bluuebunny/crossref_metadata_2025_split huggingface dataset:
Dimerization of ADARs Expands The Range of Substrates That Can Undergo A‐to‐I RNA Editing
Authors: Hundley Heather A. , Rajendren Suba , Manning Aidan C. , Takagi Yuichiro 
Published: June 2021

RNA editing alters genomically encoded information to generate the transcriptomic diversity required for normal development and proper neuronal function. The adenosine deaminase that act on RNA (ADAR) family of enzymes catalyze adenosine (A)‐to‐inosine (I) RNA editing at millions of sites in the human transcriptome. However, the molecular mechanisms that regulate editing specificity and efficiency are not understood. Using recombinant proteins, we reveal, that unlike all other A‐to‐I editing enzymes, Caenorhabditis elegans ADR‐2 has a low affinity for double‐stranded RNAs (dsRNAs). Using both in vivo and in vitro approaches, our data indicates that ADR‐2 can physically interact with the dsRNA binding protein, ADR‐1, which is also a deaminase‐deficient member of the ADAR family. Our data from RNA‐immunoprecipitation studies of ADR‐2 indicate that ADR‐1 is required for ADR‐2 to stably interact with some target mRNAs in vivo. Furthermore, biochemical editing assays from lysates expressing ADR‐1 mutant proteins indicate that both RNA binding by ADR‐1 and the protein‐protein interaction between ADR‐1 and ADR‐2 promotes RNA editing efficiency. In sum, we have identified a molecular mechanism for regulating the ability of ADAR to both bind target mRNAs and edit. As human ADARs are known to have some overlapping substrate specificities, these results raise the possibility that dimerization between ADAR family members, and perhaps protein‐protein interactions of ADARs with other dsRNA binding proteins, provides the A‐to‐I editing enzymes with a molecular mechanism to specifically recognize editing targets over the thousands of RNAs present in a cell.Support or Funding InformationAmerican Cancer Society Research Scholar Grant (15‐051‐RMC) to H.A.H. Indiana University School of Medicine Start‐up Funds to H.A.H.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A System for Global Analysis of Correlation between Protein Expression and mRNA
Authors: Johnson Kara , Zhong Sheng 
Published: June 2021

The influence of mRNA abundance on protein expression continues to be a topic of significant interest and debate. In various systems, the correlations between these molecules has been reported to be anywhere from 20–70%. Many of these studies have been conducted in complex, in vivo systems, and assayed just a fraction of the genes present due to difficulties in quantitation of many proteins simultaneously. Our approach is to examine this relationship as the result of a single translational event, in a global manner, and without the additional factors introduced by in vivo systems. We created a protein library whose constituents can be decoded by next generation sequencing using cDNA display techniques, a synthetic, bacterial based, translation system, and an expression library derived from an mRNA population. This data, along with sequencing data from the precursor mRNA library, allow for a correlation analysis across all genes contained within the mRNA library. Preliminary results indicates that in this system, ~60% of the variance in protein expression can be explained by mRNA abundance. To determine the stability of the correlation, we modified the translation sequences in the gene library and reanalyzed the protein to mRNA relationship; variations in the correlation reflect the influence of the translational sequences on the protein expression. This system can be utilized to explore the mRNA and protein relationship for wild‐type translational systems, or to evaluate the effects of modifications or synthetic sequences.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification and Functional Testing of Six Interactors of the Transmembrane Sensor Mtl1p in the Budding Yeast Saccharomyces cerevisiae
Authors: Martinez‐Matias Nelson , Rodriguez‐Medina Jose 
Published: June 2021

Protein–protein interactions (PPIs) are often central to their cellular functions. These functions are often influenced by PPIs at all levels, including structural, energy metabolism, and signaling processes among others. The latter are crucial, as signaling functions often promote cell viability. We hypothesize that cytosolic protein interacting partners of the transmembrane sensor Mtl1p in Saccharomyces cerevisiae have a role in the stress response, promoting cell viability by modulation of the signaling process through the PKC1–dependent Cell Wall Integrity (CWI) Pathway.ObjectiveThe objective of our study is to identify cytosolic interactors of Mtl1p and to assess their relative importance in promoting cell viability upon exposure to diverse environmental stresses.MethodologyUsing membrane yeast two hybrid (MYTH) assays we have identified nine full–length proteins, and six other partial clones with C–terminals of various lengths that interact physically with the cytosolic domain of Mtl1p. In addition, using Western blots and growth curves we have made functional tests of deletion mutants corresponding to five of the identified full–length interactors, FPR1, ZEO1, RPL40a, EGD2, and CPR1, and one of the truncated interactors, RAS2, to assess their relative importance in promoting cell viability under oxidative the stress produced by exposure to H2O2, and cell wall stress produced by the antifungal Caspofungin.ResultsIn CSM broth cultures at 27°C, growth of the ras2Δ and zeo1Δ strains was severely impaired by 8 hours treatment with 0.5 mM H2O2, with a 50% reduction of cell concentration yields compared to the untreated wild type strain (WT) which is our reference standard. As determined by WB analyses, the ras2Δ strain treated with 1 mM H2O2 for two hours showed reduced levels of PKC1–Slt2p accumulation, indicative of CWI Pathway signaling, when compared to both the untreated ras2Δ and WT strains. Compared to the untreated WT, all three strains exhibit severely impaired growth when treated with 10 ng/ml Caspofungin at 27°C, especially the zeo1Δ strain. Moreover, all three strains show increased levels of PKC1–Slt2p signaling when treated with 75 ng/ml Caspofungin for two hours.ConclusionsOur results show that selective deletion of Mtl1p interactors like Ras2p or Zeo1p in S. cerevisiae are required to confer stress–specific susceptibility phenotypes. We propose that Ras2p modulates PKC1‐dependent Slt2p signaling on exposure to H2O2, perhaps directly by interacting with Mtl1p or indirectly by exerting an influence on other signaling pathways through a cross‐talk mechanism.Support or Funding InformationThis work is supported in part by NIH grants from NIGMS‐INBRE P20GM103475, NIGMSRISE R25GM061838, and NIMHD‐RCMI G12 MD007600.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Bending and Wrapping of Upstream Promoter DNA on E. coli RNA Polymerase Facilitates Open Complex Formation in Transcription Initiation; A Fluorescence (FRET, PIFE) Study
Authors: McNerney Christina Lynne , Callies Katelyn , Cimperman Clare Kai , Chittur Priya , Sreenivasan Raashi , Chhabra Munish , Record M. Thomas 
Published: June 2021

Removal of the far‐upstream region of promoter DNA reduces the isomerization rate of closed complexes (CC) to open complexes (OC) of Escherichia coli RNA polymerase (RNAP) and λPR DNA by 1–2 orders of magnitude. Upstream DNA is proposed to facilitate OC formation by wrapping on RNAP and remodeling the downstream cleft to allow bending of the downstream duplex into the cleft prior to opening. Here we report stopped flow fluorescence kinetic studies of open complex formation at λPR promoter DNA labeled at far‐upstream and downstream positions with Cy3 and/or Cy5 fluorescent probes. We determine the time course of forming the CC ensemble (10 ms – 3 s at 19 C), in which the upstream and downstream duplex DNA are bent and wrapped on RNAP. Cy3 to Cy5 FRET (fluorescence resonance energy transfer) detects wrapping, and single‐dye PIFE (protein‐induced fluorescence enhancement) detects transient contacts of the far‐upstream DNA with the downstream cleft, and contacts of the downstream DNA as it is bent into the cleft. Subsequently (3 s – 400 s) we determine the time course of the isomerization of the CC ensemble to OC, in which the upstream DNA remains bent and wrapped but where contacts of the upstream DNA with the downstream cleft are released. These results are consistent with the model for facilitation of isomerization by wrapping of upstream DNA, and with previous footprinting and kinetic information.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Activation and auto‐destruction of the ubiquitin ligase Itch
Authors: Angers Annie , Ayoubi Riham , Desrochers Guillaume 
Published: June 2021

Ubiquitin ligases of the Nedd4 family are maintained inactive by intramolecular interactions (1). We reported that, like Nedd4, the closely related ubiquitin ligase Itch strongly binds to the endosomal protein LITAF through its WW domains, that recognize two PPXY motifs in the N‐terminal half of LITAF. Unlike Nedd4, Itch binding to LITAF brings the ligase to the lysosomal compartments. We thus postulated that LITAF overexpression might bring Itch to the lysosome, where it would access different substrates and target them for degradation (2). Concomitantly, LITAF binding to Itch may increase Itch ligase activity, as other PPXY motif proteins such as NDFIP and LRAD3 (3,4). Here, we set up to determine if Itch ubiquitin ligase activity is indeed up regulated by LITAF binding, and if Itch most common substrate degradation is increased in response to LITAF binding to Itch. We find that Itch, WWP1, WWP2 and Nedd4 all bind to LITAF, and that the four ligases autoubiquitylation is greatly increased upon LITAF binding, albeit to different levels. Both LITAF overexpression and addition of recombinant LITAF to crude cell lisates lead to rapid degradation of Itch, and, to a lesser extent, Nedd4. Itch substrates cJun and Endophilin degradation was also increased. In vitro ubiquitylation assays show that Itch autoubiquitylation is enhanced in the presence of LITAF, but substrate ubiquitylation was not significantly increased. In conclusion, LITAF binding strongly activate Itch autoubiquitylation, as well as that of other ligases of the Nedd4 family. The consequence of this activation is Itch proteasomal degradation, with little effect on its substrates ubiquitylation. LITAF is thus likely to be a negative regulator of Itch in cells where it is strongly expressed.Support or Funding InformationNatural Sciences and Engineering Research Council of Canada (NSERC)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural and Biochemical Analyses of Bifunctional Alcohol Dehydrogenase Enzymes from Entamoeba spp.
Authors: Gabrielle Matthew , Espinosa Avelina 
Published: June 2021

The Entamoeba lineage belongs to the Amoebozoa, one of six major divisions of eukaryotes. Entamoeba trophozoites, similar to other protists, actively capture ingest and digest bacteria thorough anaerobic pathways that process diverse energy sources. Anaerobic pathogens have evolved adaptive metabolic enzymes that differ from vertebrates, and are ideal targets for novel compounds to improve disease management. The bifunctional alcohol/ aldehyde dehydrogenase enzyme in Entamoeba histolytica (EhADH2) belongs to the ADHE iron dependent family, and is essential for trophozoite growth and survival. EhADH2 catalyzes the conversion of acetyl Co‐A to acetaldehyde and the final reduction of acetaldehyde to ethanol by its separate ADH and ALDH domains respectively. Several Entamoeba spp have homologous genes (ADHE) which might share a similar structure and function to that of the ehAdh2 gene. This study aims to clone, express, and sequence the genes that encode for ADHE in three Entamoeba spp. The sequencing identifies evolutionary relationships between the homologs. The transformation of ADHE genes into E. coli cells will be used for protein purification and kinetic assays for comparison with EhADH2. Kinetic assays will provide Km values for both substrates and information on chemical inhibitors of the ADHE proteins. Inhibitors of this enzyme have promising anti‐amoebic capabilities for drugs due to trophozoite growth dependence on ADHE. These studies will further the knowledge about the ecology and evolution of ADHE enzymes and their relevance for host survival and pathogenesis.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Determining the active site base and order of substrate addition within F420‐dependent glucose‐6‐phosphate using steady‐state and pre steady‐state kinetics and isotope effects methods
Authors: Johnson‐Winters Kayunta , Oyugi Mercy , Davis Lindsay , Bashiri Ghader , Baker Edward N. 
Published: June 2021

F420‐dependent glucose‐6‐phosphate dehydrogenase (FGD) is a Mycobacterial enzyme that catalyzes the conversion of glucose‐6‐phosphate (G6P) to 6‐phosphogluconolactone using oxidized F420. This enzyme is important within Mycobacteria tuberculosis, which is the causative agent of tuberculosis disease. Our work has focused on probing the FGD reaction mechanism using kinetic and isotope effects methods. Based upon a previous crystal structure FGD, there were two conserved amino acids, His40 and Glu109, which were we believed to function as the active site base and acid, respectively. Based upon pH profiles, we have determined that while Glu109 does serve as the active site acid, H40 does not serve as the active site base. We have identified several other amino acid candidates that could act as the active site base and will be discussed. A concurrent goal was to probe the FGD reaction mechanism using dead‐end inhibition experiments, along with solvent and substrate deuterium isotope effects studies. The dead‐end inhibition studies were conducted using the competitive inhibitor, citrate. The data revealed competitive and uncompetitive inhibition patterns for G6P and F420 respectively, thus suggesting a mechanism of ordered addition of substrates in which the F420 cofactor must first bind to FGD before G6P binding. Our proton inventory data yielded a fractionation factor of 0.37, suggesting that the single proton responsible for the observed solvent kinetic isotope effect is likely donated by Glu109 and protonates the cofactor at position N1. The steady state substrate deuterium isotope effects studies using G6P and G6P‐d1 yielded kinetic isotope effect (KIE) of 1.1 for both kcat and kcat/Km, while the pre‐steady state KIE on kobs was 1.4. Because the hydride transferred to C5 of F420 was targeted for isotopic substitution, these KIE values provide further evidence to support our findings that hydride transfer is likely not rate‐limiting in the FGD reaction.Support or Funding InformationThis work was supported by NIH Grant 1R15GM113223‐01A1 (to KJW) and the Health Research Council of New Zealand Grant HRC 12/1111 (to E.N.B.)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 new ligand Architecture derived from Purple‐Acid Phosphatase type and their interactions with Oximes: towards useful catalyst for dephosphorylating pesticides.
Authors: Le Natasha T. , Allard Marco M. 
Published: June 2021

The use of Organophosphorous compounds (OPCs) is widely spread, and their use can result in nutrient pollution in soils, waterways. OPCs are also commonly used as chemical warfare agents and are generally considered dangerous and toxic. Accidental or intentional exposure to such chemicals necessitate fast‐acting antidotes, which are currently clinically limited to some Oxime compounds. Oximes act as a Lewis base attacking the organophosphorus compound, effectively cleaving OPCs at the active sites. We are exploring novel methods of OPCs detoxification based on new complexes and architectures inspired by the enzyme Purple‐Acid phosphatase. Our complexes include an electron rich ligand donor and a non‐innocent ligand as a substitute for a second metal site. Tests were carried out using (4‐nitrophenyl) phosphate as a substitute for OPCs, and kinetics data was plotted using UV‐Vis spectroscopy. Experiments are carried out in the presence of a mild reducing agent, and different pH buffers to study their effect on the kinetics of degradation. Computational methods are explored to elucidate a plausible mechanism. These complexes were also tested in the presence of various substituted oximes and their kinetic behavior reported alongside mechanisms.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identifying the Target Protein of an Antipyocyanin Compound in Pseudomonas aeruginosa Using Photoaffinity Labeling
Authors: Moore Rebecca , Aboulhosn Kareem , Conrad Laura Miller 
Published: June 2021

Pseudomonas aeruginosa is an opportunistic Gram‐negative bacterium affecting those with weakened immune systems including burn victims, surgical implant patients and those with cystic fibrosis. Due to the rise in antibiotic resistance, our lab has developed an antivirulence approach to combat this pathogen. The current, and increasingly less effective, method is the antimicrobial approach that either kills the bacteria or prevents bacterial growth. An antivirulence strategy deactivates pathogenicity, here through the inhibition of redox‐active pyocyanin, a key virulence factor produced by the P. aeruginosa, which then allows the host's immune system to clear the infection on its own. We have developed a small molecule that disrupts production of pyocyanin through an unknown mechanism. Our primary objective is to identify the molecular target with a photoaffinity labeling (PAL) approach by incorporating a photoreactive diazirine ring on our inhibitor as well as a bioorthogonal reactive group, which is helpful for protein purification. We have successfully synthesized and characterized a key intermediate and will present our progress toward the target compound. PAL is a promising approach that has been successfully used to determine protein targets through the covalent bond formed upon irradiation with UV light in recent identification studies. By using affinity chromatography and proteomics we will to identify candidates for the molecular target that can be confirmed in further studies. We ultimately aim to characterize additional processes that control pyocyanin production. This information could provide new targets for future antivirulence therapeutics to combat this threatening pathogen.Support or Funding InformationThis research is funded by San Jose State University and CSUPERB.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Anticancer Effect of Silibinin B on Colorectal Cancer Cell Line
Authors: Horita Megan Ann , Ezekiel Uthayashanker 
Published: June 2021

Colorectal cancer is the third leading cause of cancer‐related mortality in developed countries. The most common treatment for colon and rectal cancers is surgical resection followed by therapy with anti‐neoplastic drugs, which has been shown to have toxicity and negative side effects. The purpose of this study was to identify anticancer effects of silibinin B in colorectal cancer cells. Silibinin B, the major bioactive component of Silybum marianum (milk thistle), has been used as a protective supplement against liver disease and is hypothesized to have anticancer properties. Silibinin B is known to play a key role in cell cycle arrest, apoptosis, and autophagy. We investigated the anticancer effect of silibinin B on a colorectal cancer cell line (DLD‐1). In DLD‐1 cells, viability decreased at higher concentrations of silibinin B as measured by Crystal Violet method. The cell death caused by silibinin B was mediated by apoptosis and autophagy. Western blot analysis of treated cells compared to the control exhibited an increase in autophagy markers, beclin‐1 and LC3, and apoptotic markers, cleaved caspase 3 and PARP. Future studies will focus on molecular pathways that mediate apoptosis and autophagy.Support or Funding InformationMH was supported by DeNardo Education and Research Foundation Fellowship.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Genome‐Mining in Pseudomonads Identifies a New Class of Small Molecules
Authors: Kretsch Ashley , Morgan Gina , Maria Kevin Santa , Tyrrell Jillian , Vallet‐Gely Isabelle , Li Bo 
Published: June 2021

Bioactive small molecules often play an important role in bacterial virulence. Identification and exploration of these molecules will help develop an understanding of new mechanisms for infection and potentially identify novel targets to inhibit virulence. Analysis of bacterial genomes identifies a large number of gene clusters that encode for small molecule‐synthesizing enzymes (biosynthetic gene clusters), many of which are unexplored. Through genetic manipulation of these clusters, we can discover their small molecule products and determine their biological roles.We have identified a biosynthetic gene cluster conserved in over 300 strains of Pseudomonads. Many of these strains engage in pathogenic or symbiotic relationships with a range of human, animal, and plant hosts. We have shown that deletions within this gene cluster, named the pseudomonas virulence factor (pvf), reduce fly infection by the pathogen Pseudomonas entomophila. In particular, the small molecule products of the pvf pathway are suggested to play a role in bacterial signaling and activation of virulence.Through overexpression of the pvf cluster in its native P. entomophila strain, we have discovered and characterized a number of small molecules that the pvf gene cluster is responsible for. These molecules include a class of pyrazine compounds, some of which are new to biology. We use fly infection and promoter‐reporter assays to determine the biological roles of these molecules in virulence and cell‐to‐cell signaling of P. entomophila. Pyrazines have been previously identified as volatiles in a number of bacteria, but their biological role and biosynthetic origins remain unknown.Our work provides novel insights into the biosynthesis of pyrazines. By characterizing the structures and functions of bioactive small molecules from pathogens, our work has the potential to develop an understanding for the production of these compounds and reveal novel and useful antimicrobial targets.Support or Funding InformationThis work was supported by the Burroughs Wellcome Fund Fellowship In Organic Chemistry and the University of North Carolina, Chapel Hill.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.