This is a multi-faceted project to understand the origins, function and biological roles of selected protein and peptide derived cofactors. The trihydroxyphenylalanyl quinone (TPQ)-containing copper amine oxidases are of great interest from a number of perspectives. First, they catalyze both cofactor biogenesis and substrate turnover within a single active site. How this is accomplished will be further pursued using a variety of experimental protocols that include the use of unnatural amino acids, the perturbation of the redox potential for the active site copper, the inducement of cooperativity between the two subunits per dimer, and the comparison of biogenesis intermediates for two isozymes from H. polymorpha with greatly differing substrate specificity. The location of mammalian TPQ-containing enzymes on the outer plasma membrane of endothelial tissue and adipocytes implicates these proteins in key biological functions. A program is underway that entails detailed investigation of cultured murine adipocytes to interrogate the impact of enzymatic turnover of the ectopic amine oxidase (referred to as AOC-3) on cell signaling. These studies, which involve a combination of kinetic characterization, gene profiling, and cytokine release, will be extended to include the study of adipocytes derived from ACO-3 knockout mice, as well as the impact of the co-culturing of adipocytes with macrophages. Given that AOC-3 is emerging as a logical target for anti-inflammatory drugs, human AOC-3 will be expressed and its properties compared to its murine homolog. Further, the properties of AOC-2, located in the retina, will be contrasted with AOC-3, with the goal of developing isozyme-specific inhibitors for the control of inflammation. In contrast to the protein derived TPQ, pyrroloquinoline quinone (PQQ) is formed from two amino acids located within a peptide precursor. The pathway for the production of this bacterial vitamin involves six gene products and a highly cryptic chemical mechanism. Cloning, expression, and characterization of these genes is well underway, with the goal of describing the detailed pathway for PQQ generation. Studies of this nature may provide insights for the design of novel inhibitors targeted toward pathogenic bacteria.
This work will impact public health in two major ways. First, a determination of the biosynthetic pathway for the production of the bacterial vitamin PQQ may provide insight toward the design of novel inhibitors targeting pathogenic bacteria. Second, the projected studies of the cell surface murine copper amine oxidase and their human homologs are expected to be of considerable relevance to our understanding of the role of this enzyme in the inflammatory properties that characterize diabetes (type 2) and obesity. ? ? ?
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