Browsing by Author "Scott, Brenton L."

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    Munc18b is an essential gene in mice whose expression is limiting for secretion by airway epithelial and mast cells
    (Portland Press Limited, 2012) Kim, Kyubo; Petrova, Youlia M.; Scott, Brenton L.; Nigam, Rupesh; Agrawal, Anurag; Evans, Christopher M.; Azzegagh, Zoulikha; Gomez, Alejandra; Rodarte, Elsa M.; Olkkonen, Vesa M.; Bagirzadeh, Rustam; Piccotti, Lucia; Ren, Binhui; Yoon, Joo-Heon; McNew, James A.; Adachi, Roberto; Tuvim, Michael J.; Dickey, Burton F.; Cystic Fibrosis Foundation; National Institutes of Health
    Airway mucin secretion and MC (mast cell) degranulation must be tightly controlled for homoeostasis of the lungs and immune system respectively. We found the exocytic protein Munc18b to be highly expressed in mouse airway epithelial cells and MCs, and localized to the apical pole of airway secretory cells. To address its functions, we created a mouse with a severely hypomorphic Munc18b allele such that protein expression in heterozygotes was reduced by∼50%. Homozygous mutant mice were not viable, but heterozygotes showed a ∼50% reduction in stimulated release of mucin from epithelial cells and granule contents from MCs. The defect in MCs affected only regulated secretion and not constitutive or transporter-mediated secretion. The severity of passive cutaneous anaphylaxiswas also reduced by ∼50%, showing that reduction of Munc18b expression results in an attenuation of physiological responses dependent on MC degranulation. The Munc18b promoter is controlled by INR (initiator), Sp1 (specificity protein 1), Ets, CRE (cAMP-response element), GRE (glucocorticoid-response element), GATA and E-box elements in airway epithelial cells; however, protein levels did not change during mucous metaplasia induced by allergic inflammation. Taken together, the results of the present study identifyMunc18b as an essential gene that is a limiting component of the exocytic machinery of epithelial cells and MCs.
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    Regulation of SNARE-mediated membrane fusion by Sec1/Munc18 (SM) proteins
    (2005) Scott, Brenton L.; McNew, James A.
    The intricate temporal and spatial regulation of membrane fusion is critical for all living organisms. Fusion of two opposing membranes occurs in a wide range of processes. These include intracellular transportation, cell-to-cell fusion and viral fusion. In all known cases, the SNARE proteins (Soluble NSF attachment protein receptors) (Sollner et al., 1993; Whiteheart et al., 1993) have been shown to be required for vesicular membrane fusion within cells and sufficient to drive membrane fusion in vitro (Nickel et al., 1999; Parlati et al., 1999; Weber et al., 1998). While SNAREs combine in specific combinations to drive highly specific membrane fusion, it is clear that SNARE proteins do not act independently to regulate the entire fusion process. Many regulatory proteins from different families have been identified that interact with individual SNARE proteins and SNARE complexes, yet the precise role of many of these remains unclear. One such group of regulatory proteins is the Sec1/Munc18 (SM) family. Sec1 proteins are likely to be critical players in membrane trafficking. My work has focused on the role of the yeast Sec1p in post-Golgi secretion in Saccharomyces cerevisiae. To analyze Sec1p function in vitro, I have utilized a well-characterized SNARE-mediated membrane fusion assay. For this application, conditions were optimized to allow for specific protein-protein interactions to be tested. Conditions for expression and purification of the previously elusive recombinant Sec1p are documented. In addition, an overexpressing Sec1p yeast strain was generated. Sec1p interactions with SNARE proteins that mediate post-Golgi secretion were then tested. I found that recombinant Sec1p binds strongly to the t-SNARE complex (Sso1p;Sec9c) as well as to the fully assembled ternary-SNARE complex (Sso1p;Sec9c/Snc2p), and also weakly to free Sso1p. I tested the ability of Sec1p to regulate fusion in the fusion assay. Concentration dependent stimulation of membrane fusion is observed when Sec1p is associated with the SNARE proteins. The binding and fusion data strongly argue that Sec1p directly stimulates SNARE-mediated membrane fusion. With this new information, specific binding modes of neuronal-Sec1 are currently being investigated further in yeast, Drosophila and mammalian SNARE systems.