http://hdl.handle.net/1974/816 2013-05-26T00:20:34Z http://hdl.handle.net/1974/8007 Title: NEU1 SIALIDASE AND MATRIX METALLOPROTEINASE-9 CROSS-TALK IS ESSENTIAL FOR TOLL-LIKE RECEPTOR ACTIVATION AND CELLULAR SIGNALING Authors: Abdulkhalek, SAMAR Abstract: The molecular mechanism(s) by which Toll-like receptors become activated are not well understood. For the majority of TLR receptors, dimerization is a prerequisite to facilitate MyD88-TLR complex formation and subsequent cellular signaling to activate NF-κB. However, the parameters controlling interactions between the receptors and their ligands still remain poorly defined. Previous reports have identified that neuraminidase-1 (NEU1) is an important intermediate in the initial process of TLR ligand induced receptor activation and subsequent cell function. What we do not yet understand is how NEU1 is activated following TLR ligand binding. In this thesis, the findings disclose a receptor signaling paradigm involving a process of receptor ligand-induced GPCR-signaling via neuromedin-B (NMBR) Gα-proteins, matrix metalloproteinase-9 (MMP-9) activation, and the induction of Neu1 activation. Central to this process is that NEU1–MMP-9-NMBR complex is associated with TLR-4 receptors on the cell surface of naive primary macrophages and TLR-expressing cell lines. Ligand binding to the receptor initiate GPCR-signaling via GPCR Gα subunit proteins and MMP-9 activation to induce NEU1. Activated NEU1 targets and hydrolyzes sialyl α-2-3-linked to β-galactosyl residues at the ectodomain of TLRs, enabling the removal of steric hindrance to receptor association, activation of receptors and cellular signaling. Furthermore, a novel glycosylation model is uncovered for the activation of nucleic acid sensing intracellular TLR-7 and TLR-9 receptors. It discloses an identical signaling paradigm as described for the cell-surface TLRs. NEU1 and MMP9 cross-talk in alliance with neuromedin-B receptors tethered to TLR-7 and -9 receptors at the ectodomain is essential for ligand activation of the TLRs and pro-inflammatory responses. However, the mechanism(s) behind this GPCR and TLR cross-talk has not been fully defined. Here, GPCR agonists mediate GPCR-signaling via membrane Gα subunit proteins to induce NEU1 and MMP-9 cross-talk at the TLR ectodomain on the cell surface. This molecular organizational GPCR signaling platform is proposed to be an initial processing stage for GPCR agonist-induced transactivation of TLRs and subsequent cellular signaling. Collectively, these novel findings radically redefine the current dogma(s) governing the mechanism(s) of the interaction of TLRs and their ligands, which may provide important pioneering approaches to disease intervention strategies. Description: Thesis (Ph.D, Microbiology & Immunology) -- Queen's University, 2013-04-30 12:23:42.429 2013-05-01T04:00:00Z http://hdl.handle.net/1974/7828 Title: PA5471 modulation of the Pseudomonas aeruginosa mexXY multidrug efflux pump operon repressor MexZ: Identification of important interaction residues and domains Authors: Hay, Thomas Abstract: Chemotherapeutic treatment of Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen, is substantially challenged by several membrane-spanning, multidrug-efflux pumps of the three-component RND family. Of these pumps, MexXY-OprM contributes to the intrinsic resistance of this organism by exporting clinically relevant antibiotics, most notably the ribosome-targeting aminoglycosides. Overproduction of MexXY-OprM is the most common mechanism providing pan-aminoglycoside resistance to P. aeruginosa cystic fibrosis clinical isolates. The mexXY genes are located in an operon, the expression of which is induced by ribosome-targeting antimicrobials. The mexXY operon is negatively regulated by MexZ, a repressor protein encoded by the divergently-transcribed gene mexZ. A second gene, PA5471, is also induced by ribosome-targeting antibiotics and is required for antibiotic induction of mexXY expression. One possibility is that PA5471 interacts with MexZ to alleviate repression of mexXY, thereby providing a mechanism for PA5471-dependent drug inducibility of mexXY. PA5471 interaction with MexZ was confirmed using a bacterial two-hybrid assay. To identify residues/regions of PA5471 important for interaction with MexZ, random chemical mutagenesis of the mexZ and PA5471 genes was carried out and the effects of these mutations on interaction of their protein products was assessed using the bacterial two-hybrid assay. Mutations of PA5471 that compromised interaction with MexZ included P68S, G76C, R216C, R221W, R221Q, G231D, and G252S, which occur within or in close proximity to a predicted surface-exposed α-helix of a PA5471 structural model that may contribute to the MexZ-interaction domain. Representative mutations P68S, G76C, R216C and R221W were placed into the chromosome of P. aeruginosa to assess their impact on drug-inducible mexXY expression. All of these mutations significantly reduced mexX upregulation in the presence of spectinomycin, where mutations R216C and R221W resulted in the near complete ablation of this antibiotic induction. These data suggest that PA5471 acts as a direct antirepressor of MexZ and that this interaction is key to mexXY upregulation in response to ribosome-targeting induction signals. Description: Thesis (Master, Microbiology & Immunology) -- Queen's University, 2013-02-26 13:32:39.307 2013-02-26T05:00:00Z http://hdl.handle.net/1974/7820 Title: NEURAMINIDASE-1 SIALIDASE AND MATRIX METALLOPROTEINASE-9 CROSSTALK IN ALLIANCE WITH INSULIN RECEPTORS IS AN ESSENTIAL MOLECULAR SIGNALING PLATFORM FOR INSULIN-INDUCED RECEPTOR ACTIVATION Authors: ALGHAMDI, FARAH Abstract: Molecular-targeting therapeutics directed towards growth factor receptors have become promising interventions in cancer. They include the family of mammalian receptor tyrosine kinases such as epidermal growth factor, TrkA and insulin. In particular, the insulin receptor (IR) is one of the most well-known members of the RTK family of receptors playing a role in cancer. IRs are covalently-linked heterodimers of αβ subunits on the cell membrane in the absence of insulin. The IR signaling pathways are initially triggered by insulin binding to the α subunits followed by the interaction of β subunits and ATP. The parameter(s) controlling IR activation remains unknown. Here, we report a membrane receptor signaling platform initiated by insulin binding to its receptor to induce Neu1 in live HTC-IR and MiaPaCa-2 cell lines. Microscopy colocalization and co-immunoprecipitation analyses reveal that Neu1 and MMP9 form a complex with naïve and insulin-treated receptors. Tamiflu (neuraminidase inhibitor), galardin and piperazine (broad range MMP inhibitors), MMP9 specific inhibitor and anti-Neu1 antibody blocked Neu1 activity associated with insulin stimulated live cells. Moreover, Tamiflu, anti-Neu1 antibody, and MMP9 specific inhibitor blocked insulin induced insulin receptor substrate-1 phosphorylation (p-IRS1). The previous findings reveal a molecular organizational signaling platform of Neu1 and MMP-9 crosstalk in alliance with insulin receptors. It proposes that insulin binding to the receptor induces MMP9 to activate Neu1, which hydrolyzes α-2,3 sialic acid in removing steric hindrance to generate a functional receptor. The results predict a prerequisite desialylation process by activated Neu1. A complete understanding of IR activation and the role of sialic acids in the iii signaling pathways may provide a therapeutic strategy in the prevention of different diseases such as diabetes mellitus and cancer. Description: Thesis (Master, Microbiology & Immunology) -- Queen's University, 2013-02-20 11:27:44.861 2013-02-20T05:00:00Z http://hdl.handle.net/1974/7748 Title: CHARACTERIZATION OF THE ALPHAHERPESVIRUS TEGUMENT PROTEIN US2 Authors: Kang, MING-HSI Abstract: Members of the Herpesviridae are large enveloped, double-stranded DNA viruses, whose virions are comprised of a viral genome-containing icosahedral capsid, a layer of tegument and a glycoprotein-embedded envelope. The tegument contains numerous viral proteins and cellular proteins. Most of the tegument proteins are poorly understood and require further investigation. This study focuses on one of the tegument proteins, Us2, and utilizes two model alphaherpesviruses: pseudorabies virus (PRV) and herpes simplex virus (HSV). Us2 is conserved among all alphaherpesvirus with the exception of varicella-zoster virus (VZV). The amino acid sequence of all Us2 orthologs share three N-terminal conserved regions whereas the C-terminal sequences are highly variable. PRV Us2 contains a C-terminal prenylation motif that targets Us2 to the plasma membrane. Although it is indispensable for virus growth in cell culture, deletion of Us2 gene in PRV caused an accumulation of virions in the cytoplasm of infected primary cells. Furthermore, PRV Us2 spatially regulates MAPK ERK activity by sequestering it to the plasma membrane. Inhibition of ERK kinase activity caused a delay in the release of extracellular viruses and the defect was more profound in PRV Us2-null virus infected cells. Altogether, these data suggest a requirement for ERK activity and significance of PRV Us2-ERK interaction in virus egress. To understand the mechanism of Us2-ERK interaction, PRV Us2 determinants for ERK interaction were mapped. Our data revealed that the N-terminal 214 residues are the minimal sequence of Us2 required for interaction with ERK. In addition, PRV Us2 oligomerizes and forms complexes with ERK via the ERK common docking (CD) domain that facilitates the interaction of ERK with many of its substrates. Unlike PRV Us2, HSV-2 Us2 does not have any putative membrane targeting signals. However, our data revealed that HSV-2 Us2 localizes to the plasma membrane and is lipid raft associated. In addition, HSV-2 Us2 interacts directly with ubiquitin. As ubiquitination is responsible for proteasomal degradation and is involved in endocytosis and lysosomal degradation, these findings suggest that Us2 may be involved in proteasomal degradation pathways that counteract host defenses, or participate in final envelopment in endocytic compartments by facilitating the endocytosis of viral envelope proteins. Description: Thesis (Ph.D, Microbiology & Immunology) -- Queen's University, 2013-01-21 01:25:54.103 2013-01-21T05:00:00Z