Date of Award

Spring 3-2022

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

School

Biological, Environmental, and Earth Sciences

Committee Chair

Dr. Hao Xu

Committee Chair School

Biological, Environmental, and Earth Sciences

Committee Member 2

Dr. Alex Flynt

Committee Member 2 School

Biological, Environmental, and Earth Sciences

Committee Member 3

Dr. Shahid Karim

Committee Member 3 School

Biological, Environmental, and Earth Sciences

Committee Member 4

Dr. Vijay Rangachari

Committee Member 4 School

Mathematics and Natural Sciences

Committee Member 5

Dr. Yanlin Guo

Committee Member 5 School

Biological, Environmental, and Earth Sciences

Abstract

Mast cells undergo exocytosis to release a wide array of inflammatory mediators by utilizing membrane fusion proteins-SNAREs (soluble-N-ethyl-maleimide sensitive factor attachment protein receptor) along with essential regulatory Munc18 and Munc13 proteins. Accumulating evidence in mast cell biology suggests the existence of distinct pools of mast cell mediators. However, the precise mechanism underlying the release of each mast cell mediator is not clear. To determine whether different exocytic machineries are required for differential mediator release, I used reconstitution to investigate the differential role of Munc18s in fusion machinery regulation. Munc18a and Munc18c stimulated VAMP2 and VAMP3-mediated lipid mixing, whereas Munc18b only stimulated VAMP8/Stx3/SNAP23-mediated fusion. I then investigated the role of Munc18 post-translational modification in mast cell exocytosis. In reconstitution assays, phosphomimetic mutants- Munc18aS306E/S313E and Munc18aT574E did not alter the activity or specificity of wild-type Munc18a. Munc18bT572D, on the other hand, abolished Munc18b's ability to stimulate VAMP8-dependent degranulation. I further showed that Munc18a undergo PKC-dependent phosphorylation at Ser313 in activated Rat Basophilic Leukemia (RBL-2H3) mast cells (Adhikari and Xu, 2018). These findings suggest site-specific phosphorylation regulates the interaction between Munc18 proteins and their cognate VAMPs. Furthermore, I investigated the differential requirement of vesicular SNAREs in the release of a selection of proinflammatory mediators. Using RNAi, I showed that the knockdown of VAMP8 inhibited IgE/antigen-induced release of β-hexosaminidase, histamine, and serotonin but not TNF. Knocking out VAMP3, which mediates TNF release from human synovial cells and phagocytosing macrophages, did not display any defect in the TNF exocytosis in RBL-2H3 mast cells; however, the release of β-hexosaminidase was enhanced (manuscript in preparation). Finally, I used the knockout approach to address the roles of Munc13 proteins in mast cell exocytosis. The regulated release of β-hexosaminidase, histamine, and serotonin is virtually eliminated in Munc13-4 knockout RBL-2H3 cells, but TNF release is only partially inhibited (Ayo, Adhikari, et al, 2020). Knocking out Munc13-4 homolog BAIAP3 seems to affect TNF release, although not in a statistically significant fashion. My investigation has set the stage for further dissection of the different molecular mechanisms underlying the exocytosis of distinct mast cell mediators.

ORCID ID

0000-0002-1285-3970

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