Modular Peroxidase-Based Reporters for Detecting Protease Activity and Protein Interactions with Temporal Gating.
Modular Peroxidase-Based Reporters for Detecting Protease Activity and Protein Interactions with Temporal Gating.
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DOI：
10.1021/jacs.2c08280
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发表时间：
2022-12-21
影响因子：
15
通讯作者：
Wang, Wenjing
中科院分区：
文献类型：
--
作者：
Zhou, Guanwei;Wan, Wei Wei;Wang, Wenjing
酶报告基因可以通过酶促反应放大信号并提供良好的灵敏度，因此被广泛应用于各种生物过程的研究。然而，仍然需要用于设计一系列酶报告基因的模块基序。在这里，我们报告了一个模块化的过氧化物酶为基础的基序，命名为CLAPon，具有酸碱线圈笼增强抗坏血酸过氧化物酶（APEX）。我们通过设计一系列用于检测蛋白酶活性和蛋白质-蛋白质相互作用（PPI）的报告分子来证明CLAPon的模块性。用于蛋白酶活性的CLAPon显示在烟草蚀纹病毒蛋白酶切割后荧光信号增加390倍。用于PPI检测的CLAPon（PPI-CLAPon）有两个变体，PPI-CLAPon 1.0和1. 1。PPI-CLAPon 1.0显示高亲和力PPI对的信噪比（SNR）高达107，并实现亚细胞空间分辨率成像。然而，需要具有更高灵敏度的PPI-CLAPon 1.1来检测低亲和力PPI对。PPI-CLAPon 1.0被进一步改造为具有光依赖性时间门控的报告子，称为LiPPI-CLAPon 1.0，其可以检测SNR为17的3分钟钙依赖性PPI。LiPPI-CLAPon可在特定时间窗口内进行PPI检测，具有快速APEX激活和多样化读数。最后，PPI-CLAPon 1.0被设计为具有化学门控，提供更多的通用性以补充LiPPI-CLAPon。这些基于CLAPon的报告基因设计可以广泛应用于研究涉及蛋白酶活性和PPI的各种信号传导过程，并提供了一个通用的平台来设计各种遗传编码的报告基因。
Enzymatic reporters have been widely applied to study various biological processes because they can amplify signal through enzymatic reactions and provide good sensitivity. However, there is still a need for modular motifs for designing a series of enzymatic reporters. Here, we report a modular peroxidase-based motif, named CLAPon, that features acid-base coils-caged enhanced ascorbate peroxidase (APEX). We demonstrate the modularity of CLAPon by designing a series of reporters for detecting protease activity and protein-protein interactions (PPIs). CLAPon for protease activity showed a 390-fold fluorescent signal increase upon tobacco etch virus protease cleavage. CLAPon for PPI detection (PPI-CLAPon) has two variants, PPI-CLAPon1.0 and 1.1. PPI-CLAPon1.0 showed a signal to noise ratio (SNR) of up to 107 for high-affinity PPI pairs and enabled imaging with sub-cellular spatial resolution. However, PPI-CLAPon1.1 with higher sensitivity is required for detecting low-affinity PPI pairs. PPI-CLAPon1.0 was further engineered to a reporter with light-dependent temporal gating, called LiPPI-CLAPon1.0, which can detect a 3-minute calcium-dependent PPI with a SNR of 17. LiPPI-CLAPon enables PPI detection within a specific time window with rapid APEX activation and diverse readout. Lastly, PPI-CLAPon1.0 was designed to have chemical gating, providing more versatility to complement the LiPPI-CLAPon. These CLAPon-based reporter designs can be broadly applied to study various signaling processes that involve protease activity and PPIs and provide a versatile platform to design various genetically encoded reporters.
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DOI：
10.1073/pnas.0710487105
发表时间：
2008-01-08
影响因子：
11.1
作者：
Barnea, Gilad;Strapps, Walter;Lee, Kevin J.
通讯作者：
Lee, Kevin J.
影响因子：
4
作者：
Kroning, Kayla E.;Li, Mingcheng;Shen, Jiaqi;Fiel, Hailey;Nassar, Manon;Wang, Wenjing
通讯作者：
Wang, Wenjing
影响因子：
7.7
作者：
Livingston KE;Mahoney JP;Manglik A;Sunahara RK;Traynor JR
通讯作者：
Traynor JR
影响因子：
4.6
作者：
Cabantous, Stephanie;Nguyen, Hau B.;Pedelacq, Jean-Denis;Koraichi, Faten;Chaudhary, Anu;Ganguly, Kumkum;Lockard, Meghan A.;Favre, Gilles;Terwilliger, Thomas C.;Waldo, Geoffrey S.
通讯作者：
Waldo, Geoffrey S.
影响因子：
4.8
作者：
GUREVICH, VV;DION, SB;BENOVIC, JL
通讯作者：
BENOVIC, JL