Dual Oxidases (Duox): Enzymology & Biological Function

描述(由申请人提供)：已经定义了一个双氧合酶家族(DUOX)。DUOX酶由三个结构域组成：1)与吞噬细胞呼吸爆发氧化酶催化亚基gp91Phox同源的NADPH-氧化酶结构域；2)钙结合结构域；3)过氧化物酶结构域。Duoxs位于上皮细胞(结肠、肺、甲状腺、胰腺)的质膜上，通过细胞内NADPH还原O2在细胞外形成H202发挥作用。后者被过氧化物酶结构域用来催化过氧化反应，包括细胞外基质蛋白的修饰，可能还有其他细胞外小分子的修饰。具体地说，DUOX被认为在先天性免疫、内分泌功能和癌症方面具有生物学功能。我们鉴定并克隆了h-Duox1和h-DUOX2两种人类亚型，并在包括线虫和果蝇在内的不同物种中鉴定了DUOX酶。在线虫中，我们证明了这种酶的功能是将胶原和其他细胞外基质蛋白中的酪氨酸残基化学交联，从而稳定角质层的结构。我们将检验这一假设，即DUOX酶在整个动物界的一般功能是酪氨酸和/或其他细胞外基质残基的化学修饰。遗传和生化方法将被用来在果蝇身上验证这一假说，我们提出酪氨酸修饰稳定了翅膀的结构。将利用噬菌体展示方法开发h-Duox1和h-DUOX2的过氧化物酶结构域的多肽抑制剂，并用于研究DUOX的正常生物学功能，如甲状腺激素的生物合成和天然免疫。DUOX的酶性质和钙的调节将被记录下来，并将探索结构域的跨膜拓扑。这些研究将首次提供关于这组新发现的酶的酶学、拓扑和生物学功能的基本信息，并将证明它们对细胞外基质(ECM)进行化学修饰的能力。由于ECM是转化的关键决定因素，DUOX酶可能在某些组织的癌症生物学中发挥重要作用。

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.