Regulation of Human NHE3 by Ubiquitination

项目概要 腹泻病每年导致近百万五岁以下儿童死亡。虽然其发生率是 在较富裕的国家中，腹泻率要低得多，腹泻仍然是儿科最常见的两种就诊之一 急诊室，在收容机构的老年人中也很常见。 NHE3 是主要的钠转运蛋白 位于小肠和近端结肠的刷状缘膜中。 NHE3 表达异常和 功能与急性病原体感染和炎症引起的腹泻疾病有关 肠道。该应用旨在了解人类 NHE3 的翻译后修饰。的推动力 拟议的研究来自我们最近的发现，即人类和灵长类动物的 NHE3 与人类和灵长类动物的 NHE3 不同 低等哺乳动物，包括啮齿动物和兔子。 Nedd4-2 是一种与底物相互作用的 E3 泛素连接酶 通过 PY (PPxY) 基序的蛋白质。我们发现人类 NHE3 (hNHE3) 与 Nedd4-2 相互作用，后者泛素化 hNHE3并介导内吞作用，并且hNHE3对forskolin/PKA的反应比兔子大得多 NHE3。尽管小鼠已被广泛用于了解 NHE3 的生理作用，但小鼠相对 抵抗发育性腹泻。我们将研究 hNHE3 泛素化有助于 男性急性腹泻的严重程度增加。首先，我们将比较 hNHE3 和小鼠的调节 NHE3在体内使用转基因动物。我们将研究 hNHE3 如何被内吞、回收和降解 Nedd4-2 介导的泛素化。泛素化被去泛素化所抵消。我们已经确定了几个 与 hNHE3 相互作用的推定去泛素化酶 (DUB)。我们计划研究 Nedd4-2 和 DUB 动态调节 hNHE3。我们的工作有望揭示控制的新机制 肠刷状缘 NHE3，并将 Nedd4-2 确定为治疗腹泻疾病的新靶点 由肠上皮细胞钠和水平衡异常引起。

PROJECT SUMMARY Diarrheal disease causes close to one million deaths in children under five each year. Although its incidence is much lower in the more affluent nations, diarrhea remains one of the two most common visits to pediatric emergency rooms and is also common among the institutionalized elderly. NHE3 is a major sodium transporter in the brush border membrane of the small intestine and proximal colon. Abnormal NHE3 expression and function are associated with diarrheal diseases resulting from acute pathogenic infection and inflammation in the gut. This application aims at understanding post-translational modification of human NHE3. The impetus of the proposed study comes from our recent finding that NHE3s of human and primates differ from NHE3s of lower mammals, including rodents and rabbits. Nedd4-2 is an E3 ubiquitin ligase that interacts with substrate proteins via PY (PPxY) motif. We found that human NHE3 (hNHE3) interacts with Nedd4-2, which ubiquitinates hNHE3 and mediates endocytosis, and the response of hNHE3 is much greater to forskolin/PKA than rabbit NHE3. Although mice have widely been used to understand the physiological role of NHE3, mice are relatively resistant to development diarrhea. We will examine the idea that ubiquitination of hNHE3 contributes to the increased severity of acute diarrhea in man. To begin, we will compare the regulation of hNHE3 and mouse NHE3 in vivo using transgenic animals. We will study how hNHE3 is endocytosed, recycled and degraded by Nedd4-2 mediated ubiquitination. Ubiquitination is counteracted by deubiqutination. We have identified several putative deubiquinating enzymes (DUBs) that interact with hNHE3. We plan to investigate how Nedd4-2 and DUBs dynamically regulated hNHE3. Our work is expected to reveal new mechanism for the control of intestinal brush border NHE3, and identify Nedd4-2 as a novel target for the therapeutic of diarrheal diseases caused by abnormal sodium and water balance in intestinal epithelium.