bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural Basis for NLRP6 Inflammasome Assembly and Activation
Authors: Shen Chen , Lu Alvin , Fu Tian‐Min , Wu Hao
Published: June 2021

Inflammasomes are multi‐protein complexes that detect infectious microbes and cellular stressors, and that activate inflammatory caspases for cytokine maturation and pyroptosis. NLRP6, a sensor protein in the nucleotide‐binding domain (NBD) and leucine‐rich repeat (LRR)containing (NLR) family, is shown to play multiple roles in regulation of inflammation and other innate immune signaling pathways. However, little is known about the molecular mechanism of NLRP6 inflammasome assembly and activation. Using cryo‐Electron Microscopy (Cryo‐EM) and X‐ray crystallography, we found that NLRP6 PYD domain is able to self‐assemble into filamentous structures accompanied by large conformational changes, and can recruit the adaptor protein ASC through homotypic PYD/PYD interactions. We further observed that full‐length NLRP6 assembles into wider filaments in a concentration dependent manner with a PYD core surrounded by the NBD and LRR domain. In conclusion, our results indicate a unified filamentous assembly mode of NLRP family proteins for downstream signal transduction. These findings will be of value for researchers to conceive the structural study of full‐length NLRP inflammasomes.

Support or Funding Information

This work was supported by US National Institutes of Health grants Al124491 and HD087988 (to H.W.) and the Harvard Digestive and Disease Center Grant HDDC P30 DK034854 (to T.M.F.).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Increasing Versatility of Small Angle X‐ray Scattering in Studying Biological Macromolecules
Authors: Chakravarthy Srinivas , Hopkins Jesse , Bilsel Osman , Irving Thomas
Published: June 2021

The Biophysics Collaborative Access Team (BioCAT), which operates the high‐brilliance undulator Beamline 18ID at the Advanced Photon Source, Argonne National Laboratory, is dedicated to studying biological macromolecules in solution using Small‐Angle X‐ray Scattering (SAXS). The combination of a highly brilliant X‐ray source and advanced micro‐focusing optics at BioCAT enables significant insights into the structure and dynamics of bio‐macromolecules from both equilibrium and time‐resolved SAXS experiments. The quality and interpretability of SAXS data is largely contingent on sample homogeneity. Size‐exclusion chromatography (SEC)‐SAXS, which was pioneered at BioCAT has therefore emerged as a powerful mode of data acquisition as it ensures optimal sample quality by purification immediately before exposure to x‐rays. In addition, we have also incorporated multi‐angle light scattering (MALS), dynamic light scattering (DLS), and differential refractive index (dRI) detectors in line with the SAXS camera thus enabling acquisition of a full complement of biophysical data typically required to structurally characterize biological macromolecules simultaneously with SAXS data. Demonstrably increased efficiency in terms of sample consumption and overall logistical ease have made SAXS much more accessible to non‐expert users.

The new frontier in the field is to study real‐time structural changes in biological systems as a response to very specific environmental stimuli such as a change in denaturant concentration, ionic strength, pH or ligand binding. Forefront time resolved SAXS techniques using chaotic mixing continuous flow, continuous laminar flow and stopped flow mixing are now available to a wide variety of bio‐medical projects at BioCAT primarily on account of being optimized over the past few years both for sample consumption and mixing efficiency.

Support or Funding Information

This project was supported by grant 9 P41 GM103622 from the National Institute of General Medical Sciences 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.
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 Information

This 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 Information

This 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

Introduction

Pseudomonas 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 Results

The 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.

Conclusion

TyrRS‐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 Information

National 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

INTRODUCTION

Respiratory 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.

METHODS

To 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.

RESULTS

Inhibition 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.

CONCLUSIONS

Our 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 Information

This 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 Information

This 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 Information

This 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 Information

Department of Pharmaceutical and Biomedical Sciences, Raabe College of Pharmacy, Ohio Northern University

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
C‐12 Binds to Tubulin at the Colchicine‐binding Site and Target Microtubules by Preferentially Binding to GTP‐bound Tubulin
Authors: Prassanawar Shweta Shyam , Kumari Anuradha , Guchhait Sankar K , Panda Dulal
Published: June 2021

In eukaryotic cells, microtubules are essential for cell division as they assemble into dynamic mitotic spindles which sequester and segregate the duplicated chromosomes. Microtubule‐targeting agents (MTAs) inhibit the dynamicity of the microtubules, thereby hampering chromosome segregation and ultimately inhibiting cancer cell proliferation. A natural compound combretastatin A‐4 (CA‐4), a potent MTA, is undergoing several clinical trials for the treatment of various cancers. However, its toxicity and low bioavailability present a major challenge for it to be considered as an approved drug. C‐12, a derivative of CA‐4, has been found to be more potent than CA‐4 in inhibiting proliferation of several cancer cell lines. In this work, we examined the binding characteristics of C‐12 and its effect in triple negative breast cancer (TNBC) cells. C‐12 inhibited the assembly of microtubules, in vitro, as observed by a turbidimetry assay and electron microscopy. A molecular docking study suggested that C‐12 interacts with tubulin with a binding energy of −10.53 kcal/mol and prefers the colchicine‐binding site on the tubulin dimer. This was further confirmed in a competitive assay, where podophyllotoxin, a colchicine site binder, strongly inhibited the binding of C‐12 to tubulin with an inhibitory constant (Ki) of 0.6 ± 0.2 μM. The fluorescence intensity of C‐12 increases upon binding to tubulin. Therefore, we analyzed the binding of C‐12 to tubulin using the fluorescence of C‐12. Interestingly, the compound exhibited a relatively greater affinity for GTP‐bound tubulin than the GDP‐bound tubulin. The dissociation constant (Kd) for C‐12 and GTP‐tubulin interaction was found to be 1 ± 0.8 μM which is significantly less than the Kd of C‐12 and GDP‐tubulin (9.2 ± 1.2 μM). This suggested that in cells, C‐12 might preferentially bind to the growing plus end of the microtubules. We also examined the effects of C‐12 on the cultured TNBC cells. Immunofluorescence analysis revealed that C‐12 depolymerized the interphase microtubule network and perturbed the localization of γ‐tubulin in TNBC cells. Moreover, the compound hampered chromosome congression and blocked the cells at mitosis, thereby inhibiting cell division. The results, altogether suggested that C‐12 is a potent microtubule targeting agent that binds to tubulin at the colchicine site and perturbs microtubule function in cells.

Support or Funding Information

This work is supported by TATA Innovation fellowship awarded to D.P. from Department of Biotechnology, Government of India.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Synthesis of 1,4‐disubstituted aminoanthraquinone derivatives for purification of lactate dehydrogenase
Authors: Kubista Jordan Louise
Published: June 2021

Cibacron Blue 3G‐A is a dye commonly used for the purification of various enzymes, in particular, isolating lactate dehydrogenase. Companies have Cibacron Blue available periodically but it is rather expensive. Because of these problems, an alternative dye that can be easily synthesized and has similar properties to Cibacron Blue is desired. Cibacron Blue is a dye designed to mimic natural biological ligands, such as NAD+/NADH. A suitable replacement should exhibit comparable affinity and purifying ability for targeted proteins. Cibacron Blue contains an anthraquinone core, which is a distinct feature due to its versatility. Many different functional groups can be substituted on to the anthraquinone core giving it a multitude of different properties and functions. The goal of this research is to synthesize an anthraquinone derivative that exhibits similar properties and functions as Cibacron Blue. The alternatives include aromatic anilines at the 1,4 positions of the anthraquinone to emulate Cibacron Blue's structure at the anthraquinone core and allow for attachment to a solid support. These were synthesized by treatment of 1,4‐bistosyloxyanthraquinone with two substituted anilino nucleophiles to build a library. These alternatives should mimic the adenine moiety of NAD+, which are accommodated in a hydrophobic crevice formed by five chain regions of lactate dehydrogenase. The left side of the anthraquinone is left unsubstituted since it is believed this is where it binds to the hydrophobic nucleic fold of enzymes. After the synthesis of the small library of aminoanthraquinone derivatives was completed, they are characterized. Binding affinity was tested using Fluorescence quenching. This method allows for comparisons between the binding of lactate dehydrogenase to the synthesized products and binding of lactate dehydrogenase to NADH using Kd values. The Kd value is an equilibrium dissociation constant that is used to evaluate binding affinity.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Discovering c‐di‐AMP binding proteins in Streptomyces
Authors: Lu Wei‐Shin , Bennett Jennifer A.
Published: June 2021

Streptomyces is an abundant genus of actinobacteria known for its ability to produce useful antibiotics. Streptomycetes transduce signals through a secondary messenger cyclic di‐AMP (c‐di‐AMP). This more recently discovered molecule is the only known secondary messenger to be essential to some bacteria, regulating many functions such as cell metabolism and DNA repair. Although c‐di‐AMP is an important molecule in the physiology of bacteria such as Streptomyces, not much is known about the potential c‐di‐AMP binding proteins or effector molecules in Streptomyces. The objective of this research was to discover and identify potential c‐di‐AMP binding proteins, particularly in Streptomyces coelicolor and Streptomyces scabies. Protein samples were either incubated with c‐di‐AMP capture compound and streptavidin beads or only with streptavidin beads, which acts as a control. Many isolated binding proteins were identified via trypsin digest followed by mass spectrometry.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Selective Inhibitor Design Toward CA IX For Breast Cancer Treatment Using Carbohydrate‐Based Compounds
Authors: Murray Akilah Briana , Lomelino Carrie Lynn , Li Haoxi , Horenstein Nicole , McKenna Robert
Published: June 2021

Human carbonic anhydrase IX (CA IX) is upregulated in neoplastic tissues and has been studied as a drug target for anticancer chemotherapy. Inhibition of CA IX has been shown to be therapeutically favorable in terms of reducing tumor growth. Recently, it been observed that sweeteners bind and selectively inhibit CA IX over other CA isoforms. It was found that cyclic sulfonamides, such as saccharin, inhibits CA IX with 60‐fold isoform selectivity, while disaccharides, such as sucrose, have been found bound at the opening of the active site. As fragment‐based studies show, the most potent inhibitors result from the combination of small molecules. A chemical linker would provide the spacial requirements to optimize interactions with residues in the selective pocket of the active site, improving selectivity for CA IX and preventing off target inhibition of the ubiquitously expressed isoform, CA II. Such compounds would simultaneously inhibit activity through two mechanisms: displacing the ZBS essential for catalysis and blocking the active site from substrate binding. The “tail‐approach” was done using sweeteners as fragments to develop inhibitors with higher CA IX selectivity by connecting saccharin (zinc‐binding group) to a β glucoside (tail) using a triazole linker. In this study, we present the X‐ray crystal structures of carbohydrate compounds in complex with the cancer‐related and off‐target CA isoforms, as well as the effect these compounds have on a panel of breast cancer cell lines. It was found that the carbohydrate compounds have different binding modes with compounds binding directly to the catalytic zinc in CA IX (mimic) and through a bridging water in CA II, which can account for the increased selectivity for CA IX (mimic) when compared to CA II. It was also found that the carbohydrate compounds decreased the metastatic potential of UFH‐001 breast cancer cells that are representative of triple negative breast cancer cells that have high CA IX expression. In summary, These studies will contribute to the knowledge of treating more aggressive and late stage breast cancers and identify new opportunities to increase the effectiveness of current cancer therapies.

Support or Funding Information

ABM was supported by training grant T32AI0007110 from the 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.
Development of Drug Delivery Systems to Overcome Cisplatin‐Resistance in Lung Cancer
Authors: Diaz Anthony Aponte , Correa Clarissa , Delgado Yamixa , Sánchez Anamaris Torres , Rodríguez Melissa Milian
Published: June 2021

In the US, lung cancer is the second most common cancer and the leading cause of cancer mortality in both men and women. Many cytotoxic agents for cancer chemotherapy are being developed under in vitro and in vivo studies, however, the main problem is the low therapeutic index. Another important aspect is the acquired resistance to drugs (e.g. Cisplatin) by cancer cells. The aim of this research project is the development of drug delivery systems (DDS) using bovine serum albumin (BSA) as the drug's carrier and cisplatin (CisPt) and curcumin (Curc) as the drugs. Heat and ultrasonication were used to promote partial unfolding of the protein in a nanoparticle rearrangement and intercalation of the drug moieties into the carrier protein. The DDS developed include BSA‐CisPt and BSA‐Curc. To characterize the ratio of the conjugation of these systems colorimetric assays were performed. Braford assay was used for BSA, for Curc, its intrinsic absorbance and for CisPt, o‐phenylenediamine/heat assay. The preliminary results of the DDS report a ratio of BSA‐Cisplatin 1:25 and BSA‐Curcumin 1:30. Both DDS demonstrated cytotoxicity on lung cancer cells (A549) of an IC50 in the μM range after 24h. In future experiments the DDS’ size will be determined utilizing a dynamic light scattering technique. In addition, other molecules such as folic acid and polyethylene glycol will be added to improve specificity and stability respectively. These DDS have potential to minimize drug systemic toxicity, increased bioavailability and reduce multidrug resistance.

Support or Funding Information

San Juan Bautista Institutional funds and applying to travel awards.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Heat Shock Protein 90 Inhibition in Kasumi‐1 Leukemic Stem Cells
Authors: Rosolen Matthew Jeremy , Smith MacKinzie D. , Bacon Nickolas A. , Sollars Vincent
Published: June 2021

Heat Shock Protein 90 (HSP90) is a well‐conserved chaperone protein present in all human cell types. In cancers, HSP90 protein expression is often upregulated as a result of the cancer cell's elevated metabolic rate and accumulation of mutations resulting in protein‐folding defects. Inhibition of HSP90 can be a potent anticancer strategy, and drugs that target HSP90 are currently in clinical trials. Luminespib (AUY922) is an HSP90 inhibitor that is currently undergoing clinical trials. This drug is unique in that it is one of the only HSP90 inhibitors known to target both the alpha‐ and beta‐isoforms of the protein. Various dosages of AUY922 were administered to the Kasumi‐1 acute myeloid leukemia cell line to evaluate the potency of AUY922. A Western Blot analysis was performed in three biological replicates to measure expression levels of Heat Shock Protein 70 (HSP70), a molecular partner of HSP90, that is upregulated when HSP90 is inhibited. A three‐colored flow cytometry experiment was conducted with CD34, CD38 and Live/Dead label to determine the effects of increasing dosages of AUY922 on both the overall population and specifically the stem cell population, which is thought to drive relapse. We showed that HSP90 inhibition flushes the cancer stem cell population in a dose‐responsive manner. This work may be important in furthering the discussion of drug development that targets the cancer stem cell population to promote durable remission and to prevent relapse.

Support or Funding Information

Supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence and by NIH Grant R15 CA186017.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The A‐kinase anchoring protein b‐synemin binds PKCe
Authors: Russell Mary , Prudner Bethany , Kuneli Melissa , Damron Derek , Chen Yijing
Published: June 2021

Synemin is a type IV intermediate filament (IF) protein. The two largest isoforms (α and β) are co‐expressed in a variety of cell types including muscle cells. Earlier we found that in muscle cells α‐synemin preferentially localizes to the sarcolemma and intercalated disks while β‐synemin is concentrated at the Z‐disks. Both isoforms also have an unusually long tail domain compared to most IF proteins. This domain is a site for numerous protein interactions, some unique to each isoform and some common to both. For example, in the past we found that both bind protein kinase A (PKA) and are thus also classified as A‐kinase anchoring proteins (AKAPs). AKAPs act to tether PKA to specific subcellular locations to maintain the specificity of the PKA signaling pathway. Excitingly, we have now found evidence that synemin also binds PKCɛ. Specifically, co‐immunoprecipitation studies show that PKCɛ binds to β‐synemin only upon stimulation of the β‐adrenergic pathway with isoproterenol. Additionally, western analysis using cells expressing either wild type or mutant β‐synemin indicates that, upon loss of β‐synemin anchored PKA, there is a decrease in phosphorylation of PKC substrates, including a ~22 kDa protein. Studies are ongoing to determine the identity of this protein. Based on molecular weight and known PKCɛ substrates in muscle cells we predict this protein is either troponin I (TnI) or myosin regulatory light chain 2 (RLC2). In support of RLC2, yeast‐two hybrid analysis revealed that protein‐protein interaction occurs between β‐synemin and RLC2. Thus, β‐synemin is proposed be a site of cross‐talk between PKA and PKCɛ and also may localize these kinases to the sarcomere at the substrate for PKCɛ.

Support or Funding Information

NIH R01 HL65701 (DD)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Genetic Screen for Proteins Involved in ASP Invasion of the Wing Disc of Drosophila melanogaster
Authors: Gondal Anas , Salim Hasan , Srivastava Ajay
Published: June 2021

Fruit flies (D. melanogaster) are ideal genetic model organisms that can closely replicate human genetic anomalies and characteristics. The invasion of the Air Sac Primordia (ASP), an organ analogous to the human lung, into the Wing Imaginal Disc (WID) of larval fruit flies is closely related to tumor metastasis in humans, as both processes use similar mechanisms to break down the basement membrane. By studying genes responsible for ASP invasion, researchers may be able to identify analogous genes in humans responsible for tumor metastasis. A genetic screen, using 100 protein trap lines, was conducted to find genes expressed in ASP and potentially involved in ASP invasion. The protein trap lines tagged endogenous proteins with a Green Fluorescent Protein (GFP) to indicate tissue localization of selected proteins under a fluorescent microscope. WID were isolated from 10 third instar larvae of each line and imaged using fluorescence microscopy. Eleven of the 100 lines screened exhibited GFP expression in the WID, but not in the ASP. Only one of the 65 lines exhibited GFP expression in the ASP. The gene responsible was Apontic (apt). Apontic's role in ASP invasion will be further investigated using RNA interference to knock down the gene.

Support or Funding Information

Anas Gondal*, Hasan Salim, Dr. Ajay Srivastava, and Nathan Powers

Gatton Academy of Math and Science, Department of Biology and Biotechnology Center, Western Kentucky University, Bowling Green, KY

KBRIN AREA and IDeA Grants funded by a grant from the National Institute of General Medical Sciences of the National Institutes of Health Grant # P20GM103436

Gatton Academy Research Internship Grant (RIG)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Loss of Cytoskeleton‐Associated Protein 4 (CKAP4) Expression Enhances α5β1‐mediated Bladder Cancer Cell Adhesion and Migration
Authors: Lobo Sonia , Stackhouse Elizabeth , Majernick Thomas
Published: June 2021

The transmembrane protein cytoskeleton‐associated protein 4 (CKAP4) plays an essential role in the maintenance of endoplasmic reticulum structure by anchoring the rough ER to microtubules in epithelial cells. CKAP4 has also been identified as a biologically relevant receptor for four unique ligands—tissue plasminogen activator, surfactant protein A, Dickkopf1, and antiproliferative factor. Recent data indicates that loss of CKAP4 expression leads to remodeling of the actin cytoskeleton, including disruption of actin stress fibers, and altered expression of genes known to promote cell motility and dynamic changes in the cytoskeleton. Among these genes, β1‐integrin expression was dramatically increased in response to CKAP4 silencing. Given the importance of tumor cell adhesion and migration for cancer invasion and metastasis, it is important to understand how CKAP4, through modulation of integrin functions, acts to regulate these cellular processes. To this end, we took a loss‐of‐function approach where CKAP4 expression was knocked down by means of siRNA in the human bladder carcinoma cell line T24. Western blot analysis revealed that CKAP4 knockdown induced β1‐integrin protein levels after 24 hours and continued to increase up to 96 hours after transfection with CKAP4 siRNA. Because β1‐integrin associates with α5‐integrin to form the fibronectin receptor, we investigated whether α5‐integrin protein levels are affected by CKAP4 knockdown. Western blot analysis revealed that CKAP4 knockdown induced α5‐integrin protein levels after 24 hours and continued to increase up to 96 hours after transfection with CKAP4 siRNA. Importantly, loss of CKAP4 led to increased surface expression of β1 and α5 integrin and increased cell adhesion and migration on fibronectin as measured by flow cytometry and electric cell‐substrate impedance sensing, respectively. Activation of ER stress and AMPK signaling pathways was also observed in CKAP4‐silenced bladder carcinoma cells. Our data indicate that CKAP4 knockdown mediates adhesion and migration of human bladder cancer cells through transcriptional up‐regulation of α5β1 integrin.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cardiac fibroblast lineages and extracellular matrix maturation in postnatal heart development
Authors: Yutzey Katherine
Published: June 2021

During postnatal heart development in mice, the ventricular myocardium undergoes extensive remodeling with increased organization and maturation of cardiomyocytes, fibroblasts and endothelial cells. In addition, the extracellular matrix (ECM), consisting mostly of mature fibrillar collagen is produced, providing necessary mechanical support for increased cardiac output. In adult hearts, the matricellular protein periostin (postn) is induced during cardiac injury and is required for collagen deposition in scar formation and pathologic fibrosis. During postnatal heart remodeling, postn also is expressed in cardiac fibroblasts coincident with collagen 1a1 gene expression. At the same time, fibronectin expression indicative of immature ECM permissive for cardiomyocyte proliferation is decreased. By postnatal day 30, postn expression is decreased and the collagen‐rich ECM surrounding individual cardiomyocytes is apparent. The expression of postn during postnatal heart remodeling and ECM maturation supports shared molecular regulation of cardiac fibroblasts and collagen fibrillogenesis in development and disease. However, the specific contributions of postn‐expressing cardiac fibroblasts to the initial production of the collagen‐rich ECM of the heart or to maturation and hypertrophy of adjacent cardiomyocytes are not yet known.

Support or Funding Information

This work was supported by grants from the NIH/NHLBI (P01HL069779 and R01HL142217)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.