bluuebunny/crossref_metadata_2025_split huggingface dataset:
Functional characterization of glutamate carboxypeptidase II: a multifunctional Zn‐metalloprotease
Authors: Holderby Kyrsten Faith , Gamage Hashi E. , Derham Jessica M. , Greiner Brooke R. , Mendoza Jocelyne 
Published: June 2021

Glutamate Carboxypeptidase II (GCPII) is a dimeric, transmembrane, zinc metallopeptidase produced in many organisms including humans. Human GCPII (hGCPII) is expressed in the brain, prostate, intestine, and kidney, and is therefor predicted to exhibit varying tissue‐specific hydrolase activities. The only documented GCPII hydrolase activities are the breakdown of N‐acetyl‐aspartyl‐glutamate (NAAG) – GCPII is involved in the development of neurotoxicity due to excess glutamate – and folyl‐poly‐γ‐glutamate – GCPII is involved in folate uptake across intestinal mucosal membrane. However, functions in other tissues have not been reported. A comprehensive understanding of potentially diverse tissue‐specific proteolytic and other biochemical functions of GCPII requires detailed molecular level in vivo and in vitro studies using the full‐length GCPII and suitable model organism respectively.Homology modeling and protein structure analyses identified a hGCPII ortholog in the roundworm Caenorhabditis elegans, (hereafter referred as cGCPII), thus C. elegans has been chosen for the in vivo studies. The hGCPII and the cGCPII share all essential protein structural features. The cGCPII is encoded as three paralogs (gcp‐2.1, gcp‐2.2, gcp‐2.3) compared to five paralogs in humans. Whole animal phenotypic studies carried out using the wild‐type (N2) and the mutant RB1055, which lacks the gcp‐2.1, showed a larger brood size for RB1005 than N2 and longevity assays showed similar lifespans for both strains. The N2 and RB1055 strains raised in the presence of the folate hydrolase inhibitor sulfasalazine (SSZ) and GCPII specific inhibitors 2‐(Phosphonomethyl)pentane‐1,5‐dioicacid (PMPA) or 2‐(3‐Mercaptopropyl)pentanedioic acid (2‐MPPA) and the showed significantly reduced brood size for RB1055 compared to N2. The functional roles of individual cGCPII paralogs were investigated by RNAi mediated knockout experiments. Collective results indicate that the all three cGCPII paralogs may be involved in functional complementation in C. elegans. For the in vitro studies, the full‐length hGCPII was cloned and overexpression was carried out in Pichia pastoris. Several hGCPII expressing transformants have been identified and expression optimization is underway. Parallel cloning in insect cells in process to compare the functionally important glycosylation levels in Pichia versus the insect cells in order to choose an optimal expression system. Together, the in vivo and in vitro findings will aid in further understanding of the physiological functions of GCPII.Support or Funding InformationEastern Illinois UniversityThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Elucidation of a 2.3 Å Resolution Norovirus GII.4 Protease Structure by X‐Ray Crystallography
Authors: Muzzarelli Kendall Marie , Kuiper Benjamin , Spellmon Nicholas , Brunzelle Joseph , Hackett Justin , Kovari Iulia , Yang Zhe , Kovari Ladislau 
Published: June 2021

According to a current report from the Centers for Disease Control (CDC), it is estimated that noroviruses are responsible for 685 million cases of acute gastroenteritis each year with an estimated $60 billion medically and socioeconomically. This overwhelming global burden drives the need for identification and characterization of potential norovirus drug targets to exploit for design and development. Specifically, the GII.4 norovirus genotype accounts for more than 50% of all outbreaks worldwide. Up until now, the only GII.4 model was based on an alignment of the GI.1 and GII.4 protease sequences which resulted in a sequence identity of only 67%. This high genetic diversity among human noroviruses enhances the need for an improved model of the GII.4 protease. We report the crystallization and structure elucidation of a 2.3Å resolution structure of the GII.4 norovirus protease (GII.4 NoV PR) through X‐ray crystallography. Additionally, extensive investigation and characterization of the GII.4 norovirus protease structure and mechanism were carried out through computational studies and enzyme kinetics. With the elucidation of the GII.4 norovirus protease structure, we hope to significantly improve the structure‐based design of both potent therapeutic inhibitors and potential vaccine strategies for future studies.Support or Funding InformationWayne State University School of MedicineThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Rationally designed mutations of E. coli alkaline phosphatase confer selective purine derivative binding
Authors: Malecha Mitchell Richard , Weaver Todd Michael , Grilley Daniel Patrick 
Published: June 2021

Small organic molecules, like phenylalanine and theophylline, are effective inhibitors of mammalian alkaline phosphatases, such as calf intestinal alkaline phosphatase (CIAP). However, organic compounds do not hamper E. coli alkaline phosphatase (EcAP) activity. Sequence and structural analysis of alkaline phosphatase isozymes revealed a lack of conservation at EcAP residues that may be important for organic inhibition, thereby providing a potential explanation for the contrary inhibition. By mutating these EcAP residues to mimic analogous residues in mammalian APases, uncompetitive inhibition of EcAP by a class of aromatic molecules was conferred. While variants with single mutations are unaffected by organic effectors, variants expressing multiple mutations are inhibited, suggesting a synergistic relationship essential for organic binding. Circular dichroism was utilized to verify similar stability and kinetics for each variant. Michaelis‐Menten experiments were used to identify inhibition and confirm similar pH and cofactor dependence. The importance of key residues was determined based on their ability to confer organic inhibition. Furthermore, utilization of a set of purine derivatives allowed determination of the structure‐activity relationship for inhibitor binding. Specifically, this analysis enabled identification of hydrogen bonding requirements for organic inhibition. In total, designed substitutions altering EcAP residues near the active site to mimic analogous residues in mammalian APases confers uncompetitive inhibition by specific derivatives of a class of aromatic organic molecules.Support or Funding InformationResearch generously supported by a University of Wisconsin ‐ La Crosse Undergraduate Research and Creativity grant (MRM).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Genotype and Phenotype of Caffeine Metabolism in Identical Twins
Authors: Passang Tenzin , Ye Jiayu , Kline Gabriel M. , Millard Julie T. 
Published: June 2021

A wide range of individual variability to caffeine's effects have been reported in the literature, with factors including gene variants, gender, and personal habits modulating the response to caffeine. For example, the literature suggests that a single nucleotide polymorphism (SNP) in intron 1 of the CYP1A2 gene is associated with “fast” versus “slow” caffeine metabolism. The goal of our study was to genotype and phenotype identical twins to investigate environmental versus genetic influences on caffeine metabolism. For genotyping, we are using PCR‐RFLP to determine C/A at rs762551. For phenotyping, we are using a micro‐scale solid phase extraction of caffeine from saliva, determining the concentration of caffeine via GC‐MS, and using the loss of caffeine over time to monitor the activity of CYP1A2, the principal enzyme for caffeine metabolism. Initial results suggest a good correlation of caffeine half‐lives within identical twin pairs. However, it was not possible to predict this half‐life based on rs762551 genotype. Our findings indicate a strong influence of behavioral factors such as daily caffeine consumption on CYP1A2 activity. These findings could have significance for optimizing the ergogenic effects of caffeine dosing.Support or Funding InformationInstitutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM0103423This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mitochondria‐targeting peptide from Hibiscus sabdariffa
Authors: Loo Shining , Kam Antony , Tam James P. 
Published: June 2021

Cysteine‐rich peptides (CRPs) are hyperstable, multi‐disulfide‐constrained peptides. Structurally, CRPs contain evolutionary conserved disulfided scaffolds, often with functional intercysteine loops which contribute to their multiple biological activities. Previously, we have identified Hibiscus sabdariffa as a rich source of CRPs. Hibiscus sabdariffa, commonly known as roselle, is a South‐east Asian plant. It is used as a folk medicine for treating age‐related conditions and diseases. The prototypic CRP derived from roselle is roseltide rT1, a 27‐residue cysteine‐rich peptide with six cysteine forming four intercycteine loops. We showed that Loop 1 contributes to the inhibitory activities against human neutrophil elastase. However the functions of other loops, individually or in combination have not been determined. Here we report that roseltide rT1 is a mitochondria‐targeting peptide with bioenergetic properties. We showed that roseltide rT1 interacts with the mitochondrial import receptor TOM20 for its localization to the mitochondria. We prepared the linear precursor of roseltide rT1 using a stepwise solid‐phase method followed by oxidative folding to its native state. The conformation of synthetic rT1 was confirmed using reversed‐phase high‐pressure liquid chromatography and nuclear magnetic resonance spectroscopy. Using N‐hydroxysuccinimide ester, N‐terminal biotin‐ and Cy3‐labeled roseltide rT1 were generated for biological assays. Our results showed that mitochondrial depolarization induced by mitochondria uncoupler carbonyl cyanide‐4‐(trifluoromethoxy)phenylhydrazone does not abolish the mitochondrial localization of Cy3‐rT1. In silico docking suggested that the leucine‐rich loop 2 of roseltide rT1 interacts with TOM20. Using pull‐down assay, biotin‐rT1 was shown to interact with TOM20. Using live‐cell confocal microscopy, Cy3‐rT1 was shown to co‐localize with TOM20‐GFP in stably‐transfected HEK293 cells. Additionally, the localization of Cy3‐rT1 with MitoTracker Green was reduced in TOM20‐knocked‐down HepG2 cells compared to control. Taken together, this study shows that the mitochondrial import receptor TOM20 plays an important role for the localization of roseltide rT1 to the mitochondria.Support or Funding InformationThis research was supported in part by Nanyang Technological University Internal Funding ‐ Synzyme and Natural Products (SYNC) and the AcRF Tier 3 funding (MOE2016‐T3‐1‐003).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Chemical tools for engineering glycan interactions at the stem cell‐matrix interface to promote germ layer specification
Authors: Godula Kamil 
Published: June 2021

Glycans (also known as carbohydrates, saccharides or, simply, sugars) are among the most intriguing carriers of biological information in living systems. The structures of glycans not only convey the cells' physiological state, but also regulate cellular communication and responses by engaging receptors on neighboring cells and in the extracellular matrix. Despite their structural complexity, individual glycans rarely engage their protein partners with high affinity. Yet, glycans modulate biological processes with exquisite selectivity and specificity. To correctly evaluate glycan interactions and their biological consequences, one needs to look beyond individual glycan structures and consider the entirety of the cell‐surface landscape. There, glycans are presented on protein scaffolds, or are linked directly to membrane lipids, forming a complex, hierarchically organized network with specialized functions, called the glycocalyx. In my presentation, I will describe our recent efforts in this area, placing emphasis on the applications of glycomaterials to manipulate growth factor signaling at the surfaces of embryonic stem cells to promote differentiation into the neruoectodermal and mesodermal germ layer subtypes.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Determination of Transcriptional Changes Induced by an Ovarian Cancer Targeting Peptide
Authors: Weintraub Lindsey , Rudy James , Hum‐Musser Susan , Musser Richard , Soendergaard Mette 
Published: June 2021

The fifth leading cause of cancer‐related deaths among women is ovarian cancer. Current standard treatment involve cytoreductive surgery, as well as platinum and taxane based chemotherapy. While most patients initially respond to therapy, the majority relapses with drug resistant malignancy, and ultimately succumbs to the disease. Bacteriophage (phage) display technology was previously used to discover a 15‐mer peptide (J18) with specific binding to human ovarian adenocarcinoma (SKOV‐3) cells. Based on the ovarian cancer binding properties of J18, the cytotoxic properties of the peptide was investigated using an 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay. In short, 0.25 mg/mL peptide J18, peptide p30‐1 (non‐cytotoxic), or DMSO were incubated with 103 SKOV‐3 cells for 48 h, after which MTT reagent was added. The cell viability was quantified spectrophotometrically at 560 nm. These results showed that J18 significantly (p<0.01) decreased SKOV‐3 cell viability compared to peptide p30‐1 and DMSO. Next, the mechanism behind the loss of cell viability was sought elucidated by investigation of transcriptional changes after addition of peptide J18. Specifically, genes involved in cancer progression (myc, elk‐1, smad4, hspb1, and tp53), and apoptosis (fadd, casp3, and cflar) as well as housekeeping (sdha) was chosen for analysis. In brief, SKOV‐3 cells (105) were incubated with 0.25 mg/mL J18 for 48 h, after which total RNA was extracted using the TRIzol method. Total RNA was used for cDNA synthesis with oligodT primers. Gene specific primers for selected genes were designed using Primer3 software. qPCR reactions were carried out using a SYBR Green Master Mix in optical 96‐well plates on an Applied Biosystems StepOnePlus Real‐Time qPCR System. Sdha was used as the endogenous control to correct for sample variation. The comparative quantification method deltadelta Ct (ΔΔCt) was used, and data were transformed to absolute values with 2‐ΔΔCt to obtain fold changes between treatments. Each treatment consisted of five biological replicates. Data were analyzed with ANOVA then Fisher's LSD at p<0.05. Results showed that expression of myc, elk1, smad4, hspb1, tp53, fadd, casp3, and cflar were not significantly different for peptide J18 compared to DMSO. These results indicate that the loss of cell viability mediated by peptide J18 is not associated with changes to the transcriptional levels of myc, elk‐1, smad4, hspb1, tp53, fadd, casp3, and cflar. Next, microarray analysis will be performed to investigate the changes in gene expression on the human transcriptome.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Discovery of Vascular Endothelial Growth Factor Receptor‐2 (VEGFR‐2) Inhibitors by Ligand‐based Virtual High Throughput Screening
Authors: Chelliah Selvam , Mock Charlotta D. , Mathew Omana P. , Ranganna Kasturi 
Published: June 2021

In recent years, the inhibition of angiogenesis has become a target for anti‐cancer therapeutics. Angiogenesis' role in the cancerous cells is different from that when it is employed in the physiological homeostasis of cells. As the interest of angiogenesis inhibition has grown so has, the interest in the vascular endothelial growth factor receptor‐2 (VEGFR‐2) complex that is highly expressed in many cancers such as breast, kidney. The present study reports the successful application of ligand‐based virtual high throughput screening method for the discovery of novel VEGFR‐2 inhibitors. We generated a ligand query model with pharmacophore features from the reported VEGFR‐2 inhibitors using vROCS tool and performed virtual high throughput screening. Among the 2.4 million lead like molecules of ZINC database screened, nineteen prioritized compounds were purchased from Enamine and Chembridge screening libraries and tested for VEGFR‐2 inhibitory activity using Promega's ADP‐Glo™ kinase assay. Experimental validation led to the discovery of four compounds 3, 7, 10, and 13. Staurosporine, a known kinase inhibitor was used a standard for this assay. At the beginning, all the 19 compounds were tested for VEGFR2 inhibitory activity at 50μM. The results are displayed in figure 1. The compounds 3, 7, 10, and 13 showed more than 50% inhibition are shown in red bars. These compounds were then tested for their inhibitory activity at 10 μM. Figure 2 represents the VEGFR inhibitory activity for the selected compounds at 10 μM. Docking was performed for these compounds and the predicted binding modes guide to explore the possible structural changes to get more potent VEGFR‐2 inhibitors.Support or Funding InformationThis study was made possible, in part, by Molecular Biology research infrastructure support from grant number 2G12MD007605 from the NIMHD/NIH. We thank the Openeye Inc for providing the academic license to use the virtual screening tools.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Expanding the Druggable Proteome: Ligand and Target Discovery by Fragment‐Based Screening in Cells
Authors: Parker Christopher G. 
Published: June 2021

Advances in DNA sequencing and editing technologies have revolutionized our understanding of the genetic basis of human disease. However, many disease‐relevant genes encode proteins that are poorly characterized and/or are considered “undruggable”, hindering our understanding of disease mechanisms and translating this knowledge into new therapies. Chemical probes offer a valuable way to directly interrogate the function and disease‐relevance of proteins and can also serve as valuable leads for drug development, yet most proteins in the human proteome lack small‐molecule ligands that can serve as probes. More generally, the boundaries, if any, on the ligandability, and therefore potential druggability, across the proteome remains poorly understood. In this seminar, I will describe a platform that integrates fragment‐based ligand discovery with quantitative chemical proteomics to map thousands of reversible small molecule‐protein interactions directly in cells. Many of these interactions can be site‐specifically determined and involve proteins that fall outside of traditional druggable classes. We demonstrate that this knowledge can be advanced to furnish fragment‐based ligands that affect protein activity, including inhibitors of PTGR2 and SLC25A20, proteins that play important roles in human metabolism but lacked chemical probes to study their function. Furthermore, we integrated this platform with phenotypic screening to identify ligand‐protein interactions that regulate complex cellular processes. For example, we identified small molecules that promote adipocyte differentiation by engaging the poorly characterized membrane protein PGRMC2. Fragment‐based screening in cells provides an extensive proteome‐wide portrait of native protein ligandability and facilitates the coordinated discovery of bioactive small molecules and their molecular targets.Support or Funding InformationAmerican Cancer SocietyThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
31P NMR Analysis of Divalent Magnesium Ions with ATP, ADP, AMP, and PPi in Various Buffer Systems
Authors: Donels Jordan , Greenwood Brandon , Zea Corbin 
Published: June 2021

The human body would not be able to function without the efficient relationship between the binding of adenine triphosphate (ATP) and magnesium ions. Two equivalents of magnesium ions are required per ATP for enzyme catalysis. The conformational changes that occur upon magnesium ion binding to the phosphate groups is not well understood. The metal ion binding of magnesium to ATP, ADP, AMP, and PPi are explored at various pH's (6.5, 7.0, and 7.7) and various buffers (Bicine, HEPES, and Bis‐Tris) using 31P NMR. The addition of magnesium in 0.5 equivalents to the phosphate caused a chemical shift in the 31P NPR peaks. 31P peaks broaden and decreased in intensity at half equivalence magnesium to ATP and as the magnesium concentration increased, the peaks sharpen and regain their intensity, but never reach original state or chemical shift. ADP, AMP, and PPi follow the same patterns. Chemical shifts due to buffer selection and pH was also seen and greatly impacted 31P chemical shifts.Support or Funding InformationR.W. & Mary Nelson FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.