Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the mitochondrial tryparedoxin peroxidase from<i>Leishmania braziliensis</i>

Morais, M.A.B.; Souza, Tatiana de Arruda Campos Brasil de; Murakami, Mário Tyago

doi:10.1107/s1744309113003989

Cited by 5 publications

(7 citation statements)

References 18 publications

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“…The results of the enzymatic activity in this work corroborated the predictive calculations of the subcellular localizations of the subtilisins in L . ( V ) braziliensis and are in agreement with one of the functions described for these enzymes, namely, processing TXNPx in amastigotes [30,31,32,33]. In addition, due to the presence of LbrM.15.1080 transcripts in promastigotes in the stationary growth phase in culture, and the fact that Leishmania (Leishmania) amazonensis promastigotes expressed TXNPx during their growth in culture [34], it is plausible that subtilisins can also act as maturases for promastigote proteins.…”

Section: Discussionsupporting

confidence: 80%

Serine Proteinases in Leishmania (Viannia) braziliensis Promastigotes Have Distinct Subcellular Distributions and Expression

Santos-de-Souza

Côrtes

Charret

et al. 2019

IJMS

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Serine proteinases in Leishmania (Viannia) braziliensis promastigotes were assessed in this work. This study included the investigation of the enzymatic activity of subcellular fractions obtained from benzamidine affinity chromatography, reverse transcription polymerase chain reactions, and in silico assays of subcellular localization of subtilisin. Promastigote serine proteinases showed gelatinolytic activity with molecular masses of 43 kDa to 170 kDa in the cytosolic fraction and 67 kDa to 170 kDa in the membranous fraction. Serine proteinase activities were detected using N-benzyloxycarbonyl-l-phenylalanyl-l-arginine 7-amino-4-methylcoumarin (Z-FR-AMC) and N-succinyl-l-alanine-l-phenylalanine-l-lysine 7-amino-4-methylcoumarin (Suc-AFK-AMC) as substrates in the cytosolic fraction (Z-FR-AMC = 392 ± 30 µmol.min−1 mg of protein−1 and Suc-AFK-AMC = 252 ± 20 µmol.min−1 mg of protein−1) and in the membranous fraction (Z-FR-AMC = 53 ± 5 µmol.min−1 mg of protein−1 and Suc-AFK-AMC = 63.6 ± 6.5 µmol.min−1 mg of protein−1). Enzyme specificity was shown by inhibition with aprotinin (19% to 80% inhibition) and phenylmethanesulfonyl fluoride (3% to 69%), depending on the subcellular fraction and substrate. The expression of subtilisin (LbrM.13.0860 and LbrM.28.2570) and tryparedoxin peroxidase (LbrM.15.1080) genes was observed by the detection of RNA transcripts 200 bp, 162 bp, and 166 bp long, respectively. Subsequent in silico assays showed LbrM.13.0860 can be located in the cytosol and LbrM.28.2570 in the membrane of the parasite. Data obtained here show the subcellular distribution and expression of serine proteinases, including the subtilisin-like serine proteinases in L. (V.) braziliensis promastigotes.

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Section: Discussionsupporting

confidence: 80%

Serine Proteinases in Leishmania (Viannia) braziliensis Promastigotes Have Distinct Subcellular Distributions and Expression

Santos-de-Souza

Côrtes

Charret

et al. 2019

IJMS

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“…However, the Leishmania sp. has antioxidant enzymes, such as trypanothione reductase , tryparedoxin peroxidase and iron superoxide dismutase (SOD) , which are able to scavenger ROS produced by the host cells. Specifically, SOD enzymes can be classified according to the metal cofactor bound at the active site that will ultimately confer biological activity.…”

Section: Introductionmentioning

confidence: 99%

Analysis of iron superoxide dismutase‐encoding DNA vaccine on the evolution of the Leishmania amazonensis experimental infection

Campos

Silva

Ribeiro

et al. 2015

Parasite Immunology

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The present work aimed to evaluate the immunogenicity of Leishmania amazonensis iron superoxide dismutase (SOD)-encoding DNA experimental vaccine and the protective properties of this DNA vaccine during infection. The SOD gene was subcloned into the pVAX1 plasmid, and it was used to immunize BALB/c mice. Twenty-one days after the last immunization, mice were sacrificed (immunogenicity studies) or subcutaneously challenged with L. amazonensis (studies of protection), and alterations in cellular and humoral immune responses were evaluated, as well as the course of infection. Mice only immunized with pVAX1-SOD presented increased frequencies of CD4(+) IFN-γ(+), CD8(+)IFN-γ(+) and CD8(+)IL-4(+) lymphocytes; moreover, high levels of IgG2a were detected. After challenge, mice that were immunized with pVAX1-SOD had increased frequencies of the CD4(+)IL-4(+), CD8(+)IFN-γ(+) and CD8(+)IL-4(+) T lymphocytes. In addition, the lymph node cells produced high amounts of IFN-γ and IL-4 cytokines. Increased IgG2a was also detected. The pattern of immunity induced by pVAX1-SOD partially protected the BALB/c mice from a challenge with L. amazonensis, as the animals presented reduced parasitism and lesion size when compared to controls. Taken together, these results indicate that leishmanial SOD modulates the lymphocyte response, and that the elevation in IFN-γ possibly accounted for the decreased skin parasitism observed in immunized animals.

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“…, is present in most Prx1 subfamily members, suggesting that this mechanism is conserved in typical 2-Cys Prxs. Protein Expression and Purification-The wild-type (wt) LbPrx1m and mutants were expressed in Escherichia coli BL21(DE3)⌬SlyD cells harboring plasmid pRARE2 as described (30). For protein purification, cells were resuspended in buffer A (20 mM sodium phosphate, pH 7.4, 500 mM NaCl, and 5 mM imidazole) containing 2 mM PMSF, disrupted by sonication and centrifuged at 20,000 ϫ g for 30 min (277 K).…”

mentioning

confidence: 99%

How pH Modulates the Dimer-Decamer Interconversion of 2-Cys Peroxiredoxins from the Prx1 Subfamily

Morais

Giuseppe

Souza

et al. 2015

Journal of Biological Chemistry

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Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the mitochondrial tryparedoxin peroxidase fromLeishmania braziliensis

Cited by 5 publications

References 18 publications

Serine Proteinases in Leishmania (Viannia) braziliensis Promastigotes Have Distinct Subcellular Distributions and Expression

Serine Proteinases in Leishmania (Viannia) braziliensis Promastigotes Have Distinct Subcellular Distributions and Expression

Analysis of iron superoxide dismutase‐encoding DNA vaccine on the evolution of the Leishmania amazonensis experimental infection

How pH Modulates the Dimer-Decamer Interconversion of 2-Cys Peroxiredoxins from the Prx1 Subfamily

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