Date of Award

Summer 8-2016

Degree Type

Masters Thesis

Degree Name

Master of Science (MS)

Department

Biological Sciences

Committee Chair

Hao Xu

Committee Chair Department

Biological Sciences

Committee Member 2

YanLin Guo

Committee Member 2 Department

Biological Sciences

Committee Member 3

Vijay Rangachari

Committee Member 3 Department

Chemistry and Biochemistry

Abstract

Mast cells are specialized secretory leukocytes that play diverse roles in the body, mediated by release of histamine and other pharmacologically active substances. Although offering essential protection in innate and adaptive immunity, mast cells are also essential to the progression of inflammatory diseases, including allergy and asthma, (Theoharides et al., 2012). Exocytosis of pro-inflammatory mast cell mediators in response to otherwise innocuous antigens relies on evolutionarily-conserved membrane fusion machinery. These proteins, called SNAREs are regulated by the Sec1/Munc18 (SM) protein family (Axle Lorentz, Baumann, Vitte, & Blank, 2012). Mast cells express three mammalian Munc18 isoforms (a, b, and c), which are linked to SNARE-dependent exocytosis in numerous organisms. However, the exact roles of each of these Munc18 isoforms in mast cell degranulation have not been clearly defined. In this study we investigated the functional relationship between Munc18 and eight sets of degranulation-relevant SNAREs using in vitro reconstitution. We showed that Munc18a was active in stimulating fusion mediated by VAMP2 and VAMP3, but not by VAMP7 and VAMP8. In contrast, Munc18b and Munc18c did not show any stimulatory activity. Further analysis of Munc18a function unraveled a previously unidentified role in the tethering of SNARE complexes prior to fusion, an action that is dependent on the interaction between Munc18a and N-peptide of syntaxin. Inhibition of fusion and trans-SNARE complex formation by soluble VAMP proteins did not interfere with Munc18a-mediated tethering, indicating that Munc18a-mediated tethering occurs prior to trans-SNARE complex formation. This study therefore sheds light on new roles Munc18 proteins might play to regulate mast cell degranulation.

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