bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Structure and Function of Estrogen Receptor β: Essential Roles in Gene Regulation
Authors: Dai Eddie , Liu Samuel , Kholi Khushi 
Published: June 2021

The Olathe North MSOE Center for BioMolecular Modeling SMART Team used 3‐D modeling and printing technology to examine the structure‐function relationships and regulatory roles of ERβ. Estrogens are steroid hormones synthesized from cholesterol in cells of the ovary and other tissues expressing the enzyme aromatase. These hormones are secreted into the bloodstream and modulate numerous physiological functions through gene regulation. To achieve this, estrogens pass through cell and nuclear membranes, facilitated by their lipophilic and miniscule nature. In target cells, estrogens bind to nuclear transcription factors called estrogen receptors (ERs), the two primary types being estrogen receptor alpha (ERα) and beta (ERβ). ERs typically form either homodimers with an identical molecule or heterodimers of ERα with ERβ. Estrogen binding occurs at ER's ligand binding domain (LBD), activating the ER's DNA binding domain (DBD). The ER dimer then binds to DNA at estrogen response elements (EREs) located in the promoter regions of estrogen‐responsive genes, allowing the ER to regulate the transcription of target genes. However, this process is only one of four mechanisms by which ERs function. In addition to this genomic mechanism, ERs can also function through tethered, nongenomic, and ligand‐independent mechanisms. In all these processes, ERs' role as a gene regulator is determined by the physical change in the structure of various ER domains, making it essential to analyze the ERs' protein structure. In this review, we focus on the structure and function of ERβ because of its largely unexplored physiological significance and clinical potential. ERβ is most highly expressed in the ovaries, mammary glands, prostate, testes, gastrointestinal tract, and immune system. Transcriptional regulation by ERβ is essential for ovarian follicle maturation and ovulation. Additionally, ERβ is involved in neurogenesis, vascular function modulation, cardiac myocyte maintenance, and metabolism regulation, among other physiological functions. In a pathological context, recent studies suggest ERβ is tumor suppressing while ERα increases tumor growth. Specifically, ERβ can inhibit proliferation while inducing differentiation and apoptosis in tumor cells. Thus, activation of ERβ by synthetic agonists, known as selective estrogen receptor modulators (SERM), has clinical significance concerning the treatment of breast, prostate, colon, and ovarian cancers. Here, we present a review of the latest research on ERβ in conjunction with its physical model, emphasizing ERβ's regulatory roles in human health.Support or Funding InformationThis is a SMART Team project supported through the contributions of Dr. M.A. Karim Rumi of the University of Kansas Medical Center, the Milwaukee School of Engineering, and the Olathe Medical Professions 21st Century Academy.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterizing the Role of Metal Ion Interactions in the Elasticity and Strength of Slug Glue: Steps Toward a Novel Medical Adhesive
Authors: Lazo Christopher Gallego , Smith Andrew 
Published: June 2021

Stitches are often cumbersome for patients and pose a risk of infection. Thus, there is a great interest in developing medical glues that could serve as an alternative, but current adhesives are limited. Analysis of the tough, sticky defensive secretion of the slug Arion subfuscus has guided the development of a novel medical glue, and further work characterizing the slug glue should lead to deeper insights into how to make a hydrogel glue. The glue of A. subfuscus consists of a double network of carbohydrates and proteins and is held together by sacrificial bonds. These sacrificial bonds are central to the high performance of the glue, but they have yet to be identified definitively. Calcium and magnesium seem to be most likely to form intermolecular cross‐links in the glue due to their high abundance, and the abundance of calcium‐binding proteins in the glue. The role of calcium and magnesium was tested by manipulating their interactions within the glue to observe how this impacts its elasticity. Metal ion interactions were controlled by soaking glue samples in different pH buffers and determining the resulting elasticity. The glue's elasticity was measured in cut glue samples in a tensometer. The association between metal ions and glue when under different pH buffers was measured by using an atomic absorption spectrometer to determine whether the metals stayed bound in the glue or diffused out at different pH values. We found that glue elasticity varied with pH, showing a steady decline as pH dropped below 5.5. Between 5.5 and 7.5 no significant change in elasticity was apparent. This change in stiffness was not due to dissociation of calcium and magnesium, however, as they diffused completely out of the glue within sixty minutes regardless of pH. Thus, there was no correlation between the change in elasticity with pH and the loss of these metal ions, and their hypothesized role as sacrificial bonds was not supported. Further investigation aims to look into the relationship between zinc ions or imine bonds and glue elasticity and strength.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Illuminating Protein Interactions within a Repressive Auxin Response Factor
Authors: Johnston Shelby R , Garner Reid M , Ellis Jamie P 
Published: June 2021

Auxin response factors (ARFs) are transcription factors that activate or repress gene expression in most plant species. While almost all ARFs contain three domains, the small structured C‐terminal Phox and Bem1 (PB1) domain shows the most potential for defined regulatory interactions. Each ARF PB1 domain interacts through opposing cationic and anionic surfaces to other PB1 domains, including ARFs and Aux/IAA proteins. This interaction provides a necessary pathway for hormonal regulation of transcription. From Arabidopsis thaliana, the PB1 domains of both activators ARF5 and 7 show the potential to form homo‐ and hetero‐oligomers from studies of model mutant and wild‐type proteins. To date, no similar characterization of ARF repressor PB1 domains has been performed. Our work combines genetic analysis with experimental characterization of the PB1 domain of repressor ARF1 to test for unique biophysical traits. The PB1 domains carry significant sequence diversity for potential behavioral differences. Within A. thaliana, the 20 distinct ARF PB1 domains are only 8.5% identical. ARF repressor PB1 domains differ significantly from their activating counterparts as well as show significant diversity. The repressing ARFs form clades that are distinct from each other including some within the same subclade as the activators. Analysis of wild type ARF1 has shown that ARF1 PB1 forms a mixture of trimers, tetramers, as well as the larger oligomers common to ARF5 and 7. The conserved charged residues appear to be the primary mechanism with which they form oligomers. We used the natural fluorescence of tryptophan to observe structural changes during binding. Two tryptophans are present in the intradomain interface of the beta‐grasp fold common to PB1 domains. Through fluorescence intensity and collisional quenching experiments, we quantified the relative protection of the tryptophans from their environment. Comparing ARF1 with ARF5 and 7 as well as under conditions of homo‐ and hetero‐oligomerization provided another insight to commonalities and distinctions between these regulatory proteins. The distinct traits found between an ARF repressor and homologous ARF activators may illuminate the role of sequence diversity and the evolved retention of multiple variants of ARF proteins.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Interaction of PKR and E3 protein present in vaccinia virus
Authors: Patnaik Sikta 
Published: June 2021

E3 protein from vaccinia virus is a key host‐range protein, which suppresses the innate antiviral immune response of the infected cells. In response to the viral infection, Protein Kinase R (PKR) of the host cell initiates the immune response via a cascade of reactions. E3 attacks PKR to suppress the immune response but the mechanism of PKR inhibition by E3 is unknown. Interaction studies of PKR and E3 suggested a physical interaction between the kinase domain of PKR and E3 that has biochemical relevance. E3 follows two different pathways when inhibiting inactivated PKR and activated PKR respectively. Further analysis of the interaction revealed that the inhibition of PKR activation by the E3 protein does not correlate with the dsRNA binding ability of PKR or E3.Support or Funding InformationNational Institute of Health (NIH)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Tamm‐Horsfall Protein and Properdin Biochemical and Functional Interactions
Authors: Rhodes Diana CJ 
Published: June 2021

Tamm‐Horsfall protein (THP), also known as uromodulin, is an abundant protein in normal mammalian urine that interacts with numerous cells and molecules of the immune system, including various complement proteins such as C1q and factor H. Properdin (factor P), which potentiates complement activation by stabilizing the alternative pathway C3 convertase, previously has been shown to bind to THP. In the present study, an enzyme‐linked immunosorbent assay (ELISA) was used to examine the effect of ionic strength, pH, and heparin on the binding between these two oppositely charged proteins. Additionally, since THP has been shown to act as a cofactor, like factor H, in factor I‐mediated C3b degradation, we investigated whether properdin altered this cofactor activity of THP. All ELISA buffers contained 1 mM MgCl2 and 1 mM CaCl2 because earlier studies showed a requirement for divalent cations in this reaction. As measured by ELISA, binding between THP and properdin was similar (K~ 10−8 M) in 20 mM, 60 mM, and 90 mM NaCl solutions, but no significant binding was detected in 154 mM NaCl buffers. There was a reduction in binding capacity, but not overall affinity in 120 mM NaCl buffers. Binding of properdin to immobilized THP in 60 mM NaCl was similar at pH 6.0, 7.5, and 8.5. The THP/properdin interaction was very sensitive to heparin, with 0.008 units of heparin/ml producing a 50% inhibition of binding of 2 μg/ml properdin to immobilized THP. In vitro cofactor assays analyzed on silver‐stained SDS‐PAGE gels demonstrated that while 300 ng of properdin inhibited by more than 50% the ability of 1 ng factor H to serve as a cofactor in factor‐I mediated C3b degradation, properdin appeared to increase by about 25% the ability of THP to serve as a cofactor in factor I‐mediated C3b degradation. Properdin, by itself, was not able to act as a cofactor in factor I‐mediated C3b degradation. In conclusion, THP and properdin bind more readily in lower ionic strength buffers than in higher ionic strength solutions, but these interactions are stable over a range of pH conditions typically found in urine. Heparin is very effective at inhibiting the binding between properdin to THP. On a functional level, properdin may augment THP's ability to act as a cofactor in C3b degradation, while it inhibits factor H's cofactor activity.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Does the MS2 L protein work with E. coli host factors?
Authors: Streff Kimberly J , Piefer Andrew J 
Published: June 2021

MS2 is a small single stranded RNA bacteriophage that escapes its infected Escherichia coli host through an L protein‐driven lysis mechanism. The L protein consists of 75 amino acids, with a hydrophilic N‐terminal that contains mainly basic residues and a hydrophobic C‐terminal end. Prior studies have shown that the L protein is necessary for the cell to lyse, however it is not known if L alone is sufficient for cell lysis. In our laboratory we have shown that expression of the L protein in the absence of other viral proteins causes E. coli lysis within 40 minutes of expression. Further, the actual mechanism for lysis is not completely understood and potentially requires a protein‐protein interaction with E. Coli host factors. Recent studies by Chamakura et. al [Journal of Bacteriology, 12, 199 (2017)] using a genetic screen identified DnaJ, a chaperone protein, as a host factor involved in the lysis mechanism. However, this study did not describe a direct protein‐protein interaction between L and DnaJ. Thus, the question remains whether DnaJ along with MS2 L is necessary and sufficient for lysis, or are there additional host proteins involved? In order to answer this question and investigate host protein interactions using a biochemical approach, a hemagglutinin epitope tag (HA) will be inserted into a plasmid containing the MS2 L gene and an arabinose‐inducible promoter. Once transformed into E. coli, the HA tagged L protein can be detected using a Western Blot and purified by immunoprecipitation. Using these methods, it is expected that any host protein that interacts with the L protein will be co‐immunoprecipitated. Future studies will use mass spectrometry to identify these co‐immunoprecipitated proteins.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Molecular Mechanisms of Isocitrate Dehydrogenase 1 Mutants in Driving the Oncogenic Neomorphic Reaction
Authors: Quichocho Giovanni Garrido , Xu Ruth , Luna Lucas , Matteo Diego Avellaneda , Scott Michelle , Fonbon Jeon Erik , Sohl Christal D. 
Published: June 2021

Brain tumors result in a mortality rate > 60% within 5 years of diagnosis. Mutations in cytosolic isocitrate dehydrogenase 1 (IDH1) drive over 70% of low grade gliomas and secondary glioblastomas. IDH1 catalyzes the NADP+‐dependent formation of α‐ketoglutarate (α‐KG) from isocitrate via oxidative decarboxylation. Mutation of IDH1 results in a neomorphic activity, the formation of the oncometabolite, D‐2‐hydroxyglutarate (D2HG) from α‐KG in the presence of NADPH, via reduction. The most common mutations occur at residue 132, where arginine (R132) is typically replaced by histidine (R132H). We have shown previously that replacing arginine with other amino acids resulted in a trend in catalytic efficiency, with larger, more polar residues at the 132‐position promoting the normal reaction, and smaller, more hydrophobic residues driving the neomorphic reaction. Based on previous data with R132H and R132L, we hypothesize that R132Y and R132I, having similar structures respectively, will promote similar rates of catalytic efficiency in the neomorphic reaction, and R132P will have little to no catalytic activity due to likely disruption of local secondary structure. To test this hypothesis, we generated cDNA constructs and heterologously expressed and purified R132I, R132P, and R132Y‐IDH1 from E. coli to determine how these residues affect to the normal or neomorphic reaction. Kinetic assays were performed to determine the rates of the neomorphic reaction of the IDH1 mutant in the presence of NADPH and αKG. We found that R132Y‐IDH1 was the most efficient in catalyzing the neomorphic reaction followed by R132I, and R132P‐IDH1, having the lowest catalytic activity for the neomorphic reaction. By replacing the R132 residue with a wide range of amino acids, and studying catalytic efficiency of each mutation, we will be able to discover the molecular mechanisms that facilitate this neomorphic activity and help inform drug design for selective mutant IDH1 inhibitors that are effective against a wide range of tumor‐relevant mutants.Support or Funding InformationThis work was funded by the National Institutes of Health R00 CA187594 (C.D.S.), U54CA132384 (SDSU) & U54CA132379 (UC San Diego), MARC 5T34GM008303 (SDSU), and IMSD 5R25GM058906 (SDSU), as well as the California Metabolic Research Foundation (SDSU) and SDSU startup funds (C.D.S). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.”This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Function of active site residues in the dUTPase from Epstein‐Barr Virus
Authors: Robbins Alexander 
Published: June 2021

Epstein‐Barr Virus, which causes infectious mononucleosis, contributes to many diseases such as Burkitt lymphoma and other cancers. The viral genome encodes a dUTPase, an enzyme that catalyzes the reaction of dUTP + H2O to dUMP + PPi + H+. This is an intermediate reaction involved in the formation of the thymidine nucleotide. The dUTPase found in humans is a trimeric enzyme containing three active sites while the viral enzyme is monomeric. Due to variation in the structures of the human and viral proteins, there may be differences in each enzyme's catalytic activity. Our results demonstrate the importance of a metal cofactor for activity of the viral enzyme. The role of active site residues in substrate binding and the catalysis of dUTP to dUMP is being examined by site‐directed mutagenesis. Characterization of the EBV dUTPase may facilitate the discovery of inhibitors. Inhibition of dUTPase decreases the formation of thymidine nucleotides creating a high dUTP/dTTP ratio. Uracil becomes incorporated into DNA in place of thymine which causes mutations that may be detrimental to cell survival. Selectively inhibiting the dUTPase from Epstein‐Barr Virus provides a mechanism to target cells infected with the virus without damaging uninfected cells.Support or Funding InformationUWL Undergraduate Research and Creativity GrantThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Kinetic survey of evolutionary diverse isoforms of malate dehydrogenase
Authors: Sampson Grant , Chang Nathan , Greene Erika , Provost Joseph 
Published: June 2021

Malate dehydrogenase (MDH) catalyzes the redox formation of malate to oxaloacetate (OAA). Several isoforms exist. Mitochondrial MDH plays a well‐known role in the last step of the Krebs cycle while cytosolic MDH is involved in the NADH shuttle and other metabolic pathways. Plants, fungi, and protists express MDH in the glyoxisomes, where a variant of the Krebs cycle takes place. To better understand the unique enzymatic differences between evolutionarily distinct MDH isoforms, we have synthesized C‐terminal His tagged with a protease cleavage site and codon optimized 13 different isoforms of MDH for expression in E. coli. These include MDH from Chlorobaculum parvum, a green sulfur photosynthetic bacterium which uses sulfide, sulfur or thiosulfate as electronic donors; Streptomyces avermitilis, a soil bacterium with unique behaviors including insecticidal properties; saline tolerant Haloarcula marismortui, a Halophilic red Archaeon found in the Dead sea; Aquaspirillium arcticum bacterium which is cold tolerant living under the snow and ice of the pacific northwest; as well as all forms of yeast and human MDH. The purpose of this study is to evaluate structural and functional differences between each isoform of MDH, purified using affinity chromatography. Enzyme assays were performed to determine Km/Vmax of each isoform under varying OAA and NADH concentrations and to assess the inhibitory effects of citrate. Thermal stability assays were performed to assess resilience to heat, cold, and determine optimal storage conditions. A final cladogram was constructed to represent the evolutionary diversity between MDH isoforms. This work will help uncover the shifts in primary and structural evolutionary changes with functional kinetics of each MDH isoform and support use of these enzymes in the classroom.Support or Funding InformationFunded in part by NSF IUSE DUE 1726932.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Optimization of Cell Free Protein Synthesis: Development of a Restriction Endonuclease Fitness Strategy
Authors: Tovar Sofia Aguilera , Hanlan Nicklaus , Ong Shi Qing , Wagner Jacob , Eckdahl Anthony , Edman Elise , Roland Kyla , Travieso Tatianna , Willis Caroline , Campbell A. Malcolm , Heyer Laurie J , Poet Jeffrey L , Eckdahl Todd T 
Published: June 2021

Our undergraduate researchers are interested in advancing the field of metabolic engineering for the production of useful metabolites that serve as biofuels, pharmaceuticals, or industrial chemical feedstocks. We published an approach to the metabolic engineering of bacteria called programmed evolution, which introduces variation in regulatory elements for the production of enzymes that control orthogonal metabolic pathways. Programmed evolution uses in vivo fitness modules that enable selection by employing riboswitches to transduce the production of desired metabolites into antibiotic resistance. Because of limitations imposed by in vivo programmed evolution such as crosstalk between orthogonal and native bacterial metabolism, the toxicity of some metabolites, and poor representation of large variation spaces, we want to reinvent programmed evolution as an in vitro strategy. This preliminary report describes our first steps of comparing lysates from two different strains of E. coli and using them to carry out cell‐free protein synthesis (CFPS). We also designed, constructed, and have begun testing a fitness module based on the expression of the restriction enzyme SmaI. We are working toward a proof‐of‐concept system in which combinations of DNA regulatory elements that enhance the production of caffeine demethylase are selected for their ability to produce theophylline. Theophylline production will turn on a riboswitch that leads to a burst of SmaI expression. Localized SmaI activity is expected to preferentially cleave successful control elements over unsuccessful ones, allowing purification and identification of previously unknown requirements for CFPS‐controlled metabolism. The successful development of in vitro programmed evolution would enable researchers to exploit the power of selection to discover combinations of control elements for the optimal yield of metabolites that are not easily produced in vivo.Support or Funding InformationNSF RUI grant MCB‐1613203 to Davidson College and MCB‐1613281 to Missouri Western State University, Davidson College Martin Genomics ProgramThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.