Dual Oxidases (Duox): Enzymology & Biological Function

描述（由申请人提供）：已定义了一个双氧化酶（Duox）家族。Duox酶由三个结构域组成：1）与吞噬细胞呼吸爆发氧化酶的催化亚基gp 91 phox同源的NADPH氧化酶结构域; 2）钙结合结构域;和3）过氧化物酶结构域。 Duoxs'位于上皮细胞（结肠、肺、甲状腺、胰腺）的质膜中，提出通过使用细胞内NADPH还原O2以在细胞外形成H2 O2来起作用。后者被过氧化物酶结构域用于催化涉及细胞外基质蛋白和可能的其他细胞外小分子的修饰的过氧化反应。具体而言，Duox's被认为在先天免疫、内分泌功能和癌症中发挥生物学功能。我们鉴定并克隆了两种人类同种型h-Duox 1和h-Duox 2，并在包括C.线虫和果蝇。In C.在elegans中，我们发现这种酶的功能是化学交联胶原蛋白和其他细胞外基质蛋白中的酪氨酸残基，从而稳定角质层的结构。我们将测试的假设，Duox酶在整个动物界的一般功能是酪氨酸和/或细胞外基质的其他残基的化学修饰。遗传和生物化学方法将被用来测试这一假设在果蝇，我们建议，酪氨酸修饰稳定的翅膀的结构。噬菌体展示方法将用于开发h-Duox 1和h-Duox 2的过氧化物酶结构域的肽抑制剂，并且这些将用于研究Duox的正常生物学功能，例如甲状腺激素生物合成和先天免疫。将记录Duox的酶性质和钙调节，并探索结构域的跨膜拓扑结构。这些研究将首次提供关于这组新发现的酶的酶学、拓扑学和生物学功能的基本信息，并将记录它们化学修饰细胞外基质（ECM）的能力。由于ECM是转化的关键决定因素，Duox酶可能在某些组织中的癌症生物学中起重要作用。

DESCRIPTION (provided by applicant): A family of Dual Oxidases (Duox) has been defined. Duox enzymes consist of three domains: 1) an NADPH-oxidase domain homologous to gp91phox, the catalytic subunit of the phagocyte respiratory burst oxidase; 2) a calcium-binding domain; and 3) a peroxidase domain. Located in plasma membrane of epithelial cells (colon, lung, thyroid, pancreas), Duoxs' are proposed to function by using intracellular NADPH to reduce 02 to form H202 outside the cell. The latter is used by the peroxidase domain to catalyze peroxidative reactions involving modification of extracellular matrix proteins and probably other extracellular small molecules. Specifically, Duox's are proposed to function biologically in innate immunity, endocrine function and cancer. We identified and cloned two human isoforms, h-Duox1 and h-Duox2, and have identified Duox enzymes in diverse species, including C. elegans and Drosophila. In C. elegans, we showed that the enzyme functions to chemically cross-link tyrosine residues in collagen and other extracellular matrix proteins, thus stabilizing the structure of the cuticle. We will test the hypothesis that a general function of Duox enzymes throughout the animal kingdom is the chemical modification of tyrosine and/or other residues of extracellular matrix. Genetic and biochemical methods will be used to test this hypothesis in Drosophila, where we propose that the tyrosine modifications stabilize the structure of the wing. Phage display methods will be used to develop peptide inhibitors of the peroxidase domains of h-Duoxl and h-Duox2, and these will be used to investigate normal biological functions of Duox such as thyroid hormone biosynthesis and innate immunity. Enzymatic properties and regulation by calcium of Duox will be documented, and the transmembrane topology of the domains will be explored. These studies will provide for the first time fundamental information regarding the enzymology, topology and biological functions of this newly discovered group of enzymes and will document their ability to chemically modify extracellular matrix (ECM). Because ECM is a critical determinant of transformation Duox enzymes may play an important role in cancer biology in some tissues.