Receptor glycosylation is critical in receptor-ligand interactions in immune cells, but the exact role of glycosylation in receptor activation upon ligand binding has not been elucidated. In neuronal cells, we have shown that when neurotrophic factors bind their respective Trk tyrosine kinase receptors, receptor activation and subsequent neurotrophin-mediated signaling is dependent upon the induction and activity of an endogenous sialidase enzyme. In this thesis, we report that toll-like receptor (TLR) activation upon ligand binding is similarly dependent on the induction of a cellular sialidase, which we have identified as Neu1 sialidase, which specifically targets and hydrolyses alpha-2,3-linked sialic acid residues on the receptor. Blocking Neu1 sialidase activity with specific inhibitor Tamiflu detrimentally impacts ligand-induced TLR4/MyD88 interaction, NFkappaB activation and TLR-mediated effector responses like nitric oxide and pro-inflammatory cytokine production. Diminished cytokine production is also seen in vivo in Neu1-deficient mice. We propose a mechanism for the induction of Neu1 sialidase, upon ligand binding to TLR, that involves the activation of heterotrimeric G-alpha protein-dependent G-protein coupled receptor (GPCR) signaling to activate a matrix metalloproteinase (MMP) enzyme, likely MMP-9. It is suggested that MMP-(9) targets the cell surface elastin receptor complex of Neu1/protective protein cathepsinA/elastin binding protein (EBP), which potentially catalytically activates Neu1. In addition, we report an association between Neu1 and TLR2, TLR3 and TLR4 on the plasma membrane that has not previously been described. The idea that the multiple functionality and diversity of TLRs and TLR-mediated signaling may be an immunologic paradigm capable of explaining all human disease is provocative but plausible. Certainly, the structural integrity of TLRs, their ligand interactions and activation are essential for immunological protection. Thus, understanding the molecular mechanism of Neu1 sialidase regulation of TLR activation will provide important opportunities for disease control through TLR manipulation. The future directions of this research will also open a new area of glycobiology research (the glycomics of innate immune responses) and will widen the scope for the development of novel therapeutic drugs to combat infections and inflammatory diseases. / Thesis (Ph.D, Microbiology & Immunology) -- Queen's University, 2009-01-26 12:33:32.743

No description available.

The androgen receptor (AR) is a ligand-activated transcription factor and a member of the nuclear receptor superfamily. Mutations in the androgen receptor are associated with androgen insensitivity syndrome (AIS), and a neurodegenerative disease, spinal bulbar muscular atrophy (SBMA). Most of the mutations causing AIS are loss-of-function missense mutations whereas SBMA is caused by a gain-of-function polyglutamine expansion in the N-terminal domain of the protein. Characterization of AR mutations has led to a better understanding of structure-function relationships of the AR and serves as a prototype for steroid receptors mechanisms of action. / In the first paper, we examine the role of an AR mutation in causing mild androgen insensitivity syndrome. We found that this mutation conferred reduced transactivation by AR through impaired interactions with the AR coactivator, TIF2, and impaired homodimerization. / In the second paper, we investigate the role of the AR polyGln expansion mutation in SBMA pathogenesis. Recent evidence has implicated proteolytic degradation of polyGln-expanded proteins and their subsequent intracellular aggregation in polyGn-expanded disease pathogenesis. We examined the role and composition of aggregates using fluorescently-tagged AR and found that proteolysis need not be a prerequisite for aggregation and that aggregation is not necessary for poly-Gln-induced cellular toxicity. / Finally, we characterize the novel heterodimerization of AR and ERalpha. We determined that this direct interaction has functional implications for the transactivational properties of both receptors.

No description available.

Regulators of G protein signaling (RGSs) are negative regulators of G protein coupled receptors (GPCRs). Our lab has demonstrated that yeast Saccharomyces cerevisiae is a useful system to study RGS and G protein signaling. Mammalian RGSs can be expressed in yeast and favored to interact with mammalian GPCRs as well. / Based on the observation that human RGS1 causes yeast cell growth arrest, I therefore used RGS1 expressing yeast cells to screen a mouse T cell cDNA library in order to find potential interacting proteins. From the screen, I identified a mouse sphingomyelin synthase 1 (SMS1) cDNA. By using a series of different apoptotic stimuli, such as hydrogen peroxide, osmotic stress, exogenous ceramide and its precursors, high temperature etc., SMS1 expression was found to suppress cell growth arrest and prevent viability decline, indicating that SMS1 represents an anti-apoptotic protein that functions by decreasing the intracellular level of pro-apoptotic ceramide. / Gene analysis further indicated that the SMS1 gene consists of 16 exons spread over a 256kb portion of mouse chromosome 19. It is alternatively spliced to produce 4 different transcripts (SMS1alpha1, SMS1alpha2, SMS1beta and SMS1gamma) and encode 3 different proteins (SMS1alpha, SMS1beta and SMS1gamma). Notably, I found that SMS1beta protein does not interfere with SMS1alpha anti-apoptotic function, although both of these two proteins contain the protein-protein interaction domain, sterile alpha motif (SAM), at their N-terminus. / I also carried out a study to examine GPCR-RGS interactions using the yeast expression system. Our lab had noticed that there was an extra RGS5 related protein that was detected by western blot analysis in the protein extracts prepared from yeast and HEK293 cells expressing RGS5. The size of the band was approximately 2 times the molecular weight of RGS5, indicating the possibility that RGS5 forms a dimer. To further examine this hypothesis, I, therefore, performed a series of experiments, included yeast 2 hybrid assays, to demonstrate that RGS5 does interact with itself. This is the first report that RGS can form a dimer. The implications for this finding are discussed in detail.

Since its identification in 1997 as the common receptor for Coxsackie and adenovirus (CAR) multiple lines of evidence argue in favor of CAR contributing to aspects of cell adhesion in addition to serving as a viral receptor. Nevertheless, a precise biological role for CAR remains to be identified suggesting the receptor may participate in a variety of cellular functions that reflect its tissue specific and developmentally regulated expression. This thesis elucidates aspects of CAR biology in mature striated muscle by providing studies that encompass (i) its physiological cellular/subcellular localization and expression in mature striated muscle (ii) its expression profile in human diseased skeletal muscle and (iii) the potential consequences of its sustained expression in mature striated muscle where its levels would otherwise be highly attenuated. / In non-diseased, mature striated muscle despite low and barely detectable levels of the CAR transcript (cardiac and skeletal muscle respectively), we identified CAR as a novel component of the neuromuscular junction and showed its expression to be isoform-specific in contrast to the intercalated discs, where both predominant CAR isoforms are detected. We then investigated the expression of CAR at the level of human skeletal muscle disease. From these studies we observed that in diseases characterized by active necrosis and regeneration, extrasynaptic CAR expression is detectable in regenerating fibers and co-expressed with other previously described markers of regeneration at a high degree of coincidence. Moreover, extrasynaptic CAR expression appears to be a highly reliable indicator of the regenerative process offering potential use at the diagnostic level. Following these investigations, our final studies involved assessing whether sustained CAR expression might affect the normal homeostasis in skeletal and cardiac muscle using a transgenic mouse model. We discovered that transgenic mice expressing sustained high levels of CAR (as seen in the CAR+/+ transgenics) develop a lethal necrotizing myopathy characterized by dual deficiencies in dystrophin and dysferlin, two proteins pivotal in maintaining plasmalemmal integrity, raising the possibility for a previously unrecognized cause of skeletal muscle dysfunction. / Collectively these findings argue that in non-diseased mature skeletal and cardiac muscle, CAR expression is restricted to the neuromuscular junction and cardiac intercalated discs but in diseases of skeletal muscle characterized by active necrosis and regeneration, extrasynaptic CAR expression is reexpressed at these sites of injury/repair. In addition they raise the possibility that sustained CAR expression in mature skeletal muscle may be associated with altered muscle homeostasis.

No description available.

Restenosis is the major cause of the failure of reconstruction methods to restore the blood flow in atherosclerotic arteries. Restenosis results from neointima formation and consequent constrictive remodelling. Vascular smooth muscle cell (VSMC) migration from the tunica media toward the intima is crucial in neointima genesis. The prerequisite for VSMC migratory activity is the modulation from the differentiated (contractile) to the de-differentiated (noncontractile) phenotype. VSMC phenotype change is associated with the altered expression of integrins. alpha8beta1 integrin is upregulated in cell types with contractile properties, including myofibroblasts and mesangial kidney cells. It is one of the integrins that is intensely expressed in mature VSMCs. alpha8beta1 integrin expression during vascular injury and its role in VSMC function have not been studied so far. / In this work, a rat model of carotid angioplasty was used to mimic vascular injury in humans. alpha8beta1 integrin was downregulated in the tunica media concomitantly with loss of the contractile phenotype. In vitro study revealed that it is a differentiation marker of VSMCs. To test the functional significance of the association between alpha8 integrin and the VSMC phenotype, short interference RNA was deployed to silence the alpha8 integrin gene. alpha8 integrin gene silencing heightened VSMC migratory activity as well as modulation of the VSMC phenotype in favour of the noncontractile state. In addition, alpha8 integrin overexpression induced re-differentiation of VSMCs and attenuated their migratory activity. It is, therefore, suggested that alpha8 integrin overexpression after vascular injury might control VSMC migration and neointima formation. On the other hand, alpha8 integrin gene silencing led to a reduced growth rate, which indicated a dichotomy between VSMC migration and proliferation. / In the later stages of neointima formation, constrictive remodeling plays a major role in late lumen loss. Our data demonstrated that alpha8 integrin is upregulated in the neointima during constrictive remodeling with concomitant luminal narrowing. The importance of this finding was highlighted by results showing that alpha8 integrin was required for the VSMC contractile phenotype evoked by transforming growth factor-beta (TFG-beta) and TFG-beta-induced myofibroblastic differentiation of Rat1 fibroblasts. Thus, it appears that alpha8 integrin expression blockade might reduce contractile remodeling and late lumen loss. Although the mechanism of alpha8 integrin signaling is not yet clear, our findings demonstrate that the alpha8 integrin-induced contractile phenotype is blocked by RhoA inhibitors. Furthermore, alpha8 integrin and RhoA are co-immunoprecipitated, and alpha8 integrin gene silencing reduces RhoA activity. Hence, it is postulated that alpha8-RhoA signaling might be closely intertwined. / Altogether, these studies indicate that alpha8 integrin is a contractile marker of VSMCs and a negative regulator of VSMC migration. Therefore, forced alpha8 integrin expression may be applied to reduce neointima formation. However, alpha8 integrin upregulation during constrictive remodeling concomitant with late lumen loss suggest that it could be involved in lumen narrowing. It seems likely that in therapeutic strategies to reduce restenosis the timeline of interference might be very important. Therefore, alpha8 integrin gene silencing in the later stages of neointima formation might be beneficial.

The present study has tested the hypothesis that leptin receptors are localized in caveolae and that caveolae are involved in the leptin-induced stimulation of lipolysis in 3T3-L1 adipocytes. Leptin, a peptide hormone, is secreted primarily by adipocytes and has been postulated to regulate food intake and energy expenditure via hypothalamic-mediated effects. Exposure to leptin increases the lipolytic activity in 3T3-L1 adipocytes. We isolated caveolae from 3T3-L1 adipocytes using a detergent free sucrose gradient centrifugation method. Leptin receptors were localized in the same gradient fraction as caveolin-1. Confocal microscopic studies demonstrated the colocalization of leptin receptors with caveolin-1 in the plasma membrane, indicating distribution of leptin receptors in the caveolae. We disrupted caveolae by treating cells with methyl--cyclodextrin and found that leptin induced lipolytic activity was reduced after caveolae disruption, indicating an important role of caveolae in the signaling mechanism of leptin.

No description available.