bluuebunny/crossref_metadata_2025_split huggingface dataset:
Investigating the Roles of Nap1, Taz2, and H1 on Nucleosome Assembly
Authors: Blackwell Virginia Katherine , Sarkar Prithwijit , D'Arcy Sheena 
Published: June 2021

In eukaryotic cells, DNA is packaged into the nucleus by extensive coiling, the most fundamental unit of which is the nucleosome. Nucleosomes are comprised of 146 base pairs of DNA wrapped around an octameric histone core, along with linker histone H1, controlling access to the genome. Proteins such as nucleosome assembly proteins (Naps) and p300 interact to shuttle histones to and from the nucleosome, regulating gene expression. In this project, we investigated the molecular details of interactions between Nap1 and the Taz2 domain of p300, their interactions with H1, and studied the role of these protein complexes on nucleosome dynamics. We purified Nap1, Taz2, and H1 using column chromatography, then assayed interactions between Nap1‐Taz2 and Nap1‐H1. We also performed experiments investigating the formation of a ternary complex of Nap1‐Taz2‐H1. Electrophoretic mobility shift assay (EMSA) and size exclusion chromatography with multi‐angle light scattering (SEC‐MALS) were used to study binding and determine the stoichiometry of these protein‐protein interactions. With these methods, we observed that Nap1 and Taz2 interact in vitro at an equimolar stoichiometry. We performed nucleosome assembly/disassembly assays in the presence of these protein complexes to study their effects on the nucleosome. Since Nap1, p300, and histones are a few of many ‘gatekeepers’ to our genetic code, it is critical that we determine how they function and cooperate to protect DNA. It is known that both Nap1 as well as p300 mutations contribute to cancer. While there is still much to unravel, p300 and interacting proteins are crucial to proper cell growth and division. This study furthers our understanding of nucleosome dynamics.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Rexinoid signaling promotes myogenic differentiation through a direct regulation of MyoD
Authors: Hamed Munerah , Khilji Saadia , Li Qiao 
Published: June 2021

Readout of epigenetic modification has provided us with plentiful information to thoroughly understand the regulation of genes during myogenic differentiation. While skeletal myogenesis is coordinated by sequential expression of myogenic regulatory factors including MyoD and myogenin, chromatin modifications have emerged as vital mechanisms of myogenic regulation. We have recently found that bexarotene, a retinoid X receptor (RXR)‐selective agonist, promotes the specification and differentiation of muscle lineage; however, the genome wide impact of rexinoid action on myogenic expression has not yet been investigated. Through genome wide association studies, we here determined rexinoid‐responsive residue‐specific histone acetylation at a distinct chromatin state associated with MyoD and myogenin. We also define a new mechanism of rexinoid action which is mediated by the receptor and largely reconciled through a direct regulation of MyoD expression. Thus, we provide novel molecular insights into the interplay between retinoid X receptor signaling and chromatin states associated with myogenic programs in early differentiationSupport or Funding InformationNSERCThe Cancer Research societyIRSCThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The role of lncRNA HOTAIR in the regulation of glucose metabolism
Authors: Obaid Monira , Mandal Subhrangsu S 
Published: June 2021

Glucose is a fuel for immune cells and glucose transporters have influential roles to supply glucose for immune cell functional responses. Recently we have reported that long noncoding RNA (lncRNA) HOTAIR is involved in the regulation of inflammatory and immune response. Our studies demonstrate that HOTAIR expression is induced in immune cells (macrophages) upon treatment with lipopolysaccharide (LPS). Glucose transporters have been found to be upregulated in immune cells. Our present study has correlated HOTAIR with the upregulation of the glucose transporters in the presence of LPS. Currently, we are investigating the detail mechanism how HOTAIR induces expression of glucose transporters and metabolic reprogramming. Thus, we hypothesize that HOTAIR is involved in metabolic reprogramming via increased glucose availability and modulate the macrophage inflammatory response. Understanding the role of HOTAIR in metabolic reprogramming will identify signaling pathways that would serve as novel therapeutic targets in the treatment of diabetes and obesity.Support or Funding InformationSubhrangsu S. MandalThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Altered dosage of noncoding RNAs expressed from the Dlk1‐Dio3 locus impairs skeletal muscle differentiation
Authors: Petty Christopher Anthony , Naya Francisco J 
Published: June 2021

Noncoding RNAs (ncRNAs) have emerged as key components of gene regulatory networks, yet their role in skeletal muscle diseases is still poorly understood. The maternally imprinted Dlk1‐Dio3 ncRNA locus has been shown to play an essential role in skeletal muscle development, regeneration, and metabolism. The diverse ncRNAs expressed from this locus, including over 50 microRNAs (miRNAs), several long noncoding RNAs (lncRNAs), and a cluster of C/D box snoRNAs, are thought to be transcribed as a ~200 kb polycistron initiating from the proximal promoter immediately upstream of the Gtl2 lncRNA, the most 5′ ncRNA in the locus. This project aimed to understand the importance of proper dosage of the Dlk1‐Dio3 ncRNAs in skeletal muscle differentiation. Toward this end, we utilized CRISPR‐Cas9 gene editing to generate deletions in the Gtl2 proximal promoter and the overlapping differentially methylated region (DMR) with the goal of modulating expression of the maternally expressed ncRNAs in C2C12 myoblasts, an established muscle cell line. This gene editing strategy yielded a clonal population of C2C12 myoblasts that harbored a deletion spanning a portion of the Gtl2 proximal promoter while leaving the Gtl2 promoter's TATA box intact. This CRISPR‐edited cell line showed a dramatic increase in the Dlk1‐Dio3 ncRNAs. As a result of Dlk1‐Dio3 ncRNA upregulation, this C2C12 cell line displays significantly impaired myogenic differentiation. The continued characterization of this novel C2C12 cell line will help elucidate the importance of maintaining proper dosage of the Dlk1‐Dio3 locus in skeletal muscle, which could serve as a therapeutic target for treating muscle diseases.Support or Funding InformationThis project was supported with funds from the Arnold & Mabel Beckman Foundation and the Boston University Undergraduate Research Opportunities ProgramThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Glucose‐induced dysregulation of lncRNAs in a model of chronic kidney disease
Authors: Tsotakos Nikolaos , Caldwell Eric Adam , Cerny Garret , Hartman Hannah L , Saade Namir 
Published: June 2021

Diabetic nephropathy (DN) is the leading cause for end‐stage renal disease worldwide. Despite the extensive research that has been done to identify biomarkers and novel therapeutic targets for DN, long non‐coding RNAs (lncRNAs) have not been very well studied. This study sought to identify lncRNAs that are differentially expressed in diabetic podocytes compared to normal podocytes. To do so, Human Glomerular Epithelial Cells (HGEC) were cultured in either normal or high glucose environments (5 mM and 25 mM, respectively). Differential expression of lncRNAs was detected using qPCR. HGEC cultured in high glucose levels demonstrated upregulation of MEG3, MEG8, and H19 compared to cells cultured in normal glucose. Additionally, a modest upregulation of Xist was exhibited by HGEC cultured in high glucose. No differences in MALAT1, TUG1, HOTAIR, or NEAT1 were detected. Podocalyxin, a marker of podocyte health, was used as a control. The results indicate that some lncRNAs, specifically MEG3, MEG8, Xist, and H19, are upregulated in diabetic podocytes. Further research is needed to elucidate the mechanism by which these lncRNAs contribute to the progression of DN.Support or Funding InformationThis work was supported by the Research and Development Committee of Dickinson College and by the School of Science, Engineering, and Technology of Penn State Harrisburg.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Complex Formation on Motion in DNA Binding Domains of ARFs
Authors: George Natalie A , Carroll Margaret C , Gonzalez Daniel , Ellis Jamie P 
Published: June 2021

The auxin response factor (ARF) family is a set of DNA binding transcription factors found in the model plant Arabidopsis thaliana in addition to many other food and fuel crops. Along with two other frequently conserved domains, all ARFs contain an N‐terminus DNA binding domain (DBD) that includes a B3 DNA binding motif and a dimerization subdomain. While the structure of example DBDs have been solved, motion within this domain is poorly understood. The interplay of these domains may provide selectivity in binding to variants of target DNA constructs. Our work examines the DBDs of ARF1 and ARF5, a transcription repressor and transcription activator respectively, as ARF5 is known to be more permissive than ARF1 to a larger spacing between binding sites on DNA. Our work directly tests the hypothesis that the ability to bind to a broader range of DNA targets relates to observable differences in structural flexibility. We combine spectroscopic and chromatographic methods of resolving both complex formation and molecular motion for different combinations of target DNA and DBD. We utilize fluorescence spectroscopy as it resolves protein motions and associations that occur on the same timescale as the fluorescent lifetimes (~ns). Labeled proteins can also be studied at low physiological concentrations and within an aqueous buffer. We optimized the expression and purification of the protein as well as two alternative fluorophore conjugation methods to compare thiol‐reactive fluorophore labeling to a newer technique that targets the N‐terminus. Under conditions of known dissociated versus associated states, we apply both steady‐state and time‐resolved fluorescence anisotropy to sense rotational correlation times of these proteins and distinguish subdomain motions in the presence and absence of DNA. Our work demonstrates that the N‐terminal probes are more sensitive to molecular changes likely due to its placement at the hinge between the DNA binding domain and the dimerization domain. Consistent with our hypothesis, ARF5 shifts from a highly dynamic state to a more rigid state upon complex formation while ARF1 remains more unperturbed. These differences in the available structural ensemble provide valuable insights into the structural basis of target specificity as well as the interplay of these transcriptional on‐ and off‐switches.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Phosphorylation Impact on the Behavior of Substrate‐Bound Tau
Authors: Harries Lukas , Donhauser Zachary 
Published: June 2021

Tau proteins are commonly found in neural cells and provide a variety of functions, the most notable being the regulation of microtubule stability and spacing. Tau aggregation has been implicated in several neurodegenerative diseases, namely dementia and Alzheimer's, making tau‐based therapeutics an area of high research interest. Site‐specific phosphorylation of tau has been observed to direct its behavior but can also result in a lower binding affinity of tau to microtubules, increased propensity of tau to aggregate, and cell death. As the dynamics of tau aggregation are poorly understood, more investigation is needed to elucidate tau aggregation and the effects of phosphorylation. Most of the research in this area has been centered on tau's unbound state, requiring more information regarding the dynamics of its oligomerization upon microtubule binding and cause of disassociation leading to aggregation. To study tau‐tau and tau‐microtubule interactions, we utilized a pseudohyperphosphorylated tau containing seven pseudophosphorylation changes and mimicked the surface of negatively‐charged microtubules with mica. Atomic force microscopy was used to study tau bound to mica, quantify tau‐tau intermolecular interactions, and to assess its bound structure. Changes to solution conditions, such as ionic strength or presence of aggregation inducers, were employed to alter tau's structure and its ability to oligomerize. Comparisons of resulting intermolecular interactions and bound‐tau structures between wild‐type tau and pseudohyperphosphorylated mutants are described, in an effort to address the mechanism linking phosphorylation and tau malfunction.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 Studies of GMP Reductase from Escherichia coli
Authors: Harris Darnell , Torelli Andrew T 
Published: June 2021

Guanosine Monophosphate Reductase (GMPR) is an NADPH‐dependent enzyme that catalyzes the reductive deamination of guanosine (G) monophosphate nucleotides into their corresponding inosine (I) monophosphate derivatives. GMPR plays an important role in purine salvage, re‐utilization of free intracellular bases, and contributes overall to the intracellular balance of G and adenine (A) nucleotides. As with many other enzymes in the purine salvage or biosynthesis pathways, GMPR is widespread in nature across eukaryotic and prokaryotic taxa. Given the interest in purine metabolism as a potential target for drug design, GMPR has been characterized both structurally and kinetically in different studies using GMPR derived from several species. We report progress towards solving the crystal structure of GMPR from Escherichia coli in order to extend the insight from existing studies of the E. coli GMPR kinetic and thermodynamic characteristics. Additionally, lessons learned from integrating related research activities into an undergraduate laboratory course will be presented in order to promote adoption of similar course‐based research practices at other primarily‐undergraduate institutions.Support or Funding InformationIthaca College Department of ChemistryThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Molecular mechanism for accurate dephosphorylation of RNA polymerase II during eukaryotic transcription
Authors: Irani Seema , Zhang Yan 
Published: June 2021

The phosphorylation states of the C‐terminal domain of RNA polymerase II (CTD) coordinate eukaryotic transcription by recruiting protein regulators to Pol II. The precise placement and removal of phosphate groups on the specific residues of the heptad repeat of CTD is critical for the fidelity and effectiveness of transcription. In spite of highly similar flanking residues, Ser5 dephosphorylation is carried out efficiently during transcription elongation when Ser2 phosphorylation accumulates, while Ser2 dephosphorylation occurs predominantly at the end of transcription. To understand the molecular mechanisms of how the CTD phosphatases Ssu72 and Fcp1 specifically remove CTD phosphorylation marks in the context of other phosphorylated CTD residues, we used a combination of high resolution MALDI‐TOF mass spectrometry, X‐ray crystallography, and protein engineering to analyze CTD phosphoregulation. Biochemical and bioanalytical characterization revealed that Ssu72 dephosphorylates Ser5 effectively but with much less activity towards Ser7 and Ser2 and no activity against Tyr1 and Thr4. Structural analysis demonstrated that the requirement for cis‐proline substrates of Ssu72 inhibits binding of bulky flanking residues like Tyr1 next to Ser2, which illustrates why Ser5 is the preferred substrate for Ssu72. Phosphorylation of Tyr1 even further prohibits binding of pSer2 by Ssu72, preventing the untimely dephosphorylation of Ser2 at elongation by Ssu72. Finally, we show that Fcp1 not only dephosphorylates phosphorylated Ser2 and Ser5 but also phosphorylated Tyr1. General clearance of phosphorylation marks on the CTD by Fcp1 supports its reported role in recycling Pol II for the next round of transcription. Our results reveal novel roles for Tyr1 in preventing inappropriate dephosphorylation of Ser2 by Ssu72 during transcriptional elongation and for Fcp1 in removing Tyr1 phosphorylation marks at the end of transcription.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Determining Phenotypic Consequences of EmrE Expression Under Various Environmental Stressors
Authors: Beeninga Will , Spreacker Peyton , Henzler‐Wildman Katherine 
Published: June 2021

EmrE is a multidrug resistance transporter found in the inner membrane of E. coli. EmrE has been shown to efflux many different toxic compounds, most notably quaternary ammonium compounds (QACs), such as ethidium bromide (EtBr), and other polyaromatic cations. While this protein has been extensively studied, the native substrate and function of EmrE is unknown. To understand the range of EmrE activity under various environmental conditions, we tested ethidium efflux under different salt and pH conditions. In these screens, we also noted phenotypic differences arise between different knockout strains of E. coli. Additionally, recent literature suggests that variation in carbon sources may also affect expression and/or function of multidrug efflux pumps, such as EmrE. We have screened EmrE efflux activity in E. coli grown on different carbon sources to determine the phenotypic effect for this particular pump.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 mechanism of corepressor‐selective inverse agonism of the nuclear receptor PPARγ
Authors: Kojetin Douglas , Shang Jinsai , Brust Richard , Bass Jared 
Published: June 2021

The structural mechanisms affording ligand‐induced activation of nuclear receptor transcription factor peroxisome proliferator‐activated receptor gamma (PPARγ) are well understood; however, it remains poorly understood how to design transcriptionally repressive PPARγ inverse agonists. We discovered that a simple phenyl‐to‐pyridyl substitution between two common covalent orthosteric ligands targeting PPARγ converts a transcriptionally neutral antagonist into a corepressor‐selective inverse agonist. Using X‐ray crystallography, molecular dynamics simulations, NMR spectroscopy, and mutagenesis coupled to activity assays, we discovered that hydrogen bond formation with Arg288 is essential for corepressor‐selective inverse agonism. For one corepressor‐selective inverse agonist, NMR spectroscopy revealed that PPARγ exchanges between two long‐lived conformations, including a conformation that prepopulates a corepressor‐bound state that primes PPARγ for high affinity corepressor binding. We also determined the crystal structure of corepressor‐bound PPARγ, which reveals several unique conformational features compared to the well studied active conformation. Our findings define new structural features for repressing PPARγ transcription with ligands.Support or Funding InformationThis work was supported by National Institutes of Health (NIH) grants R01DK101871, F32DK108442; and American Heart Association (AHA) award 16POST27780018 (RB)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Calmodulin Induced Changes in the Active Site of Nitric Oxide Synthase
Authors: Sanchez Austin Johnny , Esquerra Raymond , Refuerzo Ruszel 
Published: June 2021

Nitric oxide is a transient signaling molecule in mammalian physiology, known as a vasodilating neurotransmitter and effector during innate immune responses. As such, studies about the family of nitric oxide synthases (NOS) responsible for the rapid conversion of amino acid L‐arginine into nitric oxide have been of great interest. The heme domain within the NOS active site critically binds molecular oxygen which combines with L‐arginine in a series of redox reactions. NOS activity is regulated by a number of cofactors, most notably the ubiquitous regulatory protein calmodulin (CaM) which, in the presence of calcium, binds NOS and promotes nitric oxide production. Though this relationship is well‐documented, the molecular mechanism is not understood. Previously, time resolved spectroscopy has been used to probe the active sites of heme proteins and observe differences in absorption on a nanosecond time frame during changes in the catalytic pockets. Our investigation studies the kinetic effects of CaM binding NOS during the photolysis of carbon monoxide from its heme domain. CaM has been shown to directly influence the active site, promoting availability to substrates and rebinding of ligands to the NOS heme, thus enhancing enzymatic activity. A major facet of our project involves further investigation of the various domains of NOS to determine their significance in CaM‐regulated binding. To this end, we prepared a number of knockout NOS variants which, through assaying via time‐resolved spectroscopy, further elucidate the molecular mechanism of active site regulation.Support or Funding InformationNIH MARC: T34‐GM008574This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The inflammasome adapter ASC and truncated constructs containing only its Death Domains self‐assemble into different macrostructures
Authors: Sandin Suzanne , Nambayan Reinard Jeffery , Quint David , Satyadi David , de Alba Eva 
Published: June 2021

ASC (apoptosis‐associated speck‐like protein containing a CARD) is an adaptor protein that mediates in inflammasome assembly. The inflammasome is a multiprotein complex that triggers the inflammatory response. Multiple copies of a sensor protein, procaspase‐1 and ASC acting as a molecular bridge between them, self‐associate and oligomerize to form the inflammasome particle, a micrometer‐size filamentous ring in the cell cytosol. Thus, ASC self‐assembly properties and interacting capabilities via its two Death Domains (PYD and CARD) are critical for inflammasome formation. Our Transmission Electron Microscopy (TEM) studies show that full length ASC, and the truncated constructs ASCPYD and ASCCARD, form macrostructures with significantly different characteristics. In addition, we have analyzed in detail the ASCCARD–ASCCARD interaction by solution NMR and TEM, which is of particular relevance due to the current lack of information. Our analysis includes CARD mutants designed based on the NMR data.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.