Amyloid deposits result in organ dysfunction and death. Amyloidogenesis can be influenced at the level of precursor synthesis, precursor catabolism or fibrillogenesis. The least studied of these processes is fibrillogenesis, yet it may be the most feasible target for therapeutic assault. This is due to the extracellular nature of the deposit, and the commonality in all amyloid processes. All amyloid deposits consist of characteristic antiparallel cross Beta-pleated sheets, with proteoglycans, serum amyloid P component and ApoE as significant subcomponents. Although a diverse, yet select, group of protein precursors have been shown to be incorporated into amyloid, the precise molecular motif(s) that is an essential prerequisite for these unique fibrillar deposits has not been identified. We believe that the """"""""structural motif"""""""" is the presence of a cross beta-pleated sheet structure or the ability of the protein to form a cross beta-pleated sheet structure in the presence of heparan sulfate proteoglycan(s) and calcium. The mouse amyloid model is ideal to initiate our understanding of fibrillogenesis. Other systems present major problems of precursor diversity or paucity. In the mouse, serum amyloid A proteins (SAA) are the most dramatic acute phase reactants with levels increasing up to 1000-fold following inflammation. Of the two major SAA isotypes in the mouse (SAA1 and SAA2), only one (SAA2) is selectively cleared and deposited in amyloid. Our laboratory has identified that the CE/J mouse is totally amyloid resistant and that it has a single SAA1 1/2 hybrid isotype. Analyses of this isotype have allowed us to deduce that one or more of 5 amino acids hold the key to the amyloidogenicity of SAA2. To test our hypothesis. the structural and pathophysiological effects of specific alterations in the SAA1 2 cDNA with subsequent mutant protein expression will be examined. Specifically, the CE/J SAA1/2 cDNA has been cloned into an expression vector and synthesized in vitro to generate protein for analysis. Site-directed mutagenesis will be performed to identify those amino acids essential for fibrillogenesis. The structural folding of these mutants will be determined by circular dichroism, tryptophan fluorescence and correlated with their biological ability to be deposited in amyloid. Finally, transgenic mice overexpressing S A A 2 in response to administration of zinc have been generated. This would allow fibrillogenesis to be studied separate from complicating systemic inflammation.
