Browsing by Subject "caveolin-1"
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Item Effect of Vascular Heterogeneity, Aging, and Exercise Training on eNOS ? Associated Protein:Protein Interactions(2014-04-14) Luttrell, Meredith JoyEndothelial dysfunction is a major risk factor for the development of cardiovascular diseases, and aging is associated with a gradual decline in endothelial function. Furthermore, endothelial dysfunction in arteries and arterioles supplying skeletal muscle has been implicated in the decline in exercise capacity with aging. Defined as an imbalance between the production and degradation of nitric oxide (NO), limited NO bioavailability is the hallmark characteristic of endothelial dysfunction. Production of NO is controlled by the enzyme endothelial nitric oxide synthase (eNOS), which is regulated in part by post-translational protein modifications. The purpose of this research was to examine the effect of vascular heterogeneity, aging, and endurance exercise training on eNOS-associated protein:protein interactions. Caveolin-1 (Cav1) is a negative regulator of eNOS activity, so that NO production is suppressed when Cav1 is bound to eNOS. Conversely, calmodulin (CaM) and heat-shock protein 90 (Hsp90) are positive regulators of eNOS activity, thus increasing eNOS activity and NO production when either are bound to eNOS. Co-immunoprecipitation was used to determine protein:protein interactions among eNOS and its regulatory proteins throughout the arterial network, from the aorta to third order skeletal muscle arterioles. Results show that eNOS-associated protein:protein interactions vary throughout the arterial network, and Cav1:eNOS and CaM:eNOS interactions are altered with aging. Additionally, endurance exercise training has no effect on the protein:protein interactions examined. In conclusion, eNOS regulation via protein:protein interactions appears to be vessel-specific, and aging has a heterogeneous effect on protein:protein interactions throughout the arterial network.Item Secondary Structural and Functional Studies of Rotavirus NSP4 and Caveolin-1 Peptide-Peptide Interactions(2011-02-22) Schroeder, Megan ElizabethThe rotavirus NSP4 protein is the first described viral enterotoxin. Abundant data from our laboratory reveals that NSP4 binds both the N- and C-termini of caveolin- 1 (aa2-31 and 161-178, respectively). Yeast two-hybrid and peptide binding analysis mapped the caveolin-1 binding site to three hydrophobic residues within the amphipathic a-helix, enterotoxic peptide domain (aa114-135). The research studies herein utilized peptides to investigate the interaction between NSP4 and caveolin-1. Peptides were synthesized corresponding to the amphipathic a-helix and caveolin-1 binding domain of NSP4 (aa112-140) and to the N- (aa2-20 and 19-40) and C- (161-178) termini of caveolin-1, and were utilized in structural and functional studies. Fluorescence binding assays revealed that NSP4 (aa112-140) binds to the N-terminus (aa19-40) of caveolin-1 with a stronger affinity than the C-terminus (aa161-178). In addition, this assay further delineated the NSP4 binding domain on caveolin-1 to aa19-40. Secondary structural changes following NSP4-caveolin-1 peptide-peptide interactions were investigated by circular dichroism analysis. Changes in a-helix formation were observed only upon interaction of the NSP4112-140 peptide with the C-terminal caveolin-1 peptide (C-Cav161- 178). The NSP4112-140 peptide contains a potential cholesterol recognition amino acid consensus (CRAC) sequence. Therefore this peptide was examined for cholesterol binding. Results of the binding assay revealed NSP4 binds cholesterol with a Kd of 7.67 +/- 1.49nM and this interaction occurs via aa112-140. Mutation of amino acid residues within the CRAC motif resulted in weaker binding affinities between each of the corresponding mutant peptides and cholesterol. NSP4 peptides containing mutations within the hydrophobic and charged faces of the amphipathic a-helix, enterotoxic peptide and caveolin-1 binding domain of NSP4 were examined for changes in secondary structure as well as diarrhea induction in mouse pups. Circular dichroism analysis revealed that mutation of hydrophobic residues resulted in a decrease in a-helix formation, whereas mutation of acidic and basic charged residues caused little to no change in a-helical content. When tested for diarrhea induction in mouse pups, the peptides containing mutations of either the hydrophobic or basic charged residues did not cause diarrhea. Taken together, the results of this research suggest a complex interplay between NSP4 secondary structure, caveolin-1 and cholesterol binding and diarrheagenic function.Item The rotavirus nonstructural protein 4 (NSP4) interacts with both the N- and C- termini of caveolin-1(Texas A&M University, 2006-08-16) Mir, Kiran DRotavirus (RV) is an etiologic agent of viral gastroenteritis in children and infants worldwide, accounting for an estimated 500,000 deaths annually. NSP4, the first described viral enterotoxin, contributes to RV pathogenesis by mobilizing intracellular calcium through multiple mechanisms that promote abnormal ion transport and subsequent secretory diarrhea. NSP4 and the enterotoxic peptide 114-135 preferentially interact with model membranes mimicking caveolae in lipid composition and radius of curvature. Our laboratory has recently reported the colocalization and coimmunoprecipitation of NSP4 with caveolin-1, the structural protein of caveolae. Moreover, the caveolin-1 binding domain of NSP4 has been localized to the enterotoxic peptide. We now report that caveolin-1 binds NSP4 via the N- and C-termini and one terminus is sufficient for binding. A panel of caveolin-1 deletion mutants was expressed in a yeast two-hybrid assay against an NSP4 bait. Caveolin-1 mutants retaining at least one terminus were capable of binding the NSP4 bait. An in vitro binding assay confirmed the two-hybrid results and localized the NSP4 binding domains to caveolin-1 residues 2-22 and 161-178. These data support the hypothesis that caveolin-1 mediates NSP4 signaling and/or intracellular trafficking.