Proteomic amalysis in Angiotensin signaling

细胞内信号传递构成一个复杂的网络，受特定的蛋白质-蛋白质相互作用调节。因此，识别蛋白质复合体是了解细胞功能的关键。血管紧张素信号的串扰在一定程度上是通过激活Akt来调节的。Akt驱动的信号网络能够通过调节转录和翻译以及在翻译后水平上修改蛋白质来调节细胞过程。为了阐明血管紧张素信号的串扰，我们寻找了Akt1的结合分子。在此，我们尝试进行亲和纯化和蛋白质组学分析，以阐明Akt在血管紧张素II信号转导中的作用。我们探索了使用Strep-Tag方法从rAEC中分离Akt1复合体。用腺病毒载体将STEP-Akt1基因导入rAECs，用血管紧张素受体阻滞剂AngiensinII和ACE抑制剂刺激rAECs 10~30min。然后，表达带有STEP标记的Akt1的裂解物通过Strep-Tactin柱，使用去硫代生物素特异性地洗脱结合的蛋白。用变性凝胶电泳法分离纯化的蛋白质组分，胰酶消化，基质辅助激光解吸/电离飞行时间质谱仪(MALDI-TOF MS)分析，数据库搜索算法鉴定。结果，至少有50种互动伙伴被系统提纯和鉴定。其中一些蛋白质的表达水平随刺激而改变。这些包括酶、蛋白激酶、翻译调节因子、细胞骨架蛋白和假想蛋白。这些结果表明，血管紧张素信号对血管内皮细胞功能的许多生物分子反应和生理后果有影响。该方法学将为化学蛋白质组学在医学中的应用提供一种新的工具。

Intracellular signaling constitutes a complicated network, which are regulated by specific protein-protein interactions. Therefore identification of protein complexes is the key to understanding cellular functions. The crosstalk of Angiotensin signaling is mediated, in part, through the activation of Akt. The Akt-driven signaling network is capable of regulating cellular processes by modulating transcription and translation, and by modifying proteins at the posttranslational level. To elucidate the crosstalk of Angiotensin signaling, we searched for the binding molecule of Akt1. Here, we made attempts to perform affinity purification and proteomic analysis to elucidate the role of Akt in AngiotensinII signaling.We explored the use of strep-tag method for the isolation of Akt1 complexes from rAECs. The strep-Akt1 introduced into the rAECs by adenoviral vectors, and the cells were stimulated by AnguitensinII, Angiotensin receptor blocker, and ACE inhibitor for 10-30 min. Lysates expressing the strep-tagged Akt1 were then passed through a strep-Tactin column and bound proteins were specifically eluted using a desthiobiotin. The purified protein assemblies were separated by denaturing gel electrophoresis, and individual bands were digested by trypsin, analyzed by matrix-assisted laser desorption/vionization-time-of-flight mass spectrometry (MALDI-TOF MS) and identified by database search algorithms. As a result, at least 50 kinds of interaction partners were systematically purified and identified. Some of these proteins were changed the expresion level depend on stimulation. These included enzymes, protein kinases, translational regulators, cytoskeletal proteins, and hypothetical proteins. These results suggest that Angiotensin signaling has effects on the many biomolecular response and physiological consequence of vascular endothelium functions. This methodology would provide a new tool for chemical proteomics in medicinal science.