Overexpression of Mitochondrial<i>Leishmania major</i>Ascorbate Peroxidase Enhances Tolerance to Oxidative Stress-Induced Programmed Cell Death and Protein Damage

Dolai, Subhankar; Yadav, Rajesh K.; Pal, Swati; Adak, Subrata

doi:10.1128/ec.00198-09

Cited by 61 publications

(51 citation statements)

References 60 publications

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“…LmP, located at the parasite mitochondria, is involved in H 2 O 2 detoxification during parasite differentiation and infectivity (27,42). In light of its CcP activity, we searched for TcAPx-CcP localization in the different T. cruzi parasite stages.…”

Section: Discussionmentioning

confidence: 99%

Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase ( Tc APx-CcP)

Hugo

Martínez

Trujillo

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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The Trypanosoma cruzi ascorbate peroxidase is, by sequence analysis, a hybrid type A member of class I heme peroxidases [TcAPx-cytochrome c peroxidase (CcP)], suggesting both ascorbate (Asc) and cytochrome c (Cc) peroxidase activity. Here, we show that the enzyme reacts fast with H 2 O 2 (k = 2.9 × 10 7 M −1 ·s −1 ) and catalytically decomposes H 2 O 2 using Cc as the reducing substrate with higher efficiency than Asc (k cat /K m = 2.1 × 10 5 versus 3.5 × 10 4 M −1 ·s −1 , respectively). Visible-absorption spectra of purified recombinant TcAPx-CcP after H 2 O 2 reaction denote the formation of a compound I-like product, characteristic of the generation of a tryptophanyl radical-cation (Trp 233•+ ). Mutation of Trp 233 to phenylalanine (W233F) completely abolishes the Cc-dependent peroxidase activity. In addition to Trp 233•+ , a Cys 222 -derived radical was identified by electron paramagnetic resonance spin trapping, immunospin trapping, and MS analysis after equimolar H 2 O 2 addition, supporting an alternative electron transfer (ET) pathway from the heme. Molecular dynamics studies revealed that ET between Trp 233 and Cys 222 is possible and likely to participate in the catalytic cycle. Recognizing the ability of TcAPx-CcP to use alternative reducing substrates, we searched for its subcellular localization in the infective parasite stages (intracellular amastigotes and extracellular trypomastigotes). TcAPx-CcP was found closely associated with mitochondrial membranes and, most interestingly, with the outer leaflet of the plasma membrane, suggesting a role at the host-parasite interface. TcAPx-CcP overexpressers were significantly more infective to macrophages and cardiomyocytes, as well as in the mouse model of Chagas disease, supporting the involvement of TcAPx-CcP in pathogen virulence as part of the parasite antioxidant armamentarium.Trypanosoma cruzi | heme peroxidase | oxidants | virulence | kinetics

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Section: Discussionmentioning

confidence: 99%

Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase ( Tc APx-CcP)

Hugo

Martínez

Trujillo

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…TUNEL Staining-In situ DNA fragmentation was analyzed by the terminal deoxyribonucleotide transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL assay) kit (Clontech) as described previously (27). Involvement of caspases in DNA degradation was assessed by the addition of caspase inhibitors.…”

Section: Reagents-mitoprobementioning

confidence: 99%

“…Generation of mitochondrial ROS was monitored by flow cytometry with the mitochondrion-specific ROS-detecting probe MitoSOX TM Red as described previously (26). Ca 2ϩ Analysis-Intracellular free Ca 2ϩ concentrations were measured using Ca 2ϩ -specific fluorescent probe Fluo-4/AM (Molecular Probes, Eugene, OR) as described previously with minor modifications (27). 1 ϫ 10 7 cells/ml parasites were loaded with 6.0 M Fluo-4/AM for 60 min at room temperature in the presence of 5 M pluronic acid F127.…”

Section: Reagents-mitoprobementioning

confidence: 99%

“…Detection of Caspase-3/7 and Metacaspase-like Protease Activity-The detection of caspase activity by fluorochrome inhibitor of caspases (a caspase-3-and -7-specific fluorogenic inhibitor, FAM-DEVD-FMK) was based on affinity labeling of the reactive center cysteine residue of activated caspases by the FMK moiety of the fluorochrome inhibitor of caspases via the caspase-specific recognition sequence aspartic acid-glutamic acid-valine-aspartic acid (DEVD) as described previously (27). Caspase inhibitor Z-VAD-FMK was added in control reactions at 50 M for 30 min prior to the addition of ER stress inducer.…”

Section: Reagents-mitoprobementioning

confidence: 99%

“…Parasite Survival Assay-Viability of L. major during ER stress was measured by MTT (Sigma) as described previously (27). Exponentially growing promastigotes (2 ϫ 10 6 ) in M199 media were exposed to different concentrations of TM for 2.0 to 16.0 h. After treatment cells were washed with ice-cold 1ϫ PBS and incubated in fresh M199 media with 10% heat-inactivated FBS and 0.5 mg/ml MTT for 3 h. After 3 h, cells were pelleted by centrifugation (1200 ϫ g for 5.0 min).…”

Section: Reagents-mitoprobementioning

confidence: 99%

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Endoplasmic Reticulum Stress-induced Apoptosis in Leishmania through Ca2+-dependent and Caspase-independent Mechanism

Dolai¹,

Pal²,

Yadav

et al. 2011

Journal of Biological Chemistry

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100

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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

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Cytochrome c Peroxidase

Chreifi

Poulos

2017

Encyclopedia of Inorganic and Bioinorganic Chemistry

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Cytochrome c peroxidase (CcP), produced by Saccharomyces cerevisiae , is a heme peroxidase that catalyzes the reduction of H 2 O 2 to water using electrons from two molecules of ferrocytochrome c . CcP has played a unique role in the early studies of enzymology and biological electron transfer and in our understanding of how heme‐containing enzymes stabilize higher oxidation states. Recently, a heme peroxidase from the parasitic protozoan Leishmania major (LmP) was also confirmed to be a CcP. This article briefly describes our current knowledge of CcP (and LmP) biochemistry and structural biology.

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Overexpression of MitochondrialLeishmania majorAscorbate Peroxidase Enhances Tolerance to Oxidative Stress-Induced Programmed Cell Death and Protein Damage

Cited by 61 publications

References 60 publications

Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase ( Tc APx-CcP)

Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase ( Tc APx-CcP)

Endoplasmic Reticulum Stress-induced Apoptosis in Leishmania through Ca2+-dependent and Caspase-independent Mechanism

Cytochrome c Peroxidase

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