Otaka Michiro, Odashima Masaru, Izumi Yuko, Nagahara Akihito, Osada Taro, Sakamoto Naoto, Takada Makiko, Takahashi Taiji, Shimada Yuji, Tamaki Kumiko, Asaoka Daisuke, Itoh Hideaki, Watanabe Sumio
Department of Gastroenterology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-Ku, Tokyo, Japan.
Life Sci. 2009 May 8;84(19-20):664-7. doi: 10.1016/j.lfs.2009.02.009. Epub 2009 Mar 1.
Several recent studies, including ours, have indicated the importance of heat shock proteins (HSPs) in cytoprotection against cytotoxic agents and environmental stresses mediated by the chaperone function of HSPs (molecular chaperones). However, the target molecule that is recognized by HSPs in damaged cells currently remains unknown. As HSPs rapidly recognize and bind to degenerated protein in cells, target molecules of HSPs might be key molecules for the initiation and pathogenesis of cellular damage. In the present study, gastric mucosal proteins that specifically bind to the HSP70 family (HSC70) were analyzed using HSC70-affinity chromatography.
The gastric mucosa was removed from Sprague-Dawley rats after exposure to water immersion-stress for 0, 1, 3 or 5 h. Soluble fractions of each gastric mucosa were applied to the HSC70-affinity column separately. After washing off non-specific binding proteins, specific binding proteins were eluted by ATP-containing buffer. Binding proteins were analyzed by SDS-polyacrylamide gel electrophoresis. In addition, the amino acid sequence of purified proteins was also analyzed.
Specific HSC70-binding proteins with a molecular weight of 200-kDa and 45-kDa were eluted from an affinity column when gastric mucosal homogenate of 1-h stress exposure was applied. The amino acid sequencing showed that these binding proteins were cytoskeletal myosin (heavy chain) and actin, respectively.
During the pathogenesis of stress-induced gastric mucosal damage, structurally degenerated cytoskeletal myosin (heavy chain) and actin may be key or initiation molecules which structural changes were firstly recognized by molecular chaperone.
包括我们的研究在内，最近的几项研究表明热休克蛋白（HSPs）在细胞保护中对于抵抗细胞毒性剂和由HSPs的伴侣功能（分子伴侣）介导的环境应激具有重要作用。然而，目前受损细胞中被HSPs识别的靶分子仍然未知。由于HSPs能迅速识别并结合细胞内变性蛋白，HSPs的靶分子可能是细胞损伤起始和发病机制中的关键分子。在本研究中，使用HSC70亲和层析分析了与HSP70家族（HSC70）特异性结合的胃黏膜蛋白。
将Sprague-Dawley大鼠暴露于水浸应激0、1、3或5小时后，取出胃黏膜。将每个胃黏膜的可溶性部分分别应用于HSC70亲和柱。洗去非特异性结合蛋白后，用含ATP的缓冲液洗脱特异性结合蛋白。通过SDS-聚丙烯酰胺凝胶电泳分析结合蛋白。此外，还分析了纯化蛋白的氨基酸序列。
当应用1小时应激暴露的胃黏膜匀浆时，从亲和柱上洗脱得到分子量为200 kDa和45 kDa的特异性HSC70结合蛋白。氨基酸测序表明，这些结合蛋白分别是细胞骨架肌球蛋白（重链）和肌动蛋白。
在应激诱导的胃黏膜损伤发病过程中，结构退化的细胞骨架肌球蛋白（重链）和肌动蛋白可能是首先被分子伴侣识别其结构变化的关键或起始分子。
