bluuebunny/crossref_metadata_2025 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 Information

This 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 Information

MARC‐U‐STAR Program, UPR‐Mayaguez

NIH Grant number: 2T34GM008419‐26

RISE‐E‐BASE Program, UPR‐Mayaguez

NIH Grant number: 1R25GM127191‐01

This 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 Information

NIH 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 Information

University of MI Dearborn campus grants

This 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 Information

National 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 Information

NSF MCB grant 1715411; Linder Family Undergraduate Research Fellowship

This 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 Information

LK 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

Introduction

Ethanol 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.

Methods

In 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.

Results

We 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 Information

This work is supported by NIH R01DA042862, P50DA006634, F31AA025532

This 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 Information

NIH T32 Pharmacological Sciences Training Grant

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Omega‐3 polyunsaturated fatty acids protect endothelial adhesion of monocytes through FFA4 in monocytes
Authors: Lee Seung‐Jin , Im Dong‐Soon
Published: June 2021

A G protein‐coupled receptor (GPCR) named free fatty acid receptor 4 (FFA4, also known as GPR120) was found to act as a GPCR for omega‐3 polyunsaturated fatty acids (n−3 PUFA). Recently, FFA4 is known to participate in the progression of atherosclerosis, however, the underlying mechanisms are unclear. Thus, this study investigated the molecular mechanisms responsible for monocyte adhesion to the vascular endothelium, which is a key early event in macrophage foam cell formation. In ex vivo study, LPS‐induced the primary monocyte adhesion to the vascular endothelial cells were significantly attenuated by pretreatment of EPA and DHA, a representative n−3 PUFA. This EPA and DHA‐induced inhibition were blunted by primary monocytes isolated from FFA4 (−/−) mice. In in vitro study, The LPS‐induced monocyte adhesion to the endothelial cells and adhesion molecule, Mac‐1 expression were significantly attenuated by pretreatment of EPA and DHA in monocytes. This EPA and DHA‐induced inhibition was blunted by pretreatment of AH7614 (a FFA4 antagonist) and by transfection of FFA4 siRNA, suggesting a pivotal role of FFA4 on LPS‐induced monocyte adhesion to the endothelial cells and Mac‐1 expression. In addition, in promoter activity analysis and chromatin immunoprecipitation assays to identify transcription factors involved in EPA and DHA inhibited LPS‐induced Mac‐1 expression, both AP‐1 and NF‐κB played central roles to increase Mac‐1 transcription in LPS‐treated monocytes. The results of this study suggest that n‐3 PUFA protect monocyte adhesion to vascular endothelial cells and Mac‐1 expression via signaling pathways involving AP‐1 and NF‐κB by activating FFA4 in monocytes, leading to increased foam cell formation in the development of atherosclerosis.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Resolvin D1 interferes with several of the required steps for angiogenesis
Authors: Fatima Farheen , Garrett Joshua
Published: June 2021

New growth of blood vessels, angiogenesis, is essential in cancer development since the proliferation, as well as metastatic spread, of cancer cells depends on an adequate supply of oxygen and nutrients and the removal of waste products. Endothelial cells, which line the inside of the entire vasculature, play an important role in angiogenesis. Specifically, endothelial cells detach from an existing blood vessel by down‐regulating their junctional cell‐cell adhesion protein complexes, they migrate away from the existing blood vessel, and they proliferate, all in an attempt to form a new blood vessel. Resolvin D1 (RvD1) is an autocoid molecule which possesses the ability to strengthen endothelial cell‐cell adhesion leading us to hypothesize that RvD1 may inhibit some of necessary steps for angiogenesis to occur. Here we show that treatment of endothelial monolayers with RvD1 increases the amount of junctional vascular endothelial‐cadherin (VE‐Cad) and slows endothelial cell migration. We also found that RvD1 slightly decreased endothelial cell proliferation. Taken together, we found that treatment of the endothelium with RvD1 inhibits several of the essential steps required for angiogenesis to occur. This suggests that RvD1 may be a useful therapeutic in the treatment cancer as it may decrease angiogenesis, preventing cancer cell proliferation and metastasis.

Support or Funding Information

This work was supported in part through the Hartwick College Faculty research grants program.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pioglitazone Improves the Maternal High Fructose Diet‐Impaired Glycolysis and Oxidative Phosphorylation of Astrocytes
Authors: Wu Kay Li‐Hui , Wu Chih‐Wei
Published: June 2021

Accumulating reports suggest that cell metabolism in the offspring is disturbed by maternal nutritional insults, resulting in the development of metabolic diseases in the offspring. Our previous study further indicated that the spatial learning and memory is impaired in female offspring by maternal high fructose diet (HFD). However, the underlying mechanisms are largely unknown. Astrocytes, a non‐neuronal cell type in the brain known for metabolic support to neurons, elevate glycolysis and oxidative phosphorylation rates in response to energy demand arising from the high neuronal activity. In this study, we dissected the profiles of glycolysis and oxidative phosphorylation of hippocampal astrocytes obtained from female offspring of maternal regular diet (ND) and of maternal HFD. The results indicated that maternal HFD impaired astrocytic glycolysis, oxygen consumption rate and the activities of electron transport chain. Moreover, upregulation of p85 subunit of phosphatidylinositide 3‐kinases (PI3K), down‐regulation of glucose transporter 1 (GLUT1) and a decrease of glucose uptake were detected in the maternal HFD‐programmed astrocytes. An insulin sensitizer, pioglitazone, treatment effectively improved the impaired glycolysis and oxidative phosphorylation and enhanced the astrocytic ATP content. Moreover, the suppressions of GLUT1 and glucose uptake in the HFD group were reversed to the ND level. Together, these pieces of evidence suggested that maternal HFD during gestation and lactation impairs the astrocytic energy metabolism that can be normalized by promoting insulin signaling.

Support or Funding Information

This work was supported by grants from CMPRG8F0062 and CMRPG8E0263 to KLHW from the Chang Gung Memorial Hospital, Taiwan, Republic of China.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Serum albumin as a model protein in designing an undergraduate laboratory course in biochemistry
Authors: BROWNING SARAH E , ODUNUGA ODUTAYO O
Published: June 2021

A previous work on the design of an undergraduate laboratory course in biochemistry using serum albumin as model protein, presented at the EB 2017 conference was widely accepted. Based on comments and suggestions from faculty colleagues, we are extending the project. Extensions to the course include using fluorescence spectroscopy to monitor and determine the kinetic parameters of chemically induced unfolding of serum albumin. Serum albumin has desirable properties that make the protein a suitable model for the laboratory course design. The protein is highly abundant in blood plasma or serum; it is very soluble and stable in a wide range of buffer, pH and salt conditions. The structure of albumin makes it an ideal candidate for students to study protein structure‐function relationship using spectroscopic methods such as fluorescence, circular dichroism, infrared and UV‐Visible spectroscopies. Of particular note is the robustness of the course in terms of cost‐effectiveness, skill‐sets and pedagogical aims. All the data presented herein were generated and analyzed by students under the guidance of the professor. This is a classic example of using faculty research as a teaching tool.

Support or Funding Information

Welch Foundation Grant # AN‐0008

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 Protein Arginine Methyltransferase 5‐mediated Symmetric Dimethylation of Histone H4R3 as a Target of Endocrine Disruptors and its Molecular Consequences
Authors: Ghosh Krishna , Chatterjee Biji , Kanade Santosh R
Published: June 2021

Protein arginine methyltransferase 5 (PRMT5) is a type II PRMT that catalyzes the symmetric dimethylation of arginine residues of many histone and non‐histone proteins. The resultant symmetrically dimethylated arginine on histone H4 (H4R3me2s) is a critical histone mark found to be associated with repressed genes. Endocrine disruptors (EDs) are chemicals whose environmental and occupational exposure significantly affects the endocrine homeostasis and have been associated with health hazards. However, the underlying mechanisms including epigenetic alterations are not well understood. In the present study, we show that PRMT5/H4R3me2s is a target of four common environmental EDs (bisphenol A, 4‐nonylphenol, di‐2‐ethylhexyl phthalate, and cadmium) resulting in cellular proliferation in vitro. Exposure of these four EDs to the MCF‐7 or HEK293 cells exhibited a significant increase of both PRMT5 expression and global level of H4R3me2s. The western blotting and chromatin immunoprecipitation analysis suggest these chemicals coincidently increased the level of specificity protein 1 (SP1) and nuclear factor Y alpha (NFYA) and their enrichment in the PRMT5 proximal promoter. Interestingly, all the EDs exerted dose‐dependent inhibitory action on p21CDKN1A expression while cyclin D1 expression was increased. Finally, we demonstrated an enhanced selective enrichment of H4R3me2s at the proximal promoter of p21 but not cyclin D1. We propose this significant alteration in the p21 promoter might have contributed in increased recruitment of DNMT3A and HDAC1 and subsequent hypermethylation of p21 promoter CpG island and deacetylation of the promoter histones. As PRMT5 is an oncogene often overexpressed in many cancers, elevation of its activity and function by these exogenous EDs could potentially initiate cancer through epigenetic mechanisms as observed in this study.

Support or Funding Information

The authors are thankful to SERB, Central University of Kerala, and KSCSTE for financial support for the experiments.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Molecular Insights into Di‐acetyllysine Histone Recognition by the BRPF1 Bromodomain
Authors: Obi Juliet
Published: June 2021

Bromodomains are often found in chromatin‐modifying complexes, whose activity can lead to aberrant expression of genes that drive certain diseases in humans, including cancer, neurological disorders and inflammation. The bromodomain‐PHD finger protein 1 (BRPF1) is a part of the MOZ (monocytic leukemic zinc‐finger protein) HAT (histone acetyltransferase) complex implicated in chromosomal translocations known to contribute to the development of acute myeloid leukemia (AML). BRPF1 contains a unique combination of post‐translational modification (PTM) reader domains, including two plant homeodomain (PHD) fingers, a bromodomain, and a proline‐tryptophan‐tryptophan‐proline (PWWP) domain, through which it is known to modulate the HAT activity of MOZ. We hypothesized that the BRPF1 bromodomain can recognize histone ligands with multiple acetyllysine marks, and that the recognition can be modulated by PTMs adjacent to these marks. We carried out isothermal titration calorimetry (ITC), analytical ultracentrifugation (AU), and site‐directed mutagenesis experiments with the purified BRPF1 bromodomain to investigate its binding affinity to different di‐acetyllysine histone ligands. Our data reveal the BRPF1 bromodomain is able to bind several histone ligands containing di‐acetyllysine, and modifications adjacent to the acetyllysine modifications alter the binding affinity. Notably, our AU data show that the BRPF1 bromodomain binds to mono‐ and di‐acetyllysine ligands as a monomer. Our studies will further elucidate the role of the bromodomain in recruiting BRPF1 to histones, and subsequently assembling and activating the MOZ HAT complex, which has been implicated in acute myeloid leukemia.

Support or Funding Information

National Institutes of Health, National Institute of General Medical Sciences grant number R15GM104865 to KCG

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ribosomal ambiguity (ram) mutations promote 30S domain closure and thereby increase miscoding
Authors: Hoffer Eric D , Maehigashi Tatsuya , Fredrick Kurt , Dunham Christine M.
Published: June 2021

During protein synthesis, ribosomes select correct aminoacyl‐tRNAs by monitoring the nucleotide pairing between the codon on the mRNA and the anticodon of the tRNA in the decoding center. Despite the large pool of near‐cognate tRNAs, the ribosome is highly accurate with an error rate of ~10−3 – 10−5. The process of decoding by the ribosome is thought to be governed by a conformational transition in the ribosome from open to closed that occurs upon codon‐anticodon pairing within the A site. Ribosomal ambiguity (ram) mutations increase miscoding and map to both ribosomal proteins and rRNA in disparate regions, yet precisely how these mutations act has remained unclear. In this study, we solved crystal structures of Thermus thermophilus 70S ribosomes harboring 16S rRNA ram mutations G299A and G347U in the absence of A‐site tRNA (A‐tRNA) and in the presence of a near‐cognate anticodon stem‐loop. In the absence of an A‐tRNA, each of the mutant ribosomes exhibits a partially closed state. In the 70S‐G347U structure, the 30S shoulder is rotated inward and intersubunit bridge B8 is disrupted. In the 70S‐G299A structure, the 30S shoulder is rotated inward and decoding nucleotide G530 flips into the anti conformation. Both of these mutant ribosomes adopt the fully closed conformation in the presence of near‐cognate A‐tRNA, just as they do with cognate A‐tRNA. Thus, these ram mutations act by promoting the open to closed transition, albeit in somewhat distinct ways. This work reveals the functional importance of 30S shoulder movement for productive aminoacylated‐tRNA incorporation in the A site of the ribosome.

Support or Funding Information

This work was supported in part by the National Institutes of Health [grant numbers GM093278 to CMD, GM072528 to KF]. The X‐ray crystallography datasets were collected at the at the NE‐CAT beamlines (funded by the NIGMS from the NIH (P30 GM124165)), using a Pilatus detector (RR029205) and an Eiger detector (OD021527), and at the SER‐CAT beamlines (funded by its member institutions, and NIH equipment grants RR25528 and RR028976). This research used resources of the APS, a US Department of Energy Office of Science User Facility operated by Argonne National Laboratory under Contract DE‐AC02‐06CH11357 (NE‐CAT) and W‐31‐109‐Eng‐38 (SER‐CAT). Dr. Dunham is a Burroughs Wellcome Investigator in the Pathogenesis of Infectious Diseases.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural Characterization of Rosetta Designed Amino Acyl‐tRNA Synthetase Active Sites for Genetic Code Expansion
Authors: Beyer Jenna N. , Hosseinzadeh Parisa , Mehl Ryan A. , Cooley Richard B.
Published: June 2021

The site‐specific incorporation of non‐standard and novel amino acid structures enabled by Genetic Code Expansion (GCE) provides access to new avenues of protein research allowed by engineering diverse functionality into proteins. At its core, GCE requires the engineering of an orthogonal amino acyl‐tRNA synthetase/tRNA cognate pair that can function with high catalytic efficiency for a non‐canonical amino acid (ncAA). While the GCE community has generated many new amino acyl‐tRNA synthetase/tRNA pairs, these pairs have kinetics that are orders of magnitude worse than the natural translational components. A major challenge for the GCE field is identifying which amino acyl‐tRNA synthetase residues to target for engineering improved amino acyl‐tRNA synthetase/tRNA pairs.

Here we use Rosetta modeling to identify mutations in the amino acyl‐tRNA synthetase active site that will improve the efficiency of the nitroTyr amino acyl‐tRNA synthetase/tRNA pairs. Rosetta guided mutations were successful in generating improved GCE function in vivo.

Crystal structures were obtained for the Rosetta improved amino acyl‐tRNA synthetases to understand the structural basis for the enhanced activity as part of a comprehensive kinetic and structural analysis of these improved synthetases. Here, we used the 3‐nitro‐tyrosine amino acyl‐tRNA synthetase/tRNA pair as a model system for synthetase improvement and structural characterization.

We observe that the Rosetta‐improved synthetases allow a more planar 3‐nitro‐tyrosine (nitroTyr) amino acid upon binding, which is predicted to contribute to their increased activity. This Rosetta guided approach provides a third generation of more efficient nitroTyr amino acyl‐tRNA synthetase/tRNA pairs that will accelerate the study of this oxidative stress post‐translational modification and its role in human disease.

We demonstrate that Rosetta successfully identified key residues for amino acyl‐tRNA synthetase/tRNA pair optimization. This strategy can be utilized to systematically improve the efficiency of all ncAA amino acyl‐tRNA synthetase/tRNA pairs, resulting in more robust GCE technologies and facilitating new applications.

Support or Funding Information

NIH Grant #R01‐GM114653‐01, Oregon State University Summer Undergraduate Research Experience (SURE) in Science Program

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Towards a New Aminoacylation Assay for Alanyl‐tRNA Synthetases
Authors: Duplessis Kyle , Heath Jacob , McCarthy Ian , Chihade Joseph
Published: June 2021

Aminoacyl‐tRNA synthetases are crucial enzymes in protein biosynthesis, ensuring the fidelity of the genetic code by charging transfer RNAs with the appropriate amino acid. Aminoacylation is typically measured using radioactively labeled substrates, using assays that are non‐continuous, relatively expensive, and time consuming. Recently, a new assay1 was developed for tyrosyl‐tRNA synthetases in which the formation of AMP during the aminoacylation reaction is coupled to production of NADH which can be measured spectrophotometrically. To improve sensitivity, the aminoacyl‐tRNA product was regenerated through hydrolysis with a second enzymatic activity, allowing detection of aminoacylation even at low concentrations of tRNA. We have extended this assay to the alanyl‐tRNA synthetase system. Three different aminoacylation and tRNA regeneration schemes have been developed – mischarging with serine, followed by hydrolysis with the intrinsic editing domain; mischarging with glycine, followed by hydrolysis with D‐aminoacyl‐tRNA deacylase (DTD); and charging with alanine, followed by hydrolysis with a variant of DTD.

Support or Funding Information

Carleton College and the Towsley Foundation

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pathogenic Mutations in a Human Mitochondrial Enzyme Affect Protein Stability
Authors: Kennicott Hannah , McCurdy Clara , Makori Jessica , Chihade Joseph
Published: June 2021

Mutations in human mitochondrial alanyl tRNA‐synthetase have been correlated with at least two different pathologies, infantile cardiomyopathy and leukodystrophy. We have used a bacterial expression system to study the effects of these mutations on protein stability. All mutations tested lead to reduced amounts of full‐length soluble enzyme. Several render the protein insoluble, while others lead to proteolysis into soluble or insoluble fragments. Correlations between protein stability and disease state will be presented. We are currently investigating whether the addition of solubility tags will reverse these effects and facilitate the purification of full‐length mutants.

Support or Funding Information

Carleton College and the Towsley Foundation

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 Effects of N‐Terminal Acetylation on the Membrane Binding Behavior of Alpha Synuclein
Authors: Bond Judith Elizabeth , Sarver Jessica
Published: June 2021

Alpha Synuclein (asyn) is a neuronal protein that acts as a hallmark of Parkinson's Disease through fibril formation that later form Lewy bodies in the brain. The function of asyn is still relatively unclear, however it is known to bind to membranes. It is of interest to study how membrane binding is affected by N‐terminal acetylation of asyn. N‐terminal acetylation is a form of post‐translational modification. A co‐transformation in the presence of NatB in E coli was carried out, to enable the acetylated N‐terminus. In order to reduce protein modification for purification methods, such as Nickel Column Chromatography, Fast Pressure Liquid Chromatography (FPLC) was utilized. The protein was eluted from the column based on electrostatic interactions among the column, protein, and Tris‐NaCl. The fractions of interest were analyzed using an SDS‐PAGE gel, to validate the presence of the purified protein. Following purification, membrane binding behaviors will be analyzed using Electron Paramagnetic Resonance (EPR), by measuring the signal of an unpaired electron, which is introduced through site‐directed spin labeling. The spin label has a nitroxide group, which provides a three distinct sharp peaks in the EPR spectra. Through the binding of the asyn with large unilamellar vesicles (LUVs) the spectra will demonstrate a reduced peak sharpness, which indicates binding. The changes in the EPR spectra will provide insight into how the post‐translational modifications affect the ability of asyn to bind to membranes, as well as the greater impact on protein structure.

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 a Robust Direct Binding Assay for Phospholipid‐Sensing NR5A Nuclear Receptors
Authors: D'Agostino Emma Hope , Cornelison Jeffery , Mays Suzanne , Flynn Autumn , Jui Nathan , Ortlund Eric
Published: June 2021

The human NR5A nuclear receptors, steroidogenic factor‐1 (SF‐1, NR5A1) and liver receptor homolog‐1 (LRH‐1, NR5A2), are phospholipid‐sensing regulators of steroidogenesis, development, and metabolism. Their control of such diverse biological processes renders them attractive pharmacological targets for the treatment of several cancers and metabolic diseases such as obesity, type II diabetes, and non‐alcoholic fatty liver disease. However, the evaluation of candidate endogenous ligands and the development of small molecule modulators have been hindered by the lack of a robust direct binding assay. Using a structure‐guided approach, we recently developed a potent NR5A agonist (EC50 of 15 nM) targeting the ligand‐binding pocket. We have leveraged this molecule to create a high‐affinity fluorescent probe that binds SF‐1 and LRH‐1 with affinites of 12 nM and 1 nM, respectively. This probe has enabled the creation of a fluorescence polarization (FP)‐based competition assay suitable for both synthetic small molecules and mammalian phospholipids. FP is solution‐based, allowing proteins to retain their native conformation, and the competition format of the assay ensures that binding will only be detected in the ligand‐binding pocket. We have found that for a set of 25 LRH‐1 agonists derived from the previously‐reported RJW100 scaffold, affinities determined by FP correlate with potencies in a luciferase reporter assay, suggesting that in vitro binding affinity may be predictive of in‐cell activity for this class of molecule. Moreover, binding affinities reported for other NR5A agonists harboring different chemical scaffolds agree with our FP‐based measurements. Finally, we show that the previously‐reported dilauroylphosphatidylcholine agonist binds LRH‐1 with a Ki of 80 nM, suggesting that this assay may be used to evaluate candidate endogenous ligands for these receptors.

Support or Funding Information

R01 DK115213 02EHD has additional support from the National Science Foundation Graduate Research Fellowship and the Emory University NIH‐5T32GM008367‐27. SGM has additional support from F31 DK111171 03.

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