bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cloning of the Escherichia coli methyl‐accepting chemotaxis protein Tar and characterization of nickel repellent interactions
Authors: Coleman Aja Kiana , Healy Frank 
Published: June 2021

Sensory systems are vital to organismal survival and are used to interpret and detect the composition of the environment. Microorganisms such as bacteria have evolved sensory systems to detect and respond to their chemical surroundings. Escherichia coli possess transmembrane chemoreceptors known as methyl‐accepting chemotaxis proteins that bind attractant and repellent chemicals, directing swimming behavior. The E. coli chemoreceptor Tar binds the attractant aspartic acid and nickel ion repellent molecules. An important aspect of Tar is that within the periplasmic space, a region between the inner cytoplasmic membrane and the bacterial outer membrane, is a binding domain for aspartate. The binding of aspartate can initiate a stimulus‐response that ultimately affects the cell motility and migration and allows the cell to detect further changes in the concentration of aspartate and respond. Transmembrane biochemical studies have elucidated Tar‐aspartate interactions, however little is known regarding Tar‐nickel interactions. To investigate this, PCR was used to amplify the tar gene from wild‐type E. coli genomic DNA. Amplification products were ligated into the low copy number plasmid vector pACYC184 and the desired E. coli transformants were selected. Defects in nickel repellent sensing of an E. coli Δtar strain were complemented with pACYC184::tar and Tar mutants were screened utilizing a soft agar motility assay. This experimental platform will be used to identify Tar residues required for nickel binding by random mutagenesis of the periplasmic loop region of plasmid‐encoded tar. Nucleotide sequencing and biochemical studies will allow us to identify and characterize the nickel ion repellent binding interaction within the Tar periplasmic region. These results may provide insights into mechanistic differences between attractant and repellent chemosensing and intracellular signaling processes. Additionally, this work may broaden our fundamental understanding of receptor‐ligand interactions.Support or Funding InformationRonald E. McNair Scholars ProgramThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Turnip mosaic virus genome‐linked protein inhibits in vitro activity of ricin A‐chain
Authors: Domashevskiy Artem V , Singh Shaneen 
Published: June 2021

Ricin is a plant derived protein toxin produced by the castor bean plant (Ricinus communis). The Centers for Disease Control (CDC) classifies ricin as a Category B biological agent. Currently, there is neither an effective vaccine that can be used to protect against ricin exposure nor a therapeutic to reverse the effects once exposed. Here we quantitatively characterize interactions between catalytic ricin A‐chain (RTA) and a viral genome‐linked protein (VPg) from turnip mosaic virus (TuMV). VPg and its N‐terminal truncated variant, VPg1‐110, bind to RTA and abolish ricin's catalytic depurination of 28S rRNA in vitro and in a cell‐free rabbit reticulocyte translational system. RTA and VPg bind in a 1 to 1 stoichiometric ratio, and their binding affinity increases ten‐fold as temperature elevates (5 °C to 37 °C). RTA‐VPg binary complex formation is enthalpically driven and favored by entropy, resulting in an overall favorable energy, ΔG = −39.8 kJ/mol. Molecular modeling supports our experimental observations and predicts a major contribution of electrostatic interactions, suggesting an allosteric mechanism of downregulation of RTA activity through conformational changes in RTA structure, and/or disruption of binding with the ribosomal stalk. Fluorescence anisotropy studies show that heat affects the rate constant and the activation energy for the RTA‐VPg complex, Ea = −62.1 kJ/mol. The thermodynamic and kinetic findings presented here are an initial lead study with promising results and provides a rational approach for synthesis of therapeutic peptides that successfully eliminate toxicity of ricin, and other cytotoxic RIPs.Support or Funding InformationNational Institutes of Health (NIH) and National Institute of Allergy and Infectious Diseases (NIAID): 1R15AI124044This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Expression, Purification and Analysis of Recombinant Proteins Responsible for the Functional Properties of a Tough Biological Glue
Authors: Falconer Rebecca , Smith Andrew 
Published: June 2021

The slug Arion subfuscus secretes a defensive glue that is very strong despite being composed of 97% water. The overarching goal for analyzing this glue is to guide the development of a biomimetic hydrogel in hopes of replacing staples and stitches in surgical practice. Previous research has identified a group of 11 proteins (all around size 15kDa) that are unique to the glue and are integral for adhesion. The goal of this research was to express and purify recombinant versions of all of these proteins. We then tested whether the proteins could stiffen gels, and whether they formed oligomers. Ligation independent cloning was used to insert cDNA coding for each protein into a plasmid vector. The plasmid contained a 6His‐tag to facilitate purification, and the coding sequence for Protein G to decrease toxicity of the protein to the bacteria. After amplification and purification of the recombinant plasmids, the plasmids were used to transform an expression vector designed to facilitate disulfide bond formation (SHuffleT cells, NEB). After triggering overexpression, the proteins were isolated by binding to nickel resins. All eleven recombinant proteins were successfully purified in this way. Five of the proteins were analyzed. In preliminary results, they had the expected gel‐stiffening activity. Additionally, the recombinant proteins were analyzed by size‐exclusion chromatography to determine the proteins' tendency to oligomerize. The proteins primarily formed dimers at the pH of slug glue, with some trimer formation. Future work will test whether oligomerization is necessary for gel‐stiffening.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating Pal's Interaction with Peptidoglycan
Authors: Lewis Sean D , Liu Xinbei , Ward Zack , LaClair Ciara , Michel Lea Vacca 
Published: June 2021

Peptidoglycan associated lipoprotein (Pal) has been shown to exhibit a unique dual orientation in Escherichia coli (E. coli). That is, Pal has two subpopulations, one surface exposed subpopulation and one periplasmic subpopulation. The periplasmic Pal subpopulation binds tightly (but noncovalently) to peptidoglycan. Both subpopulations of Pal are thought to be released from E. coli under certain conditions, and released Pal has been shown to be implicated in the clinical condition of sepsis. This study uses site‐directed mutagenesis and ultracentrifugation to elucidate the detailed structural interaction between Pal and peptidoglycan, with the long‐term goal of understanding the role of that interaction in Pal's release from E. coli. Preliminary data suggest several key residues in Pal dictate the Pal‐peptidoglycan interaction.Support or Funding InformationRochester Institute of Technology, College of ScienceThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Probing the Effect of the Pal‐Peptidoglycan Interaction on Pal Release from Escherichia coli
Authors: Liu Xinbei , Lewis Sean D , Ward Zachary , LaClair Ciara , Michel Lea Vacca 
Published: June 2021

Peptidoglycan‐associated lipoprotein, Pal, is inserted into the outer membrane of Escherichia coli (E. coli) and other Gram‐negative bacteria. Under certain conditions, including several animal models of sepsis, Pal is released from E. coli and is thought to be involved in the clinical condition of sepsis. As its name infers, Pal is tethered to the peptidoglycan layer. We propose that the strong noncovalent Pal‐peptidoglycan interaction may have an effect on Pal's ability to be released from E. coli. We tested several site‐directed mutants of Pal with altered affinities to peptidoglycan to determine the effect of those mutations on Pal's release from E. coli. Preliminary results suggest the Pal‐peptidoglycan interaction, alone, does not dictate its release from E. coli.Support or Funding InformationRochester Institute of Technology; The Rochester Institute of Technology Research Initiative for Scientific Enhancement (RIT‐RISE)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Elucidation of the Substrate Binding Site of the Yeast Zinc Metalloprotease, Ste24
Authors: Theisen Chelsea , Hsu Erh‐Ting , Wiley Patty , Vervacke Jeffrey , Distefano Mark , Hrycyna Christine 
Published: June 2021

The Ste24 zinc metalloprotease from S. cerevisiae is an intriguing integral membrane protease whose precise mechanism of action has yet to be explored fully at a molecular level. This protease is unique in that it recognizes only isoprenylated substrates and performs two distinct cleavage reactions sequentially at structurally unrelated sites in the same substrate molecule, notably the mating pheromone a‐factor in yeast. The recent X‐ray structure of yeast Ste24 revealed unique features that define a novel class of membrane protease that does not resemble any other class of intramembrane proteases described to date. The 7 transmembrane helices of Ste24 form a voluminous water‐filled intramembrane barrel‐shaped chamber that is capped at both ends. Notably, the metalloprotease active site faces the chamber interior so that substrate entry and exit is restricted and must occur through one of four side portals in the protein. Since the structure provided only minimal mechanistic information, we use it to guide our studies to define how the farnesylated yeast mating pheromone a‐factor enters and exits the chamber, how it is recognized, and how Ste24 mediates catalysis. Herein, we aim to identify the residues that comprise the substrate binding site for isoprenylated a‐factor in Ste24. Mutant forms of Ste24 generated by site‐directed mutagenesis were characterized for protease activity by a radioactive coupled methyltransferase‐protease assay and assayed for substrate binding using a photoactivatable analog a‐factor containing a biotin moiety. Substrate binding was assessed by immunoblot analysis using Neutravidin‐HRP. Tandem MS/MS methods are being developed to accurately identify the labeled amino acids in or near the binding site of the labeled proteins. Preliminary results suggest that a region surrounding the active site, lying between two portal sites within the chamber, appear to be important for a‐factor binding for the first C‐terminal cleavage reaction. Further studies will aim to determine residues important for binding for the second proteolytic cleavage reaction.Support or Funding InformationNational Institutes of HealthThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Probing the Pal‐peptidoglycan interaction
Authors: Ward Zachary , Liu Xinbei , Lewis Sean D , LaClair Ciara , Michel Lea Vacca 
Published: June 2021

Sepsis can be caused by a bacterial infection in the human bloodstream, upon which the immune system is over‐activated causing hyper‐inflammation throughout the body. One of the molecules in Escherichia coli (E. coli) that is thought to contribute to the over‐exuberant immune response is peptidoglycan‐associated lipoprotein (PAL). PAL binds tightly to peptidoglycan in the E. coli cell, but has been shown to be released from E. coli under certain conditions. We propose that modulating the PAL‐peptidoglycan interaction would have an effect on E. coli's ability to release PAL. Here, we describe an enzyme linked immunosorbent assay in which we measure and quantify changes to the PAL‐peptidoglycan interaction. Preliminary data suggest we were able to dramatically decrease binding of PAL to peptidoglycan with a single amino acid change in PAL.Support or Funding InformationRochester Institute of TechnologyThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Exploring the functional role of Nlrp12's interaction with Hck
Authors: Zhang Yue , Okamoto Curtis 
Published: June 2021

The members of the family of Nucleotide‐binding oligomerization domain‐Like Receptor proteins (NLR) are composed of a N‐terminal domain, a central nucleotide‐binding domain, and a C‐terminal ligand‐binding domain. They are generally known to be involved in regulating NF‐kB pathway. A yeast two‐hybrid screen was performed with the pyrin plus nucleotide binding domain of NLR family pyrin domain‐containing12 protein (Nlrp12) as a bait and human leucocyte proteins a prey. It revealed that Hck, a src non‐receptor tyrosine kinase family member was the top hit. Further experiments confirmed that: 1) the C‐terminal 40 amino acids of Hck specifically binds to Nlrp12's pyrin plus nucleotide binding domains; 2) the last 30 amino acids of Hck is sufficient to bind to Nlrp12's pyrin plus nucleotide binding domains, but neither to Nlrp12's pyrin domain alone nor Nlrp12's nucleotide‐binding domain alone; and, 3) the last 40 amino acids of dephosphorylated, but not phosphorylated, Hck preferentially binds to full‐length Nlrp12. Nlrp12 co‐immunoprecipitated with Hck when both are heterologously overexpressed in 293T cells. Using the same system, Nlrp12 and Hck are co‐localized by immunofluorescence. In addition, in Raw 264.7, U937, and THP‐1 cells, endogenous Hck co‐immunoprecipitated with transfected Nlrp12. Since Csk is a tyrosine kinase of members of the src family that specifically binds to the tyrosine negative regulatory residue at the C‐terminal of src family kinases and phosphorylates Hck to inactivate it in mammalian cells, it is hypothesized that Nlrp12 competes with Csk binding to Hck, thereby activating Hck. Since Nlrp12 also inhibits the NF‐kB pathway, an alternative hypothesis is that Hck rescues cells from Nlrp12's inhibition of NF‐kB activity. An NF‐kB luciferase assay confirmed that overexpression of Hck alone does not inhibit NF‐kB, but overexpression of Hck plus Nlrp12 rescued Nlrp12's inhibition of the NF‐kB pathway.Support or Funding InformationUSC School of Pharmacy Mutual Funds 2017–2019This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Coenzyme A Persulfide Prioritizes Sulfide over Butyrate Oxidation
Authors: Landry Aaron P. , Ballou David P. , Banerjee Ruma 
Published: June 2021

The human gut microbiota metabolizes fiber remnants from the diet to produce large quantities of butyrate, the primary energy source for colonic epithelial cells. In the mitochondria of these cells, short‐chain acyl‐CoA dehydrogenase (ACADS) is a flavoenzyme that catalyzes the initial step of butyrate oxidation and relays the resulting electrons to the coenzyme Q10 pool. When ACADS is isolated from native or recombinant sources, a vibrant green color is observed in the protein that corresponds to a charge transfer complex between the FAD cofactor and coenzyme A persulfide (CoASSH). The CoASSH‐bound form of ACADS is enzymatically inactive, and previous studies demonstrated that butyrate oxidation is impaired in colonocytes exposed to hydrogen sulfide (H2S), suggesting that CoASSH may be generated under these conditions. H2S is produced at high concentrations by sulfate‐reducing bacteria in the colon, and its toxic accumulation is prevented by sulfide quinone oxidoreductase (SQR), a mitochondrial membrane‐bound flavoenzyme that oxidizes H2S to a persulfide with concomitant reduction of coenzyme Q10. SQR is remarkably promiscuous, and is capable of transferring the sulfane sulfur from H2S oxidation to a number of low molecular weight thiol acceptors. We thus hypothesized that the source of CoASSH, and therefore ACADS inhibition, is H2S oxidation via SQR activity utilizing CoA as an acceptor. We demonstrated that CoA was a competent small molecule sulfur acceptor using the steady‐state SQR assay, while the kinetics of sulfur transfer from H2S to CoA were characterized using stopped flow spectroscopy. CoASSH generated in situ by SQR led to a charge transfer complex in ACADS, providing further evidence that CoASSH is the persulfide product of the SQR reaction. As both ACADS and SQR relay electrons to coenzyme Q10, competition over the coenzyme Q10 substrate would inhibit the effective clearance of the respiratory toxin, H2S. These studies implicate the inhibition of ACADS by CoASSH as a potential modulatory mechanism to prioritize sulfide clearance by reserving the coenzyme Q10 pool for SQR.Support or Funding InformationThis work was supported in part by the National Institutes of Health (GM112455 to Ruma Banerjee and F32GM122357 to Aaron P. Landry).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Probing the SpNOX substrate binding site for NADPH vs. NADH affinity
Authors: Notice Brittany D , Scott James , Gonzalez Victoria , Thomas Sarah , Vermot Annelise , Petit‐Hartlein Isabelle , Fieschi Franck , Smith Susan M.E. 
Published: June 2021

NADPH oxidases (NOXes) are a family of eukaryotic enzymes that produce reactive oxygen species (ROS). These transmembrane enzymes move electrons from the substrate NADPH across the membrane to the final electron acceptor O2, forming the superoxide anion O2 −. SpNOX is a recently discovered prokaryotic NOX homolog that, unlike eukaryotic versions, is robust to bacterial expression and is active in detergent solution, making it a good model system for study of the NOX family. Eukaryotic NOXes use NADPH exclusively as a substrate, while SpNOX has similar affinity for either NADH and NADPH. Tyrosine 353 in SpNOX occupies the same position in space as a well‐conserved arginine residue in eukaryotic NOX; by homology to other NADPH binding sites this R should interact with both the adenine ring and the extra phosphate group of NADPH, explaining the eukaryotic NOX preference for NADPH. We have created Y353R and Y353A mutations in SpNOX to probe the effects of this position on the affinity of SpNOX for NADPH vs. NADH. These results will further define the NOX substrate binding site, crucial for drug discovery.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of a Rapid, DNA‐Based Field Test for Detection of Ranavirus, an Emerging Amphibian Disease
Authors: Angel Allyssa , Reyes Patricia , Gubler Ueli , Monsen Kirsten , Hazard Lisa , Goodey Nina 
Published: June 2021

A key problem when developing molecular diagnostic techniques is procuring target detection probes. The need of a stable and selective probe is necessary for accurate target identification in disease detection. Antibodies are widely used but they are unstable at ambient temperatures and are expensive and time consuming to develop. The use of aptamers (short ssDNA) as molecular probes provides several advantages over protein‐based methods because they are more stable, easily selected and modifiable. The need of a reliable and stable probe is necessary for accurate detection tests in the field for disease monitoring. Currently, field methods are limited in the time and expertise required to obtain results. Our objective is to develop an aptamer probe with high affinity and specificity for Ranavirus virion particles and recombinant Ranavirus major capsid protein (MCP) through an iterative process known as SELEX. We developed two SELEX methods utilizing either spin concentrators or protein affinity chromatography to successfully enrich a starting library of 1014 non‐specific aptamers to those specific to Ranavirus virion particles and MCP, respectively. After 6 rounds of selection, we have isolated 24 potential Ranavirus probes and subsequently cloned into a TOPO_BluntII vector for sequencing. Aptamer sequences will be analyzed for common structural motifs between virion specific aptamers and MCP specific aptamers, to identify possible common binding sites for these two respective targets. The binding affinities of selected aptamer probes will be measured by Surface Plasma Resonance (SPR) using a streptavidin modified biosensor chip and biotinylated aptamers. We expect binding affinities of aptamers to Ranavirus targets to be within the nanomolar range, demonstrating the aptamers high affinity and specificity. Ultimately, the selected aptamers will be incorporated as molecular probes for Ranavirus into a field usable rapid pathogen test that will surpass the limitations of current field tests.Support or Funding InformationThe New Jersey Water Resources Research Institute (NJWRRI) The PSEG Institute for Sustainability StudiesThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Electrochemical DNA Biosensors for Detection of Mannose‐capped Lipoarabinomannan
Authors: Sodia Tyler , Bonham Andrew J 
Published: June 2021

Mycobacterium Tuberculosis (TB) is one of the world's most prevalent bacterial pathogens. It is estimated that almost 10 million cases of TB emerge every year, and roughly one‐fifth of these cases are fatal. The current detection and diagnosis of TB is done primarily via two methods; the TB skin test and the TB blood tests. Neither of these tests can differentiate between latent TB infection and TB disease. In order to differentiate these states, time‐consuming sputum tests are required, which rely on culturing the mycobacterium. Designing a sensitive serologic biosensor would dramatically decrease the time line of diagnosis and therefore improve patient outcomes. One possible avenue for improved detection lies in the cell wall of TB, which includes many complex glycolipids—many of which are believed to have immunopathogenic mechanisms in physiologic pathways. Mannose‐capped lipoarabinomannan (ManLAM) is one of the most prevalent of these glycolipids, and presents a novel target as a bio‐marker for the sensitive detection of TB and related Mycobacterium strains. Here, we have utilized an existing aptamer sequence that binds to ManLAM to generate a sensitive electrochemical, DNA‐based biosensor for the detection of TB. This biosensor is able to adopt multiple different folded conformations, only one of which presents the core aptamer sequence in a state capable of binding ManLAM. An appended redox‐active tag (methylene blue) generates a measurable difference in electrochemical current upon this conformational change, providing a sensitive and quantitative measurement of ManLAM concentration. Such biosensors may ultimately allow rapid, on site, diagnosis of TB infection within the time constraints of patient‐doctor interaction.Support or Funding InformationThis work is supported by internal grant funding from the Metropolitan State University of Denver.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.