bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Effect of 1,4‐dimethylnaphthalene on the Expression of Pathogenesis‐Related Protein 4 in Solanum tuberosum.
Authors: Aljahani Abrar , Campbell Michael 
Published: June 2021

It has been shown in recent studies that 1,4‐dimethylnaphthalene (DMN) can halt cell division in tuber meristems of Solanum tuberosum. By preventing cell division, DMN can prevent sprouting and prolong storage. RNA sequence data of potatoes treated with DMN revealed that the gene encoding for pathogenesis‐related protein 4 (PR‐4) is significantly induced in response to DMN. The level of PR‐4 gene expression increased by three‐fold in the fall and winter and by six fold in the spring, which is during late storage and the tissues are no longer dormant. This demonstrates that dormant potato tubers are recalcitrant in response to DMN induction of PR‐4. The PR‐4 protein belongs to a large family of plant host proteins triggered in response to pathological situations, such as bacteria or virus invasion. Members of PR protein family have different functions. PR‐4 is specifically involved in wound response, and it has both RNase and DNase activity. Further experiments are being conducted to determine the level of gene product for PR‐4 in response to DMN. Currently, it is unclear how the expression of PR‐4 relates to the suppression of cell division in potato meristems.Support or Funding InformationSupported in part by grant from the 1,4‐Group of Meridian, IDThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of RNA Binding Protein RBM15 in m6A RNA Methylation During Megakaryocytic Differentiation
Authors: Ayala‐Lopez Nadia , Ross Robert , Halene Stephanie , Limbach Patrick , Krause Diane S. 
Published: June 2021

N6‐methyladenosine (m6A) modification of RNA regulates RNA splicing, localization, export and degradation. Loss of m6A RNA is associated with defects in stem cell differentiation and gain of m6A RNA is associated with leukemogenesis. The RNA binding protein RBM15 was recently discovered to be involved in the m6A methylation of RNA. We have shown previously that RBM15 inhibits differentiation of myeloid cell lines, and its expression is decreased in megakaryocytes compared to bone marrow hematopoietic stem cells. We hypothesize that RBM15 m6A methylates RNAs that are involved in megakaryocyte fate specification and maturation. We tested whether RBM15 overexpression affects global m6A RNA levels during megakaryocytic maturation using two human erythroleukemia (HEL) cell lines: parental control (HEL‐pcDNA) cells carrying only the Tet‐repressor gene and HEL‐RBM15 cells, which encode the RBM15 transgene tagged with hemagglutinin (HA), under the control of a tetracycline‐inducible promoter. Megakaryocytic differentiation was induced by addition of 12‐Otetradecanoylphorbol‐13‐acetate (TPA) with and without doxycycline induction of the transgene for 24 hours. Immunoprecipitation using an anti‐HA antibody to pull down the HA‐tagged RBM15 followed by a Western blot revealed that WTAP (Wilms' tumor associated protein), which is part of the N6‐methyltransferase complex, binds to HA‐tagged RBM15. Global m6A RNA methylation levels were analyzed by ELISA and confirmed by LC‐MS/MS. HEL‐pcDNA (control) m6A RNA levels did not change with any of the treatments using the ELISA or LC‐MS/MS. In contrast, overexpression of RBM15 during megakaryocytic differentiation increased m6A RNA significantly when measured by ELISA (HEL‐RBM15= 307.5±83.0% increase in m6A RNA vs. untreated (no doxycycline, no TPA); N=3]. Confirmation by LC‐MS/MS however, showed no significant changes in m6A RNA levels (HEL‐RBM15= 7.14±7.26% increase in m6A RNA compared to untreated; N=3). These data show that RBM15 associates with WTAP, one of the methyltransferase complex proteins, in a megakaryocyte cell line, and that antibody assays to detect m6A RNA should be validated by other methods such as mass spectrometry. We are currently performing rigorous identification of the factors responsible for discordance of ELISA and LC‐MS/MS measures. Additionally, we will test the hypothesis that RBM15‐induced RNA methylation is a mechanism by which RBM15 regulates megakaryocytic maturation with the ultimate goal of identifying the mechanisms of translational control at the RNA m6A methylation level that can be targeted in megakaryocytic leukemia.Support or Funding InformationNIH 2T32HL007974‐16This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A screen for novel targets casts polyphosphorylation of lysine as a common post‐translational modification
Authors: Bentley‐DeSousa Amanda , Moteshareie Houman , Holinier Charlotte , Tseng Yi‐Chieh , Bondy‐Chorney Emma , Davey Norman , Golshani Ashkan , Downey Michael 
Published: June 2021

Polyphosphates (polyP) are long chains of linked inorganic phosphates ranging from 3–1000s of residues in length. They are found in all organisms and play critical roles in a range of functions including blood clotting and bacterial virulence. Given its diverse roles, polyP is an attractive target for novel therapies. Recently, it was shown that polyP can be non‐enzymatically added to protein targets within poly‐acidic, serine, and lysine rich (PASK) motifs as a lysine post‐translational modification termed polyphosphorylation. This modification was described for two yeast proteins, Nsr1 and Top1. In yeast, polyP is initially synthesized by a polyP synthetase (VTC4) and subsequently added as a PTM non‐enzymatic. Our lab has developed a screen to assess polyphosphorylation of yeast substrates wherein we uncovered 25 novel substrates including a conserved network of proteins functioning in ribosome biogenesis. Disruption of this polyP synthesis through vtc4D deletion results in translation defects as measured by polysome profiling. Finally, we found that expression of E. coli polyP synthetase (EcPPK1) in vtc4D mutant yeast results in extreme sensitivity to rapamycin and cycloheximide, despite its ability to restore polyphosphorylation of multiple yeast targets. We propose a model wherein polyphosphorylation of unique targets regulates multiple aspects of cell growth. Moreover, regulation of polyphosphorylation in space and time is critical to preserve essential cell functions, such as ribosome function.Support or Funding InformationThe funding agencies that support this research are: University of Ottawa and CIHRThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Regulation of Cellular Proliferation by VACM‐1/CUL5 is Dependent on its Post‐translational Modifications by NEDD8
Authors: Lee Si Eun , Sundquist Skylar , Burnatowska‐Hledin Maria 
Published: June 2021

VACM‐1/CUL5 is a member of the cullin family of proteins involved in the E3 ligase complex formation and subsequent protein degradation by ubiquitin‐dependent pathways. In vivo, VACM‐1/CUL5 is expressed in endothelial cells, and when expressed in vitro it attenuates cellular growth. While the specific mechanism by which VACM‐1/CUL5 suppresses cell proliferation is unknown, its antiproliferative effect is dependent on its post‐translational modification by an ubiquitin‐like protein, NEDD8. To investigate the effect of NEDD8 on the structure and function of VACM‐1/CUL5, we have mutated its cDNA sequence at the putative neddylation sites and expressed the mutated construct in Rat Adrenal Medulla Endothelial Cells (RAMEC) and COS‐1 cells in vitro. Our results demonstrate that the antiproliferative effect of wt VACM/CUL5 was attenuated in cells transfected with the mutated VACM‐1/CUL5 cDNA. Furthermore, the mutated VACM‐1/CUL5 affected the anti‐angiogenic phenotype of wt VACM‐1 when expressed in RAMEC cells and grown on Matrigel® support. Finally, the immunoblotting results show that the mutant is modified at multiple sites by NEDD8 in vitro. These results suggest that antiproliferative effect of VACM‐1/CUL5 is regulated by its neddylation status.Support or Funding InformationThis work is supported by the Paul A. Schaap Fellowship and Hope College Department of Biology.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development and Characterization of a Model Post‐Translationsally Modified Protein Library
Authors: Major Emily K. , Magnani Roberta , Houtz Robert L. , Bradley Luke H. 
Published: June 2021

From plants to humans, calmodulin lysine methyltransferase (CaM KMT) is a highly conserved protein responsible for the trimethylation of Lys‐115 in calmodulin (CaM). Recent findings suggest that CaM trimethylation is important in a wide range of metabolic, cellular, and developmental functions. Furthermore, we used CaM KMT as a protein engineering tool to post‐translationally modify a high‐quality protein library of the CaM central linker, using a coexpression system in E. coli. We showed that trimethylation differentially altered the conformational changes of CaM associated with the binding of calcium, CaM's thermal stability, and binding specificity towards CaM‐peptide target sequences. However, the substrate specificity of CaM KMT is unknown. A total of 40 conservative and non‐conservative amino acid substitutions were designed and introduced 3 residue positions (positions P(−3) to P(+3)) flanking the target Lys‐115 trimethylation site. In vitro trimethylation assays, using bacterially expressed and purified CaM KMT with purified mutants, identified positions and putative target sequences for CaM KMT. These results suggest alternate in vivo substrates and cellular roles for CaM KMT. Furthermore, these results provide further design information towards producing an unbiased and targeted post‐translationally modified library of novel sequences, thereby providing an additional tool for designing and generating proteins with stringent protein‐target specificities.Support or Funding InformationNSF KY ESPCoR 0IA‐1355438 Bucks for Brains‐ University of Kentucky Office of Undergraduate ResearchThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulating Nociceptive Channel Trafficking by Site‐directed mutagenesis of a Calcineurin Interaction Motif
Authors: Chen Annabel 
Published: June 2021

IntroductionTransient receptor potential (TRP) channels are a family of receptor proteins regulating pain sensation. TRP receptors are activated by a variety of noxious stimuli, such as capsaicin (chili peppers), heat, and mechanical stress. Chronic TRPV1 stimulation leads to desensitization of the receptor, a process which is mediated by calcium/calmodulin dependent kinases. This attribute, however, has yet to been leveraged for treatment of chronic pain diseases. Here, we identify a C‐terminal phosphorylation site that is involved in regulating TRPV1 trafficking to the cell surface.Results and ConclusionIn dorsal root ganglion cells, confocal microscopy and western blot show that de‐phosphorylation of threonine 705 is sufficient to induce significant trafficking to the plasma membrane. Mimicking constitutive phosphorylation at that site induces the receptor to be retained on intracellular membranes. TRPV4 ‐ another member of the TRPV family ‐ also has a similar homologous region to TRPV1 that contains two adjacent threonines T739 and T740, both potentially involved in regulating TRPV4 trafficking. Confocal and western blot data show that removing the T740 phosphorylation site is sufficient to significantly increase surface trafficking. However, the phosphomimetic mutation reduced but did not eliminate surface expression. These data lead us to believe that phosphorylation at both threonines is necessary for full internalization of TRPV4 to occur. Together, these data suggest that the highly conserved Calcineurin interaction motif is an important trafficking regulation site for multiple members of the TRP family, and that manipulating key phosphorylation sites is sufficient to significantly change protein trafficking patterns.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Analysis of Spin Probe Viability for Protein Structure Investigation Using Advanced EPR Techniques
Authors: Ebersol Lauren , Bokhari Aneesa , Silakov Alexey 
Published: June 2021

An accurate depiction of protein structure and its mobility is necessary to understanding protein function and many methods exist to determine this. Obtaining protein structural characteristic by advanced electron paramagnetic resonance (EPR) spectroscopy techniques like double electron‐electron resonance (DEER) is emerging as a powerful technique that is complementary to other well established structural characterization tools. Such studies most commonly require the ligation of “spin labels” to different sites on the protein, often by a site‐specific reaction between a stable molecule containing a nitroxide radical and an amino acid residue. We seek to develop a robust set of tools for predicting structures of hard‐to‐characterize proteins by combining the distance information obtained from advanced EPR techniques with mobility information obtained from room temperature continuous wave EPR (CW EPR). Many methods of spin‐labeling a protein exist; however, the viability of these specific approaches for performing structural determination techniques like DEER has not yet been explored. The most common spin‐labeling methodology conjugates (1‐oxyl‐2,2,5,5‐tetramethylpyrroline‐3‐methyl) methanethiosulfonate (MTSSL) to a protein via a disulfide bond with a cysteine residue. To achieve this conjugation site‐specifically, all native surface‐located cysteine residues must be removed, which may not be desirable from a structural integrity standpoint. Utilizing unnatural amino acids can alleviate this issue and the spin‐label can be added to the protein via techniques such as click chemistry, a rapid cycloaddition that can occur at physiological conditions. In this work, we compare how different spin labeling techniques perform from an experimental point of view. We seek, overall, to determine a powerful and easy‐to‐use protocol for structural determination. Here, we use T4 Lysozyme as a well‐characterized protein model and compare 4‐alkyl TEMPO installed on the unnatural amino acid p‐‐azidophenylalanine (pAzF) with the aforementioned MTSSL/cysteine label. For this purpose, we use a cysteine‐free version of T4 Lysozyme and genetically encode either cysteine or pAzF to specific positions. The results presented focus on the structural rigidity of the spin labels, a key factor defining the flexibility of certain areas of the protein and the certainty of the distance determination. Moreover, as different spin probes display different spectroscopic characteristics, each spin label is evaluated in terms of DEER sensitivity and efficiency. Finally, we compare different advanced EPR techniques (DEER, DQC, relaxation enhancement) to determine the most robust in distance predictions.Support or Funding InformationThe work is funded by the Pennsylvania State University startup fund of Alexey Silakov.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Optimizing Protein Crystal Formation of a Heterocyclization Domain from Yersiniabactin Synthetase
Authors: Patterson MacKenzie , Dieu Viet , MacRae Mark , Henriquez Bryan , Soule Jessica , Gnann Andrew , Dowling Daniel P. 
Published: June 2021

The bacterium Yersinia pestis, known for causing the bubonic plague, is an organism which must scavenge free iron from its host to grow. One method of obtaining iron is by producing a siderophore, yersiniabactin (Ybt), which is a secondary metabolite synthesized by a hybrid polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS). This system utilizes three interesting heterocyclization (Cy) domains to introduce three five‐membered rings that are important for iron affinity. This research project aims to determine the molecular features of the third Cy domain (Cy3) that determine its function using X‐ray crystallography.Support or Funding InformationFunding for this research was provided in part by the Oracle Education Foundation grant to the College of Science and Mathematics and also by an ASBMB Undergraduate Research Award. Special thanks to the MIT crystallography facility. This research was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number 1R15GM123425‐01 to D.P.D.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evolutionary Conservation of Variant‐dependent Prion‐promoting Hsp40 Functions in Plants
Authors: Brown Rebecca E. , Hines Justin K. 
Published: June 2021

The J‐protein (Hsp40) Sis1 is required for the propagation of yeast prions and for the efficient curing of the prion [PSI+] by overexpression of the disaggregase Hsp104. Interestingly, some of the prion‐supporting functions of Sis1 are conserved in homologs from higher organisms: Sis1 homologs from fruit flies and humans have been shown to substitute for Sis1 in yeast in the propagation of some, but not all, yeast prions. The number and diversity of J‐proteins is greatest in the cytosol and the number of J‐protein genes in the eukaryotic genome generally scales with the overall biochemical complexity of the organism and genome size. As such, yeast has 23 J‐proteins, humans have 41, and we recently reported that the model plant Arabidopsis thaliana has 106. Not surprisingly, at least 6 functional homologs of Sis1 have been identified in A. thaliana. Here we present the results of our ongoing investigation, using yeast complementation assays and multiple prions, to examine which prion‐maintaining functions of Sis1 are conserved in various plant Sis1 homologs. Most notably, preliminary results indicate that these homologs give rise to distinct patterns of prion maintenance, indicating that the diversification of Sis1‐homolog structure has created alternative combinations of functions that affect prion‐propagation. In addition to providing information about J‐protein evolution, our investigation is expected to lead to a greater understanding of how the amino acid sequences of various J‐proteins allow for prion‐specific interactions in yeast.Support or Funding InformationThis work was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R15GM110606. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The ER Hsp70 Hspa13 Redirects an Amyloidogenic Protein to Aggregation
Authors: Genereux Joseph 
Published: June 2021

The conserved ER Hsp70 chaperone Hspa13/STCH is implicated in cancer and neurodegenerative diseases. While other Hsp70 proteins maintain proteostasis by promoting folding, degradation, or disaggregation of misfolded protein, the biological function of Hspa13 is unknown, in part because it lacks the substrate‐binding domain required for the function of other members of the Hsp70 class. We investigated the intracellular effect of Hspa13 overexpression on the kinetically stable, amyloidogenic protein transthyretin (TTR), finding that Hspa13 promotes the partitioning of TTR into high‐molecular weight, detergent‐resistant aggregates inside living cells. This function is ATP‐independent and opposed by the ER nucleotide exchange factor HYOU1, but unaffected by the primary ER Hsp70 BiP. These results suggest a role for Hspa13 in maintaining ER proteostasis by regulating the aggregation of amyloidogenic protein in the secretory pathway.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Parkinson's Disease Protein α‐synuclein Alters the Microenvironment of the Endoplasmic Reticulum in Saccharomyces cerevisiae
Authors: Haak Victoria Melina , McBride Trevor John , Haverly Meredith Julia , Austriaco Nicanor 
Published: June 2021

The protein α‐synuclein forms aggregates in human dopaminergic neurons triggering apoptosis and Parkinson's Disease (PD). To better understand the link between protein aggregate formation and cell death, we have over expressed wild type and two mutant forms, A30P and A53T, of human α‐synuclein in the budding yeast, Saccharomyces cerevisiae. Our experiments suggest that these aggregates trigger the unfolded protein response (UPR) in yeast and that different clinically relevant variants of α‐synuclein up‐regulate the UPR to different degrees. Moreover, we show that the overexpression of α‐synuclein alters the redox state and the calcium dynamics of the yeast ER. Finally, our preliminary data suggests that sub‐lethal doses of tunicamycin and β‐mercaptoethanol, drugs that trigger the UPR, may alleviate the aggregation of α‐synuclein. Our results point to possible pharmacological interventions that may lower protein aggregation in PD. [Our laboratory is supported by grant NIGMS R15 GM110578, awarded to N. Austriaco.]Support or Funding Information[Our laboratory is supported by grant NIGMS R15 GM110578, awarded to N. Austriaco.]This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.