Investigatiions on the functional regulation in the process of processing from the membrane-integrated form of HGF activator inhibitors

HGF激活剂抑制剂膜整合型加工过程中的功能调控研究

基本信息

批准号：
11680603
负责人：
DENDA Kimitoshi
金额：
$ 2.18万
依托单位：
Tokyo Institute of Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2001
项目状态：
已结题

项目摘要

Hepatocyte growth factor activator inhibitors type 1 (HAI-1) and type 2 (HAI-2) are Kunitz-type serine protease inhibitors, identified as strong inhibitors toward hepatocyte growth factor (HGF) activator. Each HAI molecule possesses the hydrophobic region at the C-terminus, suggesting that they are produced primarily in a membrane-integrated form with two Kunitz domains in its extracellular region, and ectodomain shedding releases several secreted forms. In order to clarify the physiological role of these regulatory factors, we have performed the functional analyzes of HAI-1 and HAI-2.1. Immunoblotting analysis revealed that HAI-1 is first produced in a 66 kDa membrane-integrated form, and subsequent ectodomain shedding releases two major secreted forms from the cell surface into the extracellular space, their sizes being 40/39 kDa and 58 kDa. Whereas the former containing one Kunitz domain shows strong inhibitory activity against the HGF-converting activity of HGF activator, the latte … More r containing two Kunitz domains shows markedly weaker inhibitory activity. Thus, the presence of the C-terminal Kunitz domain (Kunitz II) in the 58 kDa HAI-1 may interfere with the binding of HGF activator to the reactive site of the N-terminal Kunitz domain (Kunitz I), while elimination of this region by proteolytic processing could lead to strong binding of HGF activator to HAI-1.2. To determine roles of the Kunitz domains in the inhibitory activity of HAIs against serine proteases, we constructed various HAI mutants and examined their inhibitory activity against HGF activator. Kunitz I in each HAI molecule appears to be the functional domain for inhibiting the HGF-converting activity of HGF activator. Furthermore, the presence of two Kunitz domains affected the inhibitory activity of HAI-1 against HGF activator. These results suggest that serine protease binding sites of Kunitz I and Kunitz II are located close to each other, and proteolytic processing to generate HAI-1 with only one Kunitz domain regulates the activity of HAI-1. Identification of cognated serine proteases against Kunitz II will reveal the novel biological function of HAIs.3. In order to search for HAI secretases, we have examined protein-protein interaction in the cytoplasmic regions of HAIs using a yeast two-hybrid screening system. Several cytoplasmic proteins, including cytosleletal proteins and intracelluiar enzymes were obtained. One of them was known to be involved in suppression on apoptosis, suggesting that HAI may play important role on the regulation of death signaling (manuscripts in preparation).Further analyzes on the functional regulation in the process of the proteolytic processing of HAIs will provide a novel mechanism of the regulation on HGF activation. Less

肝细胞生长因子激活剂抑制剂 1 型 (HAI-1) 和 2 型 (HAI-2) 是 Kunitz 型丝氨酸蛋白酶抑制剂，被确定为肝细胞生长因子 (HGF) 激活剂的强抑制剂。每个 HAI 分子在其疏水性区域均具有疏水性区域。 C 末端，表明它们主要以膜整合形式产生，其胞外区域有两个 Kunitz 结构域，胞外域脱落释放出几种分泌的为了阐明这些调节因子的生理作用，我们对 HAI-1 和 HAI-2.1 进行了功能整合分析，结果表明 HAI-1 首先以 66 kDa 的膜整合形式产生。胞外域脱落将两种主要的分泌形式从细胞表面释放到细胞外空间，它们的大小分别为 40/39 kDa 和 58 kDa，而前者含有一个 Kunitz 结构域，显示出强烈的抑制活性。 HGF 激活剂的 HGF 转换活性，含有两个 Kunitz 结构域的 latte … 更多 r 显示出明显较弱的抑制活性，因此，58 kDa HAI-1 中 C 端 Kunitz 结构域 (Kunitz II) 的存在可能会干扰 HGF 激活剂的 HGF 转换活性。 HGF 激活剂与 N 末端 Kunitz 结构域 (Kunitz I) 的反应位点结合，而通过蛋白水解加工消除该区域可能会导致 HGF 的强结合为了确定 Kunitz 结构域在 HAI 对丝氨酸蛋白酶的抑制活性中的作用，我们构建了各种 HAI 突变体并检查了它们对每个 HAI 分子中似乎是 HGF 激活剂的功能结构域的抑制活性。抑制 HGF 激活剂的 HGF 转化活性此外，两个 Kunitz 结构域的存在影响了 HAI-1 对 HGF 激活剂的抑制活性。 Kunitz I 和 Kunitz II 的丝氨酸蛋白酶结合位点彼此靠近，并且仅具有一个 Kunitz 结构域的蛋白水解加工产生 HAI-1 调节 HAI-1 的活性，针对 Kunitz II 的同源丝氨酸蛋白酶的鉴定将揭示HAIs的新生物学功能。3.为了寻找HAI分泌酶，我们使用酵母双杂交体检查了HAIs细胞质区域中的蛋白质-蛋白质相互作用。获得了几种细胞质蛋白，包括细胞质蛋白和细胞内酶，其中一种已知参与细胞凋亡的抑制，表明HAI可能在死亡信号的调节中发挥重要作用（手稿正在准备中）。对 HAI 蛋白水解过程中功能调控的研究将为 HGF 激活调控提供新的机制。