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UBC Theses and Dissertations

Investigation of the role of SASH1 in the regulation of TRAF6-beta-arrestin complex formation Clayton, Ashley Rebecca


Proper activation and regulation of innate immune signaling pathways and inflammatory responses are essential to efficiently fight infection. Dysregulation of these responses, however, can result in the development of a severe hyper-inflammatory state, ultimately leading to sepsis. The endothelium has been shown to play a critical role in innate immune responses and the pathogenesis of sepsis. In the presence of Gram-negative infection, circulating LPS is recognized by TLR4 expressed on the surface of endothelial cells, mediating downstream activation of pro-inflammatory signal cascades and immune responses. SASH1 has been recently characterized as a novel scaffold protein in the regulation of endothelial TLR4 signaling. The work presented in this thesis further characterizes the role of SASH1 in the regulation of TRAF6 activation within the TLR4 pathway. LPS-induced auto-ubiquitination of TRAF6 is an essential regulatory step in the downstream activation of TLR4-mediated inflammatory signaling. Co-immunoprecipitation analyses confirmed the interaction of endogenous SASH1 with TRAF6 in an LPS-dependent manner in endothelial cells. Furthermore, SASH1 was required for LPS-induced auto-ubiquitination of TRAF6. To further investigate SASH1 function, a yeast two-hybrid approach was employed to identify novel SASH1-interacting proteins. β-arrestin 1 was identified as the top potential interactor of SASH1. Both β-arrestin 1 and β-arrestin 2 have been shown to act as negative regulators of the TLR4 pathway by impairing the oligomerization and auto-ubiquitination of TRAF6. It was speculated that SASH1 may coordinate the interaction of TRAF6 with the β-arrestins to mediate regulation of the TLR4 pathway. Reciprocal co-immunoprecipitation experiments confirmed the interaction of SASH1 with both β-arrestin 1 and β-arrestin 2. SASH1 was also found to interact with β-arrestin 1 in a complex with TRAF6. Further studies aimed to characterize the role of SASH1 in coordinating the interaction of TRAF6 with the β-arrestins. Although knockdown of SASH1 did not modulate TRAF6-β-arrestin binding, the enforced expression of SASH1 was found to specifically impair TRAF6 binding with β-arrestin 1, but not β-arrestin 2. The significance of this isoform-specific regulation remains to be determined. Overall, these studies further investigate the role of SASH1 as a critical scaffold protein in the regulation of the TLR4 pathway.

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