Protein and Peptide-Derived Redox Cofactors: Biogenesis and Function

描述(申请人提供)：这是一个多方面的项目，以了解选定的蛋白质和多肽衍生辅因子的起源、功能和生物学作用。含有三羟基苯丙氨基二酚(TPQ)的铜胺氧化酶在许多领域具有重要的应用价值。首先，它们在单一活性部位催化辅因子生物合成和底物周转。如何实现这一点将进一步探索使用各种实验方案，包括使用非天然氨基酸，扰动活性部位铜的氧化还原电位，诱导每个二聚体的两个亚基之间的协作性，以及比较具有很大不同底物特异性的两种同工酶的生物发生中间产物。哺乳动物含有TPQ的酶位于内皮组织和脂肪细胞的外质膜上，这意味着这些蛋白具有关键的生物学功能。一项需要对培养的小鼠脂肪细胞进行详细研究的计划正在进行中，以询问异位胺氧化酶(简称AOC-3)的酶促周转对细胞信号的影响。这些研究涉及动力学特征、基因图谱和细胞因子释放的组合，将扩展到包括来自ACO-3基因敲除小鼠的脂肪细胞的研究，以及脂肪细胞与巨噬细胞共同培养的影响。鉴于AOC-3正在成为抗炎药物的合理靶点，将表达人AOC-3，并将其特性与其小鼠同源物进行比较。此外，位于视网膜中的AOC-2的特性将与AOC-3进行对比，目的是开发同工酶特异性抑制物来控制炎症。与蛋白质来源的TPQ不同，吡咯喹啉醌(PQQ)是由位于多肽前体中的两个氨基酸形成的。这种细菌维生素的产生途径涉及六种基因产物和一种高度神秘的化学机制。这些基因的克隆、表达和鉴定正在进行中，目的是描述PQQ产生的详细途径。这种性质的研究可能为设计针对病原菌的新型抑制剂提供见解。公共卫生相关性：这项工作将在两个主要方面影响公共卫生。首先，确定生产细菌维生素PQQ的生物合成途径可能为设计针对病原菌的新型抑制剂提供洞察力。其次，对细胞表面小鼠铜胺氧化酶及其人类同系物的计划研究有望与我们理解这种酶在糖尿病(2型)和肥胖的炎性特征中所起的作用有相当大的相关性。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.