| Summary: | TLR4 is the most extensively studied of TLR pathways in innate immune signaling that provides the first line of defense against invading pathogens. SASH1, a large protein composed of SAM and SH3 domain, is a novel positive regulator of the pathway in endothelial cells. SASH1 acts as a scaffold protein in the TLR4 pathway by independently binding TRAF6/TAK1/ IKKβ/IKKα and regulating TRAF6 and TAK1 ubiquitination leading to LPS-induced activation of NF-κB resulting in production of pro-inflammatory cytokines. To investigate SASH1 in vivo function, SASH1 gene-trap mice were generated. These mice have a β-galactosidase reportor construct inserted into intron 14-15 resulting in a truncation of the SH3 domain, and thereby loss of the two SAM domains and TRAF6 binding motif. However, SASH1 gene-trap mice do not provide any viable homozygous adults . X-gal staining of the heterozygous SASH1 adult tissues demonstrated SASH1 transcripts to be predominantly expressed in microvascular endothelium. This thesis is the continuation of the above findings to further characterize the role of SASH1 in vitro and in vivo. Work presented here confirms the role of SASH1 as a positive regulator of the TLR4 pathway by promoting activation of NF-κB. SASH1 does not interact with the E2 ligases and IKKγ. These results further elucidate a model for SASH1 in the TLR4 pathway where the E2 ligases and IKKγ are incorporated into a complex through interaction with proteins that are assembled by SASH1 to promote the downstream signaling. SASH1 homozygous gene-trap mice die in the perinatal period and preliminary analysis shows the lung as the potential organ being affected by SASH1 disruption. Homozygous gene-trap lungs appear deflated, sink in PBS and have smaller airways compared to wild-type control. However, morphometric analysis of the lung is still required to conclusively define a lung defect. In addition, I generated SASH1-floxed embryonic stem cells to be used for generating mice with a conditionally targeted allele of SASH1 in endothelial cells to study the role of SASH1 in the endothelial response to LPS in TLR4 signaling in vivo, hence contributing to the field of innate immune signaling.
|
|---|
