Agenda
See the full research symposium agenda at this weblink: https://docs.google.com/document/d/12oBfekdhkyC6D0wd--2vkikwczb2ThMxXLaWRVruXlI/edit?usp=sharing
Abstracts
CoBRE Junior Investigator Presentations
Orion Berryman, Assistant Prof. (Chemistry/Biochemistry), A Halogen Bond Induced Triple Helicate Encapsulates Iodide.
The self-assembly of higher order anion helicates in solution remains an elusive goal. Here we present the first triple helicate to bind and encapsulate iodide in organic and aqueous media, as well as the solid state. The helicate self-assembles from three triply cationic arylethynyl strands and resembles a tubular anion channel lined with halogen bond donors. The triplex exhibits remarkable stability at elevated temperatures being held together by nine inwardly directed halogen bonds and numerous buried π surfaces. We attribute this innovation to the stringent linearity of halogen bonding, in that the divergence of halogen bond donors along a helical axis destabilizes 1:1 complexation. In contrast, hydrogen bonding ligands capacity for binding halides is less dependent on bond angle. We hypothesize that incorporating halogen bonds within a simple alternating repeat unit can induce higher-order helication.
Kasper Hansen, Assistant Prof. (BMED), Structural basis for negative allosteric modulation of NMDA receptors.
NMDA receptors mediate excitatory synaptic transmission in the central nervous system, but their dysregulation is also implicated in numerous brain disorders. We describe subunit-selective negative allosteric modulators (NAMs) that inhibit NMDA receptors by stabilizing the apo-state of the agonist binding domain, which is incapable of triggering channel gating. We identify structural determinants of NAM binding and subunit-selectivity in crystal structures, and uncover structural changes induced by NAM binding. This work provides structural and mechanistic insight to allosteric NMDA receptor inhibition, thereby facilitating the development of novel classes modulators as treatments in neurological diseases.
Andrea Stierle, Research Prof. (BMED), Directed Synthesis of More Potent Inhibitors of Molecular Pathways Associated with Epithelial Mesenchymal Transition as Novel Tools and Potential Chemotherapies.
Translating promising natural products into therapeutically relevant molecules often requires structural modifications that enhance potency and selectivity. The Stierles have a library of MMP-3 inhibitors which will provide the scaffolds for the syntheses of new analogues for further evaluation and testing. The Stierles modeled these inhibitors in well-defined crystal structures of the MMP-3 catalytic domain and assessed which ligand structural features conferred inhibition potency and/or selectivity based on potential protein-ligand interaction sites. With the X-Ray Core facility, they also expressed and crystallized the catalytic domain of MMP-3 and unsuccessfully soaked these crystals with two different inhibitors. They are now attempting to generate crystals of the complete enzyme to identify an allosteric site for these ligands.
Celine Beamer, Research Assistant Prof. (BMED), Aryl hydrocarbon Receptor (AhR) signaling modifies pseudomonas aeroginosa-regulated responses in murine dendritic cells
Currently, it is not well understood how ligands of the Aryl hydrocarbon Receptor (AhR) modify inflammatory responses triggered by bacterial agonists in dendritic cells (DCs). DCs are key antigen presenting cells controlling T cell differentiation, and activation of AhR by various ligands can induce differentiation of regulatory (Treg) or inflammatory (Th17) T cells. Here, we present novel data showing that structurally diverse AhR ligands differentially modify immunoregulatory gene expression, surface marker expression, and cytokine secretion by murine bone marrow-derived DCs (BMDCs) in response to heat killed pseudomonas aeroginosa in a ligand- and AhR-dependent manner. We are in the process of combining this data with computational docking and molecular dynamics to identify residues involved in differential signaling and immune function. We will use this data to guide small molecule design with the intent to promote a regulatory effect in DCs, and use this knowledge to develop novel therapies for inflammatory diseases.
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.
"Elevator Pitch" Student/Post-Doctoral Fellow Presentations
Mary Ellenbecker, Post-Doctoral Fellow (Voronina), Analysis of DLC-1 mediated regulation of the tumor suppressor protein GLD-1.
Authors: Mary Ellenbecker and Ekaterina Voronina
DLC-1 is a developmental regulator that controls decisions related to cell proliferation and differentiation in C. elegans. One interaction partner of DLC-1 is the tumor suppressor and RNA regulatory protein GLD-1 that promotes stem cell differentiation during development. Disruption of this gene in gld-1 mutant worms results in germline tumor formation. My results indicate that association with DLC-1 promotes the functions of GLD-1 and I am using genetic and molecular approaches to study the cooperation between DLC-1 and GLD-1. Since both DLC-1 and GLD-1 are evolutionarily conserved proteins any new insight into this cooperation will be broadly relevant.
Le Zhang, Graduate student (Ryckman), Interstrain variability in the assembly of HCMV gH/gL/gO has a multi-locus basis.
Entry of HCMV into all cell types likely involves membrane fusion mediated by gB and gH/gL/gO (trimer). Entry into select cells, such as epithelial and endothelial cells, also requires a poorly understood, non-fusion function provided by gH/gL/UL128-131 (pentamer). The UL148 protein may act as an ER-chaperone to favor the assembly of gH/gL/gO over gH/gL/UL128-131 (Li 2014 PNAS). In Zhou 2015 JVI, we showed that strain Merlin (ME) contained far less gH/gL/gO than strains TB40/e (TB) or TR, and this correlated with ME being less infectious than TB and TR, on both fibroblasts or epithelial cells. Here we report a comparison of protein expression in cells infected with TB, TR and ME. ME expressed much more of the UL128-131 proteins, but less of UL148 protein. In contrast, gH/gL levels were more comparable, but whereas TB and TR gH/gL were efficiently transported to the trans-Golgi derived virus assembly compartment, ME gH/gL remained mostly in the ER. These results are consistent with the model that UL148 acts as a chaperone to promote the assembly of gH/gL/gO, and that the dearth of gH/gL/gO in ME virions is due to both high expression of UL128-131 proteins, and low expression of UL148. To address potential effects of the 25% amino acid difference between gO encoded by ME and TR, a recombinant ME expressing the TRgO was generated (MEdeltaTRgO), and was found to be more infectious than the parental ME on both fibroblasts and epithelial cells. It is not yet clear if the increased infectivity was the result of slight changes in the amount of gH/gL/gO, or is indicative of functional differences between TRgO and MEgO. Taken together, these results suggest that variability between strains in gH/gL complexes involves differences at multiple genetic loci.
Casey Massena, Graduate student (Berryman), A Halogen Bond Induced Triple Helix Encapsulates Iodide.
Nobel laureate Roald Hoffmann once remarked that organic chemists excel at synthesizing individual molecules but struggle with molecular assemblies. My research addresses this shortcoming. At the last CBSD retreat, I presented the synthesis of a halogen bonding oligomer. Since then, I have obtained an X-ray crystal structure of the first halogen bonding triple helix. ¹H NMR, 2D NOESY, and DOSY spectroscopies have confirmed the helix’s shape persistence not only in organic solvents, but also in water and at elevated temperatures. The synthesis of a large multi-component structure with affinity for iodide is a huge step forward for organic chemistry.
Eric Schultz, Post-Doctoral Fellow (Ryckman), Mutagenesis of HCMV gH/gL distinguishes different routes of viral entry.
The diverse pathologies of HCMV correlate with the ability of the virus to infect many cell types, in various organs of the human body. An emerging model for HCMV tropism suggests that gH/gL/gO promotes gB-mediated membrane fusion for entry into all cell types, whereas gH/gL/UL128-131 is also required for infection of select cell types, working through an independent, non-fusion mechanism. We recently characterized a library of 80 HCMV gH/gL mutants using replication-defective adenovirus expression vectors. 72 of these mutants failed to support gB-mediated cell-cell fusion in transient expression experiments. The library of gH/gL mutants has now been cloned into the HCMV genome using BAC-recombination. As expected, all mutants that tested positive in cell-cell fusion experiments support HCMV replication. Interestingly, the replication of some mutants is highly restricted to cell-to-cell spread, including some that tested negative in cell-cell fusion experiments. These analyses suggest mechanistic differences between transient expression cell-cell fusion and virus entry, as well as differences between cell-free and cell-associated spread mechanisms.
Ian Chrisman, Graduate Student (Smirnov), Is ferryl a side-product or an intermediate in catalysis of L-tryptophan dioxygenation by human indoleamine 2,3-dioxygenase (hIDO1)?
hIDO1 catalyzes the addition of molecular oxygen (O2) to free L-tryptophan (L-Trp) to form N-formyl-L-kynurenine (NFK) via the heme-centered dioxygenation reaction. In literature, ferryl species have been observed by Raman spectroscopy during catalytic turnover. As well monooxygenated tryptophan species have been observed by mass spectrometry following quenching of catalysis. This data has been used to support a catalytic mechanism whereby oxygen is inserted stepwise into tryptophan. Here we present data detailing the accumulation of a neutral ferryl derivative of hIDO1, (Cpd II)•L-trp, which is formed outside of the steady-state distribution of catalytically active species. Additionally we monitor the formation of (Cpd II)•L-trp alongside NFK and demonstrate significantly different kinetics for accumulation of each. Taken together, this data suggests that formation of (Cpd II)•L-trp occurs independently of normal dioxygenase turnover.
Levi McClelland, Post-Doctoral Fellow (Sprang), Kinetic insights into Ric-8A GEF activity.
Resistance to inhibitors of cholinesterase protein, Ric-8A, acts as a chaperone for Giα in vivo and demonstrates Guanine nucleotide Exchange Factor (GEF) activity for Giα in vitro by releasing GDP from Giα:GDP to form Giα:Ric-8A which then dissociates to Giα:GTP and free Ric-8A in the presence of GTP. To date, the specific reaction mechanism by which Ric-8A functions as a GEF is poorly understood. In this work, we utilize these stopped-flow experimental techniques to elucidate the kinetics of Ric-8A GEF activity in order to gain insight into the mechanism of the Ric8A/Giα interaction.
Career Development & New Recruit Faculty Presentations
Ekaterina Voronina, Career Development Faculty (DBS), LC8 dynein light chain, a facilitator of RNA-binding proteins in C. elegans.
Our previous research identified LC8 family protein DLC-1 as a binding partner of nematode stem cell regulator FBF-2. Direct FBF-2 binding to DLC-1 is required for FBF-2 localization and biological activity. LC8 proteins promote structural organization of their partners, and we aim to characterize the interaction between FBF-2 and DLC-1. FBF-2 interacts with DLC-1 through multiple sites, and FBF-2/DLC-1 complex is significantly stabilized by these multivalent interactions. We hypothesized that DLC-1 promotes function of other post-transcriptional regulators of development, and identified a tumor suppressor GLD-1 as an additional protein that requires DLC-1-binding for activity.
Travis Hughes, New Biomedical & Pharmaceutical Sciences Dept. Faculty Recruit, Α view into the conformational ensemble of PPARγ.
Abstract not available.
Dong Wang, New Dept. of Chemistry & Biochemistry Faculty Recruit, Biomimetic, catalytic functionalization of aliphatic hydrocarbons by dinuclear M2O2 "diamond core" complexes.
Abstract not available.
Philippe Diaz, CoBRE Phase II Junior Investigator (BMED), Selective inhibition of CYP26 in the skin for the treatment of ichthyosis.
Ichthyosis is a family of rare genetic disorders characterized by persistently dry and scaly skin. Current treatments do not adequately address patient needs, presenting significant efficacy or tolerability concerns. Retinoic acid is the most efficient topical treatment for ichthyosis. However, retinoic acid induces its own clearance and adverse effects in humans. We have identified substrate-based CYP26 inhibitors acting through retinoic acid metabolism inhibition as a novel class of therapeutic agents for the treatment of ichthyosis. We will report the effects of our CYP26 inhibitors in skin cells and discuss about the therapeutic effect of this novel class of compounds.
CoBRE Core Facility Presentations & Workshops:
BCRL (Sandy Ross & Michelle Terwilliger): The BioSpectroscopy Core: Highlights from CoBRE Year 5.
The BioSpectroscopy Core Research Laboratory (BCRL) will host an interactive session for current and potential new users of the core to discuss new approaches in time-resolved spectroscopy and microscopy on Thursday, June 2 from 10-11 AM in UC 327 and an open-house in Chemistry 013 from 9-10 AM and 11 AM - 12 PM.
MCCF (Stephen Sprang & David Holley): The Molecular Computation Core Facility CoBRE Year 5 Update.
The Molecular Computation Core Facility (MCCF) will host an interactive session for current and potential new users of the core on Thursday, June 2 from 11 AM -12 PM in Skaggs 482 and an open-house in Skaggs 482 from 9-11 AM.
MXDC (Stephen Sprang & T.C. Mou): The Macromolecular X-Ray Diffraction Core Facility Year 5 Update.
The Macromolecular X-Ray Diffraction Core Facility (MXDC) will host an interactive session for current and potential new users of the core on Thursday, June 2 from 9 AM -10 AM in UC 327 and an open-house in the Interdisciplinary Science Bldg. 016 (basement) from 10 AM -12 PM.
NMR/MASS SPEC (TENTATIVE)
Abstract not available.
Poster Session Presentations
Baisen Zeng, Graduate Student (Sprang): Identification and Mutational Analysis of a Putative Gαi1 Binding Site on RIC-8A.
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 solvated upon Gαi1 binding. We hypothesize that this region is a putative protein-protein interactive surface.
Moses Leavens, Graduate Student (Bowler): Protein folding of HHR23A Ubiquitin-associated (UBA) domains.
The p62 UBA domain has been shown to contain quaternary structure in the micromolar concentration range, and has been hypothesized to play a role in modulating interaction of p62 with polyubiquitin chains. Here, HHR23A UBA domain thermodynamics and a quaternary structure study are carried out to elucidate potential competition between ubiquitin binding and UBA domain dimerization. The UBA(2) C26A variant enhances stability, no change in the UBA(1) equilibrium constant for unfolding is observed. The HHR23A UBA domains do not interact, consistent with a different mechanism other than HHR23A UBA domain homodimerization.
Jenessa Olson, Staff Technician (Voronina): The Role of Histone H3 Lysine 9 Trimethylation in Germ Cell Development.
Authors: Jenessa Olson and Ekaterina Voronina
Chromatin modifications are covalent modifications of nuclear histone proteins that can affect the transcriptional activity of DNA. We found that trimethylation of Lysine 9 in Histone H3, a transcriptional repressor, becomes prominent in late embryo germ cells of C. elegans. 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 partial sterility by defects in spermatogenesis. 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.
Joachim Veit, Graduate Student (Diaz): Novel CYP26 inhibitors modulate retinoic acid related gene expression in ichthyotic and normal human keratinocytes.
Ichthyosis, a congenital skin disorder with no cure, is often treated with retinoic acid to normalize the abnormal keratinization of the skin. Retinoic acid (RA) is primarily metabolized by CYP26 enzymes and biomarkers of its metabolism are well established. Due to the auto-induced clearance and irritation resulting from treating ichthyosis with RA, the use of RA metabolism blocking agents may better provide patients the benefits of increased RA levels in the skin. We have designed novel CYP26 inhibitors using homology modeling and synthesized a selection of compounds. By analyzing mRNA expression changes in treated keratinocytes, we show our compounds can potentiate the effects of RA.
Kelly McGrath, Graduate Student (Stierle): Structure-based drug design and Caspase-1.
Caspase-1 is one of the sentinel proteins in the inflammatory process. Once activated by the inflammasome, caspase-1 activates the proinflammatory cytokines pro-IL-1B and IL-18 which provokes the inflammatory response. When dysregulated, inflammation is associated with auto-immune and neuroinflammatory diseases, chronic pain, and certain cancers, and represents a potential target for therapeutic intervention in the treatment of these disorders. Previously, several novel caspase-1 inhibitors were isolated from extremophilic fungal species and characterized by the Stierle lab. We are currently expressing, purifying and crystallizing caspase-1 to accommodate structure-based drug design to enhance the potency and selectivity of these caspase-1 inhibitors.
Nicholas Day, Graduate Student (Voronina): The Search for Determinants of Germ Granule Localization.
Cytoplasmic complexes of RNA binding proteins called germ granules are essential for development of germ cells and reproduction. Pumilio family RNA binding protein FBF-2 is one of several proteins that associate with these germ granules. FBF-2 is responsible for maintenance of germline stem cells. Recent work has shown that the LC8 dynein light chain (DLC-1) interacts with FBF-2 and is necessary for FBF-2 localization to germ granules. We aim to identify the determinants for germ granule localization via a protein chimera approach. We test whether a DLC-1-interacting motif is sufficient to target DLC-1 partner protein to germ granules.
Feng Yi, Post-Doctoral Fellow (Hansen): Structural basis for negative allosteric modulation of GluN2A-containing NMDA receptors.
NMDA receptors mediate slow excitatory synaptic transmission and regulate synaptic plasticity in the central nervous system. Their dysregulation is implicated in psychiatric and neurological disorders. Subunit‐selective modulators that target NMDA receptor subtypes in specific brain regions or cell types could have unique therapeutic potentials. Here, we describe GluN2A-selective negative allosteric modulators (NAMs) that inhibit NMDA receptors by stabilizing the apo-state of the GluN1 agonist binding domain (ABD), which is incapable of triggering channel gating. This work provides structural and mechanistic insight to allosteric NMDA receptor inhibition, thereby facilitating the development of novel NMDA receptor modulators as treatments in neurological diseases
Levi McClelland, Post-Doctoral Fellow (Sprang): Kinetic insights into Ric-8A GEF activity.
Resistance to inhibitors of cholinesterase protein, Ric-8A, acts as a chaperone for Giα in vivo and demonstrates Guanine nucleotide Exchange Factor (GEF) activity for Giα in vitro by releasing GDP from Giα:GDP to form Giα:Ric-8A which then dissociates to Giα:GTP and free Ric-8A in the presence of GTP. To date, the specific reaction mechanism by which Ric-8A functions as a GEF is poorly understood. In this work, we utilize these stopped-flow experimental techniques to elucidate the kinetics of Ric-8A GEF activity in order to gain insight into the mechanism of the Ric8A/Giα interaction.
Le Zhang, Graduate student (Ryckman): Interstrain variability in the assembly of HCMV gH/gL/gO has a multi-locus basis.
Entry of HCMV into all cell types likely involves membrane fusion mediated by gB and gH/gL/gO (trimer). Entry into select cells, such as epithelial and endothelial cells, also requires a poorly understood, non-fusion function provided by gH/gL/UL128-131 (pentamer). The UL148 protein may act as an ER-chaperone to favor the assembly of gH/gL/gO over gH/gL/UL128-131 (Li 2014 PNAS). In Zhou 2015 JVI, we showed that strain Merlin (ME) contained far less gH/gL/gO than strains TB40/e (TB) or TR, and this correlated with ME being less infectious than TB and TR, on both fibroblasts or epithelial cells. Here we report a comparison of protein expression in cells infected with TB, TR and ME. ME expressed much more of the UL128-131 proteins, but less of UL148 protein. In contrast, gH/gL levels were more comparable, but whereas TB and TR gH/gL were efficiently transported to the trans-Golgi derived virus assembly compartment, ME gH/gL remained mostly in the ER. These results are consistent with the model that UL148 acts as a chaperone to promote the assembly of gH/gL/gO, and that the dearth of gH/gL/gO in ME virions is due to both high expression of UL128-131 proteins, and low expression of UL148. To address potential effects of the 25% amino acid difference between gO encoded by ME and TR, a recombinant ME expressing the TRgO was generated (MEdeltaTRgO), and was found to be more infectious than the parental ME on both fibroblasts and epithelial cells. It is not yet clear if the increased infectivity was the result of slight changes in the amount of gH/gL/gO, or is indicative of functional differences between TRgO and MEgO. Taken together, these results suggest that variability between strains in gH/gL complexes involves differences at multiple genetic loci.
Casey Massena, Graduate student (Berryman): A Halogen Bond Induced Triple Helix Encapsulates Iodide.
Nobel laureate Roald Hoffmann once remarked that organic chemists excel at synthesizing individual molecules but struggle with molecular assemblies. My research addresses this shortcoming. At the last CBSD retreat, I presented the synthesis of a halogen bonding oligomer. Since then, I have obtained an X-ray crystal structure of the first halogen bonding triple helix. ¹H NMR, 2D NOESY, and DOSY spectroscopies have confirmed the helix’s shape persistence not only in organic solvents, but also in water and at elevated temperatures. The synthesis of a large multi-component structure with affinity for iodide is a huge step forward for organic chemistry.
Ian Chrisman, Graduate Student (Smirnov): Is ferryl a side-product or an intermediate in catalysis of L-tryptophan dioxygenation by human indoleamine 2,3-dioxygenase (hIDO1)?
hIDO1 catalyzes the addition of molecular oxygen (O2) to free L-tryptophan (L-Trp) to form N-formyl-L-kynurenine (NFK) via the heme-centered dioxygenation reaction. In literature, ferryl species have been observed by Raman spectroscopy during catalytic turnover. As well monooxygenated tryptophan species have been observed by mass spectrometry following quenching of catalysis. This data has been used to support a catalytic mechanism whereby oxygen is inserted stepwise into tryptophan. Here we present data detailing the accumulation of a neutral ferryl derivative of hIDO1, (Cpd II)•L-trp, which is formed outside of the steady-state distribution of catalytically active species. Additionally we monitor the formation of (Cpd II)•L-trp alongside NFK and demonstrate significantly different kinetics for accumulation of each. Taken together, this data suggests that formation of (Cpd II)•L-trp occurs independently of normal dioxygenase turnover.
Eric Schultz, Post-Doctoral Fellow (Ryckman): Mutagenesis of HCMV gH/gL distinguishes different routes of viral entry.
The diverse pathologies of HCMV correlate with the ability of the virus to infect many cell types, in various organs of the human body. An emerging model for HCMV tropism suggests that gH/gL/gO promotes gB-mediated membrane fusion for entry into all cell types, whereas gH/gL/UL128-131 is also required for infection of select cell types, working through an independent, non-fusion mechanism. We recently characterized a library of 80 HCMV gH/gL mutants using replication-defective adenovirus expression vectors. 72 of these mutants failed to support gB-mediated cell-cell fusion in transient expression experiments. The library of gH/gL mutants has now been cloned into the HCMV genome using BAC-recombination. As expected, all mutants that tested positive in cell-cell fusion experiments support HCMV replication. Interestingly, the replication of some mutants is highly restricted to cell-to-cell spread, including some that tested negative in cell-cell fusion experiments. These analyses suggest mechanistic differences between transient expression cell-cell fusion and virus entry, as well as differences between cell-free and cell-associated spread mechanisms.
William Penny, Graduate Student (Ross/Palmer): Retention behavior of synthetic lipid nanodiscs in electrokinetic chromatography: correlation with octanol-water partition coefficients.
My research goal is to develop a method for the quantification of the lipophilicity of small molecules using capillary electrophoresis (CE). This method will utilize CE, electrokinetic chromatography (EKC) with an anionic nanodisc, which is a lipid-polymer pseudostationary phase (PSP). The neutral small molecule’s lipophilicity will be determined by its ability to partition between the nanodiscs and the background electrolyte (BGE) by measurement of the retention time of the analyte during an EKC run in a CE.
Mary Ellenbecker, Post-Doctoral Fellow (Voronina): Analysis of DLC-1 mediated regulation of the tumor suppressor protein GLD-1.
Authors: Mary Ellenbecker and Ekaterina Voronina
DLC-1 is a developmental regulator that controls decisions related to cell proliferation and differentiation in C. elegans. One interaction partner of DLC-1 is the tumor suppressor and RNA regulatory protein GLD-1 that promotes stem cell differentiation during development. Disruption of this gene in gld-1 mutant worms results in germline tumor formation. My results indicate that association with DLC-1 promotes the functions of GLD-1 and I am using genetic and molecular approaches to study the cooperation between DLC-1 and GLD-1. Since both DLC-1 and GLD-1 are evolutionarily conserved proteins any new insight into this cooperation will be broadly relevant.
Mark Grimes, Associate Professor (Division of Biological Sciences): Cluster-filtered network analyses of post-translational modification signaling pathways in lung cancer cell lines
Authors:
Mark Grimes3, Nicolas Fernandez1, Neil Clark1, Avi Ma’ayan1, Klarisa Rikova2, Peter Hornbeck2
1Department of Pharmacology and Systems Therapeutics, Icahn School of Medicine at Mount Sinai, Systems Biology Center, New York, NY
2Cell Signaling Technology, Danvers, MA
3Division of Biological Sciences, University of Montana, Missoula, MT
Signaling pathways involving post-translational protein modification can go awry to cause cancer, and the study of protein modifications provides both characteristic signatures and clues to the driving signaling pathways in different cancers. Recent advances in generation of modification-specific antibodies has allowed acquisition of large scale mass spectrometry data for different post-translational modifications, including phosphorylation, methylation, and acetylation. We used immunoprecipitation with modification-specific antibodies and Tandem Mass Tag (TMT) mass spectrometry to compare lung cancer cell lines to normal lung tissue, and cell lines treated with kinase inhibitor drugs. Analysis of the resulting data required special considerations because mass spectrometry produces data with a large number of missing values. We evaluated different methods for calculating statistical relationships within these data, which can grouped into three approaches that we call imputing zeros, pairwise-complete, and penalized matrix decomposition. Statistical relationships were embedded into a reduced dimension model of data structure using the machine learning algorithm, t-distributed stochastic neighbor embedding (t-SNE). Pairwise complete methods were the most effective statistical treatment that produced well-resolved t-SNE embeddings and clusters that made sense based on internal and external evaluations. A second penalized matrix decomposition and t-SNE step further resolved large clusters to produce a highly pruned co-cluster correlation network (CCCN) for strongly associated modifications. We combined the modification CCCN with protein-protein interaction (PPI) data to elucidate a cluster-filtered network that suggests a molecular signaling pathway between several receptor tyrosine kinases, transcription factors, and enzymes that modify chromatin proteins. We elucidated a pathway linking EGFR to the transcription factor, SMARCA4, and another pathway linking MET to the methyltransferase, ASH1L. These results support the hypothesis that clusters identified by statistical relationships that contain proteins known to interact with one another are likely to represent functional signaling pathways. The results suggest that extending analyses to include different post-translational modifications such as acetylation and methylation provides a link from kinases to epigenetic chromatin modifications.
Xiaobo Wang, Graduate Student (Voronina): LC8 Dynein Light Chain promotes localization and function of stem cell RNA regulator FBF-2 in C. elegans.
Authors: Xiaobo Wang1, Jenessa Olson1, Dominique Rasoloson2, Mary Ellenbecker1, Jessica Bailey1, and Ekaterina Voronina1
1Division of Biological Sciences, University of Montana, Missoula, MT 59812
2Department of Molecular Biology and Genetics, Johns Hopkins School of Medicine, Baltimore, MD 21205
The RNA binding protein FBF-2 regulates maintenance of 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 LC8 dynein light chain DLC-1 as a FBF-2 cofactor. In vitro, DLC-1 bound to FBF-2 at a noncanonical binding motif. FBF-2 interaction with DLC-1 is required for FBF-2 regulatory function and its enrichment in RNA granules. DLC-1 function contributing to FBF-2 activity and localization is independent of the dynein motor. Our findings suggest that DLC-1 facilitates stem cell maintenance by interacting with FBF-2.
Michael Kavanaugh, Professor (Biomedical & Pharmaceutical Sciences): Optical control of glutamate transport with a novel photoswitchable probe.
Abstract not available.
Sanaa AlAbbad, Graduate Student (Ross): Combined DFT/TDDFT Study of the Electronic Structure, Transe Influence, and Spectral Properties of of [RuH(CO)dcbpy(PPh3)2]+ and its Deprotonated Form.
Abstract not available.
Haotian Lei, Graduate Student (Bowler): TBA
Abstract not available.