2013 Keynote Video
2013 Poster Session
Oral Presentation & Poster Abstracts
Oral Presentation Abstracts
Oral presentation abstracts will be posted when the final presenters have been selected by the review committee.
Poster Presentation Abstracts
Poster presentation abstracts will be posted as they are submitted.
1. Thomas Mathews, "Phospholipase D regulates deoxy-ribonucleotide biosynthesis through pyrimidine metabolic intermediates," Thomas P. Mathews, Kristie L. Rose, Craig W. Lindsley, H. Alex Brown
Cancer is widely recognized as a systematic redistribution of metabolic and signaling pathways contributing to rapid cellular replication and survival. To sustain this higher rate of growth, cancer cells often upregulate the biosynthesis of central cellular building blocks necessary for replication. In recent years, these biosynthetic pathways have been found lying downstream of discrete oncogenic targets. Phospholipase D (PLD) hydrolyzes membrane-bound phospholipids to produce phosphatidic acid (PtdOH), which can in turn activate oncogenic targets, some of which directly influence metabolic regulation. In an effort to define the signaling pathways associated with PtdOH production and cancer progression, our labs have designed, developed, and characterized the most potent and subtype selective small-molecule PLD inhibitors reported in the literature to date. Using these inhibitors in a glioblastoma model cell line, U87MG cells, we found that inhibition of PLD enzymatic activity reliably decreases deoxy-ribonucleotide biosynthesis. Further analysis of metabolomic and proteomic readouts revealed that PtdOH signaling through mTOR decreases the biosynthesis of metabolites upstream of pyrimidine ring structures – one of core dNTP subunits. We found that these decreases in pyrimidine biosynthesis, from impaired mTOR signaling, are responsible for the observed reduction in dNTP levels. In this way, these studies establish a novel, cell-specific regulatory mechanism for PLD in pyrimidine biosynthesis in malignant gliomas.
2. Katie Winarski, "The anti-influenza antibody H5.3 can neutralize different H5N1 variants," Katie L Winarski, Natalie J Thornburg, James E Crowe, Jr., Benjamin W. Spiller
The influenza virus H5N1, or avian flu, is not currently directly transmissible between humans, although recent laboratory-created, respiratory droplet transmissible H5N1 strains indicate that very few point mutations are necessary to allow direct transmission in the immunologically naïve human population. We are interested in the interaction between the influenza glycoprotein hemagglutinin 5 (H5) and the anti-influenza antibody, H5.3. H5.3 was isolated from a patient in an H5N1 vaccine trial and is capable of binding multiple H5N1 strains by interacting with the H5 head domain. We have determined the structure of H5.3 Fab in complex with the H5 head domain and found H5.3 neutralizes H5N1 by inserting its CDRH3 into the receptor binding site and mimicking interactions between H5 and its receptor, sialic acid. We have also determined the structure of H5.3 Fab in complex with respiratory droplet transmissible (rdt) H5 head domain and found H5.3 binds rdt H5 in the same manner as it binds wt H5, avoiding interactions with the rdt residues. This structure reveals how receptor binding site directed antibodies can bind hemagglutinins with different receptor specificity, indicating that receptor specificity may not be critically important for vaccine development.
3. Cynthia Berry, "Optimization of a dopamine receptor 4 antagonist as a PET tracer and an in vivo tool to study cocaine addiction"
Dopamine receptors are involved in many important central nervous system processes and are indicated in diseases such as schizophrenia, attention deficit hyperactivity disorder, Parkinson’s disease, and drug addiction. Since the discovery of the five subtypes of dopamine receptors, great effort has been taken to synthesize selective ligands in order to study each receptor’s involvement in disease. The Lindsley laboratory has developed an enantioselective synthesis of a morpholine-based dopamine receptor 4 (D4) antagonist. This compound binds D4 with a Ki of 70 nM, has an IC50 of 180 nM, and is highly selective over the remaining dopamine receptors and other GPCRs. Through iterative, parallel synthesis, a structure-activity relationship study was conducted around this lead to improve binding affinity and other pharmacological properties. We hope to develop a highly selective D4 antagonist for use as a PET tracer to image D4 receptors in the brain and an in vivo tool to study cocaine addiction, which has shown evidence of treatment by D4 antagonism.
4. Matthew Surdel, "A chemical genetic approach to interrogate the heme stress response of Staphylococcus aureus," Matthew C. Surdel, Brendan F. Dutter, Devin L. Stauff, Olusegun Aranmolate, Gary A. Sulikowski, and Eric P. Skaar
Staphylococcus aureus is a pathogen contributing to significant morbidity and mortality worldwide. Within the vertebrate host, S. aureus requires heme as a nutrient iron source and as a cofactor for numerous critical processes. To satisfy these requirements, S. aureus imports host heme through dedicated systems, while retaining the ability to synthesize heme de novo. Although heme is an essential nutrient for growth, excess heme is toxic. S. aureus utilizes a two component system, the heme sensor system (HssRS), to sense and protect against heme toxicity. Upon activation, HssRS induces the expression of the heme-regulated transporter (HrtAB), an efflux pump that alleviates heme toxicity. The ability to sense and respond to heme is critical for pathogenesis, yet the mechanism of heme sensing remains unknown. Small molecules ‘8882 and ‘3981 were identified in a high-throughput screen as activators of staphylococcal HssRS. Importantly, the mechanisms through which these compounds activate HssRS differs. Whereas ‘8882 induces an increase in endogenous heme biosynthesis, ‘3981 induces HssRS independently of heme, suggesting heme is not the ligand for HssRS. Using multiple target identification strategies, we have uncovered numerous genetic requirements of the heme stress response. Notably, by utilizing a suicide strain containing a Phrt-driven relE construct, we have identified suppressor mutations preventing sensing of ‘8882 and heme. This has uncovered numerous residues within HssRS required for hrtAB activation, as well as additional genes required for sensing these molecules. In addition, a transposon screen for strains unresponsive to ‘3981 has uncovered bacterial nitric oxide synthase as crucial to the heme stress response, providing the first evidence that nitric oxide and staphylococcal heme sensing are linked. Ultimately, elucidating the mechanisms employed by S. aureus to overcome heme toxicity experienced in the host will provide a better understanding of the pathogenesis of this organism and may uncover potential therapeutic targets. Based upon the conservation of heme sensing systems across multiple medically relevant pathogens, this work may provide information applicable to heme sensing in a variety of infectious agents.
5. Isi Ero-Tolliver, "The N-Terminal Immunoglobulin Domain of Peroxidasin Is Required to Cross-Link Collagen IV," Ero-Tolliver, I.A., Colon, S., Hudson, B.G., Bhave, G.
The collagen IV sulfilimine cross-link and its catalyzing enzyme, peroxidasin, represent a dyad, conserved throughout the animal kingdom, which is critical for tissue development. Peroxidasin forms novel sulfilimine bonds between apposing methionine and hydroxylysine residues to structurally reinforce the collagen IV scaffold, a function critical for basement membrane and tissue integrity. However, the molecular mechanism underlying the unique ability of peroxidasin to cross-link collagen IV remains unclear. In this work, we demonstrate that a combination of HOBr formation and direct binding to collagen IV allows peroxidasin to reinforce basement membranes. Thus, this molecular feature accounts for the evolutionarily conserved function of peroxidasin in tissue development and integrity.
6. Carrie Shaffer, "Ring-fused 2-pyridones disrupt Helicobacter pylori type IV secretion," Carrie L. Shaffer, James A. D. Good, K. Syam Krishnan, Jennifer A. Gaddy, John T. Loh, Fredrik Almqvist, Timothy L. Cover, Maria Hadjifrangiskou
Helicobacter pylori utilize a type IV secretion system (cag T4SS) to inject the oncogenic effector protein CagA into host gastric epithelial cells. The mechanism by which T4SSs transport cargo into target cells remains poorly understood. Here, we describe two ring-fused 2-pyridones (C10 and KSK85) that significantly inhibit cag T4SS effector translocation and T4SS-dependent cellular alterations. KSK85 impedes biogenesis of T4SS-associated pili required for effector delivery, while C10 disrupts type IV secretion without perturbing pilus assembly. We provide evidence that 2-pyridones target CagA in a manner that is independent of their effects on T4SS function. In addition, we report that ring-fused 2-pyridones modulate DNA transfer through E. coli conjugative T4SSs. Thus, these chemical probes are molecular tools that enable mechanistic interrogation of diverse effector translocation processes and the unraveling of complex T4SS assembly dynamics.
7. Michael Goodman, "The Investigation of the Chemical Mechanism and Inhibition of Microsomal Prostaglandin E2 Synthase 1 (MPGES1) Michael C. Goodman and Richard N. Armstrong
Prostaglandins function as signaling molecules involved in pain, fever, and many diseases associated with chronic inflammation. The most common therapeutic treatment of inflammation involves the inhibition of the COX enzymes by nonsteroidal anti-inflammatory drugs (NSAIDs) or COX-2 selective inhibitors (coxibs). The inhibition of the COX enzymes prevents the catalytic formation of the intermediate endoperoxide compound, PGH2. With the inhibition of PGH2, the production of other downstream prostaglandins is also inhibited. Therefore, COX inhibition can result in adverse gastrointestinal and cardiovascular side effects because of the subsequent low level of various prostanoids. Microsomal Prostaglandin E2 synthase 1 (MPGES1), a member of the membrane-associated proteins in eicosanoid and glutathione metabolism (MAPEG) superfamily, is the most prominent PGE synthase expressed during inflammation and is the terminal enzyme in the PGE2 synthesis pathway. It is a promising therapeutic target for the treatment of chronic inflammation and potential drug candidates have been pursued by the pharmaceutical industry in recent years. However, little is known about the actual chemical mechanism of the enzyme. Glutathione (GSH) appears to participate as a cofactor in the reaction, though there hasn’t been any evidence to support this. In order to investigate GSH as a cofactor in the reaction, it is necessary to detect the thiolate of GSH in the active site and determine its exchange rate off of the enzyme surface. One equivalent of the thiolate is observable by difference spectroscopy. However, simple difference spectroscopy isn’t possible since the enzyme is unstable in the absence of GSH. Therefore, a solution of the enzyme in complex with GSH can be rapidly mixed with the serine analog of GSH, γ-L-glutamyl-L-serylglycine (GOH), and the kinetics of the loss in absorbance from the replacement of GS- with GO- would confirm the presence of the thiolate in the complex and its rate of dissociation from the enzyme can be determined.
8. Jessica Moore, "High Mass Resolution MALDI Protein Imaging reveals Oxidative Damage to Proteins During Staphylococcus aureus Infection," Jessica L. Moore, Jeffrey M. Spraggins, Neal D. Hammer, Kristie L. Rose, Eric P. Skaar, Richard M. Caprioli
Staphylococcus aureus is a cause of significant morbidity and mortality within the developed world. S. aureus infection causes the formation purulent inflammatory foci, called abscesses, characterized by the recruitment of neutrophils and an abundance of the host protein calprotectin. Calprotectin is composed of the calcium-binding S100 proteins S100A8 and S100A9, which are known to inhibit bacterial growth by restricting nutrient metals. Interestingly, S100 proteins are also damage-associated molecular pattern molecules (DAMPs), initiators of the inflammatory response and targets of oxidative damage. MALDI FTICR imaging mass spectrometry (IMS) allows oxidative modification of DAMPs to be regiospecifically characterized and strengthens comparisons to proteomics data using mass accuracy. This approach provides new molecular insights and oxidation-specific epitopes localized to the pathogen-host interface. Given that the abscess represents the complex nature of the host-pathogen interface, the identification of these molecules could reveal other inflammatory biomarkers.
9. Norie Sugitani, "Re-defining the DNA-Binding Domain of Human XPA," Norie Sugitani, Steven M. Shell, Sarah E. Soss, and Walter J. Chazin
Xeroderma pigmentosum complementation group A (XPA) protein plays a critical role in the repair of DNA damage via the nucleotide excision repair (NER) pathway. XPA serves as a scaffold for NER, interacting with several other NER proteins as well as the DNA substrate. The critical importance of XPA is underscored by its association with the most severe clinical phenotypes of the genetic disorder Xeroderma pigmentosum. Many of these disease-associated mutations map to the XPA98-219 DNA-binding domain (DBD) first reported ~20 years ago. Although multiple solution NMR structures of XPA98-219 have been determined, the molecular basis for the interaction of this domain with DNA is only poorly characterized. In this report, we demonstrate using a fluorescence anisotropy (FA) DNA-binding assay that the previously reported XPA DBD binds DNA with substantially weaker affinity than the full-length protein. In-depth analysis of the XPA sequence suggested that the original DBD construct lacks critical basic charge and helical elements at its C-terminus. Generation and analysis of a series of C-terminal extensions beyond residue 219 yielded a stable, soluble human XPA98-239 contstruct that binds to a Y-shaped ssDNA-dsDNA junction and other substrates with the same affinity as the full-length protein. Two-dimensional 15N-1H NMR suggested XPA98-239 contains the same globular core as XPA98-219 and likely undergoes a conformational change upon binding DNA. Together, our results demonstrate that the XPA DBD should be redefined and that XPA98-239 is a suitable model to examine the DNA binding activity of human XPA.