bluuebunny/crossref_metadata_2025_split huggingface dataset:
Crystallographic studies of human acetylcholinesterase inhibition by organophosphates and reactivation by oximes
Authors: Gerlits Oksana , Fajer Mikolai , Cheng Xiaolin , Blumenthal Donald , Taylor Palmer , Radic Zoran , Kovalevsky Andrey 
Published: June 2021

Human acetylcholinesterase (hAChE; EC 3.1.1.7) is responsible for degrading neurotransmitter acetylcholine at synapses of the nervous system. Organophosphate (OP) nerve agents and pesticides inactivate hAChE through chemical modifications of the enzyme's active center leading to a life‐threatening increase of acetylcholine (ACh) concentration. Exposure to OPs may be fatal if not treated. The current generation of oxime antidotes is not highly efficient; the rates of nucleophilic reactivation are far slower than the catalytic rate of ACh hydrolysis. Insights into the molecular structures of Torpedo and mammalian AChEs reveal the underlying limitations in enhancing reactivation rates, but limited in their ability to resolve positions of hydrogens important for understanding proton transfer in reactivation of OP‐inhibited AChEs. The only experimental method that can provide this information is macromolecular neutron crystallography.In preparation for the neutron diffraction studies we have obtained several new X‐ray structures of native and phosphonylated (with VX and/or POX) hAChE in complex with oxime reactivators, RS2‐170B and RS‐194B, at 2.15–2.45 Å resolution range. In these complexes the enzyme forms crystals in previously unseen unit cell (a=b=124.3, c=129.1 Å; space group P31) amenable to neutron crystallography. Ternary structures with RS2‐170B reveal that the oxime binds in two conformations to non‐modified and OP‐conjugated active center suggesting that conformational changes can happen inside the active center cavity for this reactivator. In one conformation the oxime moiety extends towards the catalytic Ser203 and could represent reactive orientation of the oxime. Conversely, in our structures RS‐194B binds with the oximate pointing away from Ser203 and towards the entrance to the gorge in the non‐modified active center and the entire molecule is pushed out of the active center to bind in the peripheral site in the VX‐modified structure. POX binding induces significant changes in the acyl pocket loop conformation expelling the weakly binding RS‐194B from the active site gorge completely. This alternation may hinder oxime binding for reactivation.In addition, we have determined the first room temperature (RT) structure of hAChE in complex with the oxime reactivator RS2‐170B at 2.8 Å. Detailed analysis of interactions between the reactivator and the enzyme in both 100K and room‐temperature structures will be presented.Support or Funding InformationSupported by Grant 1U01NS083451 by the COUNTERACT program from NINDSThis 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 L‐Kynureninase Orthologs during Catalysis
Authors: D'Arcy Sheena , Karamitros Christos S. , Stone Everett , Georgiou George , Murray Kyle 
Published: June 2021

The breakdown of tryptophan in eukaryotes and some bacteria can proceed via a L‐Kynurenine (Kyn) intermediate. This intermediate has three potential fates; it can be converted to kynurenic acid, hydroxylated to 3‐hydroxyl‐kynurenine (3OH‐Kyn), or hydrolyzed to L‐anthranillic acid and alanine. L‐Kynureninase (Kynase) is the aminotransferase enzyme responsible for hydrolysis of either Kyn or 3OH‐Kyn. Kynase functions as a homodimer, with one chain contributing an essential pyridoxal‐5′‐phosphate cofactor, and the other chain contributing three catalytic residues. High‐resolution structural studies show that while human and bacterial Kynase orthologs are structurally similar, they have distinct catalytic residues. Enzyme studies also identify different substrate specificities, with the human ortholog preferring 3OH‐Kyn and the bacterial ortholog preferring Kyn. The basis for this preference is beyond the catalytic residues, as simply switching active sites between orthologs fails to switch substrate preference. The goal of this study was to characterize the dynamics of Kynase enzymes during catalysis. To do this, we have used hydrogen/deuterium exchange coupled to mass spectrometry. Through a substantial series of experiments, we have compared the dynamics of several Kynase orthologs and engineered Kynase mutants. Data sets were collected for enzymes without substrate, as well as with Kyn and 3OH‐Kyn. The results identify the determinants of substrate specificity and provide insight into mechanisms of catalysis. This information is invaluable in the design of Kynase‐based enzyme therapeutics, as well as our general understanding of enzyme dynamics.Support or Funding InformationStartup funds to S. D'Arcy from The University of Texas at Dallas.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evidence for control of metabolite flux through a bacterial heme biosynthetic pathway
Authors: Celis Arianna Ixcheel , Choby Jacob , Skaar Eric , DuBois Jennifer 
Published: June 2021

Heme is an essential Fe‐containing cofactor. The final two reactions of the heme biosynthesis pathway in Gram‐positive bacteria, including several important pathogens, are unique to these organisms. The substrates for these reactions, including coproporphyrin III, Fe(II), and H2O2, are highly reactive and toxic to the cell. We hypothesized that metabolite flux via a transient protein‐protein interaction between the final two pathway enzymes, designated HemH and HemQ, might therefore be essential in these species. In this study, we showed that the UV/Vis spectral features of coproporphyrin III serve as an indicator for an in vitro protein‐protein interaction between HemH and HemQ from Staphylococcus aureus. HemH and HemQ interacted in a 5:1 HemH:Q monomer ratio specifically at high concentrations of both proteins. Interestingly, the rates of reaction for these enzymes were not affected by the presence of the other, suggesting that the role of this interaction solely supports trafficking of pathway intermediates. The ΔhemQ knock out strain of S. aureus was complemented with single amino acid mutants of HemQ, in which either the enzymatic function or putative sites of protein‐protein interaction were lost. The molecular and growth phenotypes of these mutants were examined, with the goal of confirming possible sites of protein‐protein interaction that were essential to function. Understanding the nature of interactions between these enzymes and those further upstream in the pathway could provide new means to specifically target pathogenic Gram‐positive bacteria like S. aureus.Support or Funding InformationNIH ROI GM‐090260This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Arachidonic Acid and Oxidation in the Myosin II Motor Domain
Authors: Wong Kellie , Thomas David , Moen Rebecca 
Published: June 2021

Myosin is the motor protein responsible for movement and muscle contraction in all eukaryotic cells. The catalytic motor domain of myosin II contains three functionally important subdomains: the actin‐binding cleft, the force‐generating domain, and the nucleotide binding pocket. The chemomechanical coupling in the myosin catalytic domain allows for hydrolysis of adenosine triphosphate (ATP) into adenosine diphosphate (ADP) and inorganic phosphate (Pi), producing the energy needed for the contraction of muscle. This chemical reaction of ATP hydrolysis in the nucleotide binding cleft must be accompanied by a crucial interaction with actin to allow for the proper production of mechanical force. Various physiological and pharmacological effector molecules interact with myosin, specifically in its actinbinding cleft, and affect the actomyosin functional interaction. For example, oxidation of a methionine amino acid residue in the actin‐binding cleft, M394, decreases myosin's actin‐activated ATPase activity and changes the structural dynamics of the actin‐binding cleft of myosin (1). Conversely, arachidonic acid, a bioactive lipid produced naturally in eukaryotic cells, acts as a positive allosteric effector of myosin and is postulated to bind near the actin‐binding cleft of myosin, increasing the rate at which myosin hydrolyzes ATP (2). We have investigated the combined functional and structural effects of oxidation and arachidonic acid on the myosin motor domain in a Dictyostelium myosin II model system. Changes in actomyosin functional interaction in the presence of arachidonic acid after in vitro oxidation was assessed using a myosin ATPase activity assay and an actomyosin cosedimentation assay while changes in structural dynamics of the actin‐binding cleft was investigated using electron paramagnetic resonance (EPR). The results illustrate the importance of myosin's actin‐binding cleft structural dynamics and how allosteric effectors effect actomyosin functional interaction.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dissecting the Function of IAP (Inhibitor of Apoptosis) Protein Domains in Inhibiting an Apoptotic Caspase
Authors: Storm Mackenzie Bryan , Junker Matthew 
Published: June 2021

Apoptosis (programmed cell death) is an essential process in all animals for normal development and tissue regeneration. Apoptosis also eliminates cells at risk for becoming cancerous or spreading a viral infection. Apoptosis requires the activity of intracellular caspase enzymes that are normally kept inhibited in living cells by inhibitor of apoptosis proteins (IAPs). Certain apoptosis‐promoting proteins bind IAPs to relieve caspase inhibition. IAPs contain 2–3 BIR (baculovirus IAP repeat) domains with intervening linkers. In many IAPs, residues in a linker bind and block the caspase active site but the BIR domains also influence caspase inhibition. To determine how the BIRs contribute to caspase inhibition, we compared the ability of different fragments of the Drosophila IAP DIAP1 to inhibit a Drosophila caspase using purified recombinant proteins and an in vitro caspase assay. 2 μM of a DIAP1 fragment containing the BIR1 and BIR2 domains with intervening linker inhibited the caspase by 72 ± 1%. Removing either BIR domain reduced the inhibition to 39–45 ± 10%. Neither BIR domain alone inhibited the caspase even at 6 μM. However, 6 μM added BIR1 domain interfered with the 2 μM BIR1‐linker, reducing its caspase inhibition from 56 ± 1% to 30 ± 2%. An affinity chromatography binding assay detected direct BIR1 domain binding to the caspase. This suggests that the BIR1 interfered with the BIR1‐linker by binding to and blocking a critical BIR1‐binding site on the caspase. Thus, the BIR1 likely functions within DIAP1 by helping anchor the linker to the caspase active site. The BIR2 domain did not interfere with the linker‐BIR2 nor show direct binding to the caspase, indicating that its mechanism for enhancing caspase inhibition by DIAP1 must differ from that of the BIR1 domain. The role of the BIR2 as well as of other linker residues is being further investigated.Support or Funding InformationThis research was funded by a Kutztown University Research Grant to M. Junker and a Kutztown University BEARS grant and a Kutztown University Undergraduate Research Committee Grant to M. Storm.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Detecting Protein‐Protein Interactions and Allosteric Regulation in the Corticosteroid Synthesis Pathway in Vertebrates
Authors: Kreutzmann Sydney Nikole , Olson‐Manning Carrie Nikole 
Published: June 2021

The regulation of biochemical pathways is controlled at a variety of levels, from transcriptional timing to protein localization. Here we study the corticosteroid synthesis pathway in order to understand a transition from a biochemical pathway controlled by allosteric regulation to a phenotypically equivalent pathway controlled by differential expression of paralogs and enzyme specificity.The corticosteroids aldosterone and cortisol are produced in different layers of the adrenal cortex – a phenomenon known as zonation. Most mammals have a single enzyme, CYP11B, which produces aldosterone in the exterior layer of the adrenal cortex and cortisol in the interior layers. In mammals with a single‐CYP11B, the specific production of aldosterone and cortisol in different tissues is thought to be accomplished by allosteric regulation by another enzyme, CYP11A, which is only present in the interior layer of the cortex. It is thought that the presence of CYP11A changes the function of CYP11B so that it preferentially produces cortisol instead of aldosterone.However, throughout mammalian evolution CYP11B has duplicated independently in several lineages, giving rise to two CYP11B paralogs. These species still experience zonation and produce aldosterone and cortisol in different layers of the adrenal cortex. It has been demonstrated that this zonation in species with two CYP11B enzymes is accomplished by differential expression of the paralogs in the different tissues along with the evolution of enzymatic preference for their respective products. However, it is unknown whether the interaction between CYP11B and CYP11A has been lost and whether the zonation of hormones produced is regulated solely by the two differential expression and enzymatic activity of the CYP11B enzymes.Here we used Forster Resonance Energy Transfer (FRET) High Performance Liquid Chromatography (HPLC) to determine whether CYP11B and CYP11A physically interact and whether or not that interaction would contribute to the allosteric regulation of this pathway. Our preliminary analyses demonstrate a physical interaction between CYP11B and CYP11A in a single‐CYP11B species. Further experiments are aimed to test the interaction of duplicated CYP11Bs with CYP11A. We will monitor in vivo enzymatic assays by HPLC to determine what hormones are produced in the presence of different enzyme combinations. These results will shed light on the various ways metabolic pathways are regulated.Support or Funding InformationSD BRIN P20GM103443SD EPSCOR IIA‐1355423This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of Genotype on the Ergogenic Effects of Caffeine in Collegiate Nordic Skiers
Authors: Zhang Yun , Millard Julie T. 
Published: June 2021

Despite its widespread use, caffeine's effects vary widely between individuals. Two factors that are likely to modulate response to caffeine are the rate of its metabolism and the variant of the receptor to which it binds. The activity of the liver enzyme CYP1A2, which is responsible for metabolizing caffeine, is reportedly associated with a single nucleotide polymorphism (A/C) that leads to higher activity in AA individuals than in C‐allele carriers. CYP1A2 genotype appears to affect caffeine outcomes, as evidenced by reports that slow metabolizers have increased risk of heart attack and reduced benefits of caffeine on athletic performance. The adenosine A2A receptor (ADORA2A) is the principal CNS target of caffeine, and a single nucleotide polymorphism (T/C) of ADORA2A is reported to modulate caffeine consumption habits, caffeine‐induced anxiety, and the ergogenic effects of caffeine. The goal of this project was to determine whether genotypes for caffeine metabolism and/or receptor modulate exercise performance in collegiate athletes following caffeine ingestion. Nordic skiers provided DNA samples, which were genotyped for CYP1A2 (rs762551) and ADORA2A (rs5751876). Subjects were tested for lactate threshold under three conditions: baseline, after ingestion of placebo, and after ingestion of 2 mg/kg caffeine. This work may contribute to a better understanding of the variable effects of caffeine consumption on athletic performance.Support or Funding InformationSupported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM0103423.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of a Microscopic Method to Diagnose Hemoglobin C Conditions for Use in Developing Countries
Authors: Schmidt Kayla L. , Randolph Tim R. 
Published: June 2021

Hemoglobin C is one of the second most prevalent hemoglobinopathies worldwide behind sickle cell anemia (HbS). HbC disease (HbCC) produces mild symptoms, but is a life‐threatening disorder if inherited with HbS (HbSC). HbS and HbC are most prevalent in countries underequipped to diagnose its presence through gold standard methods like electrophoresis, HPLC or isoelectric focusing. This study aims to create a simple, inexpensive method to identify the presence of Hemoglobin C using limited resources. The hypothesis is that when blood is incubated in a hypertonic salt solution, Hemoglobin C will crystalize intracellularly and become visible microscopically when stained with New Methylene Blue. The method was optimized by modifying the salt type, salt concentration, incubation time, and incubation temperature. The optimized method incubates RBCs in a 5x Dulbecco's phosphate buffered saline (DPBS) at 37°C for 4 hours. Blood samples with the HbSC genotype yielded between 600–750 HbC crystals for every 1000 RBCs counted. Future studies will include blood samples of AC, CC, SC, and AA genotypes to determine if this method will differentiate genotype.Support or Funding InformationKS is supported by DeNardo Education and Research Foundation FellowshipThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Extraction and Separation of Collagen on SDS PAGE to Determine Effect of Whitening Strips on Teeth
Authors: Mariche‐Banos David , Rotsides Photis , Keenan Kelly 
Published: June 2021

In the United States, billions of dollars are spent each year of tooth whitening products including treatments in dentist's offices as well as whitening strips which are sold over the counter. Both of these products use hydrogen peroxide as the active ingredient which has long been known to damage proteins. The outermost part of teeth is the enamel which contains several soluble proteins and the dentin underneath contains nearly 90 to 95 % collagen. The goal of this project was to extract collagen from both untreated teeth as well as those treated with whitening strips and examine the collagen on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). Teeth were placed into artificial saliva and either treated with whitening strips for one or three rounds according to manufacturer's instructions or were untreated. Following a treatment, teeth were placed in artificial saliva for 30 minutes. Following treatments, teeth were dialyzed against 10% EDTA, pH 9.0 for two weeks with several changes of EDTA solution. The teeth were then dialyzed against water for two weeks with several changes. The tooth remnants were placed in liquid nitrogen, hammered into shards and ground into a powder using a blender. The powder was suspended in 0.5 M acetic acid and is the collagen fraction (COL); it was treated with pepsin for four hours, neutralized with TRIS, centrifuged and the supernatant was the pepsin soluble collagen fraction (PSC). Gels of varying concentrations of acrylamide were prepared and COL and PSC fractions were loaded. Two modified procedures for the Coomassie Blue staining protocol were used. In one, the washing solution did not contain methanol and this caused the COL proteins to turn a pink color. In addition, the gels were stained with the Oriole fluorescent stain. In order to determine the type of collagen present, standards made from collagen I and III were also used. Results will be presented that shows the level of COL in treated and untreated teeth.Support or Funding InformationFunding was provided by the Research and Professional Development Grant from Stockton 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.
Measurement of protein and collagen in teeth before and after whitening strips
Authors: Tadros Jonathan , Giunta Nick , Keenan Kelly 
Published: June 2021

Americans spend billions of dollars each year on whitening products for the teeth. Whether it is performed in a dentist's office or with over‐the‐counter whitening strips, the active ingredient, hydrogen peroxide, is the same and has long been known to damage proteins. The goal was to measure the level of protein extracted from teeth untreated as well as those treated with one or three rounds of whitening strips. Teeth were purchased and treated according to manufacturer's instructions. In one round, teeth were dry when treated and washed with water after for 30 minutes. In another round, teeth were suspended in artificial saliva during treatment and for 30 minutes afterwards. Afterwards, the teeth were dialyzed against 10% EDTA pH 9.0 for 2 weeks. The fluid contained the soluble non collagen proteins and total protein was measured using the Lowry assay. The tooth remnants were dialyzed for two weeks against water and placed in liquid nitrogen, hammered in shards and then ground into a powder in a blender. The powder was suspended in an acetic acid, sodium chloride solution and contained collagen. The collagen was treated with pepsin for four hours to collect a pepsin soluble fraction (PSC). The amount of protein in the collagen and PSC fraction was tested using a modified Lowry assay procedure. Based on the Lowry assay, there was a significant decrease in protein content after treatment with whitening strips and the loss increased with more treatments. Based on the modified Lowry procedure, there was also a significant loss of collagen as measured by the modified Lowry procedure for both the collagen and PSC fraction. Similar losses were observed with or without the artificial saliva.Support or Funding InformationAuthors thank Stockton University for the Research and Professional Development grant to support this project.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.