrupt the initial asymmetric dimer and allow the formation of the reciprocal asymmetric dimer. Thus, some of the energy of binding to the second site is used to reorient the first asymmetric dimer, leading to a lower binding affinity and the observed negative cooperativity.     

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. Upon exposure to ER stress, the elevation of cytosolic Ca(2+) level is observed due to release of Ca(2+) from internal stores. Increase in cytosolic Ca(2+) causes mitochondrial membrane potential depolarization and ATP loss as ablation of Ca(2+) by blocking voltage-gated cation channels with verapamil preserves mitochondrial membrane potential and cellular ATP content. Furthermore, ER stress-induced reactive oxygen species (ROS)-dependent release of cytochrome c and endonuclease G from mitochondria to cytosol and subsequent translocation of endonuclease G to nucleus are observed. Inhibition of caspase-like proteases with the caspase inhibitor benzyloxycarbonyl-VAD-fluoromethyl ketone or metacaspase inhibitor antipain does not prevent nuclear DNA fragmentation and phosphatidylserine exposure. Conversely, significant protection in tunicamycin-induced DNA degradation and phosphatidylserine exposure was achieved by either pretreatment of antioxidants (N-acetyl-L-cysteine, GSH, and L-cysteine), chemical chaperone (4-phenylbutyric acid), or addition of Ca(2+) chelator (1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid-acetoxymethyl ester). Taken together, these data strongly demonstrate that ER stress-induced apoptosis in L. major is dependent on ROS and Ca(2+)-induced mitochondrial toxicity but independent of caspase-like proteases.