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1.  Cellular Entry of Human Papillomavirus Type 16 Involves Activation of the Phosphatidylinositol 3-Kinase/Akt/mTOR Pathway and Inhibition of Autophagy 
Journal of Virology  2013;87(5):2508-2517.
The mammalian target of rapamycin (mTOR) downstream of phosphatidylinositol 3-kinase (PI3K) in the growth factor receptor (GFR) pathway is a crucial metabolic sensor that integrates growth factor signals in cells. We recently showed that human papillomavirus (HPV) type 16 exposure activates signaling from GFRs in human keratinocytes. Thus, we predicted that the virus would induce the PI3K/mTOR pathway upon interaction with host cells. We detected activation of Akt and mTOR several minutes following exposure of human keratinocytes to HPV type 16 (HPV16) pseudovirions. Activated mTOR induced phosphorylation of the mTOR complex 1 substrates 4E-BP1 and S6K, which led to induction of the functional protein translational machinery. Blockade of epidermal GFR (EGFR) signaling revealed that each of these events is at least partially dependent upon EGFR activation. Importantly, activation of PI3K/Akt/mTOR signaling inhibited autophagy in the early stages of virus-host cell interaction. Biochemical and genetic approaches revealed critical roles for mTOR activation and autophagy suppression in HPV16 early infection events. In summary, the HPV-host cell interaction stimulates the PI3K/Akt/mTOR pathway and inhibits autophagy, and in combination these events benefit virus infection.
PMCID: PMC3571372  PMID: 23255786
2.  A Competitive Nucleotide Binding Inhibitor: In vitro Characterization of Rab7 GTPase Inhibition 
ACS chemical biology  2012;7(6):1095-1108.
Mapping the functionality of GTPases through small molecule inhibitors represents an underexplored area in large part due to the lack of suitable compounds. Here we report on the small chemical molecule 2-(benzoylcarbamothioylamino)-5,5-dimethyl-4,7-dihydrothieno[2,3-c]pyran-3-carboxylic acid (PubChem CID 1067700) as an inhibitor of nucleotide binding by Ras-related GTPases. The mechanism of action of this pan-GTPase inhibitor was characterized in the context of the Rab7 GTPase as there are no known inhibitors of Rab GTPases. Bead-based flow cytometry established that CID 1067700 has significant inhibitory potency on Rab7 nucleotide binding with nanomolar inhibitor (Ki) values and an inhibitory response of ≥97% for BODIPY-GTP and BODIPY-GDP binding. Other tested GTPases exhibited significantly lower responses. The compound behaves as a competitive inhibitor of Rab7 nucleotide binding based on both equilibrium binding and dissociation assays. Molecular docking analyses are compatible with CID 1067700 fitting into the nucleotide binding pocket of the GTP-conformer of Rab7. On the GDP-conformer, the molecule has greater solvent exposure and significantly less protein interaction relative to GDP, offering a molecular rationale for the experimental results. Structural features pertinent to CID 1067700 inhibitory activity have been identified through initial structure activity analyses and identified a molecular scaffold that may serve in the generation of more selective probes for Rab7 and other GTPases. Taken together, our study has identified the first competitive GTPase inhibitor and demonstrated the potential utility of the compound for dissecting the enzymology of the Rab7 GTPase as well as serving as a model for other small molecular weight GTPase inhibitors.
PMCID: PMC3440014  PMID: 22486388
Rab, Rho, Rac, Cdc42 and Ras GTPases; chemical biology; drug discovery; therapeutics; fluorescent GTP and GDP; enzyme kinetics
3.  Continuous Expression of HIF-1α in Neural Stem/Progenitor Cells 
Hypoxia-inducible factor-1 alpha subunit (HIF-1α) is a transcriptional activator mediating adaptive cellular response to hypoxia. Normally degraded in most cell types and tissues, HIF-1α becomes stable and transcriptionally active under conditions of hypoxia. In contrast, we found that HIF-1α is continuously expressed in adult brain neurogenic zones, as well as in neural stem/progenitor cells (NSPCs) from the embryonic and postnatal mouse brain. Our in vitro studies suggest that HIF-1α does not undergo typical hydroxylation, ubiquitination and degradation within NSPCs under normoxic conditions. Based on immunofluorescence and cell fractionation, HIF-1α is primarily sequestered in membranous cytoplasmic structures, identified by immuno-electron microscopy as HIF-1α-bearing vesicles (HBV), which may prevent HIF-1α from degradation within the cytoplasm. HIF-1α shRNAi-mediated knockdown reduced the resistance of NSPCs to hypoxia, and markedly altered the expression levels of Notch-1 and β-catenin, which influence NSPC differentiation. These findings indicate a unique regulation of HIF-1α protein stability in NSPCs, which may have importance in NSPCs properties and function.
PMCID: PMC3629813  PMID: 20844947
4.  Identification of a Small GTPase Inhibitor using a High-Throughput Flow Cytometry Bead-Based Multiplex Assay 
Small GTPases are key regulators of cellular activity and represent novel targets for the treatment of human diseases using small molecule inhibitors. We describe a multiplex, flow cytometry bead-based assay for the identification and characterization of inhibitors or activators of small GTPases. Six different GST-tagged small GTPases were bound to glutathione beads each labeled with a different red fluorescence intensity. Subsequently, beads bearing different GTPase were mixed and dispensed into 384-well plates with test compounds, and fluorescent-GTP binding was used as the read-out. This novel multiplex assay allowed us to screen a library of almost 200,000 compounds and identify over 1,200 positive compounds, which were further verified by dose response analyses, using 6 to 8-plex assays. After the elimination of false positive and negative compounds, several small molecule families with opposing effects on GTP-binding activity were identified. Here we detail the characterization of MLS000532223, a general inhibitor that prevents GTP-binding to several GTPases in a dose-dependent manner and is active in biochemical and cell-based secondary assays. Live cell imaging and confocal microscopy studies revealed the inhibitor-induced actin reorganization and cell morphology changes, characteristic of Rho GTPases inhibition. Thus, high throughput screening (HTS) via flow cytometry provides a strategy for identifying novel compounds that are active against small GTPases.
PMCID: PMC3433230  PMID: 20008126
Ras; Rab and Rho GTPases; actin cytoskeleton; bead-based multiplex assay; flow cytometry; fluorescent GTP binding
5.  Drug Repurposing from an Academic Perspective 
Academia and small business research units are poised to play an increasing role in drug discovery, with drug repurposing as one of the major areas of activity. Here we summarize project status for a number of drugs or classes of drugs: raltegravir, cyclobenzaprine, benzbromarone, mometasone furoate, astemizole, R-naproxen, ketorolac, tolfenamic acid, phenothiazines, methylergonovine maleate and beta-adrenergic receptor drugs, respectively. Based on this multi-year, multi-project experience we discuss strengths and weaknesses of academic-based drug repurposing research. Translational, target and disease foci are strategic advantages fostered by close proximity and frequent interactions between basic and clinical scientists, which often result in discovering new modes of action for approved drugs. On the other hand, lack of integration with pharmaceutical sciences and toxicology, lack of appropriate intellectual coverage and issues related to dosing and safety may lead to significant drawbacks. The development of a more streamlined regulatory process world-wide, and the development of pre-competitive knowledge transfer systems such as a global healthcare database focused on regulatory and scientific information for drugs world-wide, are among the ideas proposed to improve the process of academic drug discovery and repurposing, and to overcome the “valley of death” by bridging basic to clinical sciences.
PMCID: PMC3285382  PMID: 22368688
6.  Essential Roles for Soluble Virion-Associated Heparan Sulfonated Proteoglycans and Growth Factors in Human Papillomavirus Infections 
PLoS Pathogens  2012;8(2):e1002519.
A subset of human papillomavirus (HPV) infections is causally related to the development of human epithelial tumors and cancers. Like a number of pathogens, HPV entry into target cells is initiated by first binding to heparan sulfonated proteoglycan (HSPG) cell surface attachment factors. The virus must then move to distinct secondary receptors, which are responsible for particle internalization. Despite intensive investigation, the mechanism of HPV movement to and the nature of the secondary receptors have been unclear. We report that HPV16 particles are not liberated from bound HSPG attachment factors by dissociation, but rather are released by a process previously unreported for pathogen-host cell interactions. Virus particles reside in infectious soluble high molecular weight complexes with HSPG, including syndecan-1 and bioactive compounds, like growth factors. Matrix mellatoproteinase inhibitors that block HSPG and virus release from cells interfere with virus infection. Employing a co-culture assay, we demonstrate HPV associated with soluble HSPG-growth factor complexes can infect cells lacking HSPG. Interaction of HPV-HSPG-growth factor complexes with growth factor receptors leads to rapid activation of signaling pathways important for infection, whereas a variety of growth factor receptor inhibitors impede virus-induced signaling and infection. Depletion of syndecan-1 or epidermal growth factor and removal of serum factors reduce infection, while replenishment of growth factors restores infection. Our findings support an infection model whereby HPV usurps normal host mechanisms for presenting growth factors to cells via soluble HSPG complexes as a novel method for interacting with entry receptors independent of direct virus-cell receptor interactions.
Author Summary
A subset of the >120 different types of human papillomaviruses (HPVs) are the most common cause of sexually transmitted infections. Certain HPVs are also associated with approximately 5% of all cancers worldwide. Like many pathogens, HPVs bind first to heparan sulfate proteoglycans (HSPGs) on cells before moving to more specific uptake receptors. However, relatively little is known about the mechanism(s) that triggers the translocation of HPV from HSPGs to the receptors that facilitate entry. As obligate parasites, viruses have evolved numerous means to hijack host cell functions to cause infection. We report two novel mechanisms of pathogen-host interactions. First, bound HPV particles are liberated from cells in an active complex with HSPGs and growth factors rather than dissociating from the sugars to engage secondary receptors. Second, HPV uses the specificity of the associated growth factors to bridge to their cognate receptors as opposed to direct binding to a cell internalization receptor. Signals transduced during these interactions are important for HPV infection. Our study provides new insights into the transmission of a significant viral pathogen and reveals novel means whereby microbes may repurpose normal cell functions during infection of their hosts. Likewise, this work uncovers new targets for HPV prophylaxis.
PMCID: PMC3276557  PMID: 22346752
7.  Mast Cell Synapses and Exosomes: Membrane Contacts for Information Exchange 
In addition to their central role in allergy, mast cells are involved in a wide variety of cellular interactions during homeostasis and disease. In this review, we discuss the ability of mast cells to extend their mechanisms for intercellular communication beyond the release of soluble mediators. These include formation of mast cell synapses on antigen presenting surfaces, as well as cell–cell contacts with dendritic cells and T cells. Release of membrane bound exosomes also provide for the transfer of antigen, mast cell proteins, and RNA to other leukocytes. With the recognition of the extended role mast cells have during immune modulation, further investigation of the processes in which mast cells are involved is necessary. This reopens mast cell research to exciting possibilities, demonstrating it to be an immunological frontier.
PMCID: PMC3342342  PMID: 22566928
mast cell; synapse; exosome; dendritic cell
8.  Flow Cytometry for Real-Time Measurement of Guanine Nucleotide Binding and Exchange by Ras-like GTPases 
Analytical biochemistry  2008;381(2):258-266.
Ras-like small GTPases cycle between GTP-bound active and GDP-bound inactive conformational states to regulate diverse cellular processes. Despite their importance, detailed kinetic or comparative studies of family members are rarely undertaken due to the lack of real-time assays measuring nucleotide binding or exchange. Here, we report a bead-based, flow cytometric assay that quantitatively measures the nucleotide binding properties of GST-chimeras for prototypical Ras-family members Rab7 and Rho. Measurements are possible in the presence or absence of Mg2+, with magnesium cations principally increasing affinity and slowing nucleotide dissociation rate 8- to 10-fold. GST-Rab7 exhibited a 3-fold higher affinity for GDP relative to GTP that is consistent with a 3-fold slower dissociation rate of GDP. Strikingly, GST-Rab7 had a marked preference for GTP with ribose ring-conjugated BODIPY FL. The more commonly used γ-NH-conjugated BODIPY FL GTP analogue failed to bind to GST-Rab7. In contrast, both BODIPY analogues bound equally well to GST-RhoA and GST-RhoC. Comparisons of the GST-Rab7 and GST-RhoA GTP-binding pockets provide a structural basis for the observed binding differences. In sum, the flow cytometric assay can be used to measure nucleotide binding properties of GTPases in real-time and quantitatively assess differences between GTPases.
PMCID: PMC2633595  PMID: 18638444
Rab and Rho GTPases; membrane trafficking; actin remodeling; nucleotide binding and exchange; fluorescent GTP analogues
9.  Markers for Detergent-resistant Lipid Rafts Occupy Distinct and Dynamic Domains in Native Membranes 
Molecular Biology of the Cell  2004;15(6):2580-2592.
Lipid rafts isolated by detergent extraction and sucrose gradient fractionation from mast cells are enriched for the glycosylphosphatidylinositol-linked protein Thy-1, the ganglioside GM1, palmitoylated LAT, and cross-linked IgE receptors, FcεRI. This study addresses the relationship of fractionation data to the organization of raft markers in native membranes. Immunogold labeling and electron microscopy shows there is little or no colocalization of the raft markers Thy-1, GM1, and LAT with each other or with FcεRI on native membrane sheets prepared from unstimulated cells. External cross-linking of Thy-1 promotes coclustering of Thy-1 with LAT, but not with GM1. Thy-1 and LAT clusters occur on membrane regions without distinctive features. In contrast, external cross-linking of FcεRI and GM1 causes their redistribution to electron-dense membrane patches independently of each other and of Thy-1. The distinctive patches that accumulate cross-linked FcεRI and GM1 also accumulate osmium, a stain for unsaturated lipids, and are sites for coated vesicle budding. Electron microscopy reveals a more complex and dynamic topographical organization of membrane microdomains than is predicted by biochemical analysis of detergent-resistant membranes.
PMCID: PMC420084  PMID: 15034144
10.  High resolution mapping of mast cell membranes reveals primary and secondary domains of FcεRI and LAT 
The Journal of Cell Biology  2001;154(3):645-658.
In mast cells, cross-linking the high-affinity IgE receptor (FcεRI) initiates the Lyn-mediated phosphorylation of receptor ITAMs, forming phospho-ITAM binding sites for Syk. Previous immunogold labeling of membrane sheets showed that resting FcεRI colocalize loosely with Lyn, whereas cross-linked FcεRI redistribute into specialized domains (osmiophilic patches) that exclude Lyn, accumulate Syk, and are often bordered by coated pits. Here, the distribution of FcεRI β is mapped relative to linker for activation of T cells (LAT), Grb2-binding protein 2 (Gab2), two PLCγ isoforms, and the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase), all implicated in the remodeling of membrane inositol phospholipids. Before activation, PLCγ1 and Gab2 are not strongly membrane associated, LAT occurs in small membrane clusters separate from receptor, and PLCγ2, that coprecipitates with LAT, occurs in clusters and along cytoskeletal cables. After activation, PLCγ2, Gab2, and a portion of p85 colocalize with FcεRI β in osmiophilic patches. LAT clusters enlarge within 30 s of receptor activation, forming elongated complexes that can intersect osmiophilic patches without mixing. PLCγ1 and another portion of p85 associate preferentially with activated LAT. Supporting multiple distributions of PI3-kinase, FcεRI cross-linking increases PI3-kinase activity in anti-LAT, anti-FcεRIβ, and anti-Gab2 immune complexes. We propose that activated mast cells propagate signals from primary domains organized around FcεRIβ and from secondary domains, including one organized around LAT.
PMCID: PMC2196429  PMID: 11489921
microdomains; PLCγ; phosphatidylinositol 3-kinase; LAT; Gab2

Results 1-10 (10)