bluuebunny/crossref_metadata_2025_split huggingface dataset:
Protection of the Queuosine Biosynthesis Enzyme QueF from Irreversible Oxidation by a Conserved Intramolecular Disulfide
Authors: Mohammad Adeba , Ramos Adriana Bon , Lee Bobby W.K , Cohen Spencer W. , Kiani Maryam K. , Iwata‐Reuyl Dirk , Stec Boguslaw , Swairjo Manal A. 
Published: June 2021

QueF enzymes catalyze the nicotinamide adenine dinucleotide phosphate (NADPH)‐dependent reduction of the nitrile group of 7‐cyano‐7‐deazaguanine (preQ0) to 7‐aminomethyl‐7‐deazaguanine (preQ1) in the biosynthetic pathway to the tRNA modified nucleoside queuosine. The QueF‐catalyzed reaction involves the formation of a covalent thioimide intermediate with a conserved active site cysteine (Cys55) that is prone to oxidation in vivo under different conditions. Here, we report the crystal structure of a mutant of Bacillus subtilis QueF, which reveals an unanticipated intramolecular disulfide bond formed between the catalytically active Cys55 and a conserved Cys99 located near the active site. When compared to the substrate‐bound structure, this structure is more symmetric and exhibits major rearrangement of the loops responsible for substrate binding. It has been demonstrated that the disulfide does not play a catalytic role because mutation of Cys99 to Ala/Ser does not compromise enzyme activity. Inactivation of the wild‐type enzyme via peroxidation is reversible with thioredoxin, while such inactivation of the Cys99Ala/Ser mutants is irreversible. This is consistent with protection of Cys55 from irreversible oxidation by disulfide formation with Cys99. Conservation of the cysteine pair, and the reported in vivo interaction of QueF with the thioredoxin‐like hydroperoxide reductase AhpC in Escherichia coli suggest that regulation by the thioredoxin disulfide‐thiol exchange system may constitute a general protective mechanism of QueF from oxidative stress in vivo. Docking of QueF onto this enzyme complex sheds structural light on the mechanism of redox control and may inform future mutagenesis experiments.Support or Funding InformationThis project was supported by National Science Foundation grant CHE‐1309323 to D. Iwata‐Reuyl and M.A. Swairjo, National Institutes of Health (NIH) grant GM110588 to M.A. Swairjo and D. Iwata‐Reuyl, and NIH grant GM70641 to D. Iwata‐Reuyl. Use of the Stanford Synchrotron Radiation Lightsource (SSRL), Stanford Linear Accelerator Center National Accelerator Laboratory, is supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE‐AC02‐76SF00515. The SSRL Structural Molecular Biology Program is supported by the Department of Energy Office of Biological and Environmental Research, by the NIH, and the National Institute of General Medical Sciences (NIGMS) (including grant P41GM103393).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Direct Route for Asparaginyl‐tRNA Formation in B. subtilis
Authors: Wales Rebecca , Sheppard Kelly 
Published: June 2021

Attaching an amino acid to its cognate tRNA is an essential step for the fidelity of protein synthesis. Two distinct routes have evolved for ligating asparagine to tRNAAsn. In the direct route, asparagine is first synthesized by an asparagine synthetase. Asparagine is then attached to tRNAAsn by asparaginyl‐tRNA synthetase. For the indirect path, asparagine is synthesized on the tRNA. First a non‐discriminating aspartyl‐tRNA synthetase ligates aspartate to tRNAAsn and the amidotransferase GatCAB amidates the tRNA‐bound aspartate to asparagine. Bacillus subtilis encodes both routes for asparaginyl‐tRNAAsn formation. Why it acquired an asparaginyl‐tRNA synthetase is not clear. We hypothesize that it enables the organism to adapt to different environmental conditions. To test this hypothesis, we have purified the B. subtilis asparaginyl‐tRNA synthetase and asparagine synthetase B. We will report on characterizing in vitro the two enzymes for the direct route for asparaginyl‐tRNA synthesis under different physiologically relevant conditions as well as knocking out asks in the B. subtilis genome that encodes asparaginyl‐tRNA synthetase. The work will provide insight into how bacteria have adapted amino acid metabolism and use in protein synthesis for different environmental niches.Support or Funding InformationNational Science Foundation (MCB‐1615770)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Anticodon‐Binding Domain Mutations of the Bacillus subtilis Non‐discriminating Aspartyl‐tRNA Synthetase to Increase tRNA Specificity
Authors: Shi Katherine C. , Rasmussen Karli C. , Sheppard Kelly 
Published: June 2021

The fidelity of the genetic code is etablished by tRNA synthetases attaching an amino acid to the correct tRNA isoacceptor. The majority of prokaryotes lack an asparaginyl‐tRNA synthetase to ligate asparagine to tRNAAsn. Instead, these organisms possess a non‐discriminating aspartyl‐tRNA synthetase (ND‐AspRS), an AspRS with relaxed substrate specificity, that can ligate aspartate to either tRNAAsn or tRNAAsp. An amidotransferase enzyme, GatCAB, then amidates Asp‐tRNAAsn to form Asn‐tRNAAsn. In many but not all organisms encoding an asparaginyl‐tRNA synthetase to directly ligate asparagine to tRNAAsn, the aspartyl‐tRNA synthetase has evolved to be specific for tRNAAsp, a discriminating aspartyl‐tRNA synthetase (D‐AspRS). How bacterial aspartyl‐tRNA synthetase evolved specificity for tRNAAsp is not clear. Bacillus subtilis encodes a ND‐AspRS while the related Bacillus anthracis codes for a D‐AspRS. The sequence similarity between the two enzymes provides an opportunity to alter the tRNA specificity of a ND‐AspRS. Using the sequence comparison along with available structures of aspartyl‐tRNA synthetases, we have mutated the anticodon binding domain of the B. subtilis ND‐AspRS and will report how the various mutations impacted specificity for tRNAAsp. The work expands our understanding of tRNA synthetase substrate specificity and how fidelity of protein synthesis is achieved.Support or Funding InformationNational Science Foundation (MCB‐1615770)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of the Dual Pathways for B. halodurans Asparaginyl‐tRNA Formation
Authors: Schroeder Caitlin M. , Sheppard Kelly 
Published: June 2021

Protein synthesis requires the attachment of an amino acid to its cognate transfer RNA (tRNA). Two distinct pathways for attaching asparagine (Asn) to tRNAAsn are known: the direct pathway, in which Asn is directly attached to tRNAAsn by the enzyme AsnRS; and the indirect pathway, in which Asn is attached to tRNA by a non‐discriminating AspRS and GatCAB. Bacillus halodurans uses both routes for Asn‐tRNAAsn formation. To better understand why B. halodurans possesses both pathways, we have purified its AsnRS and AspRS in order to test and compare their activities under different physiologically relevant conditions such as in the presence of reactive oxygen species and changes in pH. This research will provide insight into how B. halodurans has adapted to survive in different environmental conditions.Support or Funding InformationNational Science Foundation (MCB‐1615770)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural and Functional Large Substrate Binding in Iterative Non‐ribosomal Peptide Synthesis Independent Synthesis (NIS) Enzymes
Authors: Hoffmann Katherine M. 
Published: June 2021

Prokaryotes scavenge iron using small molecule chelators called siderophores, made through a novel chemistry of peptide bond formation. One type of siderophore synthesis, increasingly associated with bacterial virulence, uses Non‐ribosomal peptide siderophore Independent Synthesis enzymes (NIS synthetases). Within a sub‐category in this family, an additional remarkable kinetic behavior exists whereby multiple bonds may be catalyzed on the same substrate (iterative.) Prior work in the field has identified a novel protein fold as well as the unique peptide bond chemistry, but no structures exist of an iterative protein. The iterative activity is correlated with broad substrate specificity, but this has never been delineated nor quantified. In fact, no iterative enzyme kinetic parameters have been satisfactorily described. We propose basic science to characterize the structure, function, and elasticity of the under‐described iterative proteins in this novel family of NIS sythetases. To do this, we will utilize the following key methodologies: (1) site‐directed mutagenesis to probe important residues; (2) isothermal titration calorimetry (ITC) to explore the thermodynamics of substrate and cofactor binding; (3) Kinetic assays to quantify and compare iterative vs. single bond formation; and (4) X‐ray crystallography to probe the structural changes of iterative substrate binding. The results of our work will be used as a foundation for future mechanism and inhibitor studies, an effort that could lead to a new class of antibiotics.Support or Funding InformationThis work was supported in part by a grant from the National Science Foundation (MCB‐1716986) and by California Lutheran University.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dynamics of Organophosphate‐Induced Structural Changes in Acetylcholinesterase Revealed by Time‐Resolved Small‐Angle X‐Ray Scattering and Inelastic Neutron Scattering
Authors: Blumenthal Donald K. , Kovalevsky Andrey , Gerlits Oksana , Fajer Mikolai , Cheng Xiaolin , Taylor Palmer , Radić Zoran 
Published: June 2021

Organophosphates (OPs), the active compounds in nerve agents and insecticides such as paraoxon (POX), exert their toxic effects by irreversibly inhibiting acetylcholinesterase (AChE). Currently available antidotes for OP toxicity can reactivate AChE, but reactivation rates are slow which can lead to irreversible nervous system damage or death. We hypothesize that better antidotes with accelerated reactivation rates can be developed by understanding the structural dynamics of OP‐AChE conjugates. Previous steady‐state small‐angle X‐ray scattering (SAXS) experiments showed that POX and certain other OPs induce quaternary structural changes in human AChE (hAChE) that were reversible by oxime antidotes. To characterize the dynamics of these OP‐induced structural changes we have used time‐resolved SAXS (TR‐SAXS) and inelastic neutron scattering (INS). Stopped‐flow TR‐SAXS experiments were performed at BL‐4 at the Stanford Synchrotron Radiation Lightsource by mixing a molar excess of POX (500 μM) with hAChE (50 μM). Scattering data were collected every 20 msec during the first 1000 msec after mixing. Changes in the solution structure of hAChE during this period occurred with kobs= 0.13(±0.01) s−1. This rate of structural change is comparable to the rate of hAChE inactivation observed using biochemical assays. INS data were collected at the Cold Neutron Chopper Spectrometer at the Spallation Neutron Source at Oakridge National Laboratory using 100 mg samples of hAChE and POX‐hAChE that were lyophilized and rehydrated in D2O. INS data collected at 120 K (to prevent translational motions) were used to compare the vibrational density states of native and POX‐conjugated hAChE. The POX‐conjugated hAChE exhibited an increased number of vibrational states, particularly those associated with regional motions such as loop dynamics, whereas global collective motions remained unchanged. Molecular dynamics simulations were used to simulate the vibrational spectra and identify the region(s) in hAChE that contributed to the differences in the INS experimental data. In aggregate, these studies provide experimental evidence for OP conjugate‐induced changes in hAChE dynamics that has to be considered in the design of antidotes for OP poisoning that have improved efficacy and patient outcomes.Support or Funding InformationThis work was supported by the CounterACT Program, National Institutes of Health Office of the Director, Grant 1U01NS083451 from NINDS, and by grants to SSRL and ORNL from the U.S. Department of Energy, and the National Institutes of Health.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Comparative Genomics and Gene Annotation of a Region of Drosophila eugracilis Chromosome 4
Authors: Gayle Ashley , Van Stry Melanie 
Published: June 2021

The common fruit fly, Drosophilia melanogaster, has around 1,300 genes in its genome. Chromosome 4 is 3.5% of its genome and contains approximately 80 genes within large heterochromatic domains. Interestingly, these genes are expressed at similar levels to genes found in euchromatin. The goal of the Genomics Education Partnership (GEP) project is to understand the gene expression within this heterochromatic environment. For the GEP project, we are identifying conserved DNA regulatory sequences around transcription start sites using the fourth chromosome from other species of Drosophila and D. melanogaster. I annotated genes located within a 35,000 base‐pair section of Drosophilia eugracilis chromosome 4 using BLASTX, Gene predictors, and RNA‐seq evidence within the UCSC Genome Browser. I annotated coding exons and splice sites for 2 genes with 3 isoforms each, Cubitis interruptus (Ci) and Ribosomal protein S3A (RpS3A) and the transcription start sites for each gene. GEP students are annotating many megabases of DNA and their pooled results are furthering an understanding of heterochromatic gene structure and regulation.Support or Funding InformationSupported by NSF IUSE #1431407 and NIH R25GM119157 to Sarah C.R. Elgin (GEP) and NSF HRD#1623340 to MVSThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Annotation of Genes on Contig 31 of the Drosophila eugracilis Chromosome 4
Authors: Hobson Joanna , Van Stry Melanie 
Published: June 2021

Drosophila melanogaster has a small fourth chromosome, the Muller F element (3.5% of the genome), that has very large heterochromatin domains which are rich in HP1. Our goal for this project is to understand the mechanisms of gene expression in this heterochromatic environment. In the Genomics Education Partnership Project, we are using comparative genomics to identify conserved DNA regulatory sequences, by comparing the D. melanogaster Muller F element to those of other fly species that diverged from D. melanogaster ~10 million years ago, aiming to find regions of homology surrounding the transcription start sites. I have annotated the genes on a 40,000 base‐pair piece from the Drosophila eugracilis chromosome four. Sequence comparisons using BLASTX, gene predictors like Gene Scans, and RNA sequence data were used to annotate coding genes in this region. I identified two genes: myo (3 isoforms) and ey (4 isoforms) and annotated protein coding exons and splice sites for each isoform. The myo amino acid sequence is identical for each isoform in D. eugracilis, with 70.3% amino acid identity compared to the D. melanogaster myo protein. The D. eugracilis ey isoforms are also similar to the D. melanogaster ey protein sequences, with a range from 73% to 78% identity to their corresponding D. melanogaster isoform. Putative Transcription Start Sites for each gene were investigated, including a search for known transcription factor binding sites. By pooling results, GEP students are annotating several megabases of DNA, and contributing to an understanding of heterochromatic genes.Support or Funding InformationSupported by NSF IUSE #1431407 and NIH R25GM119157 to Sarah C.R. Elgin (GEP) and NSF HRD#1623340 to MVSThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
PY489‐β‐Catenin Phosphorylation Involved in the Intestinal Regeneration of the Sea Cucumber H. glaberrima
Authors: Torres‐Gutierrez Vanessa , Bello Samir A. , García‐Arrarás Jose E. 
Published: June 2021

Organ regeneration is a rare phenomenon in higher vertebrates. However, members of closely related groups such as echinoderms have striking regeneration capacities. In studies of intestinal regeneration using the sea cucumber Holothuria glaberrima, recent results have shown the involvement of the Wnt signaling pathway. One important component in this intracellular cascade is β‐catenin, that, depending on its phosphorylation state, activates transcription of Wnt target genes. In one case, phosphorylation of the tyrosine residue 489 (Y489) increases transcriptional activity of the β‐catenin/Tcf complex. To determine if Y489‐β‐catenin phosphorylation is involved in intestinal regeneration, normal and regenerating intestines of H. glaberrima specimens were dissected at different post‐evisceration stages. Tissues were processed for immunohistochemistry using anti‐PY489‐β‐catenin antibody and assessed by counting the temporal and spatial appearance of labeled cells in the distal, medial and proximal ends of the mesentery and gut rudiment. In normal non‐eviscerated animals, the antibody labeled about the 14% of cells in the coelomic lining and 31.2% of cells in the luminal epithelium. In regenerating specimens, PY489‐β‐catenin was expressed at all stages of regeneration from 3‐dpe up to 14‐dpe, with a peak increment of 40.6% of cells labeled in the mesothelium of the 7‐dpe intestinal rudiment. To determine the role of activating or inhibiting the WNT/β‐catenin pathway on the expression of the PY489 labeling, we treated tissue explants with the Wnt pathway activator LiCl and inhibitor iCRT14. When compared to controls there was no significant difference in the number of PY489‐β‐catenin immunoreactive cells observed in the LiCl‐treated explants, while there was a significant increase in immunoreactive cells in the iCRT14‐treated explants. Our results suggest that the Wnt/β‐catenin signaling is activated during intestinal regeneration in H. glaberrima and that phosphorylation in the Y489 residue of β‐catenin is important for the Wnt role in intestinal regeneration. In particular, the results suggest a possible role for Wnt pathway in the dedifferentiation status of the mesothelium once the initial intestinal blastema is formed. Our study might provide the basis for the role of the Wnt signaling pathway during organogenesis in holothurians and thus provide a better understanding of general regenerative processes.Support or Funding InformationFunded by NIH R15NS081686, NSF IOS‐1252679 and the University of Puerto Rico.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Functional Role of Myc Transcript as Active Player in the Canonical Wnt Pathway During Intestinal Regeneration
Authors: Alicea‐Delgado Miosotis , Garcia Arraras Jose E. 
Published: June 2021

Molecular approaches have been widely used to explore the role of genes associated with regenerative processes in different animal models. Our own studies in the sea cucumber Holothuria glaberrima have identified several genes that appear to play a role in the regeneration of the digestive system. We have focused on two of these genes, Wnt and Myc, whose expression has been localized to the intestinal rudiment during regeneration. Myc and Wnt are interrelated, as the former is known to serve as an active player in the canonical Wnt signaling pathway. We now have quantified the levels of myc transcript at different stages of regeneration. In addition, we designed Dicer‐substrate short interfering RNAs (DsiRNAs)‐targeting Myc transcripts. DsiRNAs‐targeting Myc are electroporated in regenerating intestinal tissue explants of H. glaberrima to evaluate their effects on cell proliferation and cell dedifferentiation. Quantification of myc transcripts shows a decrease following electroporation of specific DsiRNAs when compared to explants electroporated with control DsiRNAs. Moreover, a decrease in cell proliferation is observed following the reduction in myc transcript, suggesting that Myc has a functional role on cell proliferation during the intestinal regeneration process. Instead, cell dedifferentiation does not change with the interference of the expression of Myc, suggesting that Myc does not play an important functional role on cell dedifferentiation during the intestinal regeneration process. In summary, this project not only presents data on Wnt‐Myc interactions, but presents three important technical advances: (1) the use of intestinal tissue explants, (2) the implementation of the electroporation as a transfection method and (3) the use of DsiRNAs to manipulate the gene expression in H. glaberrima explants.Support or Funding InformationFunded by NIH R15GM124595, NSF IOS‐1252679 and the University of Puerto RicoThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evolution of TLR Signaling: A Sea Anemone Model has a Single Toll‐like Receptor with Roles in Pathogen Detection, NF‐κB Signal Transduction, and Development
Authors: Messerschmidt Jonathan L. , Brennan Joseph J. , Gilmore Thomas D. 
Published: June 2021

The innate immune system is the most primitive branch of the immune system and is responsible for recognizing pathogens and eliciting responses such as inflammation and antimicrobial peptide production in many organisms. Toll‐like receptors (TLRs) are transmembrane receptors that have been characterized in organisms from insects to humans. TLRs detect molecules expressed by microbial pathogens, and then activate the NF‐κB transcription factor signaling pathway to turn on the expression of innate immune effector genes. Recent genome and transcriptome sequencing efforts have led to the identification of TLR‐like genes in organisms more basal to insects, but the biological roles of these basal TLRs are not well understood. In this project, we are characterizing a TLR‐like protein in the sea anemone Nematostella vectensis (Nv), a model organism for the phylum Cnidaria. The single Nv‐TLR harbors leucine‐rich extracellular, transmembrane, and intracellular TIR domains, consistent with TLR structures in higher organisms, including humans. In cell culture‐based reporter assays, we show that Nv‐TLR can activate the human NF‐κB signaling pathway and this pathway activation can be stimulated by the biologically relevant bacterium Vibrio coralliilyticus and purified flagellin from Salmonella typhimurium. In biochemical studies, the intracellular TIR domain of Nv‐TLR can bind to human NF‐κB signal transduction adapter proteins MAL and MyD88, and the extracellular domain of Nv‐TLR can directly bind to flagellin. In anemones, Nv‐TLR and Nv‐NF‐κB (and most intermediate TLR to NF‐κB signaling proteins) are expressed in cnidocytes, both in the body column and in a Nematostella‐specific circulating organ called the nematosome. Nv‐TLR is also required for embryonic development in N. vectensis. Ongoing studies are characterizing the biochemical aspects of the flagellin‐TLR interaction. Taken together, these studies suggest that a TLR‐to‐NF‐κB pathway is functionally conserved from cnidarians to humans. Moreover, these studies provide insight into the evolutionary basis of vertebrate innate immunity, and shed light on how simple organisms respond at the molecular level to environmental and biological stressors.Support or Funding InformationUndergraduate Research Opportunities Program (Boston University), National Science Foundation, Arnold and Mabel Beckman FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.