Interrogating Endogenous Protein Phosphatase Activity with Rationally Designed Chemosensors
Interrogating Endogenous Protein Phosphatase Activity with Rationally Designed Chemosensors
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DOI：
10.1021/acschembio.5b00506
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发表时间：
2016-01-01
影响因子：
4
通讯作者：
Stains, Cliff I.
中科院分区：
文献类型：
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作者：
Beck, Jon R.;Lawrence, Antoneal;Stains, Cliff I.
我们介绍了一种用于重新利用蛋白激酶化学传感器的通用方法，该传感器包含称为SOX的磷酸化敏感的磺胺氧基荧光团，用于从全细胞裂解物和组织匀浆中特异性地测定内源性蛋白磷酸酶的活性。作为这一方法的演示，我们设计并评估了一个直接检测蛋白质酪氨酸磷酸酶-1B(PTP1B)的化学传感器，它是人类疾病中已建立的信号转导节点。优化的传感器设计能够检测到低至6 PM(12pg)的全长重组PTP1B，并且在一组高度同源的酪氨酸磷酸酶中对PTP1B具有显著的选择性。将这种强大的活性探针与抗体的特异性结合起来，可以对胰岛素刺激后HepG2细胞产生的内源性PTP1B活性动态进行时间分析。最后，我们利用这一分析格式来描述非酒精性脂肪性肝病(NAFLD)大鼠模型中PTP1B活性的扰动，为这种疾病状态下PTP1B催化活性的升高提供了直接证据。鉴于这项检测的模块化性质，我们预计这一方法将在监测人类疾病状态下的磷酸酶活性动态方面具有广泛的实用价值。
We introduce a versatile approach for repurposing protein kinase chemosensors, containing the phosphorylation-sensitive sulfonamido-oxine fluorophore termed Sox, for the specific determination of endogenous protein phosphatase activity from whole cell lysates and tissue homogenates. As a demonstration of this approach, we design and evaluate a direct chemosensor for protein tyrosine phosphatase-1B (PTP1B), an established signaling node in human disease. The optimal sensor design is capable of detecting as little as 6 pM (12 pg) full-length recombinant PTP1B and is remarkably selective for PTP1B among a panel of highly homologous tyrosine phosphatases. Coupling this robust activity probe with the specificity of antibodies allowed for the temporal analysis of endogenous PTP1B activity dynamics in lysates generated from HepG2 cells after stimulation with insulin. Lastly, we leveraged this assay format to profile PTP1B activity perturbations in a rat model of nonalcoholic fatty liver disease (NAFLD), providing direct evidence for elevated PTP1B catalytic activity in this disease state. Given the modular nature of this assay, we anticipate that this approach will have broad utility in monitoring phosphatase activity dynamics in human disease states.