A comprehensive panel of turn-on caspase biosensors for investigating caspase specificity and caspase activation pathways.
A comprehensive panel of turn-on caspase biosensors for investigating caspase specificity and caspase activation pathways.
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用于研究 caspase 特异性和 caspase 激活途径的打开 caspase 生物传感器的综合面板。
DOI：
10.1002/cbic.201100372
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发表时间：
2011
期刊：
影响因子：
--
通讯作者：
Ghosh,Indraneel
中科院分区：
文献类型：
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作者：
Shekhawat,SujanS;Campbell,SeanT;Ghosh,Indraneel
半胱天冬酶在细胞凋亡、分化和增殖中起核心作用，并且代表用于治疗癌症和炎性病症的重要治疗靶标。为了开发新的工具来探测半胱天冬酶底物切割特异性以及系统地询问半胱天冬酶激活途径，我们构建并研究了一系列具有分裂荧光素酶激活的生物发光读出的半胱天冬酶生物传感器。我们首先询问了在广泛使用的体外翻译系统，即，兔网织红细胞裂解物（RRL）和小麦胚芽提取物（WGE）中的半胱天冬酶1-10的底物切割特异性的半胱天冬酶生物传感器的面板。商业RRL被发现是不适合调查半胱天冬酶特异性，由于内源性半胱天冬酶活性的惊人水平，而特异性概况的半胱天冬酶传感器在WGE同意非常好与传统的肽探针。利用全组生物传感器研究几种哺乳动物胞质提取物中的半胱天冬酶激活和抑制，清楚地表明它们可用于直接监测半胱天冬酶原3/7的激活或抑制。此外，完整的半胱天冬酶生物传感器组还提供了对半胱天冬酶活化途径的新见解，其中我们意外地发现了半胱天冬酶4/5对半胱天冬酶原3/7的活化。
Caspases play a central role in apoptosis, differentiation, and proliferation, and represent important therapeutic targets for treating cancer and inflammatory disorders. Toward the goal of developing new tools to probe caspase substrate cleavage specificity as well as to systematically interrogate caspase activation pathways, we have constructed and investigated a comprehensive panel of caspase biosensors with a split‐luciferase enabled bioluminescent read out. We first interrogated the panel of caspase biosensors for substrate cleavage specificity of caspase 1–10 in widely utilized in vitro translation systems, namely, rabbit reticulocyte lysate (RRL) and wheat germ extract (WGE). Commercial RRL was found to be unsuitable for investigating caspase specificity, owing to surprising levels of endogenous caspase activity, while specificity profiles of the caspase sensors in WGE agree very well with traditional peptide probes. The full panel of biosensors was utilized for studying caspase activation and inhibition in several mammalian cytosolic extracts, clearly demonstrating that they can be utilized to directly monitor activation or inhibition of procaspase 3/7. Furthermore, the complete panel of caspase biosensors also provided new insights into caspase activation pathways wherein we surprisingly discovered the activation of procaspase 3/7 by caspase 4/5.