Identification of intracellular proteins and signaling pathways in human endothelial cells regulated by angiotensin-(1-7).
Identification of intracellular proteins and signaling pathways in human endothelial cells regulated by angiotensin-(1-7).
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DOI：
10.1016/j.jprot.2015.09.020
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发表时间：
2016-01-01
影响因子：
3.3
通讯作者：
Walther T
中科院分区：
文献类型：
--
作者：
Meinert C;Gembardt F;Böhme I;Tetzner A;Wieland T;Greenberg B;Walther T
该研究旨在鉴定由心血管保护肽血管紧张素-（1-7）调节的蛋白质，并确定潜在的细胞内信号级联。用Ang-（1-7）刺激人内皮细胞1h、3 h、6 h和9 h。通过抗体微阵列评估肽对细胞内信号传导的影响，所述抗体微阵列含有针对725种蛋白质的抗体。使用生物信息学软件来鉴定受影响的细胞内信号传导途径。通过Western印迹、实时RT-PCR和免疫组织化学研究示例性地验证微阵列数据。微阵列鉴定了Ang-（1-7）刺激1h后110个、3 h后119个、6 h后31个和9 h后86个调节蛋白。受调控蛋白质以时间依赖性方式与“癌症分子机制”和“p53信号传导”等几种代谢途径高度相关。例如，E3型小泛素样修饰连接酶PIAS 2的蛋白质印迹证实了微阵列数据，并显示在Ang-（1-7）刺激后1小时和3小时降低超过50%，而不影响其mRNA。人内皮细胞PIAS 2的免疫组化研究显示，Ang-（1-7）后细胞质PIAS 2减少。Ang-（1-7）介导的PIAS 2的降低在其他类型的内皮细胞中重现。结果表明，血管紧张素-（1-7）在人内皮细胞中与细胞死亡和细胞存活相关的代谢途径中起作用。
The study aimed to identify proteins regulated by the cardiovascular protective peptide angiotensin-(1-7) and to determine potential intracellular signaling cascades. Human endothelial cells were stimulated with Ang-(1-7) for 1h, 3h, 6h, and 9h. Peptide effects on intracellular signaling were assessed via antibody microarray, containing antibodies against 725 proteins. Bioinformatics software was used to identify affected intracellular signaling pathways. Microarray data was verified exemplarily by Western blot, Real-Time RT-PCR, and immunohistochemical studies. The microarray identified 110 regulated proteins after 1h, 119 after 3h, 31 after 6h, and 86 after 9h Ang-(1-7) stimulation. Regulated proteins were associated with high significance to several metabolic pathways like “Molecular Mechanism of Cancer” and “p53 signaling” in a time dependent manner. Exemplarily, Western blots for the E3-type small ubiquitin-like modifier ligase PIAS2 confirmed the microarray data and displayed a decrease by more than 50% after Ang-(1-7) stimulation at 1h and 3h without affecting its mRNA. Immunohistochemical studies with PIAS2 in human endothelial cells showed a decrease in cytoplasmic PIAS2 after Ang-(1-7). The Ang-(1-7) mediated decrease of PIAS2 was reproduced in other endothelial cell types. The results suggest that angiotensin-(1-7) plays a role in metabolic pathways related to cell death and cell survival in human endothelial cells.