bluuebunny/crossref_metadata_2025 huggingface dataset:
Defining Molecular Calipers: Affinity and Exchange of Auxin Response Activators and Repressors
Authors: Carroll Margaret C. , Ellis Jamie P.
Published: June 2021

Auxin response factors (ARFs) regulate transcription of developmental genes in the auxin hormone pathway which affects a broad range of valuable plants including food crops and biofuel targets. Both transcription activators and repressors within the ARF family consist of three domains including a protein interaction domain, intrinsically disordered middle region and a DNA binding domain containing plant‐specific B3 domain and the ability to dimerize. The many layers of interactions, as well as a multiplicity of conserved factors within a single plant, suggest a more complex transcriptional regulation system than a simple on/off activation switch. Arabidopsis thaliana is a well‐understood model from which we chose an example transcription repressor (ARF1) and activator (ARF5). Both are present concurrently during seed embryogenesis, bind the same high‐affinity Auxin response element (AuxRE) sequence, and form homodimers in response to DNA constructs with AuxRE everted repeats (ER). We combined genomic analysis with biophysical studies to explore ARF binding and exchange on biologically relevant constructs. Through genomic analysis, we analyzed frequency and spacing of canonical and other high‐affinity AuxRE sites including both everted and direct repeats. The results reveal a much wider range of spacings between the repeats in the genome than tested in the literature with clear indications of periodicity. Utilizing a combination of fluorescence spectroscopy and gel filtration, we directly tested the association and exchange of AFR1 and ARF5 on DNA of varying repeat spacing and AuxRE sites. Control experiments indicate that fluorophore attachment does not affect dimerization or DNA binding of the proteins. FRET studies showed that dimerization clearly increases in the presence of AuxRE DNA. Varying DNA constructs indicates both binding site affinity and the spacing between sites affects the amount of dimer formed, but that a repeated sequence is not necessary to cooperatively form dimers. While ARF1 and ARF5 have different spacing preference, the high‐affinity binding site can overcoming spacing preference. The differences in dimer formation indicate that the spacing between everted repeats effects the protein's ability to bind DNA. The variation in spacing discovered in the Arabidopsis genome paired with the difference in activity with different spacings indicates that spacing motifs regulate ARF factors.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inactivation of Gene α‐1,3‐Galactosyltransferase in Bovine Aortic Smooth Muscle Cells Using CRISPR‐Cas9
Authors: Wang Xiaodan , Apple Jason , Huang Yan
Published: June 2021

People who get bites from Amblyomma americanum usually develop a severe allergic reaction to red meat. The main reason caused this allergy is because of the presence of α‐1,3‐galactosyl epitopes on the membranes of most mammalian cells. Since human beings do not create galactose‐alpha‐1,3‐galactose (alpha‐gal), the patients' immune system is free to start attacking alpha‐gal when they eat red meat. The epitope transferase coded geneα‐1,3‐galactosyltransferase (GGTA1) was already identified, and it can be knocked out to solve the allergy of eating red meat. GGTA1 gene has successfully knocked out in pigs. However, there is no evidence to show the GGTA1 gene has been knocked out in beef. In this study, we utilized bovine aortic smooth muscle cells (BAOSMC) as the module for the research of GGTA1 gene knockout.

The clustered regularly interspaced short palindromic repeats (CRISPR) systems are widely used in performing targeted genome editing in cultured cells. The use of CRISPR‐associated (Cas) systems as an RNA‐programmable DNA targeting and editing platform is followed by a synthetic single‐guide RNA (sgRNA), which can simply edit genome sequences like other tools such as transcription activator‐like effector nucleases (TALENs) and zinc‐finger nucleases (ZFNs).

We used the gRNA design tool and selected 5′‐GGCCTGACGGTTTTCGCCGT‐3′ as the target gRNA sequence from the coding DNA sequence of Bos taurus alpha‐galactosyltransferase 1 (glycoprotein). The gRNA was constructed in the pSpCas9 BB‐2A‐GFP (PX458) vector provided by GenScript USA Inc. Vectors were amplified and transfected into BAOSMC by GenePORTER2 transfection reagent when the cells were 80% confluency. Green fluorescent can be viewed after 24 hours transfection (Fig. 1). The transfection efficiency can reach about 70% to 80%.

Cells were collected in PBS at pH7.4 after 24 hours transfection. Total protein was extracted then the enzyme‐linked immunosorbent assay was used to examine the GGTA1 production. By normalized with the total protein concentration, the GGTA1 protein level in the transfected cells was 17.9 ± 7.25% lower (P< 0.05) than in the control cells, showing a significant inhibition of GGTA1 gene expression in the cells by CRISPR‐Cas9 gene edition method (Fig. 2).

Our data show that the gRNA sequence that we chose was suitable for the GGTA1 gene knockout in BAOSMC. Moreover, the CRISPR‐Cas9 system was proved can be applied in the genome editing of bovine cells.

Support or Funding Information

This study is funded by Arkansas Beef Council.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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 Information

American 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.

Objective

The 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.

Methodology

Using 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.

Results

In 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.

Conclusions

Our 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 Information

This 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 Information

Natural 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 Information

This 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 Information

This 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 Information

MH 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 Information

This 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.
Investigation of New Chemical Probes for Detection of Citrulline Using Small Molecule Model Systems
Authors: Delaney Brian , Laufenberg Brandon , Kadnikov Dmitry V.
Published: June 2021

Citrulline is an amino acid formed via post‐translational modification of arginine. Citrullination of proteins underpins development of such health problems as rheumatoid arthritis, multiple sclerosis, other autoimmune diseases, and perhaps other diseases such as Alzheimer's disease. Current probes based on phenylglyoxal exist but are lacking in stability, are difficult to synthesize, and are expensive to produce. A more viable probe that could properly detect concentrations of citrulline in the body could help with early diagnosis of patients affected by these diseases. The goal of this project is to create new, more stable probes for the detection of citrulline in the body. Currently, new reactive heads based on protected phenylglyoxal have been synthesized and their reactions with small molecules mimicking citrulline are being investigated.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Differential Acylation on Aberrant Growth Signaling by Overexpressed Gα13
Authors: White Nicholas F. , McDiarmid Gray R. , Tagliatela Alicia C. , Meigs Thomas E.
Published: June 2021

Overexpression of Gα13, a member of the G12/13 subfamily of heterotrimeric G proteins, has been implicated in cell transformation and the progression of several cancer types. The mechanism through which wildtype, overexpressed Gα13 drives aberrant growth signaling is not known. G protein α subunits are modified post‐translationally by acylation at the N‐terminus, and this addition of a palmitoyl and/or myristoyl group occurs for all mammalian Gα proteins. Gα13 was previously engineered to mutate two cysteine residues necessary for dual palmitoylation. This modification abrogated signaling to serum response factor by Gα13 in both its wildtype and constitutively activated forms. To determine whether myristoylation restores signaling to non‐acylated wildtype Gα13, the protein was modified to harbor the 6 N‐terminal amino acids from GαT, which is myristoylated but not palmitoylated. Loss of growth signaling by non‐palmitoylated, wildtype G13 was rescued by introduction of this myristoylated sequence. In addition, we used cellular fractionation to assess intracellular distribution of these Gα13 variants. Overexpressed, wildtype Gα13 showed a shift from a membrane associated fraction to a soluble pool, which correlated with a sharp increase in serum response factor signaling. Gα13 lacking any acylation sites localized entirely to the soluble fraction but exhibited no growth signaling. We have also examined the role of an N‐terminal polybasic motif in overexpressed wildtype Gα13 signaling, through amino acid substitutions in this region. To query the nucleotide bound state of the soluble Gα13 pool, we combined cell fractionation with trypsin digestion assays. Surprisingly, overexpressed wildtype Gα13 in the soluble fraction was fully degraded, suggesting lack of GTP binding. These constructs have facilitated co‐precipitation experiments to determine whether change in acylation state affects Gα13 binding to specific target proteins.

Support or Funding Information

We acknowledge support from the North Carolina GlaxoSmithKline Foundation, the C.D. Spangler Foundation, UNC Lineberger Comprehensive Cancer Center, and the UNC‐Asheville Undergraduate Research Program.

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 of Novel Fibroblast Growth Factor Receptor Signaling Components
Authors: Eichelberger Eric , Webb Jason C. , Stefinko Mariya , Stern Michael J. , Voisine Cindy , Lo Te‐Wen
Published: June 2021

Fibroblast Growth Factor Receptors (FGFRs) are a type of receptor tyrosine kinase (RTK) that phosphorylate precise tyrosine residues. FGFRs play a role in diverse developmental pathways that mediate cell proliferation, cell migration, and cell survival. We use C. elegans as a model to better understand FGFR signaling specificity. In C. elegans, the EGL‐15 FGFR is imperative for sex myoblast migration and fluid homeostasis. Defects in the processes mediated by the C. elegans EGL‐15 FGFR result in striking phenotypes that can be used to discover components of the EGL‐15 signaling pathway. For example, hyperactivation of EGL‐15 results in the excessive accumulation of clear fluid within the worm's body (the Clr phenotype). The isolation of suppressor of Clr (soc) mutants has led to the identification of many of the core components of EGL‐15 signaling. A previous soc screen identified the SEM‐5 adaptor protein that links RTK activation to the activation of the RAS/MAPK pathway.

An egl‐15 mutation, n1457 (a truncation mutation), that eliminates the known SEM‐5 binding sites (Y1009 and Y1087) on EGL‐15 does not confer a Soc phenotype indicating that a key component that links activated EGL‐15 to SEM‐5 has yet to be identified. To identify these missing components, we conducted a modified, “enhancer” soc screen in an egl‐15(n1457) background. Using CRISPR/Cas9, we are introducing mutations in the known SEM‐5 binding sites (Y1009 and Y1087) to determine if the Soc phenotype in newly identified genes is solely dependent on these binding sites. We are also using RNAi to verify the identity of a novel soc gene, cca‐1. Additional genetic analyses and whole‐genome sequencing will be used to identify the molecular identities of additional suppressors identified in our enhancer screen.

Support or Funding Information

NIH R15 (GM122001)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
O‐GlcNAcylation of the Human Kinome
Authors: Liu Xin , Han Guanghui , Renuse Santosh , Pandey Akhilesh , Zhu Heng , Hart Gerald
Published: June 2021

O‐GlcNAc, first characterized more than 30 years ago, is an O‐linked β‐N‐acetylglucosamine moiety attached to the side chain hydroxyl of a serine or threonine residue. O‐GlcNAc has been found on over 4,000 cytoplasmic and nuclear proteins. The addition of O‐GlcNAc to proteins is catalyzed by O‐GlcNAc transferase (OGT) while its removal is catalyzed by O‐linked N‐acetyl‐β‐D‐glucosaminidase (OGA). This dynamic and reversible modification is emerging as a key regulator of various cellular processes, such as signal transduction, transcription, cell cycle progression and protein‐protein interactions. O‐GlcNAc plays important roles in human diseases, such as cancer, diabetes and neurological disorders. Protein phosphorylation is a post‐translational modification that serves as a rapid and reversible means to modulate protein activity and transduce signals, the regulation of phosphorylation is a central mechanism in cell health and disease. The addition and removal of phosphorylation is through kinases and phosphates respectively, but little is known about O‐GlcNAcylation of kinases.

Kinase microarray which has 370 kinases printed in identical pair spots indicated that 297 kinases are substrates for OGT. In addition, 10 kinases were picked from kinase array for overexpression and immunopreciptation to map O‐GlcNAc site. To further confirm the results in vivo, home‐made kinase inhibitor beads were used to pull down the kinases in rat brain. 223 kinases were identified by mass spectrometry and more than 20 kinases have been identified as O‐GlcNAcylated proteins.

Support or Funding Information

Supported by NIH P01HL107153, R01DK61671 and R01GM116891. Dr. Hart receives a share of royalty received by the university on sales of the CTD 110.6 antibody, which are managed by JHU

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Developing a physical model of O‐GlcNAc transferase (OGT) in complex with TAB1
Authors: Hernandez Viviana Perez , Nguyen Sophia , Chabbra Alesa , Schmitt Lavin Emily F.
Published: June 2021

O‐GlcNAcylation is a post‐translational modification similar in importance to the mechanism of phosphorylation in its ability to affect signal transduction. This process is mediated by the enzyme, O‐GlcNAc transferase (OGT). OGT catalyzes the addition of the sugar, N‐acetylglucosamine (GlcNAc) from the carrier molecule Uridine Diphosphate N‐acetylglucosamine (UDP‐GlcNAc), to certain serine or threonine residues in target substrate proteins. The mechanisms by which OGT recognizes its protein substrates, and the details of the molecular interactions between the substrate and enzyme, are still poorly understood. The CREST (Connecting Researchers, Educators, and STudents) team at Nova Southeastern University (NSU) created a physical model of the OGT protein in complex with the fused substrate peptide from TAB1 (a transforming growth factor‐beta‐activated kinase 1 binding protein involved in a variety of signaling cascades including pro‐inflammatory pathways). Details of this protein and its fusion with a segment of the substrate peptide from TAB1 were found in the Protein Data Bank file, 5LVV, and imported into Jmol, a protein visualization software. Specific program commands were utilized in Jmol to manipulate the original file into a format that was later able to be 3‐D printed to create an instructional molecular model. Various features of the OGT fused with the TAB1 substrate peptide were highlighted. Such highlighted features include the regions upstream and downstream of the of the substrate binding sequence. The region upstream is a His6‐tag, while the region downstream is a 3xGly linker segment. Additionally, the fused substrate peptide from TAB1 was highlighted as well as the specific serine that functions as the O‐GlcNAc binding site on the TAB1 substrate. The N and C termini of the OGT were colored blue and red respectively. The overall focus of this project was to explore protein structure and design and build a physical model that illustrates key functional features of the protein. It was funded in part by NSF‐DUE 1725940 for the CREST (Connecting Researchers, Educators, and STudents) Project. Developing 3‐D molecular models in this way is a relatively inexpensive process to visually represent important biological relationships that can be useful for students, professors, and others trying to understand and explain complex molecular pathways.

Support or Funding Information

The work was funded in part by NSF‐DUE 1725940 for the CREST (Connecting Researchers, Educators, and STudents) Project.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Functional Characterization of 2QRU Based on Structural and Enzymatic Analysis
Authors: Pyne Alexandra , Sheikh Sana , D'Ausilio Morgan E.
Published: June 2021

Beginning in 2000, the National Institutes of Health's Protein Structure Initiative was a fifteen‐year program that worked to solve the structures of many proteins with known DNA sequences. The successful initiative yielded the publication of more than 500 previously uncharacterized proteins. The present study analyzes one such protein, known as 2QRU, a predicted alpha/beta hydrolase found in Enterococcus faecalis. Using modules outlined by BASIL (Biochemistry Authentic Scientific Inquiry Lab) and resources available in a high school molecular biology teaching laboratory, the specific objective of this study is to functionally characterize 2QRU based on structural analysis of the protein. In silico enzyme characterization for 2QRU was carried out using BLAST, Pfam, and Dali servers, followed by active site analysis using the PyMOL plug‐in ProMOL. ProMOL generated a list of proteins with active sites that were structurally similar to 2QRU. For each of the hits with a Levenshtein distance of zero, the predicted catalytic triad for 2QRU was composed of a Ser‐102, Asp‐219, and His‐247. Secondary structure alignments using PROMALS3D and structural alignments in PyMOL were used to compare 2QRU to the active site matches identified using ProMOL. This structural analysis suggests PDB entries 3LIP and 1YSC ‐ a triacylglycerol lipase and serine carboxypeptidase, respectively ‐ are most similar to 2QRU and share a conserved catalytic triad. However, serine carboxypeptidases have yet to be discovered in bacteria, indicating 2QRU may function as a lipase. To investigate the lipase activity of 2QRU in vitro, recombinant his‐tagged protein was isolated from E. coli. Subsequent enzymatic assays using spectrophotometric analysis will be carried out based on methods described for the characterization of Pseudomonas cepacia lipase (PCL) (PDB ID 3LIP) using the substrate p‐Nitrophenyl palmitate, an ester of palmitic acid. As a research project in a secondary school molecular biology research course, this study not only contributes to the field of bacterial lipase research, but also offers students an authentic research experience at an early stage in their scientific careers.

Support or Funding Information

This work was funded by The Pingry School.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Use of a Synchronous Collaborative Learning Environment in a Face‐to‐Face Molecular Biology Classroom
Authors: Clauson‐Kozina Cheryl , Kunzweiler Greg
Published: June 2021

As stated in “Vision and Change”, biology is an increasingly collaborative endeavor, requiring scientists to work well in teams and across disciplines. Yet, too often our classrooms are designed to be lecture‐based and don't provide sufficient opportunity for students to practice collaboration and teamwork. Here we describe a synchronous collaborative learning tool, MeTL, wherein students in face‐to‐face classrooms work together on problem sets and case studies. Rather than submitting a paper assignment at the conclusion of class time, the students instead work in an online environment that is accessible even after class has ended. Group members can see each other's work in real time and can thus provide assistance in creation of the group submission. Feedback provided to the students is viewable instantaneously, by all group members, and can be synced with Learning Management Systems. Students receive a grade for the work that was completed in addition to a scaled grade based on how well their peers felt they contributed to the activity of the day. We anticipate MeTL being useful to classrooms in many disciplines, but especially the molecular biology classroom.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sustained teaching mentoring works and benefits mentors as well as those mentored: The Promoting Active Learning and Mentoring (PALM) Network
Authors: Wolyniak Michael J. , Wick Sue , Prunuske Amy J. , Peifer Mark
Published: June 2021

Many instructors and instructors‐to‐be have heard of the value of using active learning to guide students to deep learning of course material. Some have attended workshops or several‐day immersion experiences to learn how to employ effective active learning activities. In spite of best intentions, plans to actually implement active learning techniques often collapse once an instructor gets caught up in the academic year. To address this situation, ASCB, in cooperation with other professional societies and academic groups and with NSF funding, established the Promoting Active Learning and Mentoring (PALM) Network. This program provides PALM Fellows with a one‐on‐one teaching mentoring relationship for at least one semester with a mentor experienced in active learning. Participants to date have come from various partner societies within the Network. Analysis of teaching behavior before and after mentoring provides evidence that Fellows have been able to increase their use of active learning in accord with the specific objectives they had identified. Interviews with participants indicate that Fellows gain more confidence in their ability to apply active learning principles in future coursework, and mentors appreciate the opportunity to reflect on, refine, and expand their own practice of active learning approaches.

Support or Funding Information

PALM is supported through National Science Foundation RCN‐UBE #1624200.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Opening the Doors for Nuclear Gln3 Entry In Saccharomyces cerevisiae
Authors: Tate Jennifer J. , Rai Rajendra , Cooper Terrance G.
Published: June 2021

The Gln3 and Gat1 GATA transcription activators are responsible for Nitrogen Catabolite Repression‐sensitive transcription in the yeast Saccharomyces cerevisiae. In the presence of excess, usable nitrogen, Gln3 is cytoplasmic and NCR‐sensitive transcription repressed. When cells are grown in nitrogen‐poor medium, treated with the TorC1 inhibitor, rapamycin, or glutamine synthetase inhibitor, methionine sulfoximine (Msx), Gln3 becomes highly nuclear and NCR‐sensitive transcription derepressed. In previous reports, nuclear Gln3 localization was concluded to be mediated by a single nuclear localization sequence, NLS1. Here we show that nuclear Gln3‐Myc13 localization is more complex than previously appreciated. We have identified three Gln3 sequences, other than NLS1 that are almost absolutely required for nuclear Gln3‐Myc13 localization. Two of these sequences exhibit characteristics of monopartite (K/R‐Rich NLS) and bipartite (S/R NLS) NLSs, respectively. Mutations altering these sequences are partially epistatic to a ure2Δ. In contrast, a third sequence, the Ure2 Relief Sequence (URS), exhibits no predicted NLS homology and is only necessary when Ure2 is present. Substitution of the basic amino acid repeats in the Ure2 relief sequence or phosphomimetic aspartate substitutions for the serine residues between those basic residues abolish nuclear Gln3‐Myc13 localization in response to both limiting nitrogen and rapamycin treatment. In contrast, Gln3 responses are normal in parallel serine to alanine substitution mutants. These observations suggest that Gln3 responses to specific nitrogen environments likely occur in multiple, genetically separable steps. At least one general step, i.e., that associated with the Ure2 relief sequence, appears to be prerequisite for Gln3 responses to specific stimuli, e.g., growth in poor nitrogen sources and rapamycin inhibition of TorC1.

Support or Funding Information

National Institutes of Health General Medical Sciences GM‐35642‐27

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.