CoBRE Retreat Abstracts
Fourth Annual Center for Biomolecular Structure and Dynamics (CBSD) Centers of Biomedical Research Excellence (COBRE) Research Symposum; September 11-13, 2015; Double Arrow Resort, Seeley Lake, MT.
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Oral Presentations - Invited Abstracts
Listed alphabetically by presenter's last name.
“NMR and Mass Spectrometry Year 4 Update.” September 11, 2015. Author: Earle Adams, Nuclear Magnetic Resonance Core Facility (NMR), University of Montana, Missoula, MT
Abstract: Not provided.
“Impact of AhR activation on innate lymphoid cells.” September 13, 2015. Author: Celine A. Beamer, Department of Biomedical and Pharmaceutical Sciences, University of Montana, Missoula, MT
Abstract: The aryl hydrocarbon receptor (AhR) is a highly conserved, ligand-activated, basic helix-loop-helix transcription factor that plays a critical role in maintaining the balance between inflammatory and immunosuppressive cells, although the molecular mechanisms by which the AhR does this remain to be revealed. Innate lymphoid cells (ILCs) are a newly described, heterogeneous population of cells that express the AhR, react promptly to pathogens, and contribute to inflammation and remodeling through the secretion of regulatory cytokines, such as IL-17 and IL-22. Our results show that select AhR ligands increase IL-22 expression by innate lymphoid cells (ILCs) and reduce pulmonary inflammation in vivo, and that activation by structurally diverse AhR ligands results in ligand-specific alterations in ILC3s functionality in vitro. If we can design small molecules or agents that selectively induce IL-22 production by ILC3s, it should be possible to promote this effect, and use this knowledge to develop novel therapies for respiratory diseases. Consequently, we are looking towards known and novel ligands for insights into the AhR structure-function to unlock the therapeutic potential of this system.
This work is supported by NIGMS grants P30GM103338 and P20GM103546. The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of NIEHS, NIGMS or NIH.
“Synthetic Hydrogen and Halogen bonding Oxyanion Holes.” September 13, 2015. Author: Orion Berryman, Department of Chemistry and Biochemistry, University of Montana, Missoula, MT
Abstract: Nature produces hydrogen bonding enzymes with oxyanion holes that are extremely efficient at stabilizing tetrahedral transition states for catalysis. Inspired by this approach, synthetic organocatalysts have sought similar activity and selectivity but have not yet succeeded. The rational design of organocatalysts that optimally bind transition states has been critically lacking. An improved catalyst design and understanding of how organocatalysts function will enhance catalyst activity and have far reaching implications in organic synthesis. This presentation includes crystal structures and solution studies of synthetic oxyanion hole mimics designed to activate reactions by destabilizing ground state hydrogen bonding or stabilizing transition state binding via halogen bonding.
“Protein interactions and conformational changes in fibronectin fibril formation.” September 12, 2015. Author: Klara Briknarova, Department of Chemistry and Biochemistry, University of Montana, Missoula, MT
Abstract: Fibronectin is an essential extracellular matrix (ECM) protein. It plays an important role in cell adhesion and migration and affects cell proliferation, survival and differentiation. It is secreted in a soluble form and is converted into the insoluble ECM form by cells. The molecular basis of the conversion of soluble fibronectin into insoluble fibrillar aggregates is poorly understood, and the exact arrangement of fibronectin molecules within fibrils is not known. To elucidate how molecular interactions and conformational changes lead to conversion of soluble fibronectin to insoluble fibrils, we take advantage of an experimentally accessible model system that employs a 9 kDa fragment of fibronectin, termed anastellin. Anastellin binds in vitro to soluble fibronectin and converts it to insoluble form that morphologically and functionally resembles fibronectin fibrils deposited by cells. The binding sites for anastellin in fibronectin include the second, third and eleventh type III domains (2FN3, 3FN3 and 11FN3). We determined the structures of 3FN3 and 11FN3 and investigated the interactions of 2FN3, 3FN3 and 11FN3 with anastellin. Our results suggest that anastellin binds to the beta-strand B from these FN3 domains, and some of the other inner beta-strands may also be involved in the interaction.
“Computational study of the catalytic mechanism of mammalian adenylyl cyclase.” September 12, 2015. Author: Xi Chu, Department of Chemistry and Biochemistry, University of Montana, Missoula, MT
Abstract: We propose a catalytic mechanism for Class III mammalian AC through computation. Modeling of the active site was based on a crystal structure. We compared the calculated activation free energy for thirteen reaction sequences. Comparison of the enzyme-catalyzed and nonenzymatic reactions reveals that the active site residues lower the free energy barrier for both phosphoryl transfer and proton transfer, as well as significantly shift the proton transfer equilibrium. Calculations for mutants K1065A and R1029A demonstrate that K1065 plays a significant role in shifting the proton transfer equilibrium, whereas R1029 is important for the nature of the phosphoryl transfer transition state.
“Analysis of DLC-1 mediated mRNA regulation in C. elegans.” September 11, 2015. Authors: Mary Ellenbecker, Xiaobo Wang and Ekaterina Voronina, Division of Biological Sciences, University of Montana, Missoula, MT
Abstract: Dynein light chain (DLC-1) was originally characterized as a cargo-binding light chain component of the dynein motor complex that is involved in retrograde transport of mRNA and protein. More recent studies have revealed that DLC-1 interacts with a diversity of cellular proteins and likely functions as an allosteric regulator in ribonucleoprotein complexes (mRNPs). We suggest a new role for DLC-1 as a regulator of developmentally important mRNAs that control cell division processes. Specifically, DLC-1 binding to PUF family FBF-2 protein is required for FBF-2 to localize to germ granules and function as a translational repressor. Also, we discovered that knock-down of DLC-1 results in misregulation of nos-2 mRNA expression at a different time during development. NOS-2 protein limits germ cell proliferation during embryonic development. We hypothesize that DLC-1 mediates regulation of its mRNA targets by interacting with regulatory RNA binding proteins and that DLC-1 is involved in regulating the expression of mRNA targets that have not yet been described. We used affinity purification to isolate DLC-1/mRNP complexes from worms and mRNA sequencing to identify DLC-1 associated mRNAs. Together, these data provide insights into the regulatory role(s) of DLC-1 during germ cell development and identification of other important DLC-1 regulatory targets.
“On the Elucidation of the electrostatic contribution of individual amino acids during cardiolipin binding at Cytochrome-c’s anionic site A.” September 11, 2015. Author: Margaret Elmer-Dixon, Department of Chemistry and Biochemistry, University of Montana, Missoula, MT
Abstract: During the initial stages of apoptosis, Cytochrome-c (Cytc) interacts with Cardiolipin on the inner mitochondrial membrane of the mitochrondria before its release into the cytosol where it can form the apoptosome and initiate mediated cell death. To date, several conflicting models postulating mechanisms for the Cytc-cardiolipin binding have been introduced. Further work is needed to suss out the true nature of the protein-lipid interaction. Cytc possesses three potential binding sites for cardiolipin docking. Specifically, site A is postulated to interact with cardiolipin electrostatically. Using Circular Dichroism, site A binding was monitored during cardiolipin titration. In combination with mutagenesis, isolation and investigation of specific amino acids at site A has led to the identification of the electrostatic contributors comprising the interaction site.
“Negative allosteric inhibition of NMDA receptors: Linking drug action to receptor structure.” September 13, 2015. Author: Kasper B. Hansen, Department of Biomedical and Pharmaceutical Sciences, University of Montana, Missoula, MT
Abstract: NMDA-type glutamate receptors are involved in many brain functions and neurological disorders, and a flurry of recent clinical studies has re-invigorated efforts to identify compounds that selectively modulate the individual NMDA receptor subtypes. This presentation will show data for TCN-201, which is a negative allosteric modulator selective for GluN2A subunit-containing NMDA receptors. The data will illustrate our efforts to link basic observations of drug action to detailed structural insights to the mechanism of action for TCN-201. Such insight enables rational design of more potent modulators with improved pharmacokinetic properties and therapeutic potential.
“The Molecular Computational Core Facility: CBSD Year 4.” September 11, 2015. Author: Dave Holley, Molecular Computational Core Facility (MCCF), University of Montana, Missoula, MT
Abstract: The Molecular Computational Core Facility is undergoing some exciting changes that we hope will make the MCCF more functional for researchers. Among the expected changes is a new suite of software for graphics processor (GPU)-accelerated molecular dynamics (MD). This software, when paired with a proposed new GPU cluster, will allow MD simulations of macromolecules to extend towards physiologically relevant timescales. We also hope to provide access to powerful structure-based ligand and inhibitor design software, giving researchers at UM a new strategy for rational design of small molecules. The MCCF will host workshops to help investigators integrate these new tools into their research.
Workshop: "GPU-Accelerated Molecular Dynamics using AMBER.” September 12, 2015. Author: Dave Holley, Molecular Computational Core Facility (MCCF), University of Montana, Missoula, MT
Abstract: Led by MCCF Applications Specialist Dave Holley, this workshop will give participants an introduction to GPU-accelerated molecular dynamics of macromolecules using AMBER. Specifically, we will discuss different types of analysis that can be drawn from longer timescale MD simulations, such as per-residue energy decomposition and statistical analysis of conformational movements. Following the introduction and discussion of analytical tools, we will discuss individual research needs and how we could potentially address those needs using MD.
“Elucidating RNA binding preferences of RVFV nucleocapsid protein using deep sequencing.” September 11, 2015. Author: Katie Hornak, Division of Biological Sciences, University of Montana, Missoula, MT
Abstract: Rift Valley fever virus (RVFV) is an emerging pathogen with the ability to cause serious epidemics in both livestock and humans. There is currently no effective vaccine or treatment for the disease. The viral nucleocapsid (N) protein is a promising therapeutic target. N is an RNA binding protein whose involvement is critical in several aspects of the viral replication cycle, including transcription, translation, replication, vRNA protection and encapsidation, and packaging of the viral genome into virions. Each of these functions is dependent on the ability of N protein to recognize and bind RNA. We have undertaken a variety of biochemical assays which have yielded insight into the nature and specificity of N–RNA interactions. To delve further into the importance of these interactions over the course of a viral infection, we have used crosslinking and immunoprecipitation in tandem with high throughput sequencing (CLIP-seq). CLIP-seq permits identification of hotspots where N protein interacts with the viral genome at high rates. These hit regions are hypothesized to play important roles in viral regulation and propagation. Further characterization and manipulation of these hit segments will yield insight into their mechanistic significance for the virus and could potentially serve as targets for the development of novel antiviral therapeutics.
“Matching MD hypotheses with protein NMR results.” September 12, 2015. Author: Travis Hughes, Department of Biomedical and Pharmaceutical Sciences, University of Montana, Missoula, MT
Abstract: Nuclear receptors are ligand responsive transcription factors and are thus druggable targets for conditions including metabolic and autoimmune disorders. Many current prescription drugs that target this family induce unwanted effects. Pre-clinical work indicates that ligands can induce distinct transcriptional profiles through the same nuclear receptor. A better understanding of which biophysical changes in the receptor produce the desired transcriptional profile (and functional effects) is needed for development of side-effect free drugs. Co-crystal structures of several groups of nuclear receptors bound by ligands that produce diverse functional effects are very similar. Recently, solution studies of these proteins bound to diverse ligands have revealed large differences in μs to ms movements and in hydrogen deuterium exchange kinetics at amide protons. I present here NMR, molecular dynamics simulations and other biochemical and biophysical data that indicate that ligands change the μs to ms dynamics of PPARγ (a nuclear receptor targeted by type II diabetes drugs) and that these changes correlate with a biochemical functional output. Future work will attempt correlation of biophysical measures with transcriptional profile in relevant cell types.
“Protein folding: Thermodynamics & kinetics of ubiquitin-associated domains.” September 11, 2015. Author: Moses Joseph Leavens, Department of Chemistry and Biochemistry, University of Montana, Missoula, MT
Abstract: Ubiquitin-associated (UBA) domains have been discovered in several proteins within the cell, and at least one domain plays an important functional role in cellular phenomena such as NF-κB signaling. Mutations within the UBA(2) domain of the ubiquitin-binding scaffold protein p62 are frequently observed in patients suffering from Pagat’s disease of bone. Both UBA(1) and UBA(2) nuclear magnetic resonance (NMR) structures depict three-helix bundles, yet only share approximately 20% primary sequence identity. The UBA(2) domain has been shown to dimerize at concentrations below 20 mM, and has been hypothesized to play an important functional role within the cell. Previous NMR structural analysis of UBA(1) shows that this domain is monomeric; however, matrix-assisted laser desorption/ionization mass spectrometry, thermal denaturation studies, and a fluorescent binding assay elucidate a probable shared property between the UBA(1) and UBA(2) domains, suggesting potential competition between Ubiquitin binding and UBA domain dimerization.
“Selective anion recognition in competitive media.” September 11, 2015. Author: Casey J. Massena, Department of Chemistry and Biochemistry, University of Montana, Missoula, MT
Abstract: Selective anion recognition in competitive media has presented itself as an ongoing challenge. Inspired by nature, this project attempts to exploit hydrophobic effects to preorganize a multidentate halogen bond receptor for the selective recognition of iodide.
“The Macromolecular X-Ray Diffraction Core Facility Year 4 Update.” September 11, 2015. Authors: Tung-Chung Mou, Stephen R. Sprang, Macromolecular X-Ray Diffraction Core Facility (MXDC), University of Montana, Missoula, MT
Abstract: The CBSD MXDC core provides support, expertise and infrastructure to University of Montana laboratories that use X-ray crystallography as a tool to determine the three-dimensional structures of small molecules and the biological macromolecules for their research projects. During the phase I COBRE funding period, the MXDC has increased its capabilities and capacity to support UM research groups in utilizing X-ray crystallography approach to study essential biological macromolecules, such as protein and nucleic acids, that are promising therapeutic targets for rational drug design. The MDXC continues its major infrastructure improvements aiming to provide comprehensive user access to shared major instrumentation and research training for protein expression/purification, high-throughput crystallization of macromolecules, evaluation of crystallization experiments, and measurement of diffraction data from crystals. Computational resources of the MXDC are also available for X-ray data processing, structure determination, refinement and visualization. We also routinely measure X-ray diffraction data from crystals at synchrotron sources at single and multiple wavelengths. One of the main functions of MXDC is to provide a resource for training, education, and consultation in protein expression/purification and X-ray crystallographic techniques to allow individual investigators of any experience level to initiate and successfully complete a structural biology project for their research.
Workshop: “Practical considerations for using fluorescence to study interactions and dynamics.” September 12, 2015. Authors: Sandy Ross, Michelle Terwilliger, BioSpectroscopy Core Research Laboratory (BCRL), University of Montana, Missoula, MT
Abstract: The BioSpectroscopy Core Research Laboratory Workshop will provide a platform for open-ended discussion of approaches to measurement of binding interactions and structural dynamics using fluorescence observables: emission intensity and excited-state lifetime change, spectral emission shifts, and depolarization (i.e., anisotropy). The strengths, requirements and challenges of interpreting these observables will be explored. Resources currently available within the BioSpectroscopy core and those whose development is anticipated in the near future will be described. Attendees are encouraged to consider how these resources will enhance their research questions and help them develop new questions.
“Mutagenesis of human cytomegalovirus gH/gL distinguishes functional surfaces of gH/gL/gO and gH/gL/UL128-131.” September 11, 2015. Authors: Eric P. Schultz, Jean-Marc Lanchy, Erin Ellerbeck, Brent Ryckman, Division of Biological Sciences, University of Montana, Missoula, MT
Abstract: The core, conserved function of gH/gL among herpesviruses is to promote gB-mediated membrane fusion during entry. The HCMV gH/gL can exist as either the gH/gL/gO trimer or the gH/gL/UL128-131 pentamer. To study the distinct functions of these complexes, individual mutants of gH and gL were combined to generate a library of mutant gH/gL heterodimers. The vast majority of the mutant gH/gL complexes were unable to facilitate gB-mediated membrane fusion in cell-cell fusion experiments. In contrast, all of the mutants supported the formation of functional gH/gL/UL128-131 complexes, as judged by receptor interference experiments. These results suggest that the mechanism by which gH/gL/UL128-131 promotes entry involves surfaces contained on the UL128-131 proteins, not on gH/gL.
“Dioxygen activation in human indoleamine 2,3-dioxygenase isoform-1 (hIDO1): Uncovering the mechanism of catalytic O-O bond cleavage that avoids enzyme deactivation.” September 12, 2015. Author: Valeriy Smirnov, Department of Chemistry and Biochemistry, University of Montana, Missoula, MT
Abstract: A cytokine-inducible extrahepatic human indoleamine 2,3-dioxygenase isoform-1 (hIDO1) catalyzes the first step of the kynurenine pathway. Catalytic depletion of L-tryptophan (L-Trp) by hIDO1 results in immunosuppression, weakening the host T-cell immunity and driving progression of various cancers. Here we report on steady-state and single-turnover kinetics of hIDO1, aimed at establishing the nature of the catalytically active metal-oxygen species in this enzyme. We monitor initial rates of O2 decay on a Clark-type oxygen electrode to determine the parameters for enzyme turnover at physiologically-relevant levels of dioxygen (O2) and L-Trp. Using a cytochrome b5-based activating system, we quantify the effects of O2. Additionally, we use a peroxide, meta-chloroperoxybenzoic acid (mCPBA), to cleanly generate ferryl species in hIDO1, and monitor their formation and decay by stopped-flow spectrophotometry. We discuss the role of ferryl derivatives in hIDO1 catalysis of L-Trp dioxygenation, contrasting our results with those in the published literature, where a neutral ferryl species was proposed as a catalytically active intermediate in dioxygenation of L-Trp by hIDO1.
“Directed Synthesis of More Potent Inhibitors of Molecular Pathways Associated withEpithelial Mesenchymal Transition as Novel Tools and Potential Chemotherapies.” September 13, 2015. Author: Andrea Stierle, Department of Biomedical and Pharmaceutical Sciences, University of Montana, Missoula, MT
Abstract: The Stierle laboratory is using x-ray crystallography and molecular computational methods to guide the synthesis of more potent MMP-3 inhibitors. They will test their hypothesis that potent inhibitors of enzymes that promote epithelial-mesenchymal transition (EMT) will have potent anti-proliferative activity against human cancer cell lines. The Stierles have purified and characterized a library of MMP-3 inhibitors from diverse sources that are providing the scaffolds for development of new analogues for further evaluation. Translating promising natural product leads into therapeutically relevant molecules often requires structural modifications that enhance potency, selectivity, and optimal ADME characteristics. They will describe their efforts towards their specific aim from CoBRE Year 1.
“The BioSpectroscopy Core-- Highlights from Year 4.” September 11, 2015. Authors: Michelle Terwilliger, Sandy Ross, BioSpectroscopy Core Research Laboratory (BCRL), University of Montana, Missoula, MT
Abstract: The BioSpectroscopy Core Research Laboratory continues to offer experimental services in fluorescence spectroscopy and microscopy to UM investigators for the study of biomolecular interactions and dynamics. This presentation is an overview of highlights from Year 4 of CoBRE support, including new and continued collaborative work as well as a preview of the new time-resolved confocal microscope which will be purchased in Year 5.
“Dynein light chain, an emerging facilitator of RNA-binding proteins in C. elegans.” September 12, 2015. Authors: Xiaobo Wang, Mary Ellenbecker, and Ekaterina Voronina; Division of Biological Sciences, University of Montana, Missoula, MT
Abstract: LC8 family proteins promote structural organization and stability of proteins with intrinsic disorder regions by assembling with them. We found that C. elegans LC8 protein DLC-1 is important for localization and regulatory activity of FBF-2, an RNA-binding protein required for stem cell maintenance. Mutations disrupting the interaction of FBF-2 with DLC-1 interfere with FBF-2 localization in vivo. We propose that DLC-1 facilitates regulation of numerous developmentally important mRNAs by assembling with regulatory RNA-binding proteins. We found mRNA targets of DLC-1 regulation in developing germ cells that include known targets of FBF-2 regulation as well as many others. This data suggests new candidate RNA-binding proteins that are assisted by DLC-1.
“Comparing Halogen Bonding Receptors to Their Hydrogen Bonding Counterparts.” September 11, 2015. Author: Nicholas B. Wageling, Department of Chemistry and Biochemistry, University of Montana, Missoula, MT
Abstract: Anion recognition is a growing field in organic chemistry. Many interactions can be employed to sequester anions from their surroundings: hydrogen bonding, electrostatic forces, metal complexation, and hydrophobic effects are a few examples. However, compared to the others, halogen bonding (XB) is relatively underexplored. These anion studies will illuminate details about XB, and guide us towards rationally designed receptors. As a non-covalent interaction with strict requirements, halogen bonding shows a promising future in molecular recognition and catalysis. Here is presented the results of anion titration studies, computational data, and x-ray structures of one such halogen bonding molecule and its hydrogen bonding analogue.
“M2O2 "Diamond Core": the Next Generation of Biomimetic Transition-metal Catalyst for Effective and Selective Aliphatic Hydrocarbon Functionalization." September 12, 2015. Author: Dong Wang, Department of Chemistry and Biochemistry, University of Montana, Missoula, MT
Abstract: The selective installation of functional groups onto aliphatic carbons is a key step in many metabolic transformations, and finds many uses in synthetic chemistry. While this process is catalyzed by metalloenzymes with high selectivity, great challenges exist for synthetic systems both in activating strong C-H bonds and in controlling the functional group transfer selectively onto the target carbon center. The proposed research obtain inspirations from metalloenzymes utilizing dinuclear active sites, and propose to develop M2O2 catalysts with a "diamond core" structure, where the two metals function in a cooperative manner in the catalytic cycle to achieve high efficiency and selectivity.
“Dynein subunit DLC-1 promotes localization and function of stem cell regulator FBF-2 in C. elegans.” September 11, 2015. Authors: Xiaobo Wang1, Mary Ellenbecker1, Dominique Rasoloson2, and Ekaterina Voronina1; 1Division of Biological Sciences, University of Montana, Missoula, MT; 2MBG Department, Johns Hopkins University School of Medicine/HHMI, Baltimore, MD
Abstract: The PUF family protein FBF-2 is a translational repressor necessary for maintaining germline stem cells in C. elegans. FBF-2 regulatory function depends on its localization to cytoplasmic RNA granules through mechanisms that are not completely understood. Here, we identified dynein subunit DLC-1 as a FBF-2 cofactor. We demonstrate that DLC-1 binds to FBF-2 in the in vitro pull down assay. FBF-2 interaction with DLC-1 is required for FBF-2 localization to RNA granules. This function of DLC-1 is independent of the dynein motor. Our findings suggest that DLC-1 facilitates germline stem cell maintenance by interacting with FBF-2.
Keynote Lecture: “A CRISPR immune response to viruses that infect bacteria.” September 12, 2015. Author: Blake Wiedenheft, Deptartment of Microbiology and Immunology, Montana State University, Bozeman, MT
Abstract: Viruses that infect bacteria are the most diverse and abundant biological agents on the planet, causing roughly 1023 infections every second. To defend themselves against these pervasive viral predators, bacteria have evolved sophisticated adaptive immune systems that rely on a repetitive chromosomal locus called CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat). We recently determined the X-ray crystal structure of a 405-kDa ribonucleoprotein complex that is essential for phage defense in E. coli. This X-ray structure provides a molecular blue print for understanding the mechanisms RNA-guided recognition of invading DNA targets.
Poster Presentations - Proferred Abstracts
September 11, 2015
Listed alphabetically by presenter's last name.
“Theoretical analysis of the triplet excited state of ruthenium mono-diimine and bioconjugated complexes: The effect of solvent and ligands on the photophysical properties.” Author: Sanaa AlAbbad, Department of Chemistry and Biochemistry, University of Montana, Missoula, MT
Abstract: Not provided.
“Is ferryl a side-product or an intermediate in catalysis of L-tryptophan dioxygenation by human indoleamine 2,3-dioxygenase (hIDO1)?” Authors: Ian M. Chrisman, Laura S. Dameron, Valeriy V. Smirnov*, Department of Chemistry and Biochemistry, University of Montana, Missoula, MT
Abstract: hIDO1 catalyzes the addition of molecular oxygen (O2) to L-tryptophan (L-Trp) to form N-formyl-L-kynurenine (NFK) through a heme-centered dioxygenation process. In the published literature, ferryl species has been detected by Raman spectroscopy during L-Trp conversion to NFK. Also, monooxygenated tryptophan derivatives have been identified by mass spectrometry in the quenched assay mixtures of hIDO1. These reports led to the revision of hIDO1 catalytic mechanism, proposing that O2 insertion into L-Trp is a stepwise process. Here we present our results on quantifying the activity of hIDO1 by monitoring NFK formation and O2 consumption. Our work shows evidence that monooxygenase activity of hIDO1 towards L-Trp occurs simultaneously with (yet independently of) its normal turnover as a dioxygenase.
“Impact of AhR activation on the phenotype and function of bone marrow derived dendritic cells after exposure to Pseudomonas aeruginosa.” Authors: Shelby L. Cole, Joanna M. Kreitinger, Benjamin P. Seaver, David M. Shepherd, and Celine A. Beamer, Department of Biomedical and Pharmaceutical Sciences, University of Montana, Missoula, MT
Abstract: Activation of the aryl hydrocarbon receptor (AhR) in dendritic cells (DCs) by the prototypical ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can lead to the induction of a regulatory phenotype and suppressed immune responses to bacterial ligands. Although molecules with a diverse array of chemical scaffolds are known to bind to the AhR and modulate AhR activity, the potential of these ligands to modify the phenotype and function of DCs are currently unknown. Therefore, the goal of the present study was to investigate whether molecules representative of the four families of AhR ligands (e.g. TCDD, benzo(a)pyrene (BaP), leflunomide, and YH439) alter the differentiation and function of bone marrow derived DCs in a ligand-specific and AhR-dependent manner.
Similar to TCDD, YH439 induced an immunoregulatory phenotype and suppressed immune responses to heat killed Pseudomonas Aeruginosa (HKPA) as measured by changes in the expression profiles of select cell surface markers, cytokines, and transcription factors. In contrast, Leflunomide exhibited almost no change in the expression of differentiation markers and no difference in their ability to respond to HKPA compared to control; whereas BaP showed limited induction of a regulatory phenotype and a suppressed response to HKPA. Many, but not all, of the observed changes in the phenotype and function of dendritic cells were dependent on the AhR, as assessed by deriving dendritic cells from mice expressing the low affinity receptor (AhRd) and wild-type C57Bl/6 mice. Our findings suggest that induction of an immune regulatory DC phenotype is AhR-ligand dependent, which implies that environmental contaminants may differentially modulate immunity via changes in the phenotype and function of DCs.
This work is supported by NIEHS/NIGMS grant R01-ES013784 (DMS), as well as NIGMS grants P30GM103338 and P20GM103546. The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of NIEHS, NIGMS or NIH.
“Analysis of DLC-1 mediated mRNA regulation in C. elegans.” View Abstract. Authors: Mary Ellenbecker, Xiaobo Wang and Ekaterina Voronina, Division of Biological Sciences, University of Montana, Missoula, MT
“On the Elucidation of the electrostatic contribution of individual amino acids during cardiolipin binding at Cytochrome-c’s anionic site A.” View Abstract. Author: Margaret Elmer-Dixon, Department of Chemistry and Biochemistry, University of Montana, Missoula, MT
“Identification of human SLC1 transporters that mediate transmembrane flux of D-serine.” Author: Jill Farnsworth, Department of Biomedical and Pharmaceutical Sciences, University of Montana, Missoula, MT
Abstract: ASCT1 and ASCT2 are sodium-dependent neutral amino acid transporters belonging to the solute carrier 1 (SLC1) gene family, which also includes the excitatory amino acid transporters EAAT1-5. As obligate exchangers, these transporters have the potential to regulate homeostatic flux of many neutral amino acids including L-Ala, L-Ser, and L-Cys. ASCT2 additionally mediates transport of L-Gln. Many details regarding the cellular localization and functional roles of the ASCT transporters in the central nervous system are unknown. The molecular species involved in mediating transport of D-Ser in CNS have yet to be identified despite the fact that D-Ser is well-established as a co-agonist at NMDARs and it has been suggested to play critical roles in modulating signaling and synaptic plasticity. In this study, we expressed ASCT-1 and ASCT-2 in Xenopus laevis oocytes and found that a mutation of a TM8 arginine residue conserved in EAATs leads to to broad recognition of many neutral L-amino acids. Both transporters selectively transported the D-isomer of serine with Km values around 100µM, but not other D-amino acids tested. These results suggest that ASCTs may play a role in influencing D-serine concentrations in the CNS through exchange-mediated uptake or release. Additionally, we have synthesized and explored the properties of a novel ASCT1/2 blocker that inhibits transport of D-serine through both transporters at nanomolar concentrations. This compound and other analogs may be useful for modulating D-serine concentrations in the CNS and could prove to be therapeutically relevant for such conditions as AD, stroke, and schizophrenia.
“Elucidating RNA binding preferences of RVFV nucleocapsid protein using deep sequencing.” View Abstract. Author: Katie Hornak, Division of Biological Sciences, University of Montana, Missoula, MT
“Effect of Hydrocarbon Saturation on Relaxation Rates in Membrane Lipids.” Author: Cynthia Janku, Department of Chemistry and Biochemistry, University of Montana, Missoula, MT
Abstract: Nanodiscs are a lipid bilayer model system, which can be used for studying membrane-bound proteins or lipid-protein interactions. Nanodiscs are a lipid bilayer surrounded by a membrane scaffold protein, which is a derivative of Apolipoprotein A1, a protein involved in the removal of cholesterol from the body. There are outstanding questions about how the belt protein affects the fluidity of the lipid bilayer. Using nanodiscs made of either DMPC or DOPC with 5% NBD labeled lipid, we were able to take lifetimes of the nanodiscs, which reflect the relaxation rate of the lipid bilayers. Based on our results, we conclude that an increase in saturation of the hydrocarbon tails decreases the lifetime of the lipid bilayers, thus showing a faster relaxation rate. We also concluded that an increase in temperature decreases the lifetime, and an increase in wavelength increases the time for the lipids to relax.
“Protein folding: Thermodynamics & kinetics of ubiquitin-associated domains.” View Abstract. Author: Moses Joseph Leavens, Department of Chemistry and Biochemistry, University of Montana, Missoula, MT
“Selective anion recognition in competitive media.” View Abstract. Author: Casey J. Massena, Department of Chemistry and Biochemistry, University of Montana, Missoula, MT
“The Role of Histone H3 Lysine 9 Trimethylation in Germ Cell Development.” Authors: Jenessa Olson, Ekaterina Voronina, Division of Biological Sciences, University of Montana, Missoula, MT
Abstract: Chromatin modifications are covalent modifications of nuclear histone proteins that can affect the transcriptional activity of DNA. Some chromatin modifications have been linked to specification of reproductive cells called germ cells. We found that trimethylation of Lysine 9 in Histone H3 becomes prominent in late embryo germ cells of C. elegans, which has not been previously reported. Our research has been focused on identifying the methyltransferase that generates H3K9me3 in germ cells to help determine if H3K9me3 is required for fertility.
We found set-25 mutant embryos lose H3K9me3 in all somatic cells, but maintain H3K9me3 enrichment in the germ cells of later-stage embryos. By using two complementary strategies, we tested the effects of depleting H3K9me3 during embryonic development. Our preliminary data suggest that loss of H3K9me3 correlates with defective reproduction or sterility. Since H3K9me3 modification has been shown to play a significant role in germ cell development in Drosophila and mouse, we hope our research in C. elegans will identify conserved targets of this regulatory mechanism.
“Self-Assembling Uranium Ligands: A Modular Approach to The Design and Understanding of Novel Uranyl-Organic Nanostructures.” Author: Ari Rose, Department of Chemistry and Biochemistry, University of Montana, Missoula, MT
Abstract: A huge obstacle to human progress is the need for continuous, sustainable and clean energy. As the world moves away from damaging carbon energy sources such as coal and oil, the need to improve the nuclear power system becomes evident. Closing the nuclear fuel cycle involves not only cleaner mining techniques but fast, effective environmental remediation and chelation therapy in response to accidental exposure. This project aims to design a uranium ligand technology that can be applied to all three of those areas, with particular interest in mining ocean water where uranium exists as the uranyl dication (UO22+) at a natural abundance of 3ppb. Due to the unusual geometry of UO22+, coordination is limited to the equatorial plane of uranium. This prevents the use of chelators that coordinate both equatorially and axially, and is a key consideration for the modular design approach of this project. Ligand building blocks are allowed to self- assemble around UO22+, and connect with linkers and other ligands via the reversible covalent disulfide bond. This allows complex components to self-sort to the lowest energy configuration. In this way, we use the thermodynamics of the system to screen for complexes that are both highly selective and energetically robust.
“Mutagenesis of human cytomegalovirus gH/gL distinguishes functional surfaces of gH/gL/gO and gH/gL/UL128-131.” View Abstract. Authors: Eric P. Schultz, Jean-Marc Lanchy, Erin Ellerbeck, Brent Ryckman, Division of Biological Sciences, University of Montana, Missoula, MT
“Temperature dependence of Cardiolipin and DOPC nanodiscs and their fluidity based on Time Resolved Emission Spectra.” Author: Kristian Stipe, Department of Chemistry and Biochemistry, University of Montana, Missoula, MT
Abstract: Cardiolipin is a negatively charged phospholipid that comprises roughly 20% of the lipids in the inner mitochondrial membrane. The dynamics of this membrane can influence interactions between cytochrome c and cardiolipin, which have been shown to initiate the intrinsic pathway of apoptosis. This investigation studies how the dynamics of a model membrane system will behave with a change in temperature. The nanodiscs are composed of 18:1 cardiolipin* and DOPC*, doped with either NBD-PE*. Time-Resolved Emission Spectra (TRES) of NBD-PE doped nanodiscs are used to report on head group relaxation. This method is used to provide insight into how cardiolipin affects lipid membrane dynamics.
*18:1 cardiolipin: 1',3'-bis[1,2-dioleoyl-sn-glycero-3-phospho]-sn-glycerol DOPC: (Δ9-Cis) PC 1,2-dioleoyl-sn-glycero-3-phosphocholine NBD-PE: 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl)
“Comparing Halogen Bonding Receptors to Their Hydrogen Bonding Counterparts.” View Abstract. Author: Nicholas B. Wageling, Department of Chemistry and Biochemistry, University of Montana, Missoula, MT
“Dynein subunit DLC-1 promotes localization and function of stem cell regulator FBF-2 in C. elegans.” View Abstract. Authors: Xiaobo Wang1, Mary Ellenbecker1, Dominique Rasoloson2, and Ekaterina Voronina1; 1Division of Biological Sciences, University of Montana, Missoula, MT; 2MBG Department, Johns Hopkins University School of Medicine/HHMI, Baltimore, MD
“Protein/X-Ray Core: An Awesome Collaborative Experience.” Authors: Marlene Warner, T.C. Mou, Macromolecular X-Ray Diffraction Core Facility (MXDC), University of Montana, Missoula, MT
Abstract: The CBSD Protein/X-Ray Core provides materials and expertise to assist UM laboratories to produce and purify target proteins and to crystallize and determine three-dimensional atomic crystal structure. During the Phase I COBRE funding, the comprehensive Core facility is garnering a support center for investigators and UM research projects and is fostering collaborative new projects that address important biological questions using isolated protein sample and X-ray crystallography. We offer gene-to-structure services with clones provided by user or engineered with our established collections of cloning and expression vectors and host cells for bacterial and insect expression system. The Core hosts equipment for trial and scale-up protein expression, purification and characterization from mg to mg scale. In addition, to providing access and infrastructure for crystallographery experiences, the majority of the Core users are from non-crystallography laboratories whom are interested in structure and function of their targets. Indeed, we encourage a collaborative model in which students or staff from individual investigators conducts projects with assistance and training from Core personnel. Moreover, we also offer problem-solving guidance to revise protein expression and purification protocols to improve protein quality from individual laboratories to perform biochemical and biophysical studies. In addition to the COBRE funding and institutional commitment, the Core has operated as a recharge facility with an established fee structure and even a voucher system. The objectives of the Core are to support synergies and interdisciplinary approach from UM research groups and to enhance competitiveness and productive of individual laboratories.
“Identification and Mutational Analysis of a Putative Gαi Binding Site on RIC8A.” Author: Baisen Zeng, Department of Chemistry and Biochemistry, University of Montana, Missoula, MT
Abstract: RIC-8A, acts as a soluble guanine-nucleotide exchange factor (GEF) for several G-protein alpha subunits (i, q, t, s and 13), by accelerating the rate of GDP to GTP exchange. RIC-8A binds to the Gαi1 subunit and forms a stable nucleotide free RIC-8A:Gαi1 complex which has been demonstrated to be in a highly dynamic conformational state. Hydrogen-deuterium exchange (HDX) kinetics of this complex indicates that a C-terminal region on the RIC-8A becomes less solvent exposed upon Gαi1 binding. We hypothesize that this region is a putative protein-protein interactive surface.
“Analysis of antibody epitopes of HCMV glycoproteins by fluorescence correlation spectroscopy.” Authors: Le Zhang, Brent Ryckman, Division of Biological Sciences, University of Montana, Missoula, MT
Abstract: The ectodomain of the human cytomegalovirus (HCMV) glycoprotein gH/gL is bound by either gO, or the set of UL128-131 proteins to form the complexes gH/gL/gO, and gH/gL/UL128-131. These complexes facilitate distinct stages of the entry event, and the distinction is likely due to differences in the conformation or accessibility of gH/gL surfaces. Consistent with this, immunoprecpitation experiments indicated that some antibody epitopes of gH/gL are structurally conserved between the two complexes, but others are not. The goal of this project is to conduct a more comprehensive, and quantitative analysis of gH/gL surface structures using a large panel of gH/gL antibodies. Soluble gH/gL complexes will be purified, fluorescently tagged, and analyzed by fluorescence correlation spectroscopy with or without antibody. The effect of each antibody on the diffusion of the different gH/gL complexes will allow calculation of the antibody affinity, which will provide an indication of the accessibility of that gH/gL surface on each of the two gH/gL complexes.