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Chembiochem : a European Journal of... Sep 2023'Bacterial-type' ferredoxins host a cubane [4Fe4S] cluster that enables these proteins to mediate electron transfer and facilitate a broad range of biological processes....
'Bacterial-type' ferredoxins host a cubane [4Fe4S] cluster that enables these proteins to mediate electron transfer and facilitate a broad range of biological processes. Peptide maquettes based on the conserved cluster-forming motif have previously been reported and used to model the ferredoxins. Herein we explore the integration of a [4Fe4S]-peptide maquette into a H -powered electron transport chain. While routinely formed under anaerobic conditions, we illustrate by electron paramagnetic resonance (EPR) analysis that these maquettes can be reconstituted under aerobic conditions by using photoactivated NADH to reduce the cluster at 240 K. Attempts to tune the redox properties of the iron-sulfur cluster by introducing an Fe-coordinating selenocysteine residue were also explored. To demonstrate the integration of these artificial metalloproteins into a semi-synthetic electron transport chain, we utilize a ferredoxin-inspired [4Fe4S]-peptide maquette as the redox partner in the hydrogenase-mediated oxidation of H .
Topics: Ferredoxins; Iron-Sulfur Proteins; Hydrogenase; Oxidation-Reduction; Peptides; Electron Spin Resonance Spectroscopy
PubMed: 37391388
DOI: 10.1002/cbic.202300250 -
MSystems May 2024utilizes selenocysteine- (Sec-) containing proteins (selenoproteins), mostly active in the organism's primary energy metabolism, methanogenesis. During selenium...
UNLABELLED
utilizes selenocysteine- (Sec-) containing proteins (selenoproteins), mostly active in the organism's primary energy metabolism, methanogenesis. During selenium depletion, employs a set of enzymes containing cysteine (Cys) instead of Sec. The genes coding for these Sec-/Cys-containing isoforms were the only genes known of which expression is influenced by the selenium status of the cell. Using proteomics and transcriptomics, approx. 7% and 12%, respectively, of all genes/proteins were found differentially expressed/synthesized in response to the selenium supply. Some of the genes identified involve methanogenesis, nitrogenase functions, and putative transporters. An increase of transcript abundance for putative transporters under selenium depletion indicated the organism's effort to tap into alternative sources of selenium. is known to utilize selenite and dimethylselenide as selenium sources. To expand this list, a selenium-responsive reporter strain was assessed with nine other, environmentally relevant selenium species. While the effect of some was very similar to that of selenite, others were effectively utilized at lower concentrations. Conversely, selenate and selenoamino acids were only utilized at unphysiologically high concentrations and two compounds were not utilized at all. To address the role of the selenium-regulated putative transporters, mutant strains lacking one or two of the putative transporters were tested for the capability to utilize the different selenium species. Of the five putative transporters analyzed by loss-of-function mutagenesis, none appeared to be absolutely required for utilizing any of the selenium species tested, indicating they have redundant and/or overlapping specificities or are not dedicated selenium transporters.
IMPORTANCE
While selenium metabolism in microorganisms has been studied intensively in the past, global gene expression approaches have not been employed so far. Furthermore, the use of different selenium sources, widely environmentally interconvertible via biotic and abiotic processes, was also not extensively studied before. JJ is ideally suited for such analyses, thanks to its known selenium usage and available genetic tools. Thus, an overall view on the selenium regulon of was obtained via transcriptomic and proteomic analyses, which inspired further experimentation. This led to demonstrating the use of selenium sources was previously not known to employ. Also, an attempt-although so far unsuccessful-was made to pinpoint potential selenium transporter genes, in order to deepen our understanding of trace element utilization in this important model organism.
Topics: Methanococcus; Selenium; Proteomics; Archaeal Proteins; Gene Expression Profiling; Transcriptome; Gene Expression Regulation, Archaeal; Selenoproteins
PubMed: 38591896
DOI: 10.1128/msystems.01338-23 -
The Lancet Regional Health. Southeast... Jul 2023Despite having the highest number of preterm births globally, no genomic study on preterm birth was previously published from India or other South-Asian countries.
BACKGROUND
Despite having the highest number of preterm births globally, no genomic study on preterm birth was previously published from India or other South-Asian countries.
METHODS
We conducted a genome-wide association (GWA) study of spontaneous preterm birth (sPTB) on 6211 women from India. We used a novel resampling procedure to identify the associated single nucleotide polymorphisms (SNPs) followed by haplotype association analysis and imputation.
FINDINGS
We found that 512 maternal SNPs were associated with sPTB ( < 2.51e-3), of which minor allele at 19 SNPs (after Bonferroni correction) had increased genotype relative risk. Haplotypes containing six of the 19 SNPs (rs13011430, rs8179838, rs2327290, rs4798499, rs7629800, and rs13180906) were associated with sPTB ( < 9.9e-4; Bonferroni adjusted -value <0.05). After imputation in regions around the 19 SNPs, 15 imputed SNPs were found to be associated with sPTB (Bonferroni adjusted -value <0.05). One of these imputed SNPs, rs35760881, and three other SNPs (rs17307697, rs4308815, and rs10983507) were also reported to be associated with sPTB in women belonging to European ancestry. Moreover, we found that GG genotype at rs1152954, one of the associated SNPs, enhanced risk of sPTB and reduced telomere length.
INTERPRETATION
This is the first study from South Asia on the genome-wide identification of maternal SNPs associated with sPTB. These SNPs are known to alter the expression of genes associated with major pathways in sPTB viz. inflammation, apoptosis, cervical ripening, telomere maintenance, selenocysteine biosynthesis, myometrial contraction, and innate immunity. From a public health perspective, the trans-ethnic association of four SNPs identified in our study may help to stratify women with risk of sPTB in most populations.
FUNDING
Department of Biotechnology (India), Grand Challenges India - All Children Thriving Program and Biotechnology Industry Research Assistance Council (BIRAC).
PubMed: 37492417
DOI: 10.1016/j.lansea.2023.100190 -
BioRxiv : the Preprint Server For... Nov 2023The application of ribosome profiling has revealed an unexpected abundance of translation in addition to that responsible for the synthesis of previously annotated...
The application of ribosome profiling has revealed an unexpected abundance of translation in addition to that responsible for the synthesis of previously annotated protein-coding regions. Multiple short sequences have been found to be translated within single RNA molecules, both within annotated protein-coding and non-coding regions. The biological significance of this translation is a matter of intensive investigation. However, current schematic or annotation-based representations of mRNA translation generally do not account for the apparent multitude of translated regions within the same molecules. They also do not take into account the stochasticity of the process that allows alternative translations of the same RNA molecules by different ribosomes. There is a need for formal representations of mRNA complexity that would enable the analysis of quantitative information on translation and more accurate models for predicting the phenotypic effects of genetic variants affecting translation. To address this, we developed a conceptually novel abstraction that we term Ribosome Decision Graphs (RDGs). RDGs represent translation as multiple ribosome paths through untranslated and translated mRNA segments. We termed the later 'translons'. Non-deterministic events, such as initiation, re-initiation, selenocysteine insertion or ribosomal frameshifting are then represented as branching points. This representation allows for an adequate representation of eukaryotic translation complexity and focuses on locations critical for translation regulation. We show how RDGs can be used for depicting translated regions, analysis of genetic variation and quantitative genome-wide data on translation for characterisation of regulatory modulators of translation.
PubMed: 37986835
DOI: 10.1101/2023.11.10.566564 -
Redox Biochemistry and Chemistry Dec 2023Toxic metal contaminants present in food and water have widespread effects on health and disease. Chalcophiles, such as arsenic, cadmium, and mercury, show a high...
Toxic metal contaminants present in food and water have widespread effects on health and disease. Chalcophiles, such as arsenic, cadmium, and mercury, show a high affinity to selenium and exposure to these metals could have a modulating effect on enzymes dependent on selenocysteine in their active sites. The aim of this study was to assess the influence of these metals on the activity of the selenoprotein glutathione peroxidase 1 (GPX1) in erythrocytes of 100 children residing in rural Bangladesh, where drinking water often contains arsenic. GPX1 expression, as measured using high-throughput immunoblotting, showed little correlation with GPX activity ( = 0.02, p = 0.87) in blood samples. Toxic metals and selenium measured in erythrocytes using inductively coupled plasma mass spectrometry (ICP-MS) and -reactive protein (CRP) measured in plasma, were all considered as effectors of this divergence in GPX enzymatic activity. Arsenic concentrations in erythrocytes were most influential for GPX1 activity ( = -0.395, p < 0.0001), and CRP levels also negatively impacted GPX1 activity ( = -0.443, p < 0.0001). These effects appear independent of each other as arsenic concentrations and CRP showed no correlation ( = 0.124, p = 0.2204). Erythrocyte selenium, cadmium, and mercury did not show any correlation with GPX1 activity, nor with CRP or arsenic. Our findings suggest that childhood exposure to inorganic arsenic, as well as inflammation triggering the release of CRP, may negatively affect GPX1 activity in erythrocytes.
PubMed: 37908807
DOI: 10.1016/j.rbc.2023.100015 -
Genome Research May 2024The application of ribosome profiling has revealed an unexpected abundance of translation in addition to that responsible for the synthesis of previously annotated...
The application of ribosome profiling has revealed an unexpected abundance of translation in addition to that responsible for the synthesis of previously annotated protein-coding regions. Multiple short sequences have been found to be translated within single RNA molecules, within both annotated protein-coding and noncoding regions. The biological significance of this translation is a matter of intensive investigation. However, current schematic or annotation-based representations of mRNA translation generally do not account for the apparent multitude of translated regions within the same molecules. They also do not take into account the stochasticity of the process that allows alternative translations of the same RNA molecules by different ribosomes. There is a need for formal representations of mRNA complexity that would enable the analysis of quantitative information on translation and more accurate models for predicting the phenotypic effects of genetic variants affecting translation. To address this, we developed a conceptually novel abstraction that we term ribosome decision graphs (RDGs). RDGs represent translation as multiple ribosome paths through untranslated and translated mRNA segments. We termed the latter "translons." Nondeterministic events, such as initiation, reinitiation, selenocysteine insertion, or ribosomal frameshifting, are then represented as branching points. This representation allows for an adequate representation of eukaryotic translation complexity and focuses on locations critical for translation regulation. We show how RDGs can be used for depicting translated regions and for analyzing genetic variation and quantitative genome-wide data on translation for characterization of regulatory modulators of translation.
Topics: Ribosomes; Protein Biosynthesis; RNA, Messenger; Humans; Open Reading Frames; Eukaryota
PubMed: 38719470
DOI: 10.1101/gr.278810.123 -
Zebrafish and cellular models of -Related Myopathy exhibit novel embryonic and metabolic phenotypes.BioRxiv : the Preprint Server For... Feb 2024-Related Myopathy (-RM) is a rare congenital myopathy caused by mutations of the gene characterized by axial muscle weakness and progressive respiratory insufficiency....
-Related Myopathy (-RM) is a rare congenital myopathy caused by mutations of the gene characterized by axial muscle weakness and progressive respiratory insufficiency. Muscle histopathology commonly includes multiminicores or a dystrophic pattern but is often non-specific. The gene encodes selenoprotein N (SelN), a selenocysteine-containing redox enzyme located in the endo/sarcoplasmic reticulum membrane where it colocalizes with mitochondria-associated membranes. However, the molecular mechanism(s) by which SelN deficiency causes -RM are undetermined. A hurdle is the lack of cellular and animal models that show assayable phenotypes. Here we report deep-phenotyping of SelN-deficient zebrafish and muscle cells. SelN-deficient zebrafish exhibit changes in embryonic muscle function and swimming activity in larvae. Analysis of single cell RNAseq data in a zebrafish embryo-atlas revealed coexpression between and genes involved in glutathione redox pathway. SelN-deficient zebrafish and mouse myoblasts exhibit changes in glutathione and redox homeostasis, suggesting a direct relationship with SelN function. We report changes in metabolic function abnormalities in SelN-null myotubes when compared to WT. These results suggest that SelN has functional roles during zebrafish early development and myoblast metabolism.
PubMed: 38464009
DOI: 10.1101/2024.02.26.581979 -
Molecules (Basel, Switzerland) Sep 2023Targeting thioredoxin reductase (TXNRD) with low-weight molecules is emerging as a high-efficacy anti-cancer strategy in chemotherapy. Sanguinarine has been reported to...
Benzophenanthridine Alkaloid Chelerythrine Elicits Necroptosis of Gastric Cancer Cells via Selective Conjugation at the Redox Hyperreactive C-Terminal Sec Residue of Cytosolic Selenoprotein Thioredoxin Reductase.
Targeting thioredoxin reductase (TXNRD) with low-weight molecules is emerging as a high-efficacy anti-cancer strategy in chemotherapy. Sanguinarine has been reported to inhibit the activity of TXNRD1, indicating that benzophenanthridine alkaloid is a fascinating chemical entity in the field of TXNRD1 inhibitors. In this study, the inhibition of three benzophenanthridine alkaloids, including chelerythrine, sanguinarine, and nitidine, on recombinant TXNRD1 was investigated, and their anti-cancer mechanisms were revealed using three gastric cancer cell lines. Chelerythrine and sanguinarine are more potent inhibitors of TXNRD1 than nitidine, and the inhibitory effects take place in a dose- and time-dependent manner. Site-directed mutagenesis of TXNRD1 and in vitro inhibition analysis proved that chelerythrine or sanguinarine is primarily bound to the Sec residue of the enzyme, but the neighboring Cys and remaining N-terminal redox-active cysteines could also be modified after the conjugation of Sec. With high similarity to sanguinarine, chelerythrine exhibited cytotoxic effects on multiple gastric cancer cell lines and suppressed the proliferation of tumor spheroids derived from NCI-N87 cells. Chelerythrine elevated cellular levels of reactive oxygen species (ROS) and induced endoplasmic reticulum (ER) stress. Moreover, the ROS induced by chelerythrine could be completely suppressed by the addition of -acetyl-L-cysteine (NAC), and the same is true for sanguinarine. Notably, Nec-1, an RIPK1 inhibitor, rescued the chelerythrine-induced rapid cell death, indicating that chelerythrine triggers necroptosis in gastric cancer cells. Taken together, this study demonstrates that chelerythrine is a novel inhibitor of TXNRD1 by targeting Sec and possessing high anti-tumor properties on multiple gastric cancer cell lines by eliciting necroptosis.
Topics: Humans; Benzophenanthridines; Stomach Neoplasms; Reactive Oxygen Species; Necroptosis; Alkaloids; Antineoplastic Agents; Oxidation-Reduction
PubMed: 37836684
DOI: 10.3390/molecules28196842 -
Microbial Cell Factories Jul 2023Hydrogenases (H2ases) are metalloenzymes capable of the reversible conversion of protons and electrons to molecular hydrogen. Exploiting the unique enzymatic activity of...
BACKGROUND
Hydrogenases (H2ases) are metalloenzymes capable of the reversible conversion of protons and electrons to molecular hydrogen. Exploiting the unique enzymatic activity of H2ases can lead to advancements in the process of biohydrogen evolution and green energy production.
RESULTS
Here we created of a functional, optimized operon for rapid and robust production of recombinant [NiFe] Desulfomicrobium baculatum hydrogenase (Dmb H2ase). The conversion of the [NiFeSe] Dmb H2ase to [NiFe] type was performed on genetic level by site-directed mutagenesis. The native dmb operon includes two structural H2ase genes, coding for large and small subunits, and an additional gene, encoding a specific maturase (protease) that is essential for the proper maturation of the enzyme. Dmb, like all H2ases, needs intricate bio-production machinery to incorporate its crucial inorganic ligands and cofactors. Strictly anaerobic, sulfate reducer D. baculatum bacteria are distinct, in terms of their biology, from E. coli. Thus, we introduced a series of alterations within the native dmb genes. As a result, more than 100 elements, further compiled into 32 operon variants, were constructed. The initial requirement for a specific maturase was omitted by the artificial truncation of the large Dmb subunit. The assembly of the produced H2ase subunit variants was investigated both, in vitro and in vivo. This approach resulted in 4 recombinant [NiFe] Dmb enzyme variants, capable of H evolution. The aim of this study was to overcome the gene expression, protein biosynthesis, maturation and ligand loading bottlenecks for the easy, fast, and cost-effective delivery of recombinant [NiFe] H2ase, using a commonly available E. coli strains.
CONCLUSION
The optimized genetic constructs together with the developed growth and purification procedures appear to be a promising platform for further studies toward fully-active and O tolerant, recombinant [NiFeSe] Dmb H2ase, resembling the native Dmb enzyme. It could likely be achieved by selective cysteine to selenocysteine substitution within the active site of the [NiFe] Dmb variant.
Topics: Catalytic Domain; Escherichia coli; Hydrogenase; Endopeptidases
PubMed: 37479997
DOI: 10.1186/s12934-023-02127-w -
Frontiers in Immunology 2024This study aimed to assess the impact of dietary selenoprotein extracts from (SePCH) on the growth, hematological parameters, selenium metabolism, immune responses,...
This study aimed to assess the impact of dietary selenoprotein extracts from (SePCH) on the growth, hematological parameters, selenium metabolism, immune responses, antioxidant capacities, inflammatory reactions and intestinal barrier functions in juvenile largemouth bass (). The base diet was supplemented with four different concentrations of SePCH: 0.00, 0.30, 0.60 and 1.20 g/Kg (actual selenium contents: 0.37, 0.59, 0.84 and 1.30 mg/kg). These concentrations were used to formulate four isonitrogenous and isoenergetic diets for juvenile largemouth bass during a 60-day culture period. Adequate dietary SePCH (0.60 and 1.20 g/Kg) significantly increased weight gain and daily growth rate compared to the control groups (0.00 g/Kg). Furthermore, 0.60 and 1.20 g/Kg SePCH significantly enhanced amounts of white blood cells, red blood cells, platelets, lymphocytes and monocytes, and levels of hemoglobin, mean corpuscular volume and mean corpuscular hemoglobin in the hemocytes. In addition, 0.60 and 1.20 g/Kg SePCH increased the mRNA expression levels of selenocysteine lyase, selenophosphate synthase 1, 15 kDa selenoprotein, selenoprotein T2, selenoprotein H, selenoprotein P and selenoprotein K in the fish liver and intestine compared to the controls. Adequate SePCH not only significantly elevated the activities of antioxidant enzymes (Total superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase), the levels of total antioxidant capacity and glutathione, while increased mRNA transcription levels of NF-E2-related factor 2, Cu/Zn-superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase. However, adequate SePCH significantly decreased levels of malondialdehyde and HO and the mRNA expression levels of kelch-like ECH-associated protein 1a and kelch-like ECH-associated protein 1b in the fish liver and intestine compared to the controls. Meanwhile, adequate SePCH markedly enhanced the levels of immune factors (alkaline phosphatase, acid phosphatase, lysozyme, complement component 3, complement component 4 and immunoglobulin M) and innate immune-related genes (lysozyme, hepcidin, liver-expressed antimicrobial peptide 2, complement component 3 and complement component 4) in the fish liver and intestine compared to the controls. Adequate SePCH reduced the levels of pro-inflammatory cytokines (tumour necrosis factor-α, interleukin 8, interleukin 1β and interferon γ), while increasing transforming growth factor β1 levels at both transcriptional and protein levels in the liver and intestine. The mRNA expression levels of mitogen-activated protein kinase 13 (MAPK 13), MAPK14 and nuclear factor kappa B p65 were significantly reduced in the liver and intestine of fish fed with 0.60 and 1.20 g/Kg SePCH compared to the controls. Histological sections also demonstrated that 0.60 and 1.20 g/Kg SePCH significantly increased intestinal villus height and villus width compared to the controls. Furthermore, the mRNA expression levels of tight junction proteins (zonula occludens-1, zonula occludens-3, Claudin-1, Claudin-3, Claudin-5, Claudin-11, Claudin-23 and Claudin-34) and Mucin-17 were significantly upregulated in the intestinal epithelial cells of 0.60 and 1.20 g/Kg SePCH groups compared to the controls. In conclusion, these results found that 0.60 and 1.20 g/Kg dietary SePCH can not only improve growth, hematological parameters, selenium metabolism, antioxidant capacities, enhance immune responses and intestinal functions, but also alleviate inflammatory responses. This information can serve as a useful reference for formulating feeds for largemouth bass.
Topics: Animals; Antioxidants; Catalase; Bass; Muramidase; Selenium; Cardamine; Glutathione Reductase; Hydrogen Peroxide; Intestines; Selenoproteins; RNA, Messenger; Glutathione Peroxidase; Superoxide Dismutase; Claudins
PubMed: 38318186
DOI: 10.3389/fimmu.2024.1342210