利用蛋白體學方法鑑定胃上皮細胞中磷酸化缬絡胺酸蛋白質的交互作用分子及其防止幽門螺旋桿菌誘發細胞凋亡之特性

Abstract

過去的研究結果已顯示valosin-containing protein (VCP) 和感染幽門螺旋桿菌所引起的胃癌有關。然而VCP與胃癌發展之間的關係尚未完全研究清楚，因此本論文的研究目的，是希望利用消去(subtractive )蛋白體學技術，先讓AGS 胃腺癌細胞中大量表達VCP蛋白質，接著利用免疫沉澱(immunoprecipitation)方法，得到細胞中與VCP交互作用的分子，然後進一步探討VCP其調控及胃癌發展的機制。首先，我們將免疫沉澱下來的VCP蛋白質複合體(complex)產物，經過一維電泳的展開，接著再利用液相層析偶合串聯式質譜儀(LC-MS/MS)進行分析，結果得到288個和VCP交互作用的蛋白，然後再藉由Ingenuity Pathway Analysis (IPA)軟體分析之後，得到18個和VCP交互作用的蛋白，主要都參與Akt訊息傳遞網路的的調控，於是我們利用免疫沉澱的方式，證明VCP和Akt之間的交互作用，除此之外，我們也觀察到當AGS細胞感染幽門螺旋桿菌時，會活化Akt激酶，進而促使VCP的磷酸化，同時我們也進一步分析發現， VCP的磷酸化會促使蛋白的泛素化和聚集體(aggresome)的形成，然後使一些細胞凋亡的蛋白的降解(degradation)，進而促進細胞生存，藉由這些結果，我們瞭解到VCP的磷酸化在幽門螺旋桿菌感染胃上皮細胞機制中扮演重要的角色，同時讓我們將近一步釐清VCP的磷酸化在細胞中所參予的功能，也提供一個關於幽門螺旋桿菌感染胃上皮細胞新的致病機制。

Previous studies have demonstrated that valosin-containing protein (VCP) is associated with H. pylori-induced gastric carcinogenesis. By identifying the interactome of VCP overexpressed in AGS cells using a subtractive proteomics approach, we aimed to characterize the cellular responses mediated by VCP and its functional roles in H. pylori-associated gastric cancer. VCP immunoprecipitations followed by proteomic analysis identified 288 putative interacting proteins, 18 VCP-binding proteins belonged to the PI3K/Akt signaling pathway. H. pylori infection increased the interaction between Akt and VCP, Akt-dependent phosphorylation of VCP, levels of ubiquitinated proteins, and aggresome formation in AGS cells. Furthermore, phosphorylated VCP co-localized with the aggresome, bound ubiquitinated proteins, and increased the degradation of cellular regulators to protect H. pylori-infected AGS cells from apoptosis. Our study demonstrates that VCP phosphorylation following H. pylori infection promotes both gastric epithelial cell survival, mediated by the PI3K/Akt pathway, and the degradation of cellular regulators. These findings provide novel insights into the mechanisms of H. pylori infection induced gastric carcinogenesis.