bluuebunny/crossref_metadata_2025_split huggingface dataset:
New Insights into the Assembly Mechanism of an RNA Polymerase III‐Specific Transcription Complex on a Drosophila U6 snRNA Gene Promoter
Authors: Hurlburt Ann Marie , Verma Neha , Phan Phuc , Wolfe Angela , Stumph William 
Published: June 2021

In higher eukaryotes, RNA polymerase III (Pol III) promoters at U6 snRNA genes consist of a TATA box, recognized by TFIIIB, and a proximal sequence element (PSE) recognized by the small nuclear RNA activating protein complex (SNAPc). In the fruit fly Drosophila melanogaster, DmSNAPc consists of three subunits DmSNAP190, DmSNAP50, and DmSNAP43; likewise TFIIIB also consists of three subunits, most commonly TBP, Brf1 and Bdp1. At Drosophila tRNA and 5S RNA gene promoters, TBP‐related factor 1 (TRF1) is utilized in place of TBP, but at U6 promoters the canonical TBP is utilized for Pol III transcription (Verma et al. 2013, JBC 288, 27564–27570).Site‐specific protein‐DNA photo‐cross‐linking studies of DmSNAPc and TFIIIB to U6 promoter DNA indicated that Bdp1 is in close proximity to DmSNAP43 and DmSNAP190 on the U6 promoter (Kang et al. 2016, FEBS Lett 590, 1488–1497). This suggested that the interaction between DmSNAPc and TFIIIB may be mediated, at least in part, by Bdp1. We have investigated this further by electrophoretic mobility shift assays (EMSAs). Surprisingly, we found that DmSNAPc, when bound to the U6 PSE, can recruit Bdp1 to the DNA in the absence of TBP and Brf1. Furthermore, EMSAs indicated that the DmSNAPc‐Bdp1 complex, when bound to U6 promoter DNA, can recruit TBP to form a DmSNAPc‐Bdp1‐TBP‐DNA complex of increased stability.In order to understand the protein‐protein interactions taking place, truncation mutations of Bdp1 were used. It was discovered that an area between amino acid 424 and 510 of Bdp1 is required for its recruitment by DmSNAPc. Furthermore, to investigate whether the TATA box is required for Bdp1 and TBP recruitment, the U6 TATA box was mutated to an unrelated sequence. Although mutation of the TATA box interfered with the recruitment of TBP by the DmSNAPc‐Bdp1 complex, the TATA mutation did not prevent the recruitment of Bdp1 by DmSNAPc. Interestingly, the non‐conserved amino‐terminal tail of TBP contributed to the efficiency of TBP recruitment by the DmSNAPc‐Bdp1 complex.A body of previous work from our lab has shown that DmSNAPc binds to U6 (Pol III transcribed) and U1 promoters (Pol II transcribed) in distinct conformations. Interestingly, when we switched the U6 proximal sequence element A (PSEA) to a U1 PSEA by a 5‐nucleotide change, DmSNAPc was unable to recruit Bdp1. This finding suggests that a surface of DmSNAPc that interacts with Bdp1 may be occluded when DmSNAPc binds to a U1 PSEA. It further provides a mechanism for the polymerase specificity seen when comparing the snRNA U1 and U6 genes.Support or Funding InformationThis work was supported by the National Science Foundation and by the California Metabolic Research Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Changing the Ligand Specificity of a Riboswitch from Guanine to Hypoxanthine
Authors: Garcia Jeremy George , Morse Daniel Paul 
Published: June 2021

Riboswitches are RNA elements in messenger RNAs that regulate gene expression by undergoing a ligand‐triggered conformational change. Previously, we modified several riboswitches for use as sensitive and specific sensors for their ligands. To extend the utility of our sensor design, we developed an in vitro selection strategy to isolate variants of riboswitches that have altered ligand specificity. We initially tested our strategy by attempting to change the specificity of a guanine riboswitch from guanine to hypoxanthine. Our first selection experiment produced only the original, wild‐type guanine riboswitch. In hindsight, this was not surprising since the guanine riboswitch can detect hypoxanthine (albeit poorly), and it was the most abundant RNA in our initial partially‐randomized RNA pool. For our second experiment, we reduced the abundance of the wild‐type riboswitch by increasing the degree of sequence randomization. This experiment resulted in isolating RNAs with many sequence changes and deletions that failed to detect hypoxanthine. We reasoned that these aberrant RNAs arose during PCR with Taq DNA polymerase. Here, we describe the results of a new selection experiment in which we used a proofreading DNA polymerase and a redesigned partially‐randomized RNA pool.Support or Funding InformationDefense Threat Reduction Agency (DTRA)Office of Naval Research (ONR)Chemistry Department, United States Naval AcademyThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Analyzing Protein Kinase C Interactions with Rho4 and Bud3 in the Filamentous Fungus Aspergillus nidulans
Authors: Betton Brianna , Hobson Joanna , Olsen Elisabet , Hill Terry W. , Jackson‐Hayes Loretta 
Published: June 2021

Cell division in filamentous fungi involves coalescence and subsequent constriction of a contractile actomyosin ring (CAR), as in other fungi and metazoans. In fungi, constriction of the CAR is followed by deposition of cell wall material resulting in a septum. In the model organism, Aspergillus nidulans, many proteins are involved in septation including protein kinase C (PkcA), the formin SepA, the GTPase Rho4, and its guanine nucleotide exchange factor (GEF) called Bud3. In previous work we showed that PkcA both physically and functionally interacts with SepA. The formin SepA belongs to the Diaphanous‐related family of formins that contain an N‐terminal Rho binding domain that upon binding of the appropriate Rho GTPase results in formin activation. In A. nidulans Rho4 is likely the GTPase required for SepA activation. Reports from another group have shown that Rho4 and Bud3 work as a module that is required for septation to occur. The current study focuses on the relationship of the Bud3/Rho4 partnership with PkcA and SepA during polarized growth. Using bimolecular fluorescence complementation (BiFC) and overexpressing PkcA in Bud3‐ and Rho4‐null strains, we examined the relationships between PkcA and Rho4 and PkcA and Bud3. Through BiFC we found that PkcA and Rho4 interact at septation sites. Overexpressing PkcA in a Rho4‐null mutant strain increased colony diameter and improved sporulation. However, PkcA overexpression in a Bud3‐null mutant had no effect on growth and sporulation. These results suggest that there are physical and functional interactions between PkcA and Rho4 that are important in polarized growth.Support or Funding InformationNational Science Foundation Grant 1615192 (Loretta Jackson‐Hayes, PI)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Preferential Interaction of Beryllium Ion with Carboxylate‐Rich Peptides
Authors: De Silva Bhagya , Gary Ronald K. 
Published: June 2021

Glycogen synthase kinase 3β (GSK3β) regulates a number of cellular pathways including glycogen metabolism, the Wnt signaling pathway, and cell proliferation. Hyperactivity of GSK3β has been hypothesized to be potentially related to Alzheimer's disease, bipolar disorder, type II diabetes, and some cancers, therefore GSK3β is of interest as a target for therapeutics. Lithium ion (Li+) is a classical inhibitor of GSK3β, but beryllium ion (Be2+) is approximately 1000‐fold more potent. We are interested in the inhibitory mechanism and thermodynamic characteristics for the metal‐protein binding of Be2+ and GSK3β. Be2+ is believed to inhibit GSK3β by competing for magnesium ion (Mg2+) binding sites that feature aspartic acid (D) or other acidic side chains. As a first step toward understanding how Be2+ could outcompete Mg2+ in metal‐protein interactions, we used model chelating compounds and simple peptides that might partially mimic a carboxylate‐rich binding pocket, and compared their binding properties towards Be2+ and Mg2+. Isothermal titration calorimetry (ITC) binding studies for nitrilotriacetic acid, 4,5‐imidazole dicarboxylic acid, and salicylic acid all showed higher binding affinity for Be2+ compared to Mg2+. ITC binding studies were used to compare Be2+ and Mg2+ interactions with three short peptides: DDDD, GGGG and KKKK. Under the conditions employed, Be2+ exhibited binding with the carboxylate‐rich DDDD peptide while Mg2+ displayed no binding. The neutral tetrapeptide GGGG and the polybasic tetrapeptide KKKK served as negative controls; no metal ion interactions were observed with these peptides in the ITC binding experiments. Further analysis with Be2+ and peptides GDDD and GGDD showed reduced binding affinity compared to DDDD. These results demonstrate that Be2+ has stronger affinity for negatively‐charged carboxylate‐rich peptide sequences compared to Mg2+. This strong affinity may lead to conformational changes that distinguish the inactive Be2+‐GSK3β complex from the active Mg2+‐GSK3β complex. Because GSK3β regulates multiple cellular pathways, the ideal therapeutic inhibitor would lead to selective downstream effects. Investigations using an unusual class of inhibitor like Be2+ might provide insight into the variety of modes potentially available for controlling GSK3β activity.Support or Funding InformationThis work was supported by the U.S. Army Research Laboratory and the U.S. Army Research Office under grant numbers W911NF‐15‐1‐0043 and W911NF‐15‐1‐0216.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Exploring the entry route of palmitic acid and palmitoylcarnitine into myoglobin
Authors: Chintapalli Sree V. , Anishkin Andriy , Adams Sean H. 
Published: June 2021

Our previous studies confirmed the interaction of different chain lengths of fatty acids and their derivatives (acylcarnitines) with Oxy‐ and Deoxy‐Myoglobin, using both molecular dynamic simulations and Isothermal Titration Calorimetry experiments. However, those studies were limited to testing only the binding sites predicted from comparative studies of fatty acid binding protein homologs and pre‐calculated binding predictions using AutoDock grid maps. Also, those methods could not address the dynamic processes of the ligand entry into the binding pocket of the protein and the residues involved. To analyze the entry mechanisms of the lipid ligands into myoglobin, and to assess binding specificity at the proposed binding site, we have carried out 80 different molecular dynamic simulations (total length 4.4 microseconds) of murine Oxy‐ and Deoxy‐Mb structures with either the palmitate or palmitoylcarnitine initially placed at different positions near the protein surface. The simulations indicated that both ligands quickly enter the Oxy‐Mb structure through a dynamic area called the “portal region” near the heme group, which forms the entry point for small molecule gaseous ligands like O2, CO and NO. The same phenomenon is not observed with Deoxy‐Mb where the lipid ligands move away from the protein surface, due to a quick compaction of the entry portal and the heme‐containing crevice in the Mb protein upon O2 removal. On the other hand, random starting points for the ligands on the protein surface have shown an “alternate” interaction site which is common for both Oxy‐ and Deoxy‐Mb. The latter observations strongly suggest that spontaneous and “non‐specific” binding of long‐chain fatty acids and acylcarnitines to Mb is driven by hydrophobic forces, in contrast with the “portal region” crevice being a primary and specific binding spot that can be precluded by deoxygenation. Considering the importance of lipid metabolism and trafficking in oxidative, Mb‐rich tissues such as type 1 skeletal muscle and cardiomyocytes, identifying the mechanisms of entry, binding and off‐loading of long‐chain fatty acids and acylarnitines on Mb may have important ramifications for muscle management of bioactive lipids.Support or Funding InformationUSDA‐ARS Project 6026‐51000‐010‐05SThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Understanding How Distal Residues Play a Role in Parkin Activity
Authors: Winters Jenifer , Beuning Penny , Makowski Lee , Ondrechen Mary Jo 
Published: June 2021

Partial Order Optimum Likelihood (POOL) is a machine learning method that predicts catalytically important residues based on the tertiary structure of the protein, with computed chemical and electrostatic properties of the residues as input features. POOL has predicted spatially extended active sites, where residues that are not in direct contact with the substrate still contribute to catalysis for many enzymes, including for parkin. Parkin is an E3 ubiquitin ligase protein involved in the ubiquitin‐proteosome system that mediates the targeting of proteins for degradation. Mutations in parkin lead to the accumulation of toxic substrates that damage dopaminergic neurons, causing the autosomal recessive form of Parkinson's disease (PD). POOL has predicted a dozen residues in parkin that are important for catalytic activity. Interestingly, half of these residues are positions that when mutated lead to the onset of PD. Enzyme variants were created through single‐site directed mutagenesis to further look into how these residues may play a role in catalysis through biochemical and biophysical techniques.Support or Funding InformationSupported by NSF MCB‐1517290This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Binding Association and Kinetic Characterization of DesD Reveals High Substrate Specificity and Cooperative Behavior
Authors: Tran Leslie , Hoffmann Katherine M. 
Published: June 2021

Siderophores, small iron transporting molecules, are secreted by bacteria when there is an insufficient amount of iron. These molecules have high affinity for ferric ions and often outcompete the host chelators. Siderophores synthetase are becoming increasingly associated with virulence in pathogenic bacteria as they are able to evade the human immune response system. One type of siderophore biosynthesis is through the Nonribosomal peptide synthetase Independent Siderophore (NIS) pathway. This project is interested in utilizing the NIS synthetase family's unique fold and novel chemistry as a drug target for new antibiotics.This project is focused on DesD from Streptomyces coelicolor found mostly in soil. DesD is the last enzyme in the Desferrioxamine E siderophore synthetase pathway. This enzyme is a model for the NIS synthetase family, however, while it has been studied before, there are no good results concerning kinetics. Prior literature has reported a broad substrate specificity but the binding has never been quantified. The preliminary data on binding and kinetics collected from Isothermal Titration Calorimetry (ITC) have revealed high substrate specificity via binding constants, and cooperative kinetic turnover. These baseline studies with be used in future work to quantify inhibitor effectiveness.Support or Funding InformationThis work was supported by a grant from the National Science Foundation (MCB ‐ 1716986) to K.M.H, and by California Lutheran University.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
C. elegans Avoids Toxin‐Producing Streptomyces
Authors: Miller Conrad Laura C. , Tran Alan , Tang Angelina , O'Loughlin Colleen T. , Balistreri Anthony , Chang Eric , Villa Doris Coto , Li Joy , Varshney Aruna , Matthews Sarah , Bremmer Martina , VanHoven Miri K. 
Published: June 2021

Species of the non‐motile bacterium Streptomyces protect themselves from predation by the bacteriovore Caenorhabditis elegans through the secretion of nematicides. To evade death by these agents, C. elegans has evolved the ability to detect and avoid the bacterium. We characterize fatty acid analogs, including dodecanoic acid, which elicit a fast avoidance response by C. elegans. We also detect dodecanoic acid that has been secreted by Streptomyces by derivatization and detection with high resolution mass spectrometry. By sensing these signals, C. elegans can escape the nematicidal bacteria that would otherwise be a good food source.Support or Funding InformationLaura Miller Conrad: National Institutes of Health (5SC3GM118199); Angelina Tang and Sarah Matthews: National Science Foundation (HRD‐1302873); Miri VanHoven: National Institutes of Health (NS087544, GM089595), National Science Foundation (1355202).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
P. gingivalis lipopolysaccharide‐induced exacerbation of oxidized LDL‐mediated macrophage foam cell formation is reliant on TRPV4 channels
Authors: Rahaman Shaik O. , Gupta Nabyendu , Goswami Rishov 
Published: June 2021

Although P. gingivalis, a predominant causative agent of periodontitis, has been linked to lipidladen macrophage foam cell formation, a critical process in atherogenesis, the mechanism remains elusive. Emerging data support a role for both a biochemical factor, e.g., lipopolysaccharides (LPS), and a mechanical factor, e.g., matrix stiffness, in regulation of macrophage function, vascular elasticity, and atherogenesis. We have obtained evidence that TRPV4, an ion channel in the transient receptor potential vanilloid family, and a known mechanosensor, is the likely mediator of P. gingivalis lipopolysaccharide (PgLPS)‐induced exacerbation of oxidized low‐density lipoprotein (oxLDL)‐mediated macrophage foam cell formation. Specifically, we found that: i) genetic deletion of TRPV4 or antagonism of its function by a chemical inhibitor blocked PgLPS‐induced exacerbation of oxLDL‐induced macrophage foam cell formation; ii) macrophage TRPV4 activity was increased in response to both PgLPS and pathophysiological range matrix stiffness, and iii) TRPV4 deficiency blocked PgLPS‐induced exacerbation of matrix stiffness‐induced macrophage foam cell formation. Mechanistically, we show that TRPV4 regulates PgLPS‐induced oxLDL uptake but not its cell surface binding in macrophages. Altogether, these findings identify a critical role for TRPV4 in regulating PgLPS‐induced exacerbation of macrophage foam cell formation by modulating uptake of oxLDL.Support or Funding InformationAHA (13SDG17310007), Startup grant from University of Maryland, NIH (1R01EB024556‐01), and NSF (CMMI‐1662776) grants to SOR.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
In vivo electrochemical assessment of dopamine release during conditioned withdrawal in heroin dependent rats
Authors: Pultorak Katherine , Schelp Scott , Oleson Erik 
Published: June 2021

The centers for disease control and prevention reports that heroin abuse has doubled for Americans aged 18–25. Drugs of abuse, such as heroin, are commonly thought to increase the concentration of dopamine (DA) in the nucleus accumbens core (NAcc), although their effects on phasic DA release events remains to be fully characterized. In contrast, when an addicted subject is withdrawn from a substance such as heroin, there is a clear suppression of phasic DA activity in the NAcc. Heroin withdrawal presents characteristic symptoms, most predominately increased craving for the drug, which contributes to further abuse. While withdrawal symptoms have been shown to be conditioned to previously neutral stimuli through classical conditioning, there is a lack of electrochemical evidence of conditioned withdrawal and its effects on non‐drug related stimuli. Here, we sought to explore how conditioned withdrawal in heroin addicted rats affects DA. We provided rats 23 hour access to IV heroin over 28 days. The 28 days are split into four blocks of increasing doses ranging from 0.06 mg/kg to 6 mg/kg to more accurately simulate the progression of heroin addiction. During the 28 day history, circadian changes in heroin, food, and water intake were altered and overall consumption of heroin increased. We then, investigated how conditioned withdrawal affects cue‐evoked dopamine release in the NAcc using fast scan cyclic voltammetry (FSCV) during conditioned suppression of food maintained responding. Here, animals were first trained to respond for food under an FR1 schedule for 3 consecutive days where food availability was predicted by a cue‐light placed above the lever. This was followed by 3 consecutive days of conditioned withdrawal training where a tone was conditioned to naloxone‐precipitated withdrawal. On test day, animals responded for food under normal conditions, but the withdrawal associated tone was presented concurrently with cue‐light onset during 50% of trials. By simultaneously presenting the withdrawal paired tone with the food predictive cue light we observed a substantial suppression of DA concentration at the cue light relative to non‐paired trials. These data demonstrated that conditioned suppression of food maintained responding induced by conditioned heroin withdrawal is accompanied by a transient suppression in dopamine concentrationSupport or Funding InformationFunding for the project was provided by NSF grant IOS‐1557755, NIH grant R03DA038734, Boettcher Young Investigator Award and NARSAD Young Investigator Award to EBO and an institutional UROP to KJP.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Observing Signaling Dynamics at Endogenous Levels Using a New Class of Biosensors FluoSTEP
Authors: Zhang Jason Zhaoxing , Tenner Brian , Huang Bo , Mehta Sohum , Zhang Jin 
Published: June 2021

Intracellular signaling is highly spatiotemporally regulated with specific kinetically‐distinct microdomains. Fluorescent biosensors such as FRET‐based kinase activity reporter, second messenger indicators, and GTPase activation biosensors have facilitated the elucidation of the spatiotemporal regulation for various signaling protein networks; however, overexpression of these biosensors could perturb endogenous signaling. This is especially problematic for GTPase activation biosensors as they contain the GTPase within the sensing domain, thus, causing artificially high GTPase signaling activity. To overcome these challenges, we designed a novel class of fluorescent biosensors named Fluorescent Sensors Targeted to Endogenous Proteins (FluoSTEPs) that utilizes a split fluorescent protein approach to target a specific protein and reconstitute a functional sensor. We have shown that this design is generalizable for various FRET–based biosensors. Furthermore, combining this design with CRISPR knock‐in technology, we demonstrated the application of FluoSTEP in studying cAMP and PKA signaling in the native clathrin‐coated pits and Sec61B microdomains. With the capability of reporting endogenous signaling in the native context of a living cell, FluoSTEP will be powerful tools to investigate spatiotemporal regulation of signaling enzymes such as kinases and GTPases.Support or Funding InformationNIH R01 DK073368 and R35 CA197622 (to JZ), NSF GRFP (to JZZ)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.