Designing protease sensors for real-time imaging of trypsin activation in pancreatic cancer cells.
Designing protease sensors for real-time imaging of trypsin activation in pancreatic cancer cells.
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DOI：
10.1021/bi802289v
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发表时间：
2009-04-21
期刊：
影响因子：
2.9
通讯作者：
Yang, Jenny J.
中科院分区：
文献类型：
--
作者：
Chen, Ning;Zou, Jin;Wang, Siming;Ye, Yiming;Huang, Yun;Gadda, Giovanni;Yang, Jenny J.
急性胰腺炎是由细胞内胰蛋白酶原激活引起的一种严重的、可能致命的疾病。尽管能够监测蛋白酶激活和抑制的蛋白酶探针的发展极大地促进了蛋白酶检测，但活细胞中蛋白酶活性的实时定量测量仍然是一个挑战，胰蛋白酶原激活的细胞区室的鉴定尚无结论。在这里，我们报告了一种通过将酶裂解位点嫁接到单个增强型绿色荧光蛋白（EGFP）的发色团光学变化的敏感位置来开发胰蛋白酶传感器的新策略。我们设计的胰蛋白酶传感器对蛋白酶的激活和抑制具有快速的动力学响应，具有较大的比例光信号变化。此外，它具有很强的特异性，因为在凝血酶、组织蛋白酶B、胰蛋白酶和组织纤溶酶原激活剂等其他蛋白酶中没有观察到酶促分裂。此外，开发的胰蛋白酶传感器使我们第一次能够实时观察到在没有酶原颗粒的胰腺癌细胞系MIA PaCa-2中，小蛋白对胰蛋白酶原的激活。这些开发的蛋白酶传感器将有助于提高对蛋白酶激活机制和位置的理解，并进一步提供具有治疗作用的蛋白酶抑制剂的筛选。
Acute pancreatitis is a serious and potentially fatal disease caused by intracellular trypsinogen activation. Although protease detection has been greatly facilitated by the development of protease probes capable of monitoring protease activation and inhibition, real-time quantitative measurement of protease activity in living cells remains a challenge, and the identification of the cellular compartment for trypsinogen activation is inconclusive. Here we report a novel strategy for developing trypsin sensors by grafting an enzymatic cleavage site into a sensitive location for optical change of chromophore in a single enhanced green fluorescent protein (EGFP). Our designed trypsin sensor exhibits rapid kinetic responses for protease activation and inhibition with a large ratiometric optical signal change. In addition, it has strong specificity, as enzymatic cleavage is not observed with other proteases such as thrombin, cathepsin B, tryptase, and tissue plasminogen activator. Moreover, the developed trypsin sensor allows us for the first time to observe, in real time, trypsinogen activation by caerulein in the pancreatic cancer cell line, MIA PaCa-2 without zymogen granules. These developed protease sensors will facilitate improved understanding of mechanisms and locations of protease activation and further provide screening of protease inhibitors with therapeutic effects.
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DOI：
10.1073/pnas.97.24.13126
发表时间：
2000-11-21
影响因子：
11.1
作者：
Raraty, M;Ward, J;Petersen, OH
通讯作者：
Petersen, OH
影响因子：
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作者：
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通讯作者：
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影响因子：
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作者：
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通讯作者：
Deperthes, D
影响因子：
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作者：
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Zoli, Wainer
影响因子：
4.1
作者：
Zou, J;Ye, YM;Yang, JJ
通讯作者：
Yang, JJ