幽門螺旋桿菌感染之胃上皮細胞引起缬絡胺酸蛋白質的磷酸化及聚集體之形成並促進抗細胞凋亡之產生

Abstract

幽門螺旋桿菌係是導致胃癌之重要因子，雖然因幽門螺旋桿菌所導致胃癌的關聯性早已建立，但由幽門螺旋桿菌所造成胃癌的分子機制尚未明確。先前，本實驗室利用免疫沉澱及蛋白質體學的方式，發現當幽門螺旋桿菌感染胃腺癌上皮細胞(AGS)時，有許多與癌症相關的蛋白質表現量亦為提高。其中以缬絡胺酸蛋白質(Valosin-containing protein)的表現差異特別顯著，此結果與病理組織切片染色觀察到缬絡胺酸蛋白質表現量的上升亦有符合。本實驗目的是探討當幽門螺旋桿菌感染胃腺癌上皮細胞時，缬絡胺酸蛋白質在其中所扮演的角色，並且分析這些與缬絡胺酸蛋白質有交互作用的蛋白質。在當幽門螺旋桿菌感染細胞後，經免疫沉澱及蛋白質體學的實驗下，我們鑑定到了176個與缬絡胺酸蛋白質有交互作用的蛋白，我們併用生物資訊軟體(IPA)分析。分析結果顯示，與缬絡胺酸蛋白質有交互作用的蛋白建構成一個網路，這個網絡與細胞存活、細胞增生、癌症生成，等生理作用有關。更進一步分析，此網絡與絲氨酸/蘇氨酸激酶(AKT singaling)信息傳遞，有高度的關聯。我們更進一步在實驗中發現當幽門螺旋桿菌感染胃腺癌上皮細胞時，絲氨酸/蘇氨酸激酶會被磷酸化，活化後的絲氨酸/蘇氨酸激酶會磷酸化缬絡胺酸蛋白質，缬絡胺酸蛋白質的磷酸化，會促使促細胞凋亡分因子、細胞週期抑制蛋白的降解。藉由共軛焦螢光顯微鏡的觀察，幽門螺旋桿菌感染胃腺癌上皮細胞時，經泛素修飾蛋白質與缬絡胺酸蛋白質形成聚集體，此行為促使凋亡因子及細胞週期抑制蛋白的降解，進而造成細胞的存活及癌化。

Helicobacter pylori is an important risk factor of gastric cancer. Although many H. pylori virulence factors have been reported, the pathogenic mechanism by which H. pylori infection causes gastric cancer remains unclear. Previously, by using proteomics approaches and immunohistochemistry, we found that the protein expression level of many cancer-related factors were elevated in AGS cells after H. pylori infection, and one important factor was valosin-containing protein (VCP). By identification the interactome of over-expressed VCP in AGS cells through a proteomics approach, we aimed to characterize the cellular responses mediated by VCP and its functional roles in H. pylori-associated gastric cancer. 176 interacting proteins were identified through via approach, and with the assistance of IPA analysis to search for significantly enriched signaling pathways, we focused on proteins involved in cell survival, proliferation and cancer development among these candidates. We found AKT as a key interacting protein of VCP in H. pylori-induced gastric cancer. We observed that H. pylori infection enhances the interaction between AKT and VCP, and also an AKT-dependent phosphorylation of VCP in AGS cells. Interestingly, the AKT-dependent VCP phosphorylation protects AGS cells from apoptosis, which is governed by phosphotidylinositol 3-kinase (PI3K) and also degradation of apoptotic factor or cell cycle inhibitor. Confocal microscopy indicated a co-localization between VCP and ubiquitinated cell regulators upon AKT stimulation. In addition, our current data indicated that VCP may involve in aggresome formation, possibly for their ubiquitinated protein degradation. Together, these data identified VCP as an important target in the AKT-mediated signaling of cell survival.