Department of Chemical and Biomolecular Engineering, University of Houston, Houston, Texas, USA.
Department of Electrical and Computer Engineering, University of Houston, Houston, Texas, USA.
Biotechnol Bioeng. 2022 Jan;119(1):199-210. doi: 10.1002/bit.27966. Epub 2021 Nov 3.
Ligand inducible proteins that enable precise and reversible control of nuclear translocation of passenger proteins have broad applications ranging from genetic studies in mammals to therapeutics that target diseases such as cancer and diabetes. One of the drawbacks of the current translocation systems is that the ligands used to control nuclear localization are either toxic or prone to crosstalk with endogenous protein cascades within live animals. We sought to take advantage of salicylic acid (SA), a small molecule that has been extensively used in humans. In plants, SA functions as a hormone that can mediate immunity and is sensed by the nonexpressor of pathogenesis-related (NPR) proteins. Although it is well recognized that nuclear translocation of NPR1 is essential to promoting immunity in plants, the exact subdomain of Arabidopsis thaliana NPR1 (AtNPR1) essential for SA-mediated nuclear translocation is controversial. Here, we utilized the fluorescent protein mCherry as the reporter to investigate the ability of SA to induce nuclear translocation of the full-length NPR1 protein or its C-terminal transactivation (TAD) domain using HEK293 cells as a heterologous system. HEK293 cells lack accessory plant proteins including NPR3/NPR4 and are thus ideally suited for studying the impact of SA-induced changes in NPR1. Our results obtained using a stable expression system show that the TAD of AtNPR1 is sufficient to enable the reversible SA-mediated nuclear translocation of mCherry. Our studies advance a basic understanding of nuclear translocation mediated by the TAD of AtNPR1 and uncover a biotechnological tool for SA-mediated nuclear localization.
配体诱导蛋白能够精确且可逆地控制过客蛋白的核转位，其应用范围广泛，从哺乳动物的基因研究到针对癌症和糖尿病等疾病的治疗方法。目前的转位系统的一个缺点是，用于控制核定位的配体要么有毒，要么容易与活体内的内源性蛋白级联发生串扰。我们试图利用水杨酸 (SA)，一种在人类中广泛使用的小分子。在植物中，SA 作为一种激素发挥作用，可以介导免疫，并被非致病性相关蛋白 (NPR) 蛋白感知。尽管人们已经认识到 NPR1 的核转位对于促进植物中的免疫至关重要，但 Arabidopsis thaliana NPR1（AtNPR1）的的确切亚结构域对于 SA 介导的核转位是有争议的。在这里，我们利用荧光蛋白 mCherry 作为报告蛋白，利用 HEK293 细胞作为异源系统，研究 SA 诱导全长 NPR1 蛋白或其 C 端转录激活（TAD）结构域核转位的能力。HEK293 细胞缺乏包括 NPR3/NPR4 在内的辅助植物蛋白，因此非常适合研究 SA 诱导的 NPR1 变化的影响。我们使用稳定表达系统获得的结果表明，AtNPR1 的 TAD 足以实现 mCherry 的可逆 SA 介导的核转位。我们的研究推进了对 AtNPR1 的 TAD 介导的核转位的基本理解，并揭示了一种用于 SA 介导的核定位的生物技术工具。
