Con Sullivan

Date of Award

5-2006

Level of Access Assigned by Author

Campus-Only Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry and Molecular Biology

Advisor

Carol H. Kim

Second Committee Member

Robert E. Gundersen

Third Committee Member

Keith W. Hutchinson

Abstract

Mechanistic studies aimed at describing the way lower vertebrates like zebrafish utilize Toll-like receptors have not been extensively undertaken. In this dissertation, zebrafish TRIF is shown to signal in a manner that differs from what has been shown in mammals. Zebrafish TRIF is smaller than mammalian homologs and possesses significant sequence gaps in its N- and C-termini, calling into question whether it can interact with proteins deemed essential to signal transduction in mammals. Through co-immunoprecipitation, zebrafish TRIF was shown not to interact with zebrafish TRAF6 but was shown to interact with RIPl and TBKl. Overexpression of full-length zebrafish TRIF activates NF-KB and type I interferon (IFN); however, the way zebrafish TRIF signals differs from mammalian homologs. Through overexpression of mutants, it was determined that zebrafish TRIF relies upon its TIR domain and C-terminus for both NF-KB and IFN signaling. It was observed that an N-terminal deletion of TRIF exhibited the most potent IFN upregulation, calling into question TBKl's in zebrafish TRIF signaling. Taken together, the data indicate that NF-KB signaling is RHIM domain-dependent and IFN signaling is TBK1-independent. This dissertation also explored the role of TLR4 signaling in zebrafish. Lipopolysaccharide (LPS), the ligand that activates TLR4 pathways in mammals, induces interleukin 1 and tumor-necrosis factor - alpha in zebrafish; however, this response must be regulated by an alternative LPS receptor. Zebrafish TLR4a (zfTLR4a) and TLR4b failed to respond to LPS stimulation, as measured by NF-KB activation. zfTLR4a and zfTLR4a, unlike mammalian TLR4s, also cannot be made constitutively active, which argues for their potential roles as co-receptors. zfTLR4a-GFP and zfTLR4b-GFP exhibited alternative localization patterns, indicating that, despite their sequence similarities, they may play alternative roles in the cell. Unlike mammals, zebrafish cannot be induced to express IFN by LPS. The functional inability of TLR4 to recognize LPS and thereby activate NF-KB and IFN has not been replaced by the other TLRs. The discovery, through analysis of synteny across vertebrates, that the TIR domain containing adaptor TRAM, which is essential for TLR4-mediated LPS induction of IFN, is not present in nonmammals may also contribute to the absence of LPS-inducible IFN activation in zebrafish.

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