bluuebunny/crossref_metadata_2025_split huggingface dataset:
Validation Studies of CRF Receptor 1 as a Target for AD
Authors: Nguyen Kevin Hoang , Ellisman Maya , Patanapirom Jasmine , Sarsoza Floyd , Rissman Robert 
Published: June 2021

Alzheimer's disease (AD) is the most common form of dementia and is defined by two neuropathologic hallmarks: senile plaques and neurofibrillary tangles, respectively composed of β‐amyloid (Aβ) and phosphorylated tau protein. Very few incidences of AD are developed from genetic mutations. A majority of cases arise sporadically, and are thought to result from the interplay of genetic and environmental factors. Corticotropin‐releasing factor (CRF) signaling in AD, when exacerbated by stress, has been shown to influence AD pathology. With an aging population of veterans and individuals with stress‐related neuropsychiatric disorders (i.e. PTSD, depression, anxiety, etc.), uncovering the link between this stress pathway and AD pathogenesis can help to develop therapeutics to relieve the emotional and economic burden created by AD. Rodent models of this disease have found that stress exposure and CRF signaling are linked to the pathogenesis of AD. In this study, we explored whether therapeutic targets that modulate the CRF system (i.e. the CRFR1‐selective antagonist R121919) could impact the behavioral and pathological endpoints seen in AD mice. AD mice (PSAPP) were aged to 6 and 12 months in the presence or absence of restraint stress and/or R121919. Drug‐treated PSAPP mice had significantly reduced Aβ levels, plaque accumulation, and cognitive impartment, relative to the control cohorts. Collectively, our data provide evidence that CRFR1 modulation may be mechanistically linked to the neuropathogenesis of AD.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Differential pattern of expression for endothelial cell markers in the microvessels of different brain regions.
Authors: Mbagwu Smart , Marti Christiane , Blanc Pauline , Mantel Pierre‐Yves , Filgueira Luis 
Published: June 2021

BackgroundThe role of microvascular endothelial cells in the brain is of importance in both healthy and diseased conditions. Various studies have reported heterogenicity in the phenotypical, morphological and molecular characteristics of endothelial cells in the different organs of the body. There is still not much known about the heterogenicity of the endothelial cells of the human brain microcirculation. This study was aimed at characterizing qualitatively the pattern of expression for endothelial cell markers for the microvessels within different regions of the brain.MethodsParaffin sections from different anatomical regions (precentral and postcentral gyrus, hippocampus, rhinal and visual cortex) of human formalin fixed brains (n=3) were stained immunohistochemically for endothelial cell markers, including CD31, claudin 5, von Willebrand Factor, E‐ selectin and P‐selectin.ResultsThe expression patterns of the markers was heterogenous in the different microvessels within the regions of the brain studied. The capillaries had the most positive expression among the different biomarkers especially for VWF and CD31 while the arterioles were most positive for the E‐Selectin marker. The density of of the stained microvessels were more in the white matter compared to the gray matter with the visual, rhinal and hippocampal regions showing high densities of the microvessels.ConclusionThe expression pattern of the different endothelial markers is heterogenous for the microvasculature within the different anatomical regions of the brain. This results indicate that the heterogenicity in microvascular endothelial cells contribute to functional differences in the different brain regions.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Limited Proteolysis Analysis of a DEAD‐box Protein and its Domain Truncated Variants
Authors: Arnold Megan , Garcia Ivelitza 
Published: June 2021

RNA helicases play a crucial role in virtually all aspects of RNA metabolism, and although they share a highly conserved structure, the enzymes exhibit a wide variety of biochemical activity. One family of RNA helicases is DEAD‐box proteins, and while their functions are diverse, the one defining activity of these proteins is their ability to hydrolyze ATP in the presence of single‐stranded or double‐stranded RNA.1 ATPase activity leads to changes in RNA affinity, enabling ligand dissociation and cyclic rebinding to promote correct RNA folding in the cell. For example, Rok1p is a yeast DEAD‐box protein essential in ribosomal RNA folding, and is characterized by a catalytic core where ATP and RNA bind, as well as two peripheral domains.2 The peripheral domains (N‐terminal domain or NTD, and the C‐terminal domain, or CTD), are proposed to regulate ATPase activity as well as provide a region for RNA specificity. Thus, peripheral domains could play a role in regulating the overall structure of the protein and its mechanism of RNA refolding. To monitor peripheral effects, the structural changes of Rok1p in the presence and absence of a ligand were analyzed using limited proteolysis under various conditions. These results were subsequently compared to the truncated variant, Rok1p‐ΔCTD. This comparative analysis determined the structural role of the NTD, and to some extent, the CTD. Time dependent proteolysis suggests that the protein is more dynamic at higher temperatures. In the presence of RNA or a nucleotide, the level of structure fluctuation increases. The observed level of conformational changes is minimal yet has dramatic effects on the ATPase activity of the protein. Specifically, the results show that the protein does not occupy a fully extended core state, which accounts for the unique catalytic activity of Rok1p.Support or Funding InformationThe Thomas Lord Charitable Trust FellowshipThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of Non‐Active Site Amino Acid Residues on Inhibitor Selectivity
Authors: Eck Tyler , Gubler Ueli , Goodey Nina 
Published: June 2021

Enzyme inhibitors are important in biological processes and as pharmaceutical drugs, the cornerstone of modern medicine. It is necessary to build a comprehensive picture of the binding event between the inhibitor and its enzyme target to effectively design pharmaceutical drugs and predict the binding of known drugs to new targets. It is currently known that inhibitor selectivity is in part determined by the amino acid residues within the “active site”, which is the site/cavity in the enzyme where the inhibitor binds. Less well understood is the role of residues situated away from (distal to) the active site and how they contribute to inhibitor selectivity, which refers to the fact that inhibitor molecules often bind to several members of an enzyme family but not all. By understanding the features that determine inhibitor binding within an enzyme family, it is possible to design better inhibitors and predict possible new targets for a given inhibitor. Using Bacillus stearothermophilus dihydrofolate reductase (Bs DHFR) as a model system, this project studies how amino acids distal from the active site affect inhibitor binding and selectivity. Previous studies in our lab have indeed predicted and experimentally verified three clusters of amino acids in Bs DHFR that are distal to the active site but impact inhibitor binding. Here, random mutations are being introduced individually into these clusters. Mutants will be screened for DHFR activity using a functional assay based on ability to synthesize thymidine. DHFR coding genes in selected colonies will be sequenced. Genes with functional amino acid mutations will be used to produce the corresponding DHFR enzyme variants. To ensure that the variants are active, kcat and KM values will be determined. To investigate the impact of the mutations on inhibitor selectivity, KD values will be determined for multiple inhibitors including Methotrexate (MTX), Trimethoprim (TMP), and Pyrimethamine (PMT). Analysis of these results will show how and how much distal residues affect Bs DHFR's inhibitor selectivity.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Oxidative Susceptibility of Calmodulin Cardiac Arrhythmia Mutants
Authors: Moen Rebecca Jane , McCarthy Megan , Hinde Amanda , Thomas David Dale 
Published: June 2021

The congenital arrhythmia disorder catecholaminergic polymorphic ventricular tachycardia (CPVT) is linked to missense mutations within the ubiquitous calcium‐sensing protein calmodulin (CaM). CaM plays various important roles in cellular and biological processes. In cardiomyocytes, CaM is critical for regulating excitation‐contraction coupling by binding calcium as well as binding a diverse set of targets including the ryanodine receptor (RyR2) to regulate intracellular calcium levels. CaM is a unique protein, not only in its ability to bind four calcium ions through its N‐ and C‐terminal lobes but also due to its high number of methionine amino acid residues within its primary sequence. These methionine residues are solvent‐exposed upon binding calcium and facilitate CaM binding its targets. However, the location of these methionine residues make CaM extremely sensitive to oxidative modification by reactive oxygen species (ROS). ROS can directly interact with CaM, causing reversible oxidation of the methionine side chain to a methionine sulfoxide or irreversible oxidation to methionine sulfone. CaM also contains a potential PEST sequence, an internal signal for rapid proteolytic degradation. We have investigated the oxidative and proteolytic susceptibility of CPVT cardiac arrhythmia CaM mutants with particular focus on methionine oxidation.Support or Funding InformationNIH R37 AG026160This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Crossroad control of cell proliferation and apoptosis by PEA‐15 phosphorylation homeostasis and allosteric regulation of protein conformations and interactions
Authors: Wei Yufeng , Crespo Sergio , Hassan Sherouk 
Published: June 2021

Cell fate is determined by the relative tendency of cell proliferation and cell death. PEA‐15 (phosphoprotein enriched in astrocytes, 15 kD), a small, non‐catalytic, death‐effector domain (DED) containing protein, regulates both proliferation and apoptosis through protein‐protein interactions. PEA‐15 is widely expressed in different tissues and highly conserved among mammals, and the overall expression level of PEA‐15 is almost constant in most tissues. PEA‐15 contains two phosphorylation sites on the C‐terminal tail, Ser104 and Ser116, and the phosphorylation states of these two serine can vary significantly depending on cell and tissue types and/or cellular environment and conditions. Unphosphorylated PEA‐15 inhibits extracellular signal‐regulated kinase (ERK)‐dependent cell proliferation, while the doubly phosphorylated protein binds to Fas‐associated death domain (FADD) and blocks death receptor mediated apoptosis. Using advanced nuclear magnetic resonance (NMR) techniques, we previously reported a surprising conformational change of PEA‐15 DED upon interaction with ERK2. In addition, DED conformation is significantly modulated by the phosphorylation of the C‐terminal serine residues, which switches binding specificity from ERK1/2 to FADD. Based on our most recent results, we propose the phosphorylation homeostasis model, in which that the balance between phosphorylated and unphosphorylated PEA‐15 is strictly regulated in different cell types and tissues and controls the cell fate, any disruption of the delicate balance could lead to various diseases, such as cancers and neurodegenerative diseases. To test this hypothesis, we recently conducted quantitative Western blot to detect the phosphorylation states of PEA‐15 in various primary cells and cell lines, as well as the effects of PEA‐15 phosphorylation on the activation of caspases in the cells. We have also transfected wildtype, S104A/S116A (unphosphorylated mimic), and S104D/S116D (phosphorylated mimic) PEA‐15 into these cells. Using novel in‐cell NMR technique, we studied the conformation of these proteins within the cellular contents, where all its natural interactions are present. The effects of transfected proteins on apoptosis and activation of caspases have also been examined.Support or Funding InformationNIH AREA R15CA179410This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
n‐π* and Other Atomic Level Interactions of Formamides with Nucleobases and Base Analogs in Water
Authors: Karim Rezwana , Cheng Xian , Record M. Thomas 
Published: June 2021

To understand the molecular origins of favorable amide‐nucleobase interactions and predict or interpret effects of amides on nucleic acid processes, we quantify the preferential interactions of formamide and N‐methylated formamides with nucleobases and base analogs differing in amount and composition of water‐accessible surface area (ASA) by solubility assays. These formamide series solutes have much larger amide sp2C ASA than the alkylurea solutes studied previously. Using an established additive, ASA‐based analysis, we interpret these thermodynamic results to determine intrinsic strengths of interaction of each amide with unit area of the five types of nucleobase unified atoms (sp2C, sp3C; sp2N, sp3N; sp2O). All formamides interact favorably with all nucleobases and base analogs studied. “One‐way” analysis of results to date indicate favorable interactions of these formamides with all five types of unified atoms of nucleobases, and set the stage for a “two‐way” atom‐atom analysis quantifying interactions of the four atoms of amides (aliphatic sp3C; amide sp2C, sp2N and sp2O) with five atoms on nucleobase (aromatic ring sp2C and sp2N, methyl sp3C, amino sp3N, carbonyl sp2O). In particular, this “two‐way” analysis will quantify the favorable n‐π* interactions of nucleobase carbonyl O with amide sp2C and of formamide sp2O with nucleobase heterocyclic ring sp2C to compare with the very favorable n‐π* interactions of amide sp2O with homocyclic aromatic ring sp2C reported previously. Progress toward these goals will be reported.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
α‐synuclein inhibits Snx3‐retromer‐mediated retrograde recycling of iron transporters in a S. cerevisiae model of Parkinson's disease
Authors: Witt Stephan N. , Nagarajan Sureshbabu N. , Liu Zhengchang , Hemphill Wayne O. , Shi Runhua , Uversky Vladimir N. , Patel Dhaval 
Published: June 2021

We probed the role of alpha‐synuclein (α‐syn) in modulating sorting nexin 3 (Snx3)‐retromer‐mediated recycling of iron importers in the budding yeast Saccharomyces cerevisiae. Fet3/Ftr1 comprises the high affinity iron importer in yeast. Fet3 is a membrane‐bound multicopper ferroxidase, whose ferroxidase domain is orthologous to human ceruloplasmin. The ferroxidase domain of Fet3 oxidizes external Fe+2 to Fe+3, and the Fe+3 ions then channel through the Ftr1 permease into the cell. When the concentration of external iron is low (< 1 μM), Fet3/Ftr1 is maintained on the plasma membrane by retrograde endocytic‐recycling; whereas, when the concentration of external iron is high (> 10 μM), Fet3/Ftr1 is endocytosed and sorted to the vacuole for degradation. We discovered that α‐syn expression phenocopies the high iron condition: under the low iron condition (< 1 μM), α‐syn inhibits Snx3‐retromer‐mediated recycling of Fet3/Ftr1 and consequently the complexes are sorted to the vacuole for degradation. Because α‐syn has a propensity to bind to negatively charged lipids and vesicles, we asked whether α‐syn might disrupt recycling by interfering with the association of retromer components with early endosomes. Using fluorescence microscopy, we found that α‐syn blocks the association of retromer components Snx3‐mCherry and Vps17‐mCherry molecules with early endosomes. Early endosomes are enriched in phosphatidylinositol‐3‐monophosphate (PI3P), and Snx3 and Vps17 each possess a phox homology domain that selectively binds to PI3P. We tested the hypothesis that α‐syn blocks the binding of Snx3 to vesicles enriched PI3P in a liposome‐protein flotation assay using synthetic liposomes containing phosphatidylcholine, phosphatidylserine, and PI3P (70:25:5) and found that α‐syn inhibits the association of the purified phox homology domain of Snx3 to the synthetic vesicles. Overall, we show that α‐syn disrupts endocytic recycling by interfering with retromer (Fig. 1), and that α‐syn accelerates the sorting to the vacuole of Fet3/Ftr1 complexes that emanate from the plasma membrane. This work raises the possibility that α‐syn can disrupt iron homeostasis in cells by interfering with the trafficking of iron transporters.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Discovery of Protein Phosphatase 2A Substrates
Authors: Wang Xinru , Page Rebecca , Wolfgang Peti 
Published: June 2021

The protein ser/thr phosphatases (PSPs) dephosphorylate 1000s of key biological targets, making them attractive therapeutic targets. The serine/threonine protein phosphatases 1 and 2B (PP1; PP2B or calcineurin/CN) bind substrates and regulatory proteins using short functional sequences known as Short Linear Motifs (SLiMs). These SLiMs are found within intrinsically disordered regions (IDRs) of substrates and regulatory proteins and mediate specific protein‐protein interactions. While tremendous progress had been made in identifying where and how SLiMs bind PP1 and CN, very little is known about how substrates are specifically recruited to PP2A, a validated cancer drug target. Here we describe three structures of the first known PP2A‐SLiM interaction (B56:pS‐RepoMan, B56:pS‐BubR1, and B56:pSpS‐BubR1), which reveal that this PP2A‐specific SLiM is defined as LSPIxE. We also show that the affinity of this SLiM for B56 is modulated both by residues immediately adjacent to the SLiM and its phosphorylation, both of which provide the fine‐tuning needed to ensure signaling pathway fidelity. Finally, by using this newly discovered motif, we identify nearly 100 proteins that we predict are novel PP2A:B56 regulators/substrates, substantially expanding the PP2A interactome. We are now leveraging this data to identify new PP2A‐specific SLiMs. Further, we are expanding this approach to discover new SLiMs that are specific for other PSPs, especially PP1 and CN. Together, these data are providing a powerful approach not only for dissecting PSP interaction networks in cells but also for targeting PSP diseases, such as cancer.Support or Funding InformationNIH: R01GM098482 NIH: R01NS091336This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of a cell‐based assay for Tau strain discrimination
Authors: Angeles‐Perez Lidiette M. , Vaquer‐Alicea Jaime , Manon Victor , Diamond Marc 
Published: June 2021

Alzheimer's Disease and other related tauopathies are characterized by the accumulation of the microtubule‐associated protein Tau into fibrillary deposits. Interestingly, there is evidence supporting amyloid structures, or strains, formed as a consequence of Tau aggregation are unique to each pathology, suggesting different conformations play an important role in the development of specific tauopathies. The potential critical role of Tau in disease progression, together with the isolation of 19 different Tau strains, led us to work in developing a panel of cell‐based fluorescence resonance energy transfer (FRET) biosensors. Each biosensor carried a different mutation on Tau's aggregation‐prone repeat domain, based on the hypothesis that each mutant would have unique interactions with Tau strains and, thus, alter Tau aggregation kinetics within the cell. We hoped that by studying these differences in FRET signal we would be able to discriminate between distinct amyloid conformations. In order to do this, we first created the biosensors by transducing HEK 293 cells with lentivirus coding Tau's repeat domain with specific mutations, fused to both, cyan florescent protein (CFP) or yellow florescent protein (YFP). These biosensors were later treated with different sources of Tau seeds and FRET flow cytometry was used to describe their seeding activity. Experiments with the 19 strains in our Tau library and the engineered biosensors revealed noticeable differences in percent FRET positivity, such that discrimination was achieved for most of the strains. Interestingly, the highest seeding activity among all the biosensors was obtained from strain 15 (DS15), even at low total protein concentrations. Additionally, biosensors expressing the wild‐type four‐repeat domain of Tau had higher percent FRET positivity only when seeded with strains derived from Corticobasal Degeneration (CBD) patients. Furthermore, treatment of our panel of biosensors with brain homogenates of multiple tauopathy patients correlated with our previous results and revealed more distinct patterns of FRET behavior. Together, these results suggest that measuring proteopathic seeding activity while using our panel of biosensors is a sensitive and reliable tool to discriminate between different amyloid conformations, which could potentially lead the development of improved diagnostic methods for neurodegenerative disorders.Support or Funding InformationThis research project was supported by the NIH‐Maximizing Access to Research Careers (Grant # 5T34GM0072821‐38), the Summer Undergraduate Research Fellowship at UT Southwestern Medical Center and the Center for Alzheimer's and Neurodegenerative Diseases at UT Southwestern Medical Center.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
“The Importance of a Phenolic Group in the Substrates of Iodotyrosine Deiodinase”
Authors: Quinones Carla Michelle , Kozyryev Anton , Rokita Steven 
Published: June 2021

Iodotyrosine deiodinase (IYD) is an enzyme that is responsible for recycling iodide from monoiodotyrosine (I‐Tyr) and diiodotyrosine (I2‐Tyr), which are formed as side‐products during the biosynthesis of thyroid hormones. Recently, a gene encoding IYD was found in bacterium Haliscomenobacter hydrossis (hhIYD), but the function of hhIYD in this organism is still unknown. This bacterium does not generate thyroid hormone and is not known to require iodide salvage. HhIYD can bind and deiodinate 2‐iodophenol, but not as efficiently as I‐Tyr. We wanted to know how broad the substrate specificity for this enzyme is; thus, the goal of the project is to learn how important the OH‐group of I‐Tyr is for catalysis. Since aryl amine and hydroxyl groups have common properties, we synthesized the aniline derivative of I‐Tyr. Both OH‐ and NH2‐groups are electron donating substituents to the phenyl ring and can participate in hydrogen bonding. Moreover, we can propose a similar mechanism for deiodination of both I‐Tyr and its aniline derivative. We expressed and purified hhIYD. The binding of the aniline derivative to hhIYD was measured using fluorescence quenching, but no binding was observed even at a ligand concentration as high as 1370 μM. We also tested deiodination of the aniline derivative of I‐Tyr by hhIYD, but no deiodination was detected above turnover number of 7.05 × 10−5 s−1. We expect that this project will help to identify the full catalytic potential of IYD and its role in nature.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.