Regulation of Human NHE3 by Ubiquitination

项目摘要 腹泻病每年造成近100万5岁以下儿童死亡。虽然它的发病率是 在较富裕的国家，腹泻仍然是儿科最常见的两种疾病之一， 急诊室，也是常见的机构老人。NHE 3是一种主要的钠转运蛋白 在小肠和近端结肠的刷状缘膜中。NHE 3表达异常， 功能与急性病原性感染和炎症引起的呼吸道疾病有关， 内脏本申请旨在理解人NHE 3的翻译后修饰。的推动 这项拟议的研究来自于我们最近的发现，即人类和灵长类动物的NHE 3与人类的NHE 3不同。 低等哺乳动物，包括啮齿动物和兔子。Nedd 4 -2是一种E3泛素连接酶， 通过PY（PPxY）基序的蛋白质。我们发现人NHE 3（hNHE 3）与Nedd 4 -2相互作用， hNHE 3介导内吞作用，并且hNHE 3对forskolin/PKA的反应比兔大得多 NHE3。虽然小鼠已被广泛用于了解NHE 3的生理作用，但小鼠相对较弱。 对腹泻有抵抗力。我们将研究hNHE 3的泛素化有助于 开始，我们将比较hNHE 3和小鼠的调节作用， 使用转基因动物的体内NHE 3。我们将研究hNHE 3是如何内吞，回收和降解， Nedd 4 -2介导的泛素化。泛素化被去泛素化抵消。我们发现了几个 与hNHE 3相互作用的推定的去泛素化酶（DUB）。我们计划调查Nedd 4 -2和 DUBs动态调节hNHE 3。我们的工作有望揭示新的控制机制， 肠刷状缘NHE 3，并将Nedd 4 -2鉴定为治疗结肠炎疾病的新靶点 由肠上皮细胞中的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.