bluuebunny/crossref_metadata_2025_split huggingface dataset:
Examining Fmoc‐amino acid derivatives as selective inhibitors of butyrylcholinesterase
Authors: Ramirez Jennifer , Gonzalez Jeannette , Schwans Jason 
Published: June 2021

The design of enzyme inhibitors to act as drugs or as substrates for biocatalysts remains a challenge in biochemistry, pharmacology, and biotechnology. To mitigate the effects of Alzheimer's disease (AD), a neurodegenerative disease characterized by progressive memory loss and the sixth leading cause of death in the United States, numerous enzyme inhibitors have been developed. Activity of the enzyme butyrylcholinesterase (BChE) increases in Alzheimer's patients, and this increase is suggested to lead to a depletion of the neurotransmitter, acetylcholine. To counter this increase in activity BChE‐specific inhibitors have been developed to manage the progression of AD. We hypothesized 9‐fluorenylmethyloxycarbonyl (Fmoc) amino acids could provide a new class of inhibitors, as these compounds features found in BChE inhibitors currently available as pharmaceuticals. The readily available chemistry makes amino acids an attractive scaffold for identifying BChE inhibitors. We found Fmoc‐Leu‐O− as an effective and selective BChE inhibitor with an inhibition constant (KI) value of 115 μM. A series of Fmoc‐Leu‐O− derivatives with side chain modifications were then evaluated. The Fmoc‐amino acids were purchased or synthesized from the corresponding amino acid. The KI value for each compound was determined using kinetics assays and UV‐absorbance spectroscopy. For the four Fmoc‐Leu‐O− derivatives tested, Fmoc‐neopentylglycine‐O− showed the greatest potency with an inhibition constant of 36 μM, ~three‐fold lower than the original compounds. A series of Fmocdi‐, tri‐, tetra‐, and pentapeptides were synthesized by solid phase peptide synthesis. The products were purified by HPLC and characterized by NMR and mass spectrometry. Our initial results identified the dipeptide (Fmoc‐Leu‐Leu‐O−) as an effective inhibitor with KI value of 28 μM. We are currently investigating if longer Fmoc‐Leu‐O− peptides and other Fmoc‐peptides are potent and selective BChE inhibitors. The results explore a new class of potent and selective BChE inhibitors and may identify features important for binding in the enzyme active site.Support or Funding InformationThis project was supported by the National Institute of General Medical Sciences, National Institutes of Health (NIH) under award numbers T34GM008074, UL1GM118979, and RL5GM118978, and the National Science Foundation (NSF; MRI CHE‐1337559). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH or NSF.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Small Molecule Allosteric Modulation of Protein Tyrosine Kinases in Live Cells
Authors: Lasansky David , Bienick Matthew , Ghosh Indraneel 
Published: June 2021

The protein kinase and phosphatase enzymes within a cell catalyze the addition or removal of a phosphate group onto a protein respectively, and previous research has shown their involvement in nearly all cell‐signaling pathways. A consequence of this is that the disruption of the function of a particular kinase or phosphatase may lead to the development of diseases such as cancer. A major goal of chemical biology is to develop methods that allow for a detailed understanding of cell‐signaling pathways. However, current methods for studying the roles of these enzymes in signaling such as CRISPR‐Cas9 and siRNA for gene knockout suffer from potential compensatory mechanisms in the cell. Our research has led to the development of a potentially general approach for controlling enzyme activity via insertion of small peptide chains (BH3 helices) which bind selectively to a protein partner (Bcl‐xL) (Figure 1) for allosteric inhibition of kinase activity. Kinase activity can then be modulated by the addition of a specific small molecule that disrupts BH3/Bcl‐xL interactions thus providing specific control over turning on a single type of protein kinase within a cell. This approach is distinct compared to an approach by Professor Shokat that allows turning off activity of individual protein kinases in cells. We have shown success with this model in live cells when applied to the tyrosine‐protein kinase c‐Src, and our current project focuses on applying the model to other homologous tyrosine‐protein kinases. If successful, this research will provide greater insight into the role of specific kinases in cell‐signaling pathways, and thus allow for the development of genetic and small molecule therapeutics targeted towards dysfunctional kinases.Support or Funding InformationThis work was supported by the National Institutes of Health [1R01GM115595‐01] and the National Science Foundation [CHE‐1506091].This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Engineering Starch Kinases For Increased Biofuel Production Efficiency
Authors: Rondon Alberto , Emanuelle Shane 
Published: June 2021

The growth of the bioethanol industry over the last decade has been a result of the growing demand for gasoline fuels, which in the United States constitutes at least 10% by volume, and the implementation of higher Renewable Fuel Standards (RFS). Bioethanol is produced by the fermentation of simple sugars by yeasts and as a result starch feedstocks must undergo pretreatment steps. The various mechanical, thermal and chemical treatments used to degrade starch are both expensive and energy intensive. Alternatively, plants use a reversible phosphorylation mechanism to solubilize starch and access stored glucose for nocturnal metabolism. Starch is formed by linear α‐1,4 linked glucose units and branching glucose via α‐1,6 bonds which form insoluble helical structures. Starch kinases phosphorylate the crystalline layers, disrupting the helical structures, and exposing the polymer to hydrolysis by amylases. This mechanism could be adopted during biofuels production to decrease the need for solubilizing treatments. Starch kinases are characterized by two distinct domains: carbohydrate binding modules (CBMs) and a dikinase domain connected via a peptide linker. The CBM functions to locate the kinase on the starch substrate, which is subsequently phosphorylated by the kinase domain. In order to expand the substrate affinity of a kinase, we fused the CBM from a Neurospora crassa starch‐active polysaccharide monooxygenase to multiple dikinase domains. We defined the melting temperature of the chimeric proteins using differential scanning fluorimetry and found that the chimeric proteins had increased stability. Using [33P‐β‐]ATP, we radiolabeled starch with the chimeric proteins and found that they exhibited at least 7‐fold greater activity than wild type dikinase using maize, barley, and potato starch as the substrate.Support or Funding InformationNSF IIA‐1355438 (R. Andrews), NSF MCB‐1252345 (M. Gentry)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Designed Metal‐Mediated Protein Dimerization
Authors: Maniaci Brian J. , Love John J. 
Published: June 2021

The specificity and stability of protein/protein interactions is of vital interest to the pharmaceutical and biotechnological industries. Incorporation of metal‐binding sites at target protein interfaces may be one approach to improve the specificity and affinity of naturally occurring and designed protein‐protein interactions. The main goal for this research was to generate a metal‐mediated protein interface and study the effects of interfacial modification on protein‐protein affinity. Our approach recently resulted in the creation of a high‐affinity, zinc‐mediated protein homodimer.Using a metal‐templated approach we engineered novel metal binding sites to generate a high affinity interaction using the β1 domain of Streptoccocal Protein G (Gβ1). Metal coordination enables focused surface redesign, allowing for the preservation of naturally occurring or designed interfaces. Incorporation of metal binding sites generates concerted interactions allowing for enhanced affinity and specificity for protein interactions. Our protein complex is driven by metal coordination through histidine side‐chains, allowing for a metal‐dependent interaction.Complex formation and assembly was evaluating using size exclusion chromatography and x‐ray crystallography. To accurately measure the molecular weight of the protein variants we have used size‐exclusion chromatography with multi‐angle light scattering (SEC‐MALS). This analytical technique first separates proteins based on size, and the molecular weight is determined from scattered laser light. The Gβ1 monomer and metal‐mediated dimers had molecular weight of 6.2 and 12.2–13.1 kDa, respectively. Crystal structure of the dimeric proteins proved the target design was achieved and that zinc was bound at the designed interface.We have also engineered a library of rationally designed proteins that contain mutations in proximity to the metal binding sites. The goal is to exploit and explore key thermodynamic parameters that are essential for protein complex formation. We measured the binding affinity of the metal‐mediated protein‐protein interactions using a fluorescence polarization competition assay. The measured affinities were stronger than 100 nM, which is both physiologically relevant and provides additional support for the design of high‐affinity metal‐mediated protein‐protein interactions.Support or Funding InformationThis work was supported by the Department of Defense and by the California Metabolic Research Foundation.Brian is a recipient of an Arne N. Wick Pre‐doctoral Research Fellowship from the California Metabolic Research Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Targeting the Myristoylation of FRS2α Inhibits FGF/FGFRs‐mediated Oncogenic Signaling and Tumor Progression
Authors: Cai Houjian , Li Qianjin 
Published: June 2021

Objective of the studyFibroblast growth factor (FGF)/fibroblast growth factor receptor (FGFR) signaling facilitates tumor initiation and progression. Although there are currently approved inhibitors and agents in clinical trials targeting FGFR kinase activity that show therapeutic benefit, over‐expression and/or mutations of FGFRs eventually confer drug‐resistance and bypass the mechanisms of kinase inhibitors in a variety of cancer types. We hypothesize that a compound blocks the myristoylation of FRS2α, thus suppressing its interaction with FGFRs/FGFRsDRM and downstream proteins. As a result, it inhibits oncogenic signaling and tumor progression mediated by FGF/FGFRs or FGFRsDRM. The goal of this study is to investigate if the novel lead compound inhibits FGF/FGFRs oncogenic signaling and overcomes FGFRsDRM mediated drug resistance.Statement of MethodsWe used CRISPR/Cas9 mediated gene knockout to demonstrate that FRS2α is essential for FGF/FGFR signaling. The myristoylation of FRS2α was detected by Click chemistry. Additionally, inhibition of cell proliferation, migration, invasion assays and cell cycle analysis were analyzed in numerous cancer cells. Prostate regeneration assay and xenograft tumor models were applied for in vivo studies.Summary of the resultsWe demonstrate that loss of myristoylation of FRS2α, a scaffold protein key in FGFRs signaling, inhibits FGF/FGFRs‐mediated oncogenic signaling and FGF10‐induced tumorigenesis. Additionally, a previously synthesized myristoyl‐CoA analog, B13, which targets the activity of N‐myristoyltransferases, suppresses FRS2α myristoylation, and modulates the phosphorylation and localization of FRS2α at the cytoplasmic membrane. B13 exhibited an inhibitory effect on oncogenic signaling induced by wild type FGFRs and their drug resistant mutants (FGFRsDRM). The single agent or in collaboration with an FGFR inhibitor suppressed FGF induced wild type FGFR or FGFRDRM‐initiated PI3K and/or MAPK signaling, subsequently inhibiting cell proliferation and migration in a variety of cancer cell types by inducing cell cycle arrest. Finally, B13 significantly inhibited growth of xenograft tumors without pathological toxicity to the liver, kidney, or lung in vivo.ConclusionOur study demonstrates that B13, a myristoyl‐CoA analog, inhibits FGF/FGFR‐mediated oncogenic signaling and tumor progression. This study provides a novel therapeutic approach for inhibiting cancer progression mediated by FGF/FGFRs and their drug resistant mutants.Support or Funding InformationNIH (R01CA172495) and DOD (W81XWH‐15‐1‐0507)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Seasonal Changes in the Central Nervous System of the Arctic Ground Squirrel
Authors: Frare Carla , Lokken Elisabeth , Drew Kelly 
Published: June 2021

Hibernation is a seasonal adaptive strategy characterized by metabolic suppression and a decrease in body temperature (Tb). The Arctic Ground Squirrel (AGS) is an obligatory hibernator; the onset of hibernation is linked to the circannual rhythm even when AGS are housed at constant environmental conditions. This phenomenon suggests an endogenous circannual clock, which mechanism is unknown. We test the hypothesis that sleep‐awake pathways are differentially activated between summer and winter season. Summer season was defined by the lack of torpor bouts; winter season was defined by the presence of periodic torpor bouts. AGS, implanted with body temperature transmitter, were maintained at constant environmental conditions: 2°C and 4L:20D. Euthermic AGS, in winter and summer, were perfused with 4% paraformaldehyde and brains removed for immunohistochemical analysis. Free‐floating immunohistochemistry was used to localize active nuclei as indicated by cFos‐immunoreactivity (mouse anti‐cFos 1:20,000, Millipore). Data analysis was performed in R. Wake active neurons are localized in the Perifornical Nucleus and the Tuberomamillary Nucleus. Both nuclei show higher activation, measured as higher cFos+ count, in summer than winter. A lower wakefulness activity in winter suggests an increase in sleep propensity, supporting that hibernation is characterized by an increase in sleep. In addition the Raphe Pallidus, a region controlling thermoregulation, shows a lower activation in winter compared to summer. This result correlates with the lower euthermic body temperature of AGS in winter compared to summer. In conclusion, winter AGS are characterized by a seasonal decreased in wakefulness and thermogenic nuclei activity; these changes may underlie the mechanisms of the circannual endogenous clock.Support or Funding InformationNIH R03 NS081637 NSF IOS‐1258179 Alaska INBRE P20GM103395 NIH TL4GM118992This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Correlating Enzymes to Antimicrobial Resistance in the Protein Data Bank
Authors: Salcedo Priscilla M. , Burley Stephen 
Published: June 2021

Antibiotic resistant bacteria are predicted to be the leading cause of death worldwide, with an expected death rate of 10 million people, annually, by 2050. It is vital that antimicrobial resistance (AMR) be understood at the molecular level. The Protein Data Bank (PDB) archive holds over 130,000 3D structures of macromolecular machines that can be analyzed using the RCSB PDB website at RCSB.org. Studying these 3D structures enables scientific research and education in a variety of fields, including drug discovery, nanotechnology, green energy, and disease. In this study, PDB enzymes related to antibiotic inhibitors were identified and further analyzed using additional bioinformatics including The Comprehensive Antibiotic Resistance Database (CARD). CARD holds up to date clinically relevant antibiotic related information. CARD identified 4,688 Genbank IDs (protein accession codes) that are related to AMR. These codes were then put into the Uniprot website which holds protein sequences. As a result, 696 macromolecules related to AMR were found in the PDB. I specifically researched Metallo Beta Lactamases (MBL) because have a large correlation to AMR and have been studied in great detail, with many examples in the PDB. Prominent enzymes in this category include the New Delhi Beta Lactamase which can cleave most if not all antibiotics. This particular “superbug” was found to cleave penicillin, cephalosporin, and carbapenem antibiotics. Using 3D visualizations of beta lactamase enzymes a correlation between zinc ions and spectrum of antibiotic resistance was found. For example, if an enzyme had two zinc ions instead of one it could inhibit broad spectrum antibiotics. In conclusion, the findings of this research presented here will be used to develop novel AMR tab at RCSB.org that will provide antimicrobial details of enzymes that were unavailable before. In the near future a phylogenetic tree of AMR related enzymes and classification details will be implemented.Support or Funding InformationRCSB PDB is funded by a grant from the National Science Foundation (DBI‐1338415), the National Institutes of Health, and the US Department of Energy.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Resuscitation with Fresh Whole Blood Improves Long Term Restoration of Muscle Strength following Hemorrhage and Limb Ischemia‐Reperfusion Injury
Authors: Walters Thomas J. , Roe Janet L. , Aurora Amit 
Published: June 2021

BackgroundExtremity vascular injuries are commonplace in battlefield trauma and often result in ischemia‐reperfusion injury (I‐R). Fluid resuscitation following hemorrhage is often required. We compared the impact of resuscitation with colloid, Hextend™ (HEX), and fresh whole blood (FWB) on markers of microvascular damage and long‐term muscle function. Using a rodent model of combined hemorrhage and tourniquet induced I‐R injury, we tested the hypotheses that, compared to resuscitation with HEX, FWB 1) mitigates microvascular damage; and, 2) results in greater return of long‐term muscle strength.MethodsSprague Dawley rats were randomly assigned to HEX or FWB resuscitation groups and two time‐points (2 and 28 days; n = 8/group/time‐point). Anesthetized rats underwent arterial hemorrhage (42.5% blood vol.; 100% lethal without resuscitation) followed by 3 hours of tourniquet application. Subsequently, rats were resuscitated (15mL/kg body wt). At two days post‐injury, muscles of interest were harvested, weighed, and frozen for: histology; analysis for markers of inflammation, growth and angiogenic factors; and gene expression for markers of microvascular damage. Plasma was assayed for inflammatory markers. Muscle edema was calculated (wet:dry weight). At 28 day post‐injury, in vivo contractile properties of the tibialis anterior (TA) muscle were obtained, as well as muscle weight.ResultsIn all animals, the MAP reached a nadir of ~40 mm Hg following hemorrhage. At 2 days, muscle edema was similarly pronounced in both HEX and FWB groups. Expression of Connexin 43 and VE cadherin was significantly higher with FWB than HEX, suggesting microvascular damage with HEX resuscitation (p = 0.04). Corroborating this outcome was the significantly higher tissue level expression of VEGF and angiogenic factors (TCK‐1 and Neuropilin‐2) with FWB compared to HEX (p ≤ 0.05). Compared to HEX, FWB resuscitation showed an increased expression of IFN‐γ and IL‐4 cytokines (p = 0.004). At 28 days post‐injury, in vivo muscle strength (peak tetanic torque) was 16% greater in FWB than in HEX (p = 0.03). This coincided with a 15% greater muscle mass in FWB versus HEX (p=0.07).ConclusionsCompared to HEX, resuscitation with FWB mitigated damage to the microvasculature and improved return of muscle strength. The impact of FWB on mitigating microvascular damage may be a likely reason for the observed difference of muscle strength in the long term. Collectively, these results demonstrate that FWB resuscitation may provide benefits and possibly contribute to improved limb salvage. While FWB did provide a benefit, large improvements in muscle healing will require additional treatment strategies aimed at improving muscle regeneration.Support or Funding InformationU.S. Army Medical Research and Materiel CommandThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of gravidity on grasshopper oxygen delivery
Authors: Kirkton Scott D. , Altman Elizabeth K. , Zembrzuski Deanna 
Published: June 2021

Although the insect tracheal system is extremely efficient, life history changes can reduce oxygen delivery. For example, intermolt growth results in compressed tracheae, lower tracheal volumes, and reduced femoral air sac ventilation rates. During gravidity, the egg mass of Schistocerca americana grasshoppers can reach up to 40% of their body mass. We investigated whether this increase in egg mass compressed tracheae, lowered tracheal volumes, and reduced ventilation rates in the abdomen and femur. Using micro‐dissections and live video analysis, we examined the tracheal system of thirty‐six female grasshoppers. We found that abdominal compression rates correlated with the inflation of proximal and distal femoral air sacs. In addition, abdominal compressibility decreased with gravidity, suggesting lower tidal volumes. However, there was a positive correlation between gravidity and tracheal compression rate. Furthermore, initial micro computed tomography findings also suggest that gravid females have reduced tracheal volumes. These findings suggest that gravid females compensate for reduced tidal and tracheal volumes by increasing tracheal ventilation rates to improve oxygen delivery.Support or Funding InformationThis research was funded in part by NSF award 1531850 to SDK.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.