Protein expression and degradation are fundamental to cell function and physiological status of organisms. Interfering with protein expression not only provides powerful strategies to analyze the function of proteins but also inspires effective treatment methods for diseases caused by protein dysfunction. Recently, harnessing the power of the ubiquitin-proteasome system for targeted protein degradation (TPD) has become the focus of researches. Over the past two decades, TPD technologies, such as E3 ligase modification, PROTACs, and the Trim-Away method, have successfully re-oriented the ubiquitin-proteasome pathway and thus degraded many pathogenic proteins and even "undruggable" targets. However, A low-cost, convenient, and modularized TPD method is currently not available. Herein, we proposed a synthetic biology TPD method, termed Predator, by integrating the classic function of E3 ligase Trim21 and the expression of a bifunctional fusion protein that links Trim21 and the target protein, which leads to the formation of a ternary complex inside mammalian cells and therefore induce the ubiquitination and subsequent proteasome-dependent degradation of the target protein. We first proved this concept by using nanobody and scFv as the targeting module for the Predator system to degrade free GFP and membrane protein ErbB3, respectively. Then, we give an example of how the engineered Predator system can be developed towards biomedical solutions in the context of diabetes mellitus. Ligands-receptor interaction and adenovirus-mediated gene delivery were introduced to the Predator system, and we found this bifunctional fusion protein, in which glucagon was selected to function as the targeting module, downregulated the endogenous glucagon receptor (GCGR) and attenuated glucagon-stimulated glucose production in primary hepatocytes. Although preliminarily, our results showed that this Predator system is a highly modularized and convenient TPD method with good potential for both fundamental researches and clinical usage.

中文翻译：

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捕食者：靶向蛋白质降解的新方法

蛋白质表达和降解是细胞功能和生物生理状态的基础。干扰蛋白质表达不仅提供了分析蛋白质功能的有力策略，而且激发了针对蛋白质功能障碍引起的疾病的有效治疗方法。近年来，利用泛素-蛋白酶体系统的功能进行靶向蛋白质降解（TPD）已成为研究的重点。在过去的二十年中，TPD技术（例如E3连接酶修饰，PROTAC和Trim-Away方法）已成功地重新定向了泛素-蛋白酶体途径，从而降解了许多致病蛋白，甚至降解了“不可负担的”靶标。但是，目前没有低成本，方便且模块化的TPD方法。在这里，我们提出了一种合成生物学TPD方法，通过整合E3连接酶Trim21的经典功能和连接Trim21与目标蛋白的双功能融合蛋白的表达，将其称为“捕食者”，从而导致哺乳动物细胞内部三元复合物的形成，从而诱导泛素化和随后的蛋白酶体依赖性靶蛋白的降解。我们首先通过使用纳米抗体和scFv作为Predator系统的靶向模块分别降解游离GFP和膜蛋白ErbB3来证明了这一概念。然后，我们给出一个示例，说明如何在糖尿病的情况下将工程化的Predator系统开发为生物医学解决方案。配体-受体相互作用和腺病毒介导的基因传递被引入到Predator系统中，我们发现了这种双功能融合蛋白，其中选择了胰高血糖素作为靶向模块，下调了内源性胰高血糖素受体（GCGR）并减弱了胰高血糖素刺激的原代肝细胞中的葡萄糖生成。尽管初步，但我们的结果表明，该Predator系统是高度模块化且方便的TPD方法，对于基础研究和临床应用均具有良好的潜力。