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Hemerythrin is required for Aeromonas hydraphlia to survive in the macrophages of Anguilla japonica.Genetics and Molecular Research : GMR May 2016Survival in host phagocytes is an effective strategy for pathogenic microbes to spread. To understand the mechanisms of Aeromonas hydrophila survival within host...
Survival in host phagocytes is an effective strategy for pathogenic microbes to spread. To understand the mechanisms of Aeromonas hydrophila survival within host macrophages, a library of mini-Tn10 transposon insertion mutants was constructed. The M85 mutant, whose survival in host macrophages was only 23.1% of that of the wild-type (WT) strain, was utilized for further study. Molecular analysis showed that a 756-bp open reading frame (ORF) (GenBank accession No. CP007576) in the M85 mutant was interrupted by mini-Tn10. This ORF encodes for a 183-amino acid protein and displays the highest sequence identity (99%) with the hemerythrin (Hr) protein of A. hydrophila subspecies hydrophila ATCC 7966. The survival of the WT, M85 mutant, and complemented M85 (Hr) strains were compared in host macrophages in vitro, and the results showed that M85 exhibited defective survival, while that of M85 (Hr) was restored. To investigate the possible mechanisms of A. hydrophila survival in host macrophages, the expression of Hr under hyperoxic and hypoxic conditions was evaluated. The results revealed that the expression of this protein was higher under hyperoxic conditions than under hypoxic conditions, which indicates that Hr protein expression is sensitive to O2 concentration. Hydrogen peroxide sensitivity tests further suggested that the M85 mutant was more sensitive to oxidative stress than the WT and M85 (Hr) strains. Taken together, these results suggest that the Hr protein may act as an O2 sensor and as a detoxifier of reactive oxygen species, and is required for A. hydrophila survival within host macrophages.
Topics: Aeromonas hydrophila; Amino Acid Sequence; Anguilla; Animals; Bacterial Proteins; Cell Movement; Hemerythrin; Macrophages; Virulence
PubMed: 27173333
DOI: 10.4238/gmr.15028074 -
BMC Plant Biology Apr 2022CHY zinc-finger and RING finger (CHYR) proteins have been functionally characterized in plant growth, development and various stress responses. However, the genome-wide...
BACKGROUND
CHY zinc-finger and RING finger (CHYR) proteins have been functionally characterized in plant growth, development and various stress responses. However, the genome-wide analysis was not performed in wheat.
RESULTS
In this study, a total of 18 TaCHYR genes were identified in wheat and classified into three groups. All TaCHYR genes contained CHY-zinc finger, C3H2C3-type RING finger and zinc ribbon domains, and group III members included 1-3 hemerythrin domains in the N-terminus regions. TaCHYR genes in each group shared similar conserved domains distribution. Chromosomal location, synteny and cis-elements analysis of TaCHYRs were also analyzed. Real-time PCR results indicated that most of selected 9 TaCHYR genes exhibited higher expression levels in leaves during wheat seedling stage. All these TaCHYR genes were up-regulated after PEG treatment, and these TaCHYRs exhibited differential expression patterns in response to salt, cold and heat stress in seedling leaves. The growth of yeast cells expressing TaCHYR2.1, TaCHYR9.2 and TaCHYR11.1 were inhibited under salt and dehydration stress. Moreover, gene ontology (GO) annotation, protein interaction and miRNA regulatory network of TaCHYR genes were analyzed.
CONCLUSIONS
These results increase our understanding of CHYR genes and provide robust candidate genes for further functional investigations aimed at crop improvement.
Topics: Bread; Gene Expression Regulation, Plant; Genome, Plant; Multigene Family; Phylogeny; Plant Proteins; Seedlings; Stress, Physiological; Triticum; Zinc
PubMed: 35443615
DOI: 10.1186/s12870-022-03589-7 -
European Review For Medical and... Jun 2016The hemerythrin-like domain of F-box and leucine-rich repeat protein 5 (FBXL5), an E3 ubiquitin ligase subunit, has critical roles in the regulation of cancer cells...
OBJECTIVE
The hemerythrin-like domain of F-box and leucine-rich repeat protein 5 (FBXL5), an E3 ubiquitin ligase subunit, has critical roles in the regulation of cancer cells metastasis and chemoresistance by targeting diverse substrates for ubiquitin-mediated destruction.
MATERIALS AND METHODS
Here, we report that FBXL5 interacts with Rho GDP dissociation inhibitor 2 (RhoGDI2) and attenuates RhoGDI2-induced cisplatin resistance in gastric cancer cells. By utilizing immunoprecipitation (IP) coupled with mass spectrometry assay, we found that FBXL5 regulated gastric cancers migration via cortactin destruction.
RESULTS
Depletion of FBXL5 enhances cisplatin resistance of gastric cancer cells through Erk and p38 activation. However, FBXL5 did not affect the abundance and stability of RhoGDI2. Instead, FBXL5 was rapidly degraded in response to cisplatin treatment in RhoGDI2-overexpressing gastric cancer cells.
CONCLUSIONS
Collectively, our data suggested the existence of a FBXL5-RhoGDI2 negative feedback loop in RhoGDI2-induced cisplatin resistance in gastric cancer cells, implicating FBXL5 as a novel and promising therapeutic target for RhoGDI2-induced cisplatin resistance gastric cancers.
Topics: Cisplatin; Cortactin; Drug Interactions; Drug Resistance, Neoplasm; F-Box Proteins; Humans; Stomach Neoplasms; Ubiquitin-Protein Ligase Complexes; rho Guanine Nucleotide Dissociation Inhibitor beta
PubMed: 27383304
DOI: No ID Found -
Plant Signaling & Behavior Aug 2016BRUTUS (BTS) is a hemerythrin (HHE) domain containing E3 ligase that facilitates the degradation of POPEYE-like (PYEL) proteins in a proteasomal-dependent manner....
BRUTUS (BTS) is a hemerythrin (HHE) domain containing E3 ligase that facilitates the degradation of POPEYE-like (PYEL) proteins in a proteasomal-dependent manner. Deletion of BTS HHE domains enhances BTS stability in the presence of iron and also complements loss of BTS function, suggesting that the HHE domains are critical for protein stability but not for enzymatic function. The RING E3 domain plays an essential role in BTS' capacity to both interact with PYEL proteins and to act as an E3 ligase. Here we show that removal of the RING domain does not complement loss of BTS function. We conclude that enzymatic activity of BTS via the RING domain is essential for response to iron deficiency in plants. Further, we analyze possible BTS domain structure evolution and predict that the combination of domains found in BTS is specific to photosynthetic organisms, potentially indicative of a role for BTS and its orthologs in mitigating the iron-related challenges presented by photosynthesis.
Topics: Arabidopsis; Arabidopsis Proteins; Hemerythrin; Iron; Iron Deficiencies; Ubiquitin-Protein Ligases
PubMed: 27359166
DOI: 10.1080/15592324.2016.1204508 -
Journal of the American Chemical Society Aug 2017Flavo-diiron proteins (FDPs) are non-heme iron containing enzymes that are widespread in anaerobic bacteria, archaea, and protozoa, serving as the terminal components to...
Flavo-diiron proteins (FDPs) are non-heme iron containing enzymes that are widespread in anaerobic bacteria, archaea, and protozoa, serving as the terminal components to dioxygen and nitric oxide reductive scavenging pathways in these organisms. FDPs contain a dinuclear iron active site similar to that in hemerythrin, ribonucleotide reductase, and methane monooxygenase, all of which can bind NO and O. However, only FDP competently turns over NO to NO. Here, EPR and Mössbauer spectroscopies allow electronic characterization of the diferric and diferrous species of FDP. The exchange-coupling constant J (H = JS·S) was found to increase from +20 cm to +32 cm upon reduction of the diferric to the diferrous species, indicative of (1) at least one hydroxo bridge between the iron ions for both states and (2) a change to the diiron core structure upon reduction. In comparison to characterized diiron proteins and synthetic complexes, the experimental values were consistent with a dihydroxo bridged diferric core, which loses one hydroxo bridge upon reduction. DFT calculations of these structures gave values of J and Mössbauer parameters in agreement with experiment. Although the crystal structure shows a hydrogen bond between the iron bound aspartate and the bridging solvent molecule, the DFT calculations of structures consistent with the crystal structure gave calculated values of J incompatible with the spectroscopic results. We conclude that the crystal structure of the diferric state does not represent the frozen solution structure and that a mono-μ-hydroxo diferrous species is the catalytically functional state that reacts with NO and O. The new EPR spectroscopic probe of the diferric state indicated that the diferric structure of FDP prior to and immediately after turnover with NO are flavin mononucleotide (FMN) dependent, implicating an additional proton transfer role for FMN in turnover of NO.
Topics: Catalytic Domain; Electron Spin Resonance Spectroscopy; Ferric Compounds; Flavoproteins; Iron; Models, Molecular; Quantum Theory; Spectroscopy, Mossbauer; Thermotoga maritima
PubMed: 28756660
DOI: 10.1021/jacs.7b06546 -
Plant Physiology Sep 2017HEMERYTHRIN MOTIF-CONTAINING REALLY INTERESTING NEW GENE AND ZINC-FINGER PROTEIN1 (OsHRZ1) is a putative iron-binding sensor. However, it is unclear how OsHRZ1...
HEMERYTHRIN MOTIF-CONTAINING REALLY INTERESTING NEW GENE AND ZINC-FINGER PROTEIN1 (OsHRZ1) is a putative iron-binding sensor. However, it is unclear how OsHRZ1 transmits signals. In this study, we reveal that POSITIVE REGULATOR OF IRON HOMEOSTASIS1 (OsPRI1) interacts with OsHRZ1. A loss-of-function mutation to increased the sensitivity of plants to Fe-deficient conditions and down-regulated the expression of Fe-deficiency-responsive genes. Yeast one-hybrid and electrophoretic mobility shift assay results suggested that OsPRI1 binds to the and promoters. In vitro ubiquitination experiments indicated that OsPRI1 is ubiquitinated by OsHRZ1. Cell-free degradation assays revealed that the stability of OsPRI1 decreased in wild-type roots but increased in the mutant, suggesting OsHRZ1 is responsible for the instability of OsPRI1. The seedlings were insensitive to Fe-deficient conditions. When the mutation was introduced into mutants, the double mutant was more sensitive to Fe deficiency than the mutant. Additionally, the expression levels of Fe-deficiency-responsive genes were lower in the double mutant than in the mutant. Collectively, these results imply that OsPRI1, which is ubiquitinated by OsHRZ1, mediates rice responses to Fe deficiency by positively regulating and expression as part of the signal transduction cascade.
Topics: Down-Regulation; Gene Editing; Homeostasis; Iron; Iron Deficiencies; Loss of Function Mutation; Oryza; Phenotype; Plant Leaves; Plant Proteins; Plant Roots; Plant Shoots; Promoter Regions, Genetic; Protein Interaction Mapping; Seedlings; Signal Transduction; Transcription Factors; Two-Hybrid System Techniques; Ubiquitination
PubMed: 28751317
DOI: 10.1104/pp.17.00794 -
International Journal For Parasitology.... Dec 2020Copper is a trace metal that is necessary for all organisms but toxic when present in excess. Different mechanisms to avoid copper toxicity have been reported to date in...
Copper is a trace metal that is necessary for all organisms but toxic when present in excess. Different mechanisms to avoid copper toxicity have been reported to date in pathogenic organisms such as Cryptococcus neoformans and Candida albicans. However, little if anything is known about pathogenic protozoans despite their importance in human and veterinary medicine. Naegleria fowleri is a free-living amoeba that occurs naturally in warm fresh water and can cause a rapid and deadly brain infection called primary amoebic meningoencephalitis (PAM). Here, we describe the mechanisms employed by N. fowleri to tolerate high copper concentrations, which include various strategies such as copper efflux mediated by a copper-translocating ATPase and upregulation of the expression of antioxidant enzymes and obscure hemerythrin-like and protoglobin-like proteins. The combination of different mechanisms efficiently protects the cell and ensures its high copper tolerance, which can be advantageous both in the natural environment and in the host. Nevertheless, we demonstrate that copper ionophores are potent antiamoebic agents; thus, copper metabolism may be considered a therapeutic target.
Topics: Adenosine Triphosphatases; Amoeba; Antioxidants; Brain; Copper; Humans; Naegleria fowleri
PubMed: 33096396
DOI: 10.1016/j.ijpddr.2020.10.001 -
Applied and Environmental Microbiology Dec 2018" Methanoperedens nitroreducens" is an archaeon that couples the anaerobic oxidation of methane to nitrate reduction. In natural and man-made ecosystems, this archaeon...
" Methanoperedens nitroreducens" is an archaeon that couples the anaerobic oxidation of methane to nitrate reduction. In natural and man-made ecosystems, this archaeon is often found at oxic-anoxic interfaces where nitrate, the product of aerobic nitrification, cooccurs with methane produced by methanogens. As such, populations of " Methanoperedens nitroreducens" could be prone to regular oxygen exposure. Here, we investigated the effect of 5% (vol/vol) oxygen exposure in batch activity assays on a " Methanoperedens nitroreducens" culture, enriched from an Italian paddy field. Metagenome sequencing of the DNA extracted from the enrichment culture revealed that 83% of 16S rRNA gene reads were assigned to a novel strain, " Methanoperedens nitroreducens Verserenetto." RNA was extracted, and metatranscriptome sequencing upon oxygen exposure revealed that the active community changed, most notably in the appearance of aerobic methanotrophs. The gene expression of " Methanoperedens nitroreducens" revealed that the key genes encoding enzymes of the methane oxidation and nitrate reduction pathways were downregulated. In contrast to this, we identified upregulation of glutaredoxin, thioredoxin family/like proteins, rubrerythrins, peroxiredoxins, peroxidase, alkyl hydroperoxidase, type A flavoproteins, FeS cluster assembly protein, and cysteine desulfurases, indicating the genomic potential of " Methanoperedens nitroreducens Verserenetto" to counteract the oxidative damage and adapt in environments where they might be exposed to regular oxygen intrusion. " Methanoperedens nitroreducens" is an anaerobic archaeon which couples the reduction of nitrate to the oxidation of methane. This microorganism is present in a wide range of aquatic environments and man-made ecosystems, such as paddy fields and wastewater treatment systems. In such environments, these archaea may experience regular oxygen exposure. However, " Methanoperedens nitroreducens" is able to thrive under such conditions and could be applied for the simultaneous removal of dissolved methane and nitrogenous pollutants in oxygen-limited systems. To understand what machinery " Methanoperedens nitroreducens" possesses to counteract the oxidative stress and survive, we characterized the response to oxygen exposure using a multi-omics approach.
Topics: Anaerobiosis; Archaeal Proteins; Bioreactors; Carboxylic Ester Hydrolases; DNA, Archaeal; Ecosystem; Flavoproteins; Gene Expression Regulation, Archaeal; Glutaredoxins; Hemerythrin; Metagenome; Methane; Methanosarcinales; Nitrates; Oxidation-Reduction; Oxidative Stress; Oxygen; Peroxidase; Peroxiredoxins; Phylogeny; RNA, Ribosomal, 16S; Rubredoxins; Sequence Analysis; Thioredoxins; Up-Regulation; Wastewater; Water Purification
PubMed: 30291120
DOI: 10.1128/AEM.01832-18 -
Journal of the American Chemical Society Apr 2012Flavodiiron proteins (FDPs) play important roles in the microbial nitrosative stress response in low-oxygen environments by reductively scavenging nitric oxide (NO)....
Flavodiiron proteins (FDPs) play important roles in the microbial nitrosative stress response in low-oxygen environments by reductively scavenging nitric oxide (NO). Recently, we showed that FMN-free diferrous FDP from Thermotoga maritima exposed to 1 equiv NO forms a stable diiron-mononitrosyl complex (deflavo-FDP(NO)) that can react further with NO to form N(2)O [Hayashi, T.; Caranto, J. D.; Wampler, D. A; Kurtz, D. M., Jr.; Moënne-Loccoz, P. Biochemistry 2010, 49, 7040-7049]. Here we report resonance Raman and low-temperature photolysis FTIR data that better define the structure of this diiron-mononitrosyl complex. We first validate this approach using the stable diiron-mononitrosyl complex of hemerythrin, Hr(NO), for which we observe a ν(NO) at 1658 cm(-1), the lowest ν(NO) ever reported for a nonheme {FeNO}(7) species. Both deflavo-FDP(NO) and the mononitrosyl adduct of the flavinated FPD (FDP(NO)) show ν(NO) at 1681 cm(-1), which is also unusually low. These results indicate that, in Hr(NO) and FDP(NO), the coordinated NO is exceptionally electron rich, more closely approaching the Fe(III)(NO(-)) resonance structure. In the case of Hr(NO), this polarization may be promoted by steric enforcement of an unusually small FeNO angle, while in FDP(NO), the Fe(III)(NO(-)) structure may be due to a semibridging electrostatic interaction with the second Fe(II) ion. In Hr(NO), accessibility and steric constraints prevent further reaction of the diiron-mononitrosyl complex with NO, whereas in FDP(NO) the increased nucleophilicity of the nitrosyl group may promote attack by a second NO to produce N(2)O. This latter scenario is supported by theoretical modeling [Blomberg, L. M.; Blomberg, M. R.; Siegbahn, P. E. J. Biol. Inorg. Chem. 2007, 12, 79-89]. Published vibrational data on bioengineered models of denitrifying heme-nonheme NO reductases [Hayashi, T.; Miner, K. D.; Yeung, N.; Lin, Y.-W.; Lu, Y.; Moënne-Loccoz, P. Biochemistry 2011, 50, 5939-5947 ] support a similar mode of activation of a heme {FeNO}(7) species by the nearby nonheme Fe(II).
Topics: Hemerythrin; Iron-Binding Proteins; Nitric Oxide; Oxidoreductases; Spectroscopy, Fourier Transform Infrared; Spectrum Analysis, Raman
PubMed: 22449095
DOI: 10.1021/ja301812p -
Proceedings of the National Academy of... Aug 1992A general method is described for substitution of Mn(II) and Co(II) into the diiron sites of hemerythrin and myohemerythrin. Characterizations of these metal-substituted...
A general method is described for substitution of Mn(II) and Co(II) into the diiron sites of hemerythrin and myohemerythrin. Characterizations of these metal-substituted proteins show that their structures closely resemble those of the native proteins. In particular, the four-helix bundle structure appears to be maintained. The apomyohemerythrin retains most of the native helix content but is considerably less stable to denaturation than are the metal-containing proteins. The relative affinities of M(II) for apohemerythrin--namely, Co greater than Fe greater than Mn--parallel the stabilities of the M2myohemerythrins to denaturation by guanidinium chloride. These results indicate that for myohemerythrin (i) the majority of the helical structure found in the native protein does not require incorporation of M(II) and (ii) stabilization of the native structure relative to the fully unfolded structure appears to be due predominantly to M(II)-protein interactions, at least for M = Fe and Co. Incorporation of M(II) also generates unfolding cooperativity in myohemerythrin. This cooperativity can be attributed to interhelical interactions, which are prevented in the apoprotein by solvation of the seven metal ligand residues. The results are consistent with a minimal model for folding/unfolding of myohemerythrin and hemerythrin subunits consisting of the sequential equilibria, N in equilibrium with I in equilibrium with D, between native, intermediate, and fully unfolded states, respectively. The properties of apomyohemerythrin make it a candidate for the intermediate state, I.
Topics: Amino Acid Sequence; Binding Sites; Calorimetry; Cations, Divalent; Cobalt; Hemerythrin; Iron; Manganese; Protein Conformation; Protein Denaturation
PubMed: 1496001
DOI: 10.1073/pnas.89.15.7065