scholarly journals Entamoeba histolytica Adaption to Auranofin: A Phenotypic and Multi-Omics Characterization

Antioxidants ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1240
Author(s):  
Yana Shaulov ◽  
Lotem Sarid ◽  
Meirav Trebicz-Geffen ◽  
Serge Ankri
Keyword(s):  
Entamoeba Histolytica ◽  
Pathogenic Bacteria ◽  
Nitrosative Stress ◽  
Redox Homeostasis ◽  
Thioredoxin Reductase ◽  
Cytoskeletal Proteins ◽  
Wild Type ◽  
Genes Encoding ◽  
Upregulated Genes ◽  
Important Enzyme

Auranofin (AF), an antirheumatic agent, targets mammalian thioredoxin reductase (TrxR), an important enzyme controlling redox homeostasis. AF is also highly effective against a diversity of pathogenic bacteria and protozoan parasites. Here, we report on the resistance of the parasite Entamoeba histolytica to 2 µM of AF that was acquired by gradual exposure of the parasite to an increasing amount of the drug. AF-adapted E. histolytica trophozoites (AFAT) have impaired growth and cytopathic activity, and are more sensitive to oxidative stress (OS), nitrosative stress (NS), and metronidazole (MNZ) than wild type (WT) trophozoites. Integrated transcriptomics and redoxomics analyses showed that many upregulated genes in AFAT, including genes encoding for dehydrogenase and cytoskeletal proteins, have their product oxidized in wild type trophozoites exposed to AF (acute AF trophozoites) but not in AFAT. We also showed that the level of reactive oxygen species (ROS) and oxidized proteins (OXs) in AFAT is lower than that in acute AF trophozoites. Overexpression of E. histolytica TrxR (EhTrxR) did not protect the parasite against AF, which suggests that EhTrxR is not central to the mechanism of adaptation to AF.

scholarly journals Entamoeba histolytica adaption to auranofin: a phenotypic and multi-omics characterization

2021 ◽  
Author(s):  
Yana Shaulov ◽  
Lotem SARID ◽  
Meirav Trebicz-Geffen ◽  
Serge Ankri
Keyword(s):  
Entamoeba Histolytica ◽  
Pathogenic Bacteria ◽  
Nitrosative Stress ◽  
Thioredoxin Reductase ◽  
Cytoskeletal Proteins ◽  
Wild Type ◽  
Protozoan Parasites ◽  
Genes Encoding ◽  
Upregulated Genes ◽  
Important Enzyme

Auranofin (AF), an antirheumatic agent, targets mammalian thioredoxin reductase (TrxR), an important enzyme controlling redox homeostasisis, AF is also very effective against a diversity of pathogenic bacteria and protozoan parasites. Here, we report about the resistance of the parasite Entamoeba histolytica to 2 microM of AF that has been acquired by gradual exposure of the parasite to increasing amount of the drug. AF adapted E.histolytica trophozoites (AFAT) has an impaired growth, cytopathic activity and they are more sensitive to oxidative stress (OS), nitrosative stress (NS) and metronidazole (MTZ) than wild type (WT) trophozoites. Integrated transcriptomics and redoxomics analyses showed that many upregulated genes in AFAT, including genes encoding for dehydrogenase and cytoskeletal proteins, have their product oxidized in wild type trophozoites exposed to AF (acute AF trophozoites) but not in AFAT. We also showed that the level of reactive oxygen species (ROS) and oxidized proteins (OXs) in AFAT is lower than that of acute AF trophozoites. Overexpression of E.histolytica TrxR (EhTrxR) did not protect the parasite against AF which suggests that EhTrxR is not central is the mechanism of adaptation to AF.

scholarly journals Rsp5p, a New Link between the Actin Cytoskeleton and Endocytosis in the Yeast Saccharomyces cerevisiae

2002 ◽  
Vol 22 (20) ◽  
pp. 6946-6948 ◽  
Author(s):  
Joanna Kamińska ◽  
Beata Gajewska ◽  
Anita K. Hopper ◽  
Teresa ˙Zołądek
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Actin Cytoskeleton ◽  
Cytoskeletal Proteins ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Ww Domains ◽  
Ubiquitin Protein Ligase ◽  
Growth Defects ◽  
Genes Encoding ◽  
Cytoskeleton Dynamics

ABSTRACT Rsp5p is an ubiquitin-protein ligase of Saccharomyces cerevisiae that has been implicated in numerous processes including transcription, mitochondrial inheritance, and endocytosis. Rsp5p functions at multiple steps of endocytosis, including ubiquitination of substrates and other undefined steps. We propose that one of the roles of Rsp5p in endocytosis involves maintenance and remodeling of the actin cytoskeleton. We report the following. (i) There are genetic interactions between rsp5 and several mutant genes encoding actin cytoskeletal proteins. rsp5 arp2, rsp5 end3, and rsp5 sla2 double mutants all show synthetic growth defects. Overexpressed wild-type RSP5 or mutant rsp5 genes with lesions of some WW domains suppress growth defects of arp2 and end3 cells. The defects in endocytosis, actin cytoskeleton, and morphology of arp2 are also suppressed. (ii) Rsp5p and Sla2p colocalize in abnormal F-actin-containing clumps in arp2 and pan1 mutants. Immunoprecipitation experiments confirmed that Rsp5p and Act1p colocalize in pan1 mutants. (iii) Rsp5p and Sla2p coimmunoprecipitate and partially colocalize to punctate structures in wild-type cells. These studies provide the first evidence for an interaction of an actin cytoskeleton protein with Rsp5p. (iv) rsp5-w1 mutants are resistant to latrunculin A, a drug that sequesters actin monomers and depolymerizes actin filaments, consistent with the fact that Rsp5p is involved in actin cytoskeleton dynamics.

Impairment of NF-κB activation and modulation of gene expression by calpastatin

2000 ◽  
Vol 279 (3) ◽  
pp. C709-C716 ◽  
Author(s):  
Fei Chen ◽  
Laurence M. Demers ◽  
Val Vallyathan ◽  
Yongju Lu ◽  
Vincent Castranova ◽  
...  
Keyword(s):  
Morphological Change ◽  
Bronchial Epithelial Cell ◽  
Cytoskeletal Proteins ◽  
Regulatory Proteins ◽  
Human Genes ◽  
Genes Encoding ◽  
Associated Proteins ◽  
Proteolytic Activities ◽  
Modulation Of Gene Expression ◽  
Upregulated Genes

To address the involvement of the calpain system in both basal and silica-induced nuclear factor (NF)-κB activation, several human bronchial epithelial cell lines were established in which an intracellular inhibitor of calpain, calpastatin, was stably expressed. Reduced basal and silica-induced inhibitor (IκBα) degradation and NF-κB activation were observed in cells stably overexpressing calpastatin. In addition, the cells in which calpain was constitutively inhibited by the overexpression of calpastatin exhibited a notable morphological change. Whereas empty vector-transfected cells displayed a morphology indistinguishable from that of parental cells, cells overexpressing calpastatin exhibited a mosaic morphological change with reduced formation of lamella 30 min after the cells were seeded. Genefilter microarray experiments, in which 3,965 human genes can be evaluated for their expression at the same time, showed that calpastatin downregulated genes encoding several membrane-associated proteins or nuclear proteins and upregulated genes of collagen α2, DAZ, and mitochondrial capsule selenoprotein. These results suggest that, in addition to their proteolytic activities on cytoskeletal proteins and other cellular regulatory proteins, calpain-calpastatin systems can also affect the expression levels of genes encoding structural or regulatory proteins.

scholarly journals Phycobilisome breakdown effector NblD is required to maintain the cellular amino acid composition during nitrogen starvation

2021 ◽  
Author(s):  
Vanessa Krauspe ◽  
Stefan Timm ◽  
Martin Hagemann ◽  
Wolfgang R. Hess
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Essential Role ◽  
Nitrogen Starvation ◽  
Redox Homeostasis ◽  
Metabolic Homeostasis ◽  
Wild Type ◽  
Small Protein ◽  
Small Proteins ◽  
Genes Encoding

Small proteins are critically involved in the acclimation response of photosynthetic cyanobacteria to nitrogen starvation. NblD is the 66-amino-acid effector of nitrogen-limitation-induced phycobilisome breakdown, which is believed to replenish the cellular amino acid pools. To address the physiological functions of NblD, the concentrations of amino acids, intermediates of the arginine catabolism pathway and several organic acids were measured during the response to nitrogen starvation in the cyanobacterium Synechocystis sp. PCC 6803 wild type and in an nblD deletion strain. A characteristic signature of metabolite pool composition was identified, which shows that NblD-mediated phycobilisome degradation is required to maintain the cellular amino acid and organic acid pools during nitrogen starvation. Specific deviations from the wild type suggest wider-reaching effects that also affect such processes as redox homeostasis via glutathione and tetrapyrrole biosynthesis, both of which are linked to the strongly decreased glutamate pool, and transcriptional reprogramming via an enhanced concentration of 2-oxoglutarate, the metabolite co-regulator of the NtcA transcription factor. The essential role played by NblD in metabolic homeostasis is consistent with the widespread occurrence of NblD throughout the cyanobacterial radiation and the previously observed strong positive selection for the nblD gene under fluctuating nitrogen supply. Importance Cyanobacteria play important roles in the global carbon and nitrogen cycles. In their natural environment, these organisms are exposed to fluctuating nutrient conditions. Nitrogen starvation induces a coordinated nitrogen-saving program that includes the breakdown of nitrogen-rich photosynthetic pigments, particularly phycobiliproteins. The small protein NblD was recently identified as an effector of phycobilisome breakdown in cyanobacteria. In this study, we demonstrate that the NblD-mediated degradation of phycobiliproteins is needed to sustain cellular pools of soluble amino acids and other crucial metabolites. The essential role played by NblD in metabolic homeostasis explains why genes encoding this small protein are conserved in almost all members of cyanobacterial radiation.

Pap1p-dependent upregulation of thioredoxin 3 and thioredoxin reductase genes from the fission yeast under nitrosative stress

2012 ◽  
Vol 58 (2) ◽  
pp. 206-211 ◽  
Author(s):  
Min-Sik Park ◽  
Hyeon-Jung Kim ◽  
A Rum Park ◽  
Kisup Ahn ◽  
Hye-Won Lim ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Nitrosative Stress ◽  
Thioredoxin Reductase

scholarly journals Expression and function of the cdgD gene, encoding a CHASE–PAS-DGC-EAL domain protein, in Azospirillum brasilense

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
José Francisco Cruz-Pérez ◽  
Roxana Lara-Oueilhe ◽  
Cynthia Marcos-Jiménez ◽  
Ricardo Cuatlayotl-Olarte ◽  
María Luisa Xiqui-Vázquez ◽  
...  
Keyword(s):  
Azospirillum Brasilense ◽  
Type Strain ◽  
Wild Type Strain ◽  
Soil Conditions ◽  
Wild Type ◽  
Gene Encoding ◽  
Wheat Roots ◽  
Genes Encoding ◽  
In The Wild ◽  
Plant Growth Promoting

AbstractThe plant growth-promoting bacterium Azospirillum brasilense contains several genes encoding proteins involved in the biosynthesis and degradation of the second messenger cyclic-di-GMP, which may control key bacterial functions, such as biofilm formation and motility. Here, we analysed the function and expression of the cdgD gene, encoding a multidomain protein that includes GGDEF-EAL domains and CHASE and PAS domains. An insertional cdgD gene mutant was constructed, and analysis of biofilm and extracellular polymeric substance production, as well as the motility phenotype indicated that cdgD encoded a functional diguanylate protein. These results were correlated with a reduced overall cellular concentration of cyclic-di-GMP in the mutant over 48 h compared with that observed in the wild-type strain, which was recovered in the complemented strain. In addition, cdgD gene expression was measured in cells growing under planktonic or biofilm conditions, and differential expression was observed when KNO3 or NH4Cl was added to the minimal medium as a nitrogen source. The transcriptional fusion of the cdgD promoter with the gene encoding the autofluorescent mCherry protein indicated that the cdgD gene was expressed both under abiotic conditions and in association with wheat roots. Reduced colonization of wheat roots was observed for the mutant compared with the wild-type strain grown in the same soil conditions. The Azospirillum-plant association begins with the motility of the bacterium towards the plant rhizosphere followed by the adsorption and adherence of these bacteria to plant roots. Therefore, it is important to study the genes that contribute to this initial interaction of the bacterium with its host plant.

scholarly journals Alopecia in Harlequin mutant mice is associated with reduced AIF protein levels and expression of retroviral elements

2020 ◽  
Author(s):  
Maik Hintze ◽  
Sebastian Griesing ◽  
Marion Michels ◽  
Birgit Blanck ◽  
Lena Wischhof ◽  
...  
Keyword(s):  
Hair Growth ◽  
Transcriptional Regulators ◽  
Mutant Mice ◽  
Wild Type ◽  
Protein Levels ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Keratinocyte Cell ◽  
Apoptosis Inducing Factor ◽  
Hair Cortex

AbstractWe investigated the contribution of apoptosis-inducing factor (AIF), a key regulator of mitochondrial biogenesis, in supporting hair growth. We report that pelage abnormalities developed during hair follicle (HF) morphogenesis in Harlequin (Hq) mutant mice. Fragility of the hair cortex was associated with decreased expression of genes encoding structural hair proteins, though key transcriptional regulators of HF development were expressed at normal levels. Notably, Aifm1 (R200 del) knockin males and Aifm1(R200 del)/Hq females showed minor hair defects, despite substantially reduced AIF levels. Furthermore, we cloned the integrated ecotropic provirus of the Aifm1Hq allele. We found that its overexpression in wild-type keratinocyte cell lines led to down-regulation of HF-specific Krt84 and Krtap3-3 genes without altering Aifm1 or epidermal Krt5 expression. Together, our findings imply that pelage paucity in Hq mutant mice is mechanistically linked to severe AIF deficiency and is associated with the expression of retroviral elements that might potentially influence the transcriptional regulation of structural hair proteins.

scholarly journals HGG-26. H3G34V MUTATION AFFECTS GENOMIC H3K36 METHYLATION IN PEDIATRIC GLIOMA

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii348-iii348
Author(s):  
Tina Huang ◽  
Andrea Piunti ◽  
Elizabeth Bartom ◽  
Jin Qi ◽  
Rintaro Hashizume ◽  
...  
Keyword(s):  
Western Blot ◽  
Allelic Frequency ◽  
Glioma Cell Line ◽  
Fluorescent Imaging ◽  
Wild Type ◽  
Gene Expressions ◽  
Pediatric Glioma ◽  
Genes Encoding ◽  
Normal Human

Abstract BACKGROUND Histone H3.3 mutation (H3F3A) occurs in 50% of cortical pediatric high-grade gliomas. This mutation replaces glycine 34 with arginine or valine (G34R/V), impairing SETD2 activity (H3K36-specific trimethyltransferase), resulting in reduced H3K36me on H3G34V nucleosomes relative to wild-type. This contributes to genomic instability and drives distinct gene expressions associated with tumorigenesis. However, it is not known if this differential H3K36me3 enrichment is due to H3G34V mutant protein alone. Therefore, we set to elucidate the effect of H3G34V on genomic H3K36me3 enrichment in vitro. METHODS Doxycycline-inducible short hairpin RNA (shRNA) against H3F3A was delivered via lentivirus to established H3G34V mutant pediatric glioma cell line KNS42, and H3G34V introduced into H3.3 wild type normal human astrocytes (NHA). Transfections were confirmed by western blot, fluorescent imaging, and flow cytometry, with resulting H3.3WT and H3K36me3 expression determined by western blot. H3.3WT, H3K36me3, and H3G34V ChIP-Seq was performed to evaluate genomic enrichment. RESULTS Complete knockdown of H3G34V was achieved with DOX-induced shRNA, with no change in total H3.3, suggesting disproportionate allelic frequency of genes encoding H3.3 (H3F3A and H3F3B). Modest increase in H3K36me3 occurred after H3F3A-knockdown from KNS42, suggesting H3G34V alone impacts observed H3K36me3 levels. Distinct H3K36me3 genomic enrichment was observed with H3G34V knock-in. CONCLUSIONS We demonstrate that DOX-inducible knockdown of H3F3A in an H3G34V mutant pediatric glioma cells and H3G34V mutation transduction in wild-type astrocytes affects H3K36me3 expression. Further evaluation by ChIP-Seq analysis for restoration of wild-type genomic H3K36me3 enrichment patterns with H3G34V knockdown, and mutant H3K36me3 patterns with H3G34V transduction, is currently underway.

scholarly journals S-Nitrosothiol Signaling Is involved in Regulating Hydrogen Peroxide Metabolism of Zinc-Stressed Arabidopsis

2019 ◽  
Vol 60 (11) ◽  
pp. 2449-2463 ◽  
Author(s):  
Zs Kolbert ◽  
� Moln�r ◽  
D Ol�h ◽  
G Feigl ◽  
E Horv�th ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Plant Cells ◽  
Thioredoxin Reductase ◽  
Protein S ◽  
Protein Nitration ◽  
Wild Type ◽  
Accumulation Capacity ◽  
Zn Tolerance ◽  
Activity Loss ◽  
New Evidence

Abstract Accumulation of heavy metals such as zinc (Zn) disturbs the metabolism of reactive oxygen (e.g. hydrogen peroxide, H2O2) and nitrogen species (e.g. nitric oxide, NO; S-nitrosoglutathione, GSNO) in plant cells; however, their signal interactions are not well understood. Therefore, this study examines the interplay between H2O2 metabolism and GSNO signaling in Arabidopsis. Comparing the Zn tolerance of the wild type (WT), GSNO reductase (GSNOR) overexpressor 35S::FLAG-GSNOR1 and GSNOR-deficient gsnor1-3, we observed relative Zn tolerance of gsnor1-3, which was not accompanied by altered Zn accumulation capacity. Moreover, in gsnor1-3 plants Zn did not induce NO/S-nitrosothiol (SNO) signaling, possibly due to the enhanced activity of NADPH-dependent thioredoxin reductase. In WT and 35S::FLAG-GSNOR1, GSNOR was inactivated by Zn, and Zn-induced H2O2 is directly involved in the GSNOR activity loss. In WT seedlings, Zn resulted in a slight intensification of protein nitration detected by Western blot and protein S-nitrosation observed by resin-assisted capture of SNO proteins (RSNO-RAC). LC-MS/MS analyses indicate that Zn induces the S-nitrosation of ascorbate peroxidase 1. Our data collectively show that Zn-induced H2O2 may influence its own level, which involves GSNOR inactivation-triggered SNO signaling. These data provide new evidence for the interplay between H2O2 and SNO signaling in Arabidopsis plants affected by metal stress.

scholarly journals act Operon Control of Developmental Gene Expression in Myxococcus xanthus

2002 ◽  
Vol 184 (4) ◽  
pp. 1172-1179 ◽  
Author(s):  
Thomas M. A. Gronewold ◽  
Dale Kaiser
Keyword(s):  
Fruiting Body ◽  
Myxococcus Xanthus ◽  
The Other ◽  
Loss Of Function ◽  
Wild Type ◽  
Developmental Gene ◽  
Developmental Gene Expression ◽  
Mutant Strains ◽  
Genes Encoding ◽  
Signal Production

ABSTRACT Cell-bound C-signal guides the building of a fruiting body and triggers the differentiation of myxospores. Earlier work has shown that transcription of the csgA gene, which encodes the C-signal, is directed by four genes of the act operon. To see how expression of the genes encoding components of the aggregation and sporulation processes depends on C-signaling, mutants with loss-of-function mutations in each of the act genes were investigated. These mutations were found to have no effect on genes that are normally expressed up to 3 h into development and are C-signal independent. Neither the time of first expression nor the rate of expression increase was changed in actA, actB, actC, or actD mutant strains. Also, there was no effect on A-signal production, which normally starts before 3 h. By contrast, the null act mutants have striking defects in C-signal production. These mutations changed the expression of four gene reporters that are related to aggregation and sporulation and are expressed at 6 h or later in development. The actA and actB null mutations substantially decreased the expression of all these reporters. The other act null mutations caused either premature expression to wild-type levels (actC) or delayed expression (actD), which ultimately rose to wild-type levels. The pattern of effects on these reporters shows how the C-signal differentially regulates the steps that together build a fruiting body and differentiate spores within it.

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