The gene history of zebrafish tlr4a and tlr4b is predictive of their divergent functions.

Mammalian immune responses to LPS exposure are typified by the robust induction of NF-kappaB and IFN-beta responses largely mediated by TLR4 signal transduction pathways. In contrast to mammals, Tlr4 signal transduction pathways in nontetrapods are not well understood. Comprehensive syntenic and phylogenetic analyses support our hypothesis that zebrafish tlr4a and tlr4b genes are paralogous rather than orthologous to human TLR4. Furthermore, we provide evidence to support our assertion that the in vivo responsiveness of zebrafish to LPS exposure is not mediated by Tlr4a and Tlr4b paralogs because they fail to respond to LPS stimulation in vitro. Zebrafish Tlr4a and Tlr4b paralogs were also unresponsive to heat-killed Escherichia coli and Legionella pneumophila. Using chimeric molecules in which portions of the zebrafish Tlr4 proteins were fused to portions of the mouse TLR4 protein, we show that the lack of responsiveness to LPS was most likely due to the inability of the extracellular portions of zebrafish Tlr4a and Tlr4b to recognize the molecule, rather than to changes in their capacities to transduce signals through their Toll/IL-1 receptor (TIR) domains. Taken together, these findings strongly support the notion that zebrafish tlr4a and tlr4b paralogs have evolved to provide alternative ligand specificities to the Tlr immune defense system in this species. These data demonstrate that intensive examination of gene histories when describing the Tlr proteins of basally diverging vertebrates is required to obtain fuller appreciation of the evolution of their function. These studies provide the first evidence for the functional evolution of a novel Tlr.

Dioxin alters the human low-density and very low-density lipoprotein structure with evidence for specific quenching of Trp-48 in apolipoprotein C-II.

The intercellular transport of cholesterol and triglycerides via lipoproteins interacting with their receptors is a critical component in human lipid metabolism. The delivery of cholesterol to cells is accomplished primarily through low-density lipoproteins (LDLs), while the transport of fatty acids to adipose and muscle tissue is accomplished primarily through the actions of very low-density lipoproteins (VLDLs). Disruption of lipoprotein structure leading to impaired binding between these lipoproteins and their obligate receptors is a known risk factor for cardiovascular disease. Because of recent investigations linking 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure in humans with coronary artery disease, investigations have been carried out by fluorescence and circular dichroism to evaluate conformational changes in LDL and VLDL structure upon binding of TCDD. These studies demonstrate that, at a molar ratio of three TCDD molecules to one lipoprotein molecule, TCDD binds and disrupts the secondary and tertiary lipoprotein structure. Circular dichroism studies show that residues within the inner core of apoC-II, which compose a four-alpha-helix bundle when this apolipoprotein is associated with VLDL, are directly affected upon binding TCDD. Fluorescence also indicates the specific interaction of Trp-48 within apoC-II upon TCDD binding. We found that the TCDD/apoC-II complex suffers a 5-fold reduction in its ability to bind lipoprotein lipase compared to untreated apoC-II. The interaction of TCDD with LDL markedly altered the secondary structure of apoB reducing its alpha-helical content. These cumulative responses in lipoprotein structure may impair the LDL and VLDL cellular uptake leading to a buildup of serum lipoproteins and fats thus hastening the development of coronary artery disease.