siRNA-mediated identification and regulation of the ternary SNARE complex mediating mast cell degranulation

The identification of mast cell protein targets that mediate disease or disease progression is critical for the development of novel therapeutics for the treatment of allergy/asthma and autoimmune disease. Mast cells are granulocytes that emanate from myeloid progenitors in the bone marrow and play a critical role in innate immunity as vital sentinel cells that combat invading microorganisms through the release of a plethora of inflammatory mediators. Dysregulation of mast cell function leads to allergic disease and autoimmune disease that affect millions of people in the United States alone. Within cells, the transport and fusion of inflammatory mediator-laden vesicles to the membrane in granulocytes and their subsequent exocytosis is mediated by a family of evolutionarily-conserved proteins known as the SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors). The expression and functional roles of the SNARE family protein members in mast cell degranulation has not been elucidated. Here, we evaluate mast cell SNARE expression profiles at the mRNA and protein levels and determine the candidate SNARE proteins mediating mast cell degranulation in RBL-2H3 cells. We use immunoprecipitation to characterize the pertinent ternary SNARE complexes that interact following cell activation. We demonstrate that these complexes are critical for mast cell degranulation by utilizing RNA interference (siRNA) to knockdown mRNA and protein levels of the individual constituent SNARE proteins during FcepsilonRI receptor-activated rat RBL-2H3 mast cell activation. We use SNARE gene overexpression studies to confirm the phenotypes discovered using siRNA. We have identified specific SNARE proteins and complexes mediating mast cell degranulation in RBL-2H3 cells. Further, we have established sets of specific siRNA oligomers to begin the development of novel nucleic acid therapeutics to treat allergic and autoimmune disease.