Novel Defensins of Human Epithelia

描述(申请人提供)：抗菌肽的发现揭示了动物宿主防御中一个以前未被认识到的成分。我们和其他人发现，哺乳动物粘膜上皮细胞表达防御素多肽，为它们提供了参与粘膜宿主防御的能力。在人的小肠中，防御素HD5和HD6在称为Paneth细胞的分泌上皮细胞中高度表达。我们小组和其他小组的研究为潘氏细胞防御素肽在肠道中提供强大的关键防御功能的假设提供了强有力的支持。我们的研究还发现，HD5作为一种修饰的前肽储存在Paneth细胞中。我们鉴定了Paneth细胞胰酶是一种将proHD5加工成管腔中活性抗菌剂的蛋白酶。这些数据表明，Paneth Call Trypsin的蛋白分解活性可能是调节肠道宿主防御的中心。 我们的假设是：1)HD5(可能还有HD6)以糖基化的前肽形式储存在Paneth细胞中，为潜在的抗生素活性和降低对宿主细胞的毒性提供了一种机制；2)HD6在小肠中与HD5具有部分重叠(或协同)的抗菌功能；3)一旦激活，Paneth细胞的胰蛋白酶活性就会揭示潜在的抗菌物质，并激活肠细胞上的蛋白酶激活受体-2(PAR-2)。在三个特定目标中，我们将确定人ProHD-5组织形式的翻译后结构修饰(目标1)；建立HD-6组织和管腔形式的结构并测定其体外抗菌活性(目标2)；鉴定Paneth细胞胰蛋白酶异构体，确定它们的关键活性，包括ProHD-5和-6的加工，其他潜在抗菌剂的激活和PAR-2的激活，并确定Paneth细胞胰蛋白酶原激活的机制(目标3)。拟议的研究可能为抗击肠道感染性和炎症性疾病的新的治疗目标和方法提供见解。

DESCRIPTION (provided by applicant): The discovery of antimicrobial peptides has unveiled a previously unrecognized component of animal host defense. We and others have discovered that mammalian mucosal epithelial cells express defensin peptides, providing them with the capacity to participate in mucosal host defense. In the human small intestine, defensins HD5 and HD6 are highly expressed in secretory epithelial cells called Paneth cells. Investigations by our group, and those of others, provide strong support for the hypothesis that Paneth cell defensin peptides provide a robust, critical defensive function in the intestine. Our studies have discovered also that HD5 is stored in Paneth cells as a modified propeptide. We identified that Paneth cell trypsin is the protease that processes proHD5 to the active antimicrobial present in the lumen. These data suggest that the proteolytic activity of Paneth call trypsin might be central to the regulation of enteric host defense. It is our hypothesis that 1) HD5 (and possibly HD6) is stored in Paneth cells as a glycosylated propeptide, providing a mechanism for both latent antibiotic activity and decreased toxicity to host cells, 2) HD6 has partially overlapping (or synergistic) antimicrobial functions with HD5 in the small intestine, and 3) once activated, Paneth cell trypsin activity unmasks latent antimicrobials and activates protease-activated receptor-2 (PAR-2) on enterocytes. In three specific aims, we will determine the posttranslational structural modifications of the tissue form of human proHD-5 (aim 1); establish the structure of tissue and luminal forms of HD-6 and determine their in vitro antimicrobial activity (aim 2); characterize Paneth cell trypsin isoforms, identify their key activities including processing of proHD-5 and -6, activation of other latent antimicrobials and activation of PAR-2, and identify the mechanism of Paneth cell protrypsin activation (aim 3). The proposed investigations may provide insights to novel therapeutic targets and approaches to combat infectious and inflammatory disease of the intestine.