bluuebunny/crossref_metadata_2025_split huggingface dataset:
Modulating the Effect of Electrostatic Steering for Protein‐Protein Interactions via Circular Permutation
Authors: Chen Xingyu , Gao Meng , Huang Yongqi 
Published: June 2021

Assembly of protein complexes proceeds via nonspecific encounter complexes whose formation is facilitated by long range electrostatic interactions. Mutation of charged residues and change of salt concentration are two strategies widely applied to modulate the effect of electrostatic steering. Furthermore, electrostatic interactions between two proteins may be also influenced by their conformations. Consequently, the electrostatic steering effect during binding of an unfolded protein to its target may be different from that during binding of a folded protein to its target. In this study, we employed sequence circular permutation and molecular dynamics simulations to investigate the influence of protein conformation on the encounter process. We used the well‐studied barnase/barstar complex as a model system and the coarse‐grained structure‐based Gō‐model and Debye‐Hückel function to describe interactions. Binding simulations were performed at two different temperatures such that the WT and permutated barnase were folded at the lower temperature and they were unfolded at the higher temperature. The ensembles of encounter complex and the binding rate constants were analyzed for WT barnase as well as seven permutants. Our results showed that the ensembles of encounter complex of the unfolded barnase were much diverse than those of the folded barnase. We also found that circular permutation modified the structure ensemble of unfolded barnase, resulting in difference among their ensembles of encounter complex and binding rates. Our results suggest that the protein binding process can be engineered by modulating the protein conformation ensemble.Support or Funding InformationThis work was supported by National Natural Science Foundation of China (Grant number: 21603121) and Hubei University of Technology.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of eNOS ser‐600 Phosphorylation: A Link to the Cell Cycle
Authors: Patel Kevin , Lecher Julia , Chrestensen Carol 
Published: June 2021

Nitric oxide (NO) is a gaseous, free‐radical signaling molecule produced by nitric oxide synthases (NOS). NO has a variety of cellular and physiological functions ranging from maintaining cardiovascular homeostasis to neurotransmission. In low concentrations NO is deemed physiologically crucial and in high concentrations NO is understood to be pathophysiological. The role of NO in the cell cycle is less understood, it is likely cell type specific and concentration dependent, in many cases low levels of NO promote proliferation and prevent apoptosis; at high levels NO has been shown to arrest cells and cause apoptosis. Endothelial nitric oxide synthase (eNOS) is one of three isoforms in the NOS family, it is the most regulated of the isoforms. Regulations include protein‐protein interactions and posttranslational modifications. The most commonly studied and well understood posttranslational modification of eNOS is phosphorylation. This work builds on our laboratories discovery of the ser‐600 phosphorylation site in the auto‐inhibitory loop of eNOS. Our work confirms this site is modulated in cells and links it to the cell cycle using immunofluorescence, flow cytometry, western blot analysis, in vitro kinase assays, and in vitro eNOS activity assays.Support or Funding InformationThis work was supported by NIH grant # R15 GM110634‐01A1 to Dr. Carol Chrestensen.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect Of Loading Method On Metabolism Of Foreign Molecules In Intact Cells Of Dictyostelium Discoideum
Authors: Chadha Rahuljeet (RJ) , Kalminskii Grigorii , Tierney Allison J. , Kovarik Michelle L. 
Published: June 2021

Loading foreign molecules inside living cells allows one to monitor cellular metabolism, track various metabolic pathways and alter activities of the cell. However, the effects of loading methods on the fate of these foreign molecules are poorly understood. In this experiment, we examined three promising loading methods including pinocytosis, electroporation and myristoylation to insert a peptide substrate reporter in cells of Dictyostelium discoideum. These methods were optimized to maximize cell viability, and factors such as loading efficiency, loading duration, localization and uniformity were characterized. Capillary electrophoresis was used to determine how each loading method affected the enzyme activity on the peptide substrate reporter. It was observed that these loading methods had strong effects on the stability and degradation of the peptide. Myristoylation was found to be the most effective method at transporting the peptide into cells while electroporation produced the most uniform loading within cells suggesting cytoplasmic loading. The peptide was degraded most rapidly after loading by myristoylation (half‐life = 12 ± 1 min), while pinocytic loading produced slightly slower degradation (half‐life = 19 ± 2 min). Phosphorylation of the peptide was only observed in cells loaded with electroporation. This experiment suggests that the choice of loading method influences the stability and metabolism of foreign molecules in cells. Our future work includes investigating the specificity of different peptide substrate reporters in Dictyostelium lysates.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Primary cilia on LECs play a crucial role in lymphatic vasculature development and remodeling
Authors: Paulson Delayna Marie , Leddy Rebecca , Ward Cody , Latterell Mackenzie , Peterson Chase , Ford Khalil , Ekle Courtney , Pazour Gregory , Fink Darci 
Published: June 2021

Primary cilia are non‐motile organelles which project from the cell surface and function to receive and process molecular signals. We have recently discovered that lymphatic endothelial cells (LECs) display primary cilia in vitro and in vivo in both developmental and inflammation/wound healing contexts. We have induced primary cilia formation in both immortalized mouse and primary adult human LECs by serum starvation protocols and detected them by immunofluorescence microscopy for ciliary markers ADP ribosylation factor like GTPase 13B (ARL13B) and acetylated alpha tubulin. We have used the mouse corneal suture model to create in vivo conditions of inflammation and wound recovery and found that primary cilia incidence both in LECs and globally in cornea is dependent on inflammatory status. Primary cilia assembly and transport of ciliary cargo is governed by a set of protein complexes made up of intraflagellar transport (IFT) proteins. To ascertain the role of intraflagellar transport in lymphatic development, we developed a conditional knockout (KO) mouse model of IFT20. At the gross level, E16.5 IFT20 KO mice embryos show hydrops, polydactyly, and craniofacial abnormalities. These embryos also possess a malformed, red blood cell‐filled lymphatic vessel (LV) phenotype. Using two photon microscopy of whole mount embryonic skin, we have found the IFT20 KO embryos to have higher lymphatic vessel density and larger LV diameter in comparison to negative littermate controls. We have found that the difference in LV diameter is not due to increased LEC proliferation. Differences in dorsal skin lymphatic vessel patterning across the midline have also been identified in the IFT20 KO. We have also confirmed decreased incidence of primary cilia in IFT20 KO embryonic skin using spinning disk confocal microscopy. Ongoing studies in these embryos are designed to characterize differences in smooth muscle actin coverage of collecting LVs, intercellular LEC junction protein expression and organization, and lymphatic valve formation and possible insufficiency. We have also generated a lymphatic‐specific conditional deletion mouse model of IFT20 loss and are working to understand whether the LV phenotypes present in the global IFT20 KO mouse are due to LEC‐intrinsic defects or to secondary effects on lymphatic vasculature due to defects in other cell types following loss of IFT20. The discovery of primary cilia on LECs may fundamentally change what is known about how extracellular signals are processed by lymphatic endothelium to direct development and remodeling of the lymphatic vasculature.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 Novel Interaction between Regulator of G‐protein Signaling (RGS10) and STIM2 Identifies New Mechanism for the Regulation of COX‐2 in Microglia
Authors: Wendimu Menbere Y. , Alqinyah Mohammed , Freeman Paul , Vella Stephen , Moreno Silvia NJ , Hooks Shelley 
Published: June 2021

Regulator of G‐protein Signaling 10 (RGS10) mediates strong anti‐inflammatory and neuroprotective effects. RGS10 is highly enriched in microglia where it suppresses the expression of many proinflammatory genes, including the key inflammatory enzyme cyclooxygenase 2 (COX‐2). The neuroprotective and anti‐inflammatory effects of RGS10 position it as a potential therapeutic target in neurodegenerative diseases, including Parkinson's and Alzheimer's diseases. Our previous work demonstrated that microglial RGS10 strongly suppresses lipopolysaccharide (LPS)‐induced COX‐2 and tumor necrosis factor alpha (TNFα) expression, and this suppression is independent of its canonical G protein‐targeted mechanism. In this study, we further define the G protein‐independent anti‐inflammatory mechanism of RGS10 in microglia, focusing on the regulation of COX‐2. We hypothesized that novel binding partners link RGS10 to LPS‐stimulated inflammatory signaling pathways. Using an unbiased RGS10 co‐immunoprecipitation approach coupled with LC‐MS/MS, we identified the endoplasmic reticulum (ER)‐localized calcium sensor STIM2 as a major RGS10 interacting protein. STIM2 is an essential component of the store‐operated calcium entry (SOCE) machinery, which functions to detect Ca2+ depletion from the ER, and subsequently activate extracellular Ca2+ entry through plasma membrane Orai channels. Orai‐mediated Ca2+ entry is implicated in regulating inflammatory signaling in microglia through activation of the Ca2+‐dependent phosphatase, calcineurin, and its downstream pro‐inflammatory transcription factor target, NFAT. We have validated the RGS10‐STIM2 interaction in microglia and defined the specificity of interaction and its regulation by calcium and G protein signaling. To delineate the role of STIM2 and downstream calcineurin signaling in mediating the anti‐inflammatory effect of RGS10, we determined the effect of STIM2 knockdown and calcineurin inhibition on LPS‐stimulated COX‐2 expression in wildtype and RGS10 knockout microglial cells. Our findings demonstrate that LPS‐stimulated COX‐2 amplification in RGS10 knockout microglial cells is blocked by inhibition of either STIM2 expression or calcineurin activity. Furthermore, the increased inflammatory signaling observed in RGS10 knockout microglia correlates with enhanced SOCE, as measured by Ca2+ imaging using a fluorescent calcium indicator. Collectively, these results suggest that RGS10 strongly suppresses LPS‐stimulated inflammatory signaling in microglia by inhibiting STIM2‐induced calcium entry and the subsequent activation of calcineurin‐dependent inflammatory gene expression.Support or Funding InformationNational Institute of Neurological Disorders and Stroke (NINDS)[Grant NS101161]This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Using CYP3A4 NMR derived structural information to mitigate time‐dependent inhibition due to diltiazem metabolism
Authors: Wilderman Ross , Halpert James 
Published: June 2021

Human CYP3A4 metabolizes a significant fraction of clinically relevant drugs. CYP3A4‐mediated metabolism of the vasodilator diltiazem leads to time‐dependent inhibition (TDI) and clinically relevant drug‐drug interactions. We hypothesized that introduction of steric bulk into the CYP3A4 active site by site‐directed mutagenesis will disrupt active site contacts, alter diltiazem orientation in the CYP3A4 active site, and decrease TDI, as previously seen with midazolam. A model of diltiazem in the CYP3A4 active site was produced using distance restraints generated from longitudinal T1 NMR relaxation experiments. CYP3A4‐ligand interactions were also monitored using spectral binding titrations. Evaluation of diltiazem‐mediated CYP3A4 TDI was performed using the reporter substrate 7‐ethoxy‐4‐(trifluoromethyl)coumarin. The diltiazem model revealed that the dimethylamino group that undergoes CYP3A4‐mediated N‐demethylation is oriented toward the heme iron with the aromatic ring of the benzothiazepine core near active site residues I369 and L373, the ester moiety near L216, and the p‐hydroxyphenyl group near S119. Diltiazem caused a Type II spin shift upon binding to CYP3A4, indicating interaction of the amine with the heme iron. Substitution of I369 with a larger residue decreased CYP3A4 TDI, and an NMR derived model of diltiazem in complex with CYP3A4 I369W revealed that the p‐hydroxyphenyl group was toward the heme iron instead of the dimethylamino group. In summary, a hybrid NMR‐computational approach was used to rapidly generate a model of diltiazem in complex with CYP3A4. Introduction of steric bulk into the CYP3A4 active site by mutagenesis altered the orientation of diltiazem and mitigated TDI. The next step is to add steric bulk to the cognate part of the compound. These results indicate that NMR‐derived models of protein‐ligand interactions provide a structure‐based approach for decreasing metabolic liabilities of CYP enzyme substrates.Support or Funding InformationThis work was supported by internal funding from the University of Connecticut to James Halpert.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sphingosine‐1‐phosphate Enhances Mitochondrial Integrity and Endothelial Barrier Function Following Hemorrhagic Shock
Authors: Alves Natascha Guimaraes , Yuan Sarah , Breslin Jerome 
Published: June 2021

Excessive microvascular permeability is a serious complication involved in traumatic injury and inflammatory diseases. Sphingosine‐1‐phosphate (S1P) has known endothelial barrier‐protective properties, and has been shown to ameliorate microvascular leakage in a model of combined alcohol intoxication and hemorrhagic shock and resuscitation (HSR). However, the mechanisms of S1P‐induced barrier protection during HSR are unknown. In the current study, we tested the hypothesis that S1P could enhance endothelial barrier function during hemorrhagic shock by preserving the integrity of junction proteins and the endothelial glycocalyx, and protecting mitochondrial function. We used an established in vivo rat model of conscious HSR and assessed microvascular leakage, endothelial glycocalyx integrity, and mitochondrial function by intravital microscopy. Assessment of adherens junction protein localization was done by immunofluorescence confocal microscopy after HSR. Cultured rat intestinal microvascular endothelial cell (RIMEC) monolayers were used to test the ability of S1P to protect against glycocalyx shedding and endothelial barrier dysfunction caused by direct disruption of mitochondrial integrity due to inhibition of mitochondrial complex III. Our in vivo results show that S1P protects against HSR‐induced hyperpermeability, preserves the expression of adherens junctional proteins, and protects against glycocalyx degradation. S1P treatment during HSR also protects against mitochondrial membrane depolarization. Besides that, S1P protects RIMECs against mitochondrial dysfunction‐induced endothelial barrier dysfunction and glycocalyx degradation by blocking complex III inhibition. Our results show that S1P protects against HSR‐induced mitochondrial dysfunction in endothelial cells, which in turn improves the structure of the endothelial glycocalyx after HSR and allows for better junctional integrity, preventing excess microvascular permeability.Support or Funding InformationSupported by NIH grants R01HL098215, R01GM120774, R01HL070752 and R01GM097270.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
TGFβ1 Regulates KIM‐1 and Caveolin‐1 Expression through the Smad3 and ERK1/2 Signaling Pathways in Renal Cancer Cells
Authors: Zhang Yuanyuan , Chen Xiaoming , Zhao Xueying 
Published: June 2021

Kidney injury molecule‐1 (KIM‐1) is a type I transmembrane glycoprotein that is rapidly upregulated at the gene and protein level following various types of injury to the kidney. KIM‐1 expression is increased not only in acute and chronic kidney injury diseases but also in renal cell carcinoma. It may play an important role in carcinoma process. Caveolin‐1, the principal component of caveolae, is regarded as an important inhibitor of multiple signaling molecules. The cytokine transforming growth factor‐β1 (TGF‐β1) has been extensively studied in tumor biology and is believed to serve a variety of functions in tumor progression. In this study, we investigated the regulatory mechanism of TGF‐β1 on KIM‐1 and caveolin‐1 expression in 769‐P cells, a human renal cell adenocarcinoma cell line. Western blot analysis indicated that TGF‐β1 treatment resulted in a downregulation of KIM‐1 and an upregulation of caveolin‐1 in 769‐P cells. Specific knockdown of Smad3 with siRNAs significantly increased basal KIM‐1 expression and attenuated TGF‐β1‐induced KIM‐1 down regulation, which was associated with increased activation of extracellular signal‐regulated kinase (ERK) 1/2 and signal transducer and activator of transcription 3 (STAT3) signaling pathways. Interestingly, pre‐treatment of 769‐P cells with ERK inhibitor U0126 abolished TGF‐β1‐induced caveolin‐1 upregulation and partially restored KIM‐1 protein level. We also observed a significant decrease in KIM‐1 in 769‐P cells administered with caveolin‐1 scaffolding domain peptides. Taken together, our data demonstrate that TGF‐β1 may differentially regulate KIM‐1 and caveolin‐1 expression in renal cancer cells via both Smad3 and ERK1/2 signaling pathways.Support or Funding InformationNIH SC1DK112151; NIH/NCRR/RCMI 8G12MD007602 & 8U54MD007588This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pretreatment With Water Kefir Promotes A Decrease In Ulcer Development In An Ethanol‐Acidified Ulcer Model
Authors: Brasil Girlandia Alexandre , Andrade Moraes Flavia S. , Prucoli Falsoni Raiana M , Resende Mahira Sabino , Andrade Tadeu Uggere , Lima Ewelyne Miranda 
Published: June 2021

Gastric ulcer are one of the most prevalent diseases in the world, the treatment are expensive and produce many side effects, so the research of new therapies are valuable. To evaluate the gastroprotective and antioxidant effects of pretreatment with water kefir (WK) on ulcer induction with HCL/ethanol. All pretreatments were maintained for 14 days. Male mice (C57BL/J6) were separated into five groups: the control (C) group received vehicle without ulcer induction; the ulcerated (U) group received vehicle; the lansoprazole (L) group received 30 mg/kg/day lansoprazole; the water kefir (WK15 and WK30) groups received WK at a dose of 0.15 or 0.30 ml/kg/day, respectively. Gastroprotection was measured by ulcer area, ulcer index and ulcer reduction percentage. The antioxidant effects were quantified by measuring advanced oxidized protein products (AOPPs), superoxide dismutase (SOD), and catalase activity in the stomach.Pretreatment with WK at both doses exerted gastroprotection against HCL/ethanol ulcer induced similar to pretreatment with lansoprazole (ulcer area: C: 14477±6204; U: 127578±33260; L: 39958±13921; K15: 79357±23814; K30: 63252±22435 μm2. p<0.05). In addition, WK promoted decreased protein oxidation (C: 0.018±0.004; U: 0.036±0.004; L: 0.021±0.004; WK15: 0.014±0.006; WK30: 0.023±0.003 μM of chloramine T/mg of protein) and increased SOD activity (C: 21.75±8.16, U: 2.91±1.63, L: 27.27±2.97, WK15: 28.79±4.84, and WK30: 28.25±8.69 USOD/mg protein) and catalase activity (C: 5.73±1.63, U: 0.78±0.23, L: 4.29±1.21, WK15: 4.28±0.74, and WK30: 3.56±1.23 ΔEmin/mg protein−1).We conclude that pretreatment with water kefir for 14 days promotes increased antioxidant enzyme activity, preventing gastric lesions against HCl/ethanol ulcer model by maintaining antioxidant performance in gastric tissue.Support or Funding InformationThis work was partially financed by FAPES (#72662319) and UVV.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.