bluuebunny/crossref_metadata_2025_split huggingface dataset:
Expression and purification of Class D flavin dependent monooxygenase N‐oxygenases
Authors: Truong Sharon , Rungreang Pukjera Jena , Bergonio Jessica Sanez 
Published: June 2021

Members of the Class D flavin monooxygenase family are found in numerous natural product biosynthetic pathways, including those of valanimycin and daunorubicin, two medicinally useful natural products. Our lab is interested in understanding catalysis by this enzyme family. We are investigating several representative Class D monooxygenases, with the specific goal of understanding structure‐function relationships in their active sites. Our ultimate goal is to enable rational design of the enzymes in this family. The research presented here summarizes our efforts towards expression, purification and initial biochemical characterization of nitrososynthase from Streptomyces peucetius (DnmZ), and two isobutylamine N‐hydroxylase homologs (from Streptomyces setae, vlmH6, and Streptomyces viridochromogenes, vlmH7). These three Class D monooxygenases catalyze a biosynthetic step common to multiple natural products – flavin‐dependent hydroxylation of a primary amine. Streptomyces setae vlmH was successfully overexpressed in E. coli cells. The highest levels of protein were obtained when cultures were induced with 0.5 mM IPTG and incubated at 18°C. DnmZ is likewise expressed in E. coli with an optimal IPTG concentration and induction temperature of 0.1mM and 25°C, respectively. Both proteins will be purified by nickel affinity chromatography in preparation for preliminary characterization.Support or Funding InformationThis research was made possible by funding through the National Institutes of Health (NIH) grant #1SC3GM122652.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, timing and catalytic mechanism of NosN, a class C radical SAM methylase involved in the biosynthesis of nosiheptide's side‐ring system
Authors: Wang Bo , LaMattina Joseph W , Booker Squire J 
Published: June 2021

Nosiheptide is a bicyclic thiopeptide natural product that exhibits potent antibiotic activity against a number of clinically important Gram‐positive pathogens. It is biosynthesized from a ribosomally produced core peptide that is then modified extensively to contain a number of thiazole rings and dehydrated amino acids, as well as other modifications. It also contains a unique side‐ring structure composed of a 3,4‐dimethylindolic acid bridge connected to the side chains of Glu6 and Cys8 of the core peptide via ester and thioester linkages, respectively. In addition to the core peptide, encoded by the nosM gene, the biosynthesis of the side‐ring structure requires the actions of NosI, J, K, L, and N. NosN is annotated as a class C radical S‐adenosylmethionine (SAM) methylase, but little is known about how the enzyme functions or what its true substrate is. By designing, synthesizing and testing small peptides as substrate mimics, we show that the true function of NosN is to transfer a C1 unit from SAM to C4 of 3‐methyl‐2‐indolic acid (MIA) with concomitant formation of a bond between the carboxylate of Glu6 of the core peptide and the nascent C1 unit. In addition, using full‐length peptides, we show that NosN completes installation of the side‐ring structure immediately after NosK appends MIA to the core peptide. Using a substrate mimic containing a rigid structure, we also identify and characterize two reaction‐based adducts containing SAM fused to C4 of MIA The two SAM‐adducts are derived from a consensus radical‐containing species proposed to be the key intermediate in our proposed catalytic mechanism of NosN.Support or Funding InformationThis work was supported by NIH (GM‐122595 and AI‐133318 to S.J.B.). S.J.B. is an investigator of the Howard Hughes Medical Institute.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cloning and Characterization of the Tyrosyl‐S and Tyrosyl‐Z tRNA Synthetases from Pseudomonas aeruginosa
Authors: Hughes Casey A , Gorabi Varesh , Bullard James M 
Published: June 2021

IntroductionPseudomonas aeruginosa, a Gram‐negative opportunistic pathogen, is a leading cause of nosocomial infections. The ability of this bacteria to form resilient biofilms on implanted medical devices results in high mortality rates. Patients with cystic fibrosis (CF) are especially at risk as P. aeruginosa colonizes the lungs of these patients forming biofilms which leads to chronic infections and is a leading cause of death. Aminoacyl‐tRNA synthetases (aaRSs) catalyze the covalent attachment of amino acids to their cognate tRNAs. These enzymes are essential for protein synthesis in bacteria and serve as validated targets for the development of new anti‐infectives. Unlike in other bacteria, the P. aeruginosa genome contains two genes (tyrS and tyrZ) which encode two distinct TyrRS enzymes, which are very different at the amino acid level containing only 41.1/26.8 percent similar/conserved residues.Methods and ResultsThe gene encoding P. aeruginosa TyrRS‐Z was cloned and overexpressed in E. coli cells, and purified to homogeneity. Amplification of tyrS by PCR was unsuccessful, therefore the gene sequence was optimized for codon expression and synthetically synthesized. Cloning of the optimized gene resulted in expression of large amounts of TyrRS‐S. Both forms of TyrRS were active in aminoacylation. The kinetic parameters for the interaction of these enzymes with their three substrates (tRNA, ATP, tyrosine) were determined using timed aminoacylation and ATP:PPi exchange assays. Both forms of TyrRS were developed into screening platforms using scintillation proximity assay (SPA) technology. Using this assay, a synthetic compound library from ChemDiv, Inc. containing over 2000 distinct compounds was screened to detect compounds with the ability to inhibit the function of TyrRS. A number of inhibitory compounds were confirmed and are currently being characterized for the ability to inhibit both the enzymatic activity (IC50) of both forms of TyrRS and growth (MIC) of a panel of problematic pathogens in culture. They are also being analyzed to determine the potential mechanism of inhibition relative to substrate binding, global mode of action of inhibition of growing cultures of bacteria, as well as toxicity to human cell cultures.ConclusionTyrRS‐S and TyrRS‐Z from P. aeruginosa were cloned, expressed, characterized and used for development of screening platforms to identify compounds that have the potential for development as antibacterial agents against drug resistant pathogenic organisms.Support or Funding InformationNational Institutes of Health (grant number: 1SC3GM098173)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Disruption of Rho GTPase Prenylation by Statins Inhibits Respiratory Syncytial Virus
Authors: Malhi Manpreet , Norris Michael J , Duan Wenming , Moraes Theo J , Maynes Jason T 
Published: June 2021

INTRODUCTIONRespiratory syncytial virus (RSV) is a leading cause of acute lower respiratory tract infections necessitating hospitalization in the pediatric population. Despite the significant healthcare burden associated with RSV, few options are available to prevent or treat infection. To address the lack of therapeutic strategies for RSV, we developed a high content screening platform to identify small molecule inhibitors of the virus. Following a screen of 2400 compounds, statins were found to significantly reduce RSV infection rates. Here we show that statin‐mediated disruption of isoprenoid biosynthesis and Rho GTPase prenylation blocks late events within the life cycle of RSV, including protein trafficking and viral assembly.METHODSTo screen for inhibitors of RSV, human laryngeal epidermoid carcinoma (HEp‐2) cells were infected with recombinant RSV engineered to express green fluorescent protein (GFP). Automated image analysis algorithms were used to quantify infection rate by measuring the proportion of GFP‐fluorescent cells. Statin‐induced changes to the life cycle of RSV were determined by immunofluorescence microscopy and temporal quantification of viral fusion and nucleoproteins. Effects on protein prenylation by statins and RSV were evaluated via click chemistry using azide‐modified isoprenoids with a biotin‐alkyne detection reagent. Rho GTPase activity was quantified by Western blot following affinity purification of active, GTP‐bound RhoA or Rac1.RESULTSInhibition of RSV by statins was dose‐dependent with an effective concentration (EC50) of 1 μM. Viral events occurring >12 hours post‐infection, including protein trafficking and assembly, were inhibited by statins, whereas early events, such as viral entry, were unaffected. Viral trafficking and assembly are thought to be mediated by Rho GTPases, a group of prenylated proteins which modulate dynamics of the actin cytoskeleton. We found that RhoA‐specific inhibition by Rhosin (50 μM) moderately reduced RSV infection rate (26 ± 6% reduction), but Rac1‐specific inhibition by EHT1864 (20 μM) elicited a profound effect (90 ± 2% reduction). Critically, RSV infection promoted Rac1 prenylation and activity, while statin treatment blocked Rac1 prenylation and reduced active, GTP‐bound protein by 2.5‐fold.CONCLUSIONSOur results demonstrate the importance of protein prenylation to RSV infection and perturbation of this process by statins. Inhibition of Rac1 prenylation and activity by statins serves to block viral protein trafficking and assembly. Based on our findings, we propose repurposing statins as a treatment for RSV as these drugs potently inhibit the virus, are US Food and Drug Administration approved and have favorable clinical safety profiles.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Assessing Lipid Activation of Human P‐Glycoprotein for ATP Hydrolysis Analyses in the Presence of Novel Inhibitors
Authors: Okwuone Dakota D. D. , Oliveira Maisa C. , Farokhnia Roxana C. , Jensen Madeline K. , Edgar Jennifer M. , Wise John G. , Vogel Pia D. 
Published: June 2021

P‐glycoprotein (P‐gp) belongs to the ATP‐Binding Cassette (ABC) transporter family of protein. This class of membrane proteins is strongly linked with multidrug resistance (MDR) in tumors where overexpression of these proteins often nullifies the effects of chemotherapeutics by removing the therapeutics from the cells. In hopes of creating co‐therapeutics to re‐sensitize tumors to chemotherapeutics, we previously used high‐throughput in silico ligand docking studies and identified drug‐like compounds that reversed MDR in human cancer cells in culture. The effects on P‐gp ATPase hydrolysis activity were used as an initial biochemical screen for inhibitor effectiveness followed by assessments of MDR‐reversal in various P‐gp overexpressing cancer cell lines. For the initial biochemical analyses, P‐glycoprotein (MDR3) from mouse was used, which is closely related to the human (MDR1) protein. To evaluate the mechanism of inhibition also in the human P‐protein, we have isolated human P‐gp from the yeast Pichia pastoris using mixed detergent micelles. Lipid activation of ABC transporters has been shown previously to be a vital step in ensuring best enzyme activity for the analysis of such proteins. In our pursuit to optimize the P‐gp ATPase activity, we have varied amounts and combinations of different lipids including phosphatidylcholine (PC), E. coli lipids and cholesterol and assessed the resulting ATPase hydrolysis activities in coupled enzyme assays. Preliminary results showed P‐gp activity in the presence of PC was significantly higher than when E. coli lipids were used. Here we report our efforts to achieve the best protein:lipid ratios as well as assess various techniques to achieve the high ATP hydrolysis activity of P‐gp to test our newly found drug‐like molecules in their effectiveness as P‐gp inhibitors.Support or Funding InformationThis work was supported by NIH NIGMS [R15GM094771‐02] to PDV and JGW, SMU University Research Council, SMU Hamilton Undergraduate Research Scholars and Undergraduate Research Assistantship Programs, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and a private gift from Ms. Suzy Ruff of Dallas, Texas. The authors would like to thank Dr. Ina L. Urbatsch, Texas Tech University Health Science Center, for providing the P. pastoris cells expressing the human wild‐type MDR1 used in this work.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Induction of Apoptosis by Peptide J18 in Ovarian Cancer Cells
Authors: Oldenburg Kimberly , Wieskamp Matthew , Soendergaard Mette 
Published: June 2021

Ovarian cancer is the fifth leading cause of cancer‐related deaths for women, which is correlated to a low rate of early‐stage detection. Currently, standard treatment involves cytoreductive surgery and chemotherapy. However, patients often present with recurrent and resistant disease leading to overall low 5‐year survival rates of approximately 40%.Bacteriophage (phage) display technology was previously used to identify peptide J18 that binds specifically to human ovarian adenocarcinoma (SKOV‐3) cells. In addition, J18 was shown to reduce cell viability via the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) reduction assay. In order to further investigate cytotoxic effects of peptide J18, induction of apoptosis was measured by a caspase 3/7 assay. Briefly, SKOV‐3 cells were grown to 90% confluency on 96‐well plates in McCoy's 5A cell medium supplemented with 10% fetal bovine serum and 50 μg/mL gentamicin at 37°C and 5% CO2. Cells were then treated with 0.25 mg/mL peptide J18, 0.25 mg/mL peptide N35 (negative control), 100 nM paclitaxel, or dimethyl sulfoxide (DMSO; vehicle) for 12 h, after which 100 μL Apo‐ONE Caspase‐3/7 Reagent (Promega, WI) was added and incubated at 37°C for 3 h. The activity of caspase 3/7 was then measured fluorescently (λex=499 nm, λem=521 nm) using a microplate reader (SpectraMax Gemini EM, Molecular Devices, CA). The relative fluorescent units (RFU) was measured to be 409.7±96.0, 560.6±84.1, 652.5±149.1, and 648.0±47.5 (mean±standard deviation) for DMSO, N35, J18, and paclitaxel, respectively. Results showed that J18 and paclitaxel significantly (p<0.05) increased caspase 3/7 activity compared to DMSO (control), while N35 (negative control) was unchanged. Further, there was no significant difference (p>0.5) between J18 and paclitaxel at the concentrations measured. These results indicate that J18 reduces cell viability in human ovarian adenocarcinoma cells by apoptosis, and that the peptide may be utilized in the treatment of the disease.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Purification of Recombinant Zika Virus NS3 Helicase for the Identification of an Antiviral Drug
Authors: Westemeier Emily , Pina Ivett , Hamann Mark T , Hamann Michael J 
Published: June 2021

The Zika Virus is linked to microcephaly in unborn children, leading to development issues or death. Zika is an emerging disease within the United States but has spread throughout Asia and Africa for several decades. In order to slow or stop the spread of the disease, both antiviral medications and vaccines need to be formulated against the virus. One logical target for potential antiviral drug development is the NS3 helicase of the Zika virus because of its unique function in viral replication. In order to screen for potential lead compounds that can inhibit NS3, a plasmid encoding a maltose‐binding fusion protein of the NS3 helicase was generated, and used to purify the protein from E. coli BL21. The protein will be used in drug identification experiments where natural product extracts will be incubated with the protein and bound ligands identified by mass spectrometry. MBP‐NS3 will also be tested for helicase activity via a chemiluminescence assay with a digoxigenin‐tagged RNA substrate. In these experiments, the activity of the helicase will be detected by a decrease in the luminescence within a plate‐based assay. The assay will be used to validate potential lead compounds for their ability to inhibit helicase activity. Overall, this work is aimed at not only identification of a possible antiviral for Zika virus, but identifying compounds that target a wider array of ssRNA flaviviruses encoding structurally similar helicases.Support or Funding InformationThis work was supported by Bemidji State University Biology Department and the College of Business, Mathematics, and Science. Support was also provided through the Neilson Foundation, Bemidji, MN and Regenerative Medicine Minnesota (RMM‐2017‐EP‐04).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Butelase: Linkage‐specific Ligase
Authors: Tam James P 
Published: June 2021

Site‐specific ligation is enormously useful to enable selective bonding of chemicals, polymers, peptides, and proteins to form new compounds. Recent advances in chemistry, biochemistry and molecular biology have provided novel methods to staple peptide and non‐peptide bonds under physiological conditions. An example is the family of linkage‐specific, peptide asparaginyl‐specific ligases (PAL). A prototypic member of PAL is butelase 1 isolated from the butterfly pea (Bunga Telang). Butelase 1, catalyzing the reverse reaction of an asparaginyl endopeptidase, is a stand‐alone and ATP‐independent ligase [1]. Importantly, butelase 1 is C‐terminus‐specific for Asn/Asp (Asx), traceless, and accepts a tripeptide Asx‐His‐Val with the dipeptide His‐Val as the leaving group. Butelase 1 accepts most N‐terminal amino acids with D‐or L‐configuration. It exhibits unmatched kinetics with catalytic efficiencies of up to 1,340,000 M−1 s−1 and is >10,000 times faster than other known ligases. Our recently published work showed that butelase 1 is useful for both intra‐ and intermolecular ligation. It efficiently cyclizes or ligates various peptides and proteins ranging in size from 8 to >300 amino acids. Thus, the high catalytic efficiency and broad substrate specificity of butelase 1 could augment new applications, both in in vitro and in vivo systems for basic and translational research. Here, I will present our latest results on Asx‐specific ligases [2–6], their applications, and live‐cell labeling to explore new frontiers in biochemical, medical and material sciences.Support or Funding InformationThis research was supported in part by Nanyang Technological University Internal Funding ‐ Synzymes and Natural Products (SYNC) and the AcRF Tier 3 funding (MOE2016‐T3‐1‐003).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ligand docking suggests isomer specific catechin induced SIRT‐1 activation
Authors: Brenneman Martin , Mahfouz Tarek , Stockert Amy 
Published: June 2021

Cinnamon has been proposed to have multiple metabolic effects ranging from anti‐hyperglycemic effects to regulation of basal metabolic rates. Polyphenols such as resveratrol, caffeic acid, aspalathin, and quercetin have been shown to have similar effects through various pathways. Cinnamon extract contains many polyphenolic compounds with structural similarities to the studied polyphenols and as such may exhibit effects via the same biochemical pathways. Cinnamon extract contains a number of catechin isomers, polyphenolic molecules closely related to those stated previously. Due to these similarities, we propose that catechin may act through the pathways proposed for other polyphenols. Resveratrol has been shown to exert effects through modulation of SIRT‐1, a deacetylase enzyme involved in chromatin remodeling and control of gene expression. Our research primarily involved docking of the four catechin isomers into the allosteric binding site of the SIRT‐1 enzyme that resveratrol has been shown to act through. The two R isomers, (−)‐epicatechin (2R,3R) and (+)‐catechin (2R,3S), did not align with our assigned baseline values. However, these molecules cannot be ruled out for biological activity. The two S isomers of catechin, (−)‐catechin (2S,3R) and (+)‐epicatechin (2S,3S), showed favorable scores and positioning similarity to resveratrol. It was also observed through cooperative binding studies that (+)‐epicatechin (2S,3S) has the potential to be an activator of SIRT‐1. Other possible mechanisms of catechin include those of the other polyphenolic compounds. Quercetin has been shown to have effects via two major mechanisms. The first is by promoting glucose uptake in liver cells via upregulation of GLUT‐2 and GLUT‐4 gene expression. The other pathway involves down‐regulation of GLUT‐2 in intestinal microvilli to decrease absorption. Finally the effects of aspalathin have been shown to regulate GLUT‐4 translocation to the plasma membrane in L6 myocytes.Support or Funding InformationDepartment of Pharmaceutical and Biomedical Sciences, Raabe College of Pharmacy, Ohio Northern UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.