bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of a newly identified polymorphic variant (M71V) of ABCG2 multidrug transporter
Authors: Homolya Laszlo , Bartos Zsuzsa , Zambo Boglarka , Mozner Orsolya , Sarkadi Balazs 
Published: June 2021

The ABCG2 is a membrane transporter protein, which expels endo‐ and xenobiotics from the cells. Since its overexpression in tumor cells can confer resistance to multiple drugs, ABCG2 is also known as a multidrug transporter. Uric acid is one of the relevant transported physiological substrates of ABCG2, thus, dysfunction of this transporter can lead to hyperuricaemia or gout. In a previous work, using expression level screening in red blood cells from hyperuricaemic patients and normal subjects, a new polymorphic variant (M71V) of ABCG2 has been identified. Our recent study focuses on the cell biological characterization of the ABCG2‐M71V variant, investigating its expression, transport properties, localization, and cellular trafficking in comparison with that of the wild type and the gout‐associated Q141K ABCG2 variants. In addition to classical biochemical approaches, we applied advanced cell biological method to follow the dynamics of cellular routing of the ABCG2 variants. Our result demonstrated that ABCG2‐M71V has a trafficking defect, exhibits predominant intracellular localization and shows lower expression level, but its transport function remained mainly preserved. Treatment of the cells with pharmacological chaperons, such as 4‐phenyl butyrate and colchicine, can enhance the delivery of this ABCG2 variant to the cell surface. In summary, our results imply that the M71V substitution in the ABCG2 transporter may result in an unstable protein, which gets partially degraded; however, rescuing this ABCG2 variant from degradation by pharmacological chaperons can recover the impaired functionality caused by this polymorphism.Support or Funding InformationThis research has been supported by the National Research, Development and Innovation Fund (Hungary) (grant numbers: OTKA_K 115375 to B.S., OTKA_K 128123 to L.H., and FIEK_16‐1‐2016‐0005 to L.H. and B.S.), as well as by the Momentum Program of the Hungarian Academy of Sciences (LP2012‐025).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hydrogen Sulfide (H2S) Limits Amyloid Development in Hen Egg White Lysozyme (HEWL) as a Function of Concentration
Authors: Ojeda Indra Gonzalez , Ruiz Tatiana Quiñones , Alomar Manuel Rosario , Lednev Igor K , Garriga Juan López 
Published: June 2021

Amyloid fibrils are conformations of misfolded proteins with a stable beta‐sheet structure. These structures aggregate in various parts of the human body and disrupt the healthy functioning of vital organs, causing diseases known as amyloidosis. Lysozyme is a bacteriolytic enzyme that is synthesized in macrophages, gastrointestinal cells, and hepatocytes and it is commonly found in human saliva and tears; when this protein misfolds, its amyloidosis can have devastating effects on the health, causing sicca syndrome (dry eyes and mouth), rheumatoid arthritis and even renal failure. Here, it is shown that by adding hydrogen sulfide (H2S) to hen egg white lysozyme (HEWL) the formation of amyloid fibers is inhibited. This inhibition results in small spherical aggregates of unordered protein that exhibit almost no cytotoxicity. This effect was further investigated by exposing HEWL to various concentrations of H2S and exploring the protein's conformational changes. This was done through atomic force microscopy (AFM), non‐resonance Raman spectroscopy and circular dichroism (CD). The results show that when H2S was present, the protein did not undergo fibrillation and instead kept a secondary structure alike its native structure, with this effect increasing as a function of H2S concentration. Additionally, it is seen that HEWL does not form fibers and instead organizes itself in spherical aggregates and that these new aggregates have a higher abundance of trisulfide bonds (S‐S‐S). This fact provides valuable information about the mechanism through which H2S might inhibit the pathogenic fibrillation. Further studies are being conducted to unravel the exact mechanism through which hydrogen sulfide prevents the aggregation of lysozyme; However, the effects observed open the door to a possible preventive therapy for deadly amyloidosis disorders.Support or Funding InformationMARC‐U‐STAR Program, UPR‐MayaguezNIH Grant number: 2T34GM008419‐26RISE‐E‐BASE Program, UPR‐MayaguezNIH Grant number: 1R25GM127191‐01This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural Basis for Improved Proteolytic Stability and Target Affinity of Disulfide engineered Human Kunitz‐type Serine Protease Inhibitors
Authors: Coban Matt , Cohen Itay , Shahar Anat , Sankaran Banumathi , Hockla Alexandra , Caulfield Thomas R. , Radisky Evette S. , Papo Niv 
Published: June 2021

Serine protease inhibitors of the Kunitz‐bovine pancreatic trypsin inhibitor (BPTI) family, such as tissue factor pathway inhibitor‐1 Kunitz domain 1 (KD1TFPI1) and amyloid precursor protein inhibitor (APPI), are biological regulators of proteolysis and serve as important scaffolds for developing therapeutics. Mesotrypsin is a unique human protease with cancer‐promoting properties which rapidly cleaves and inactivates serine protease inhibitors; because of this, it is a challenging therapeutic target and an excellent model for developing therapeutic inhibitors with improved proteolytic resistance. We hypothesize that stabilization of the Kunitz inhibitor binding loop by disulfide bond anchoring to the scaffold can reduce proteolytic susceptibility and improve binding efficacy. Here we have engineered a new disulfide bond into both KD1TFPI1 and an APPI variant previously engineered for mesotrypsin affinity. The new disulfide bond anchors the binding loop to the scaffold, resulting in reduced mesotrypsin proteolytic cleavage by 17‐fold and 3‐fold, respectively. To gain insight into the structural basis for improved proteolytic stability, we crystallized mutant KD1TFPI1 and APPI with mesotrypsin and solved these novel structures. We then performed all‐atom molecular dynamics simulations using these mesotrypsin‐mutant inhibitor complexes compared to models lacking the engineered disulfide bond. Our simulation results support our hypothesis that the cause of decreased proteolysis rates is dampened conformational fluctuations in the binding loop and minimized motion at the enzyme‐inhibitor interface. We also performed simulations using unbound inhibitors to show that stabilization of the binding loop to states similar to the bound configuration correlates with stronger binding affinity. Our disulfide engineering approach is potentially a useful tool in biopharmaceutical development for mesotrypsin, other serine proteases, and possibly for other enzyme targets.Support or Funding InformationNIH grant R01CA154387 to E.S.R., European Research Council grant 336041 to N.P., US‐Israel Binational Science Foundation grant BSF2015134 to E.S.R. and N.P. Xray diffraction data were measured at the Berkeley Center for Structural Biology at the Advanced Light Source and at the European Synchrotron Radiation Facility.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Improved Purification of Apo and Holo Riboflavin Binding Protein from Chicken and Turkey Egg Yolk
Authors: Doctrove Quinlan Arnold , Alsafi Zahra , Smith Sheila Rose , Benore Marilee Ann 
Published: June 2021

Riboflavin is a necessary vitamin required for metabolic processes, and is a member of the B vitamin family (B2). This vitamin is transported throughout tissues via Riboflavin Binding Proteins (RBP). In oviparous species, such as chickens and turkeys, these RBP deliver Riboflavin to the developing egg white and egg yolk. The protein has also been identified in other species, including mammals. Egg RBP is a glyco‐phospho‐protein, structurally stable to temperature and pH variation, and is structurally intriguing.Purification of the protein from egg yolk is challenging due to the high concentration of fats. We improved a traditional purification method using chicken eggs and a novel species of turkey egg using a modified purification protocol.Support or Funding InformationUniversity of MI Dearborn campus grantsThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Monothiol Glutaredoxins Grx3/4 and the BolA Protein Bol2 Modulate Iron Sensing and Regulation in Yeast S. cerevisiae
Authors: Albetel Angela‐Nadia , Li Haoran , Outten Caryn E 
Published: June 2021

An elaborate cascade of iron‐sulfur cluster‐dependent cellular interactions are employed in the yeast S. cerevisiae in order to maintain adequate iron levels. Currently available data indicate that under iron deplete conditions, the two paralogous transcription factors, Aft1 and Aft2, are primarily localized in the nucleus, and activate the transcription of iron uptake and transport genes. Under iron replete conditions, Aft1/2 undergo conformational changes and nucleocytoplasmic shuttling upon interacting with CGFS monothiol glutaredoxins Grx3 and Grx4 and the BolA protein Bol2, resulting in subsequent deactivation of the iron regulon. Iron‐sulfur clusters have been chosen by nature as signaling molecules to accomplish the inhibition of Aft2 (and presumably Aft1), as the dimerization occurs via direct ligation of a [2Fe‐2S] cluster acquired from the [2Fe‐2S] cluster‐bridged Grx3/4‐Bol2 heterodimer. However, the mechanistic details of iron regulation at the molecular level in the baker's yeast are not fully understood, and defining the functional interactions of each component in the iron signaling pathway remains to be elucidated. We are currently using complementary biophysical and molecular genetic methods to probe the iron‐sulfur cluster‐dependent interaction between [2Fe‐2S]2+‐Grx3/4 and Bol2, and [2Fe‐2S]2+‐Grx3/4‐Bol2 and Aft2 to gain a better understanding of their in vivo functions. The results indicate rapid interaction between [2Fe‐2S]2+‐Grx3 and Bol2, followed by a fast iron‐sulfur cluster transfer to Aft2, with second order rates above previously reported rates for reactions involving iron‐sulfur clusters. Furthermore, we are showing that mutations in the amino acid residues ligating the iron‐sulfur cluster in the [2Fe‐2S]2+‐Grx3‐Bol2 heterodimer, or in the CxC iron‐sulfur cluster binding motif of Aft2 have a significant impact on the rate of interaction and cluster transfer, respectively. Since several key proteins in this pathway are conserved in humans and essential for viability, exploiting the yeast system to define their functional and physical interactions will provide a fundamental understanding of their roles in human iron metabolism.Support or Funding InformationNational Institute of General Medical Sciences R01 (Grant GM100069 to CEO)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 Putative Kinases with a Solved Structure but Unknown Function from the Protein Data Bank
Authors: Dollen Julia C. , Duplan Amanda , Hall Bonnie L. 
Published: June 2021

The protein databank (PDB) contains 146,000 structures, with approximately 4300 of those currently classified as having “unknown function”. Of those, about 2500 are expected to be enzymes, based on structures with known function(s) in the PDB. The Biochemistry Authentic Scientific Inquiry Lab (BASIL) project has defined a series of in silico and in vitro modules that allow prediction and confirmation of function(s) for these proteins. To date, BASIL has focused primarily on putative hydrolases, especially the NUDIX hydrolases. We are adapting the BASIL modules for use with a novel category of proteins, putative kinases (EC 2.7.X.X). Kinases were selected because of their abundance in humans (over 500 members of the kinase superfamily) and their involvement in almost every aspect of cellular function. Utilizing ProMol and the Mechanism and Catalytic Site Atlas (M‐CSA), we are defining units and test motifs for identification of putative kinases with unknown function within the PDB. These putative kinases are being further characterized utilizing a variety of in silico tools, including BLAST, Pfam, and Dali. In parallel, we are optimizing bacterial protein overexpression of the kinase(s), affinity chromatography purification of the protein(s), and the in vitro kinase assays. This should allow experimental confirmation of the kinase function(s) identified computationally. To support expansion of these modules to concurrent analyses by multiple students, we are designing reliable controls and a consistent workflow for the computational and in vitro analyses of kinase function. Overall, adaptation of the BASIL modules is allowing us to design a method for student characterization of multiple putative kinases simultaneously.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mapping regulatory interactions in Proline‐rich tyrosine kinase 2 by cross‐linking
Authors: McClure Natalie A. , Loving Hanna S. , Underbakke Eric S. 
Published: June 2021

Pyk2 is a non‐receptor tyrosine kinase enriched at the post‐synaptic density of glutamatergic synapses. Activated by Ca2+influx, Pyk2 participates in the translation of synaptic activity into signaling cascades that tune synaptic strength. Pyk2 is a multi‐domain protein kinase with a regulatory FERM domain. Structures have been reported for isolated Pyk2 domains, yet the architectures of the auto‐inhibited Pyk2 and Pyk2 activation complex remain unclear. We employed a cysteine cross‐linking strategy to define the inter‐domain interactions responsible for kinase regulation. Cysteine residues were engineered at putative interfaces in both kinase and FERM domains. Rates of cysteine disulfide bond formation were measured using mass spectrometry (MS). Ultimately, cross‐linking results were integrated with hydrogen/deuterium exchange MS to develop a model of the auto‐inhibited architecture.Support or Funding InformationNSF MCB grant 1715411; Linder Family Undergraduate Research FellowshipThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structures and gating mechanism of human TRPM2
Authors: Wang Longfei , Fu Tianmin , Xia Shiyu , Wu Hao 
Published: June 2021

Transient receptor potential (TRP) melastatin 2 (TRPM2) is a cation channel associated with numerous diseases. We determined the cryo‐EM structures of human TRPM2, alone, with ADPR, and with ADPR and Ca2+. The C‐terminal NUDT9H, in contrast to previously proposed felixible linking, forms both intra‐ and inter‐subunit interactions with the N‐terminal TRPM homology region (MHR1/2/3) in the apo state, but undergoes conformational changes upon ADPR binding, resulting in rotation of MHR1/2 and disruption of the inter‐subunit interaction. Ca2+ binding further engages transmembrane helices and the conserved TRP helix to cause conformational changes at the MHR arm and the lower gating pore to potentiate channel opening. These findings explain the molecular mechanism of concerted TRPM2 gating by ADPR and Ca2+and provide insights into the gating mechanism of other TRP channels.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Repression of COUP‐TFII by proinflammatory cytokines contributes to endometriotic lymphangiogenesis
Authors: Li Wan‐Ning , Hsiao Kuei‐Yang , Wang Chu‐An , Chang Ning , Wu Meng‐Hsing , Tsai Shaw‐Jenq Sean 
Published: June 2021

Endometriosis is a common gynecological disease that affects 8–10% women of reproductive age. It is characterized as the presence of endometriotic lesions outside the uterine cavity and causes severe symptoms in patients such as pelvic pains, dyspareunia, and even infertility. Greater angiogenic and lymphangiogenic processes have been found in ectopic lesions. However, the underlying mechanism is largely unknown. In the present study, we found VEGF‐C is highly secreted by endometriotic stromal cells. Bioinformatic and molecular characterization reveals that elevation of VEGF‐C in endometriotic stromal cells is mediated by derepression of chicken ovalbumin upstream promoter‐transcription factor II (COUP‐TFII) dependent transcriptional regulation. Further investigation demonstrates that level of COUP‐TFII is suppressed by proinflammatory cytokines such as interleukin‐1β and tumor necrosis factor‐α. More importantly, we show, for the first time, that functional VEGF‐C can be transported by exosomes, a 30–100 nm‐size extracellular vesicles derived from the endosomal compartments, to enhance the tube formation abilities of lymphatic endothelial cells. Blockage of VEGF‐C signaling by a highly selective inhibitor of VEGFR‐2/3, Lenvatinib, abolishes loss of COUP‐TFII‐mediated lymphangiogenesis in endometriosis both in in vitro and in vivo models. Herein, we unveil the novel mechanism of VEGF‐C transportation by extracellular vesicles to communicate between endometriotic cells and lymphatic endothelial cells and demonstrate the pathophysiological process of VEGF‐C in the progression of endometriosis.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Probiotic Effects of Yeasts on Virulence of Candida Species
Authors: Kunyeit Lohith , Rao Reeta P , Anu‐Appaiah KA 
Published: June 2021

Antifungal resistance and life‐threatening systemic infections of Candida species pose a major therapeutic challenge. As an alternative approach to combat this wild spread opportunistic fungal infection, we are investigating the probiotic effects of two yeasts Saccharomyces cerevisiae (strain KTP) an Issatchenkia occidentalis (strain ApC) against pathogenic Candida species. These yeasts were isolated from toddy and apple juice respectively. Their ability to prevent virulence traits such as adhesion, morphological transition and biofilm formation were studied. In this study, we focused on Candida albicans, Candida tropicalis, Candida krusei, Candida glabrata and Candida parapsilosis as test organisms. Probiotic effects were assessed using in vitro (simulated gastrointestinal conditions), ex vivo (Caco‐2 cell line) and in vivo (Caenorhabditis elegans) models. The results indicate that yeasts S. cerevisiae and I. occidentalis exhibited higher tolerance to simulated gastrointestinal juices as compared to Saccharomyces cerevisiae var. boulardii, a commercialised probiotic yeast. In the presence of S. cerevisiae and I. occidentalis, pathogenic fungi of the Candida species showed decreased virulence traits of surface adhesion and filamentation as compared to probiotic control strain S. boulardii. Finally, in vivo studies using C. elegans infection assay suggests that the novel probiotic yeasts protect the animal during infection. Together these results reveal the potential biotherapeutic potential of these novel yeasts against Candida infections.Support or Funding InformationLK received a fellowship under the program DST‐INSPIRE, Govt. of India and Fulbright‐Nehru doctoral program, USIEF, India. This work is partially supported by NIH‐NCCIH 1R15AT009926‐01 grant to RPR.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ethanol Dose‐Dependently Changes 5HT1A Receptor Trafficking in Neuroblastoma 2A Cells
Authors: Adams Kristen L , Luessen Deborah J , Sun Haiguo , McCool Brian A , Chen Rong 
Published: June 2021

IntroductionEthanol exposure elicits robust changes in the trafficking and function of many neurotransmitter G protein‐coupled receptors (GPCRs). However, the molecular mechanisms remain elusive. Using serotonin 5‐HT1A receptors (5‐HT1ARs) as a model system, the goal of this study was to examine the effect and mechanism of acute ethanol exposure on constitutive 5‐HT1AR internalization and recycling. The Rab family of proteins has been implicated as critical modulators of endosomal trafficking involved in receptor internalization and recycling. More specifically, Rab5 is implicated in GPCR internalization and commonly used as a protein marker for internalization into the early endosomes whereas Rab11 is associated with GPCR recycling. Thus, the second goal was to investigate the molecular mechanism underlying ethanol‐induced alterations in constitutive 5‐HT1AR internalization and recycling by determining the endocytic pathway of 5‐HT1AR.MethodsIn the present study, we used a neuroblastoma 2a cell line stably expressing 5‐HT1ARs (N2A‐5HT1ARs). Cells were cultured in growth medium containing ethanol (15–75 mM) for 18 hrs prior to experiments. Immunocytochemistry and confocal microscopy were utilized to examine 5‐HT1AR surface expression, constitutive 5‐HT1AR internalization and recycling, and co‐localization of 5‐HT1AR with Rab proteins. Western blotting was carried out to measure HA‐tagged 5‐HT1AR, Rab5 and Rab11 protein levels in ethanol‐ and vehicle‐treated cells.ResultsWe found that N2A‐5HT1AR cells treated with 30 or 75 mM of ethanol for 18 hrs significantly reduced 5‐HT1AR surface expression compared to vehicle treatment without altering 5‐HT1AR total protein levels in N2A‐5HT1AR cells. To determine the mechanism underlying reduced surface 5‐HT1AR expression, the effect of acute ethanol exposure on constitutive 5‐HT1AR internalization and recycling was determined. Additionally, the endocytic fate of 5‐HT1AR in Rab5 or Rab11‐positive endosomes were assessed to understand the molecular mechanisms governing ethanol‐induced alteration in trafficking of 5‐HT1AR.This study demonstrates for the first time that acute ethanol exposure alters the constitutive trafficking of 5‐HT1ARs which may be mediated by Rab proteins. Alcohol‐associated alterations in GPCR trafficking may contribute to alcohol tolerance and dependence.Support or Funding InformationThis work is supported by NIH R01DA042862, P50DA006634, F31AA025532This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Antiproliferative Natural Product Superstolide/ZJ‐101 Affects Cellular Adhesion in 3D Spheroid Model
Authors: Sanchez Phillip R , Head Sarah A , Jin Zhendong , Liu Jun O 
Published: June 2021

The natural product known as Superstolide/ZJ‐101 has anti‐proliferative activity across a multitude of human cancer cell lines with an IC50 in the range of 30–100 nanomolar. Structure activity relationships have improved this activity to the single‐digit nanomolar range in 2D viability assays. Additionally, phenotypic assays have identified a strong cytostatic rather than cytotoxic effect. Recently ZJ‐101 has been tested in a 3D spheroid model of triple‐negative breast cancer to reveal a novel phenotype of cellular adhesion disruption. This effect has also been observed in 2D culture, indicating that both cell‐cell and cell‐substrate adhesion are similarly affected. Taken together, the anti‐adhesion phenotype could suggest a new type of cytostatic therapeutic which dissolves intractable tumors and prevents their migration, invasion and metastasis.Support or Funding InformationNIH T32 Pharmacological Sciences Training GrantThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.