Proteomic amalysis in Angiotensin signaling

血管紧张素信号传导的蛋白质组学分析

• 批准号: 17390068
• 负责人: IWAO Hiroshi
• 金额: $ 10.09万
• 依托单位: Osaka City University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-17390068/
• 关键词: Angiotensin; network analysis; proteome; Akt; シグナル伝達; 循環器・高血圧; アンジオテンシンII; 薬理学; シグナル; 血管; プロテオーム解析; TAPタグ

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.

细胞内信号转导是一个复杂的网络，受特定的蛋白质间相互作用的调控。因此，蛋白质复合物的鉴定是理解细胞功能的关键。血管紧张素信号传导的串扰部分地通过Akt的激活来介导。Akt驱动的信号网络能够通过调节转录和翻译以及通过在翻译后水平修饰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Akt 1的结合分子。本研究尝试通过亲和纯化和蛋白质组学分析来阐明Akt在血管紧张素II信号转导中的作用，并探索了利用strep-tag法从rAECs中分离Akt 1复合物的方法。通过腺病毒载体将strep-Akt 1导入rAEC，并通过血管紧张素II、血管紧张素受体阻断剂和ACE抑制剂刺激细胞10-30 min。然后将表达strep-taged Akt 1的裂解物通过strep-Tactin柱，并使用脱硫生物素特异性洗脱结合的蛋白质。纯化的蛋白质组装体通过变性凝胶电泳分离，并且单个条带通过胰蛋白酶消化，通过基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）分析，并通过数据库搜索算法鉴定。结果，至少有50种相互作用伙伴进行了系统的纯化和鉴定。其中一些蛋白质的表达水平随着刺激的变化而变化。这些包括酶、蛋白激酶、翻译调节因子、细胞骨架蛋白和假设蛋白。这些结果表明，血管紧张素信号转导对血管内皮功能的许多生物分子反应和生理后果具有影响。该方法为药物化学蛋白质组学研究提供了新的工具。

项目成果

Coronary flow velocity reserve measurement in three major coronary arteries using transthoracic doppler echocardiography

• DOI: 10.1111/j.1540-8175.2006.00206.x
• 发表时间: 2006-04-01
• 期刊: ECHOCARDIOGRAPHY-A JOURNAL OF CARDIOVASCULAR ULTRASOUND AND ALLIED TECHNIQUES
• 影响因子: 1.5
• 作者: Murata, E;Hozumi, T;Yoshikawa, J
• 通讯作者: Yoshikawa, J

Critical role of angiotensin II in excess salt-induced brain oxidative stress of stroke-prone spontaneously hypertensive rats

• DOI: 10.1161/01.str.0000163084.16505.e3
• 发表时间: 2005-05-01
• 期刊: STROKE
• 影响因子: 8.3
• 作者: Kim-Mitsuyama, S;Yamamoto, E;Iwao, H
• 通讯作者: Iwao, H

Granulocyte-colony stimulating factor augments neovascularization induced by bone marrow transplantation in rat hindlimb ischemia

• DOI: 10.1254/jphs.fp0040966
• 发表时间: 2005-09-01
• 期刊: JOURNAL OF PHARMACOLOGICAL SCIENCES
• 影响因子: 3.5
• 作者: Takagi, Y;Omura, T;Yoshikawa, J
• 通讯作者: Yoshikawa, J

Pharmacogenomics of caediovascular pharmacology

心血管药理学的药物基因组学

• 发表时间: 2006
• 期刊: Nitiyakurishi 128
• 作者: Tanaka;T.;Oka;Y;Iwao;H
• 通讯作者: H

Angiotensin blockade inhibits osteoponin expression in noninfarcted myocardium after myocardial infarction

血管紧张素阻滞抑制心肌梗死后非梗死心肌中骨钙素的表达

• 发表时间: 2005
• 期刊: J Pharmacol Sci 98
• 作者: Kusuyama;T.;Yoshiyama;M.;Omura;T.;Nishiya;D.;Enomoto;S.;Matsumoto;R.;Izumi;Y.;Akioka;K.;Takeuchi;K.;Iwao;H.;Yoshikawa;J.
• 通讯作者: J.