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American Journal of Physiology. Renal... Nov 2014Peptidyl arginine deiminase (PAD)4 is a nuclear enzyme that catalyzes the posttranslational conversion of arginine residues to citrulline. Posttranslational protein...
Peptidyl arginine deiminase (PAD)4 is a nuclear enzyme that catalyzes the posttranslational conversion of arginine residues to citrulline. Posttranslational protein citrullination has been implicated in several inflammatory autoimmune diseases, including rheumatoid arthritis, colitis, and multiple sclerosis. Here, we tested the hypothesis that PAD4 contributes to ischemic acute kidney injury (AKI) by exacerbating the inflammatory response after renal ischemia-reperfusion (I/R). Renal I/R injury in mice increased PAD4 activity as well as PAD4 expression in the mouse kidney. After 30 min of renal I/R, vehicle-treated mice developed severe AKI with large increases in plasma creatinine. In contrast, mice pretreated with PAD4 inhibitors (2-chloroamidine or streptonigrin) had significantly reduced renal I/R injury. Further supporting a critical role for PAD4 in generating ischemic AKI, mice pretreated with recombinant human PAD4 (rPAD4) protein and subjected to mild (20 min) renal I/R developed exacerbated ischemic AKI. Consistent with the hypothesis that PAD4 regulates renal tubular inflammation after I/R, mice treated with a PAD4 inhibitor had significantly reduced renal neutrophil chemotactic cytokine (macrophage inflammatory protein-2 and keratinocyte-derived cytokine) expression and had decreased neutrophil infiltration. Furthermore, mice treated with rPAD4 had significantly increased renal tubular macrophage inflammatory protein-2 and keratinocyte-derived cytokine expression as well as increased neutrophil infiltration and necrosis. Finally, cultured mouse kidney proximal tubules treated with rPAD4 had significantly increased proinflammatory chemokine expression compared with vehicle-treated cells. Taken together, our results suggest that PAD4 plays a critical role in renal I/R injury by increasing renal tubular inflammatory responses and neutrophil infiltration after renal I/R.
Topics: Acute Kidney Injury; Amphetamines; Animals; Humans; Hydrolases; Male; Mice, Inbred C57BL; Protein-Arginine Deiminase Type 4; Protein-Arginine Deiminases; Reperfusion Injury
PubMed: 25164081
DOI: 10.1152/ajprenal.00243.2014 -
Scientific Reports Feb 2016Streptonigrin is a potent antitumor antibiotic, active against a wide range of mammalian tumor cells. It was reported that its biosynthesis relies on...
Streptonigrin is a potent antitumor antibiotic, active against a wide range of mammalian tumor cells. It was reported that its biosynthesis relies on (2S,3R)-β-methyltryptophan as an intermediate. In this study, the biosynthesis of (2S,3R)-β-methyltryptophan and its isomer (2S,3S)-β-methyltryptophan by enzymes from the streptonigrin biosynthetic pathway is demonstrated. StnR is a pyridoxal 5'-phosphate (PLP)-dependent aminotransferase that catalyzes a transamination between L-tryptophan and β-methyl indolepyruvate. StnQ1 is an S-adenosylmethionine (SAM)-dependent C-methyltransferase and catalyzes β-methylation of indolepyruvate to generate (R)-β-methyl indolepyruvate. Although StnR exhibited a significant preference for (S)-β-methyl indolepyruvate over the (R)-epimer, StnQ1 and StnR together catalyze (2S,3R)-β-methyltryptophan formation from L-tryptophan. StnK3 is a cupin superfamily protein responsible for conversion of (R)-β-methyl indolepyruvate to its (S)-epimer and enables (2S,3S)-β-methyltryptophan biosynthesis from L-tryptophan when combined with StnQ1 and StnR. Most importantly, (2S,3S)-β-methyltryptophan was established as the biosynthetic intermediate of the streptonigrin pathway by feeding experiments with a knockout mutant, contradicting the previous proposal that stated (2S,3R)-β-methyltryptophan as the intermediate. These data set the stage for the complete elucidation of the streptonigrin biosynthetic pathway, which would unlock the potential of creating new streptonigrin analogues by genetic manipulation of the biosynthetic machinery.
Topics: Antineoplastic Agents; Bacterial Proteins; Chromatography, High Pressure Liquid; Escherichia coli; Mass Spectrometry; Methyltransferases; Plasmids; S-Adenosylmethionine; Stereoisomerism; Streptomyces; Streptonigrin; Tryptophan
PubMed: 26847951
DOI: 10.1038/srep20273 -
Infection and Immunity Jul 2018Pneumococcal conjugate vaccines (PCV) elicit opsonophagocytic (opsonic) antibodies to pneumococcal capsular polysaccharides (PPS) and reduce nasopharyngeal (NP)...
Pneumococcal conjugate vaccines (PCV) elicit opsonophagocytic (opsonic) antibodies to pneumococcal capsular polysaccharides (PPS) and reduce nasopharyngeal (NP) colonization by vaccine-included serotypes. However, nonopsonic antibodies may also be important for protection against pneumococcal disease. For example, 1E2, a mouse IgG1 monoclonal antibody (MAb) to the serotype 3 (ST3) PPS (PPS3), reduced ST3 NP colonization in mice and altered ST3 gene expression Here, we determined whether 1E2 affects ST3 gene expression during colonization of mice by performing RNA sequencing on NP lavage fluid from ST3-infected mice treated with 1E2, a control MAb, or phosphate-buffered saline. Compared to the results for the controls, 1E2 significantly altered the expression of over 50 genes. It increased the expression of the operon, which encodes an iron uptake system, and decreased the expression of , which encodes a protein critical for resistance to oxidative stress. 1E2-mediated effects on ST3 required divalent binding, as Fab fragments did not reduce NP colonization or alter ST3 gene expression. , 1E2 induced dose-dependent ST3 growth arrest and altered and expression, whereas an opsonic PPS3 MAb, 5F6, did not. 1E2-treated bacteria were more sensitive to hydrogen peroxide and the iron-requiring antibiotic streptonigrin, suggesting that 1E2 may increase iron import and enhance sensitivity to oxidative stress. Finally, 1E2 also induced rapid capsule shedding , suggesting that this may initiate 1E2-induced changes in sensitivity to oxidative stress and gene expression. Our data reveal a novel mechanism of direct, antibody-mediated antibacterial activity that could inform new directions in antipneumococcal therapy and vaccine development.
Topics: Animals; Antibodies, Bacterial; Antibodies, Monoclonal; Bacterial Capsules; Female; Gene Expression; Mice; Mice, Inbred C57BL; Nasopharynx; Oxidative Stress; Pneumococcal Vaccines; Streptococcus pneumoniae
PubMed: 29735523
DOI: 10.1128/IAI.00300-18 -
Scientific Reports Jun 2020Elevated levels of plasma alpha1-antitrypsin (AAT) correlate with a poor prognosis of various cancers. Herein, we investigated effects of exogenous AAT on non-small lung...
Elevated levels of plasma alpha1-antitrypsin (AAT) correlate with a poor prognosis of various cancers. Herein, we investigated effects of exogenous AAT on non-small lung cancer cell lines with high (H1975) and very low (H661) baseline expression of SERPINA1 gene encoding AAT protein. Comparison of cells grown for 3 weeks in a regular medium versus medium supplemented with 2 mg/ml of AAT revealed that in the presence of AAT cells acquire better proliferative properties, resistance to staurosporine (STS)-induced apoptosis, and show higher expression of CLU, a pro-tumorigenic gene coding clusterin protein. Similarly, the co-administration of STS with AAT or addition of AAT to the cells pre-treated with STS abrogated effects of STS in both cell lines. Following experiments with H1975 cells have shown that AAT blocks critical steps in STS-induced cell death: inhibition of AKT/MAPK pathways, and activation of caspase 3 and autophagy. AAT does not inhibit apoptosis-triggered by chloroquine (inhibitor of autophagy) or streptonigrin (inducer of p53 pathway). The anti-apoptotic effects of AAT were unaffected by lipopolysaccharide (LPS). However, AAT induced TLR4 levels and enhanced LPS effects on the production of IL-6, a tumor-promoting cytokine. Our data provide further evidence that AAT plays a significant role in the tumorigenesis.
Topics: Apoptosis; Autophagy; Cell Movement; Cell Proliferation; Cytokines; Drug Resistance, Neoplasm; Enzyme Inhibitors; Humans; Lipopolysaccharides; Lung Neoplasms; Protective Agents; Serine Proteinase Inhibitors; Staurosporine; Tumor Cells, Cultured; alpha 1-Antitrypsin
PubMed: 32533048
DOI: 10.1038/s41598-020-66825-w -
Environmental Microbiology Sep 2021Xanthomonas oryzae pv. oryzae (Xoo) causes a serious disease of rice known as bacterial leaf blight. Several virulence-associated functions have been characterized in...
Xanthomonas oryzae pv. oryzae (Xoo) causes a serious disease of rice known as bacterial leaf blight. Several virulence-associated functions have been characterized in Xoo. However, the role of important second messenger c-di-GMP signalling in the regulation of virulence-associated functions still remains elusive in this phytopathogen. In this study we have performed an investigation of 13 c-di-GMP modulating deletion mutants to understand their contribution in Xoo virulence and lifestyle transition. We show that four Xoo proteins, Xoo2331, Xoo2563, Xoo2860 and Xoo2616, are involved in fine-tuning the in vivo c-di-GMP abundance and also play a role in the regulation of virulence-associated functions. We have further established the importance of the GGDEF domain of Xoo2563, a previously characterized c-di-GMP phosphodiesterase, in the virulence-associated functions of Xoo. Interestingly the strain harbouring the GGDEF domain deletion (ΔXoo2563 ) exhibited EPS deficiency and hypersensitivity to streptonigrin, indicative of altered iron metabolism. This is in contrast to the phenotype exhibited by an EAL overexpression strain wherein, the ΔXoo2563 exhibited other phenotypes, similar to the strain overexpressing the EAL domain. Taken together, our results indicate a complex interplay of c-di-GMP signalling with the cell-cell signalling to coordinate virulence-associated function in Xoo.
Topics: Bacterial Proteins; Cyclic GMP; Gene Expression Regulation, Bacterial; Plant Diseases; Virulence; Xanthomonas
PubMed: 34240791
DOI: 10.1111/1462-2920.15664 -
Journal of Toxicology and Environmental... 2018The functional characterization of marine macroalgae toward their potential to strength genome protection is still scarce. Hence, the aim of this study was to assess the...
The functional characterization of marine macroalgae toward their potential to strength genome protection is still scarce. Hence, the aim of this study was to assess the antigenotoxic potential of Ulva rigida, Fucus vesiculosus, and Gracilaria species in Drosophila melanogaster following dietary exposure and adopting the somatic mutation and recombination test (SMART). All macroalgae displayed a genoprotection activity, namely against an exogenous challenge (streptonigrin). The action against subtler endogenous pressures was also noted indicating that supplementation level is a critical factor. Gracilaria species provided ambivalent indications, since 10% of G. vermiculophylla inhibited the egg laying and/or larvae development, while 10% of G. gracilis promoted spontaneous genotoxicity. The effects of U. rigida were modulated (in intensity) by the growing conditions, demonstrating higher genoprotection against streptonigrin-induced damage when grown in an aquaculture-controlled system, while the effectiveness against spontaneous genotoxicity was more apparent in specimens grown under wild conditions. In contrast, F. vesiculosus did not produce significant differences in its potential under varying growing conditions. Overall, these findings shed some light on the macroalgae ability toward genome protection, contributing to the development of algaculture industry, and reinforcing the concept of functional food and its benefits.
Topics: Animal Feed; Animals; Diet; Dietary Supplements; Drosophila melanogaster; Fucus; Gracilaria; Larva; Mutagenicity Tests; Mutagens; Protective Agents; Seaweed; Streptonigrin; Ulva
PubMed: 30156999
DOI: 10.1080/15287394.2018.1507856 -
Scientific Reports Jan 2017Streptonigrin methylesterase A (StnA) is one of the tailoring enzymes that modify the aminoquinone skeleton in the biosynthesis pathway of Streptomyces species. Although...
Streptonigrin methylesterase A (StnA) is one of the tailoring enzymes that modify the aminoquinone skeleton in the biosynthesis pathway of Streptomyces species. Although StnA has no significant sequence homology with the reported α/β-fold hydrolases, it shows typical hydrolytic activity in vivo and in vitro. In order to reveal its functional characteristics, the crystal structures of the selenomethionine substituted StnA (SeMet-StnA) and the complex (S185A mutant) with its substrate were resolved to the resolution of 2.71 Å and 2.90 Å, respectively. The overall structure of StnA can be described as an α-helix cap domain on top of a common α/β hydrolase domain. The substrate methyl ester of 10'-demethoxystreptonigrin binds in a hydrophobic pocket that mainly consists of cap domain residues and is close to the catalytic triad Ser185-His349-Asp308. The transition state is stabilized by an oxyanion hole formed by the backbone amides of Ala102 and Leu186. The substrate binding appears to be dominated by interactions with several specific hydrophobic contacts and hydrogen bonds in the cap domain. The molecular dynamics simulation and site-directed mutagenesis confirmed the important roles of the key interacting residues in the cap domain. Structural alignment and phylogenetic tree analysis indicate that StnA represents a new subfamily of lipolytic enzymes with the specific binding pocket located at the cap domain instead of the interface between the two domains.
Topics: Antibiotics, Antineoplastic; Bacterial Proteins; Carboxylic Ester Hydrolases; Catalytic Domain; Escherichia coli; Molecular Conformation; Molecular Dynamics Simulation; Sequence Analysis, Protein; Streptonigrin; Substrate Specificity
PubMed: 28074848
DOI: 10.1038/srep40254 -
Microorganisms May 2024is an emerging human pathogen that has a high rate of incidence in immunocompromised individuals. We have found a putative secondary metabolite pathway within , which...
is an emerging human pathogen that has a high rate of incidence in immunocompromised individuals. We have found a putative secondary metabolite pathway within , which may be a key factor in its pathogenesis. This novel pathway is encoded in a gene cluster spanning MAB_0284c to 0305 and is related to pathways, producing the secondary metabolites streptonigrin and nybomycin. We constructed an in-frame deletion of the MAB_0295 () gene and tested it in our animal model. We have previously shown that tadpoles, which have functional lungs and T cells, can serve as a reliable comparative model for persistent infection and pathogenesis. Here, we report that tadpoles intraperitoneally infected with the ∆ mutant exhibit early decreased bacterial loads and significantly increased survival compared with those infected with WT . ∆ mutant also induced lower transcript levels of several pro-inflammatory cytokines (, , , ) than those of WT in the liver and lungs. In addition, there was impaired macrophage recruitment and decreased macrophage infection in tadpoles infected with the ∆ mutant, by tail wound inoculation, compared to those infected with the WT bacteria, as assayed by intravital confocal microscopy. These data underline the relevance and usefulness of tadpoles as a novel comparative animal model to identify genetic determinants of immunopathogenesis, suggesting a role for this novel and uncharacterized pathway in pathogenesis and macrophage recruitment.
PubMed: 38930501
DOI: 10.3390/microorganisms12061120 -
Oncology Reports Nov 2017Tumor necrosis factor α (TNFα) and TNF-related apoptosis-inducing ligand (TRAIL) can trigger the extrinsic apoptosis pathway. Our previous study indicated that...
Tumor necrosis factor α (TNFα) and TNF-related apoptosis-inducing ligand (TRAIL) can trigger the extrinsic apoptosis pathway. Our previous study indicated that connexin32 (Cx32) inhibited streptonigrin-induced intrinsic apoptosis via the epidermal growth factor receptor (EGFR) pathway. However, whether Cx32 can exert effects on the extrinsic apoptosis pathway through EGFR signaling remains unclear. In the present study, we investigated the role of Cx32 in extrinsic apoptosis induced by treatment with TNFα + cycloheximide (CHX) or afatinib in human cervical cancer (CaCx) cells. In stable inducible Cx32-transfected HeLa cells (HeLa-Cx32), Cx32 expression was induced by treatment with doxycycline (Dox). Furthermore, C-33A cells, which natively express high levels of Cx32, were used as a cell model for knockdown of Cx32 with siRNA. To determine the non-junctional function of Cx32 in apoptosis, 18α-glycyrrhetinic acid (18α-GA), a gap junction intracellular communication (GJIC) inhibitor, was used. Our results showed that Cx32 could inhibit apoptosis induced by TNFα + afatinib with or without the GJIC inhibitor. In clinical cervical tissue samples, we found that the expression of survivin was markedly higher in CaCx than in normal cervix tissue, which was in accordance with the expression of Cx32 in our previous study. In HeLa-Cx32 cells, we also found that Cx32 upregulated the expression of Cox-2. In addition, Cx32 upregulated EGFR expression in low-density culture (lacking GJ formation). Cx32 could also promote the expression of EGFR, phospho-STAT3 and phospho-ERK in HeLa-Cx32 cells following TNFα treatment. After knocking down Cx32 in C-33A cells, the expression levels of survivin and TNFα were downregulated. The present study verifies that Cx32 exerts an inhibitory effect on extrinsic apoptosis in CaCx cells, and suggests that Cx32 may regulate the progression and micro-environment of CaCx cells.
Topics: Afatinib; Apoptosis; Connexins; Cycloheximide; Doxycycline; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; HeLa Cells; Humans; Inhibitor of Apoptosis Proteins; Phosphorylation; Quinazolines; Survivin; Tumor Necrosis Factor-alpha; Uterine Cervical Neoplasms; Gap Junction beta-1 Protein
PubMed: 28901517
DOI: 10.3892/or.2017.5950 -
Infection and Immunity Jun 2017(group A [GAS]) is an obligate human pathogen responsible for a broad spectrum of human disease. GAS has a requirement for metal homeostasis within the human host and,...
(group A [GAS]) is an obligate human pathogen responsible for a broad spectrum of human disease. GAS has a requirement for metal homeostasis within the human host and, as such, tightly modulates metal uptake and efflux during infection. Metal acquisition systems are required to combat metal sequestration by the host, while metal efflux systems are essential to protect against metal overload poisoning. Here, we investigated the function of PmtA (erR-regulated etal ransporter ), a P-type ATPase efflux pump, in invasive GAS M1T1 strain 5448. We reveal that PmtA functions as a ferrous iron [Fe(II)] efflux system. In the presence of high Fe(II) concentrations, the 5448Δ deletion mutant exhibited diminished growth and accumulated 5-fold-higher levels of intracellular Fe(II) than did the wild type and the complemented mutant. The 5448Δ deletion mutant also showed enhanced susceptibility to killing by the Fe-dependent antibiotic streptonigrin as well as increased sensitivity to hydrogen peroxide and superoxide. We suggest that the PerR-mediated control of Fe(II) efflux by PmtA is important for bacterial defense against oxidative stress. PmtA represents an exemplar for an Fe(II) efflux system in a host-adapted Gram-positive bacterial pathogen.
Topics: Adenosine Triphosphatases; Animals; Bacterial Proteins; Gene Expression Regulation, Bacterial; Homeostasis; Humans; Hydrogen Peroxide; Iron; Membrane Transport Proteins; Mice; Mice, Transgenic; Mutation; Oxidative Stress; Streptococcal Infections; Streptococcus pyogenes; Streptonigrin
PubMed: 28373352
DOI: 10.1128/IAI.00140-17