Engineering Single Pan-Specific Ubiquibodies for Targeted Degradation of All Forms of Endogenous ERK Protein Kinase,ACS Synthetic Biology

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Engineering Single Pan-Specific Ubiquibodies for Targeted Degradation of All Forms of Endogenous ERK Protein Kinase
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2021-08-16 , DOI: 10.1021/acssynbio.1c00357
Erin A Stephens ₁ , Morgan B Ludwicki ₂ , Bunyarit Meksiriporn ₃ , Mingji Li ₂ , Tianzheng Ye ₂ , Connor Monticello ₃ , Katherine J Forsythe ₄ , Lutz Kummer ₅ , Pengbo Zhou ₆ , Andreas Plückthun ₅ , Matthew P DeLisa _{1,

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Affiliation

Ubiquibodies (uAbs) are a customizable proteome editing technology that utilizes E3 ubiquitin ligases genetically fused to synthetic binding proteins to steer otherwise stable proteins of interest (POIs) to the 26S proteasome for degradation. The ability of engineered uAbs to accelerate the turnover of exogenous or endogenous POIs in a post-translational manner offers a simple yet robust tool for dissecting diverse functional properties of cellular proteins as well as for expanding the druggable proteome to include tumorigenic protein families that have yet-to-be successfully drugged by conventional inhibitors. Here, we describe the engineering of uAbs composed of human carboxyl-terminus of Hsc70-interacting protein (CHIP), a highly modular human E3 ubiquitin ligase, tethered to differently designed ankyrin repeat proteins (DARPins) that bind to nonphosphorylated (inactive) and/or doubly phosphorylated (active) forms of extracellular signal-regulated kinase 1 and 2 (ERK1/2). Two of the resulting uAbs were found to be global ERK degraders, pan-specifically capturing all endogenous ERK1/2 protein forms and redirecting them to the proteasome for degradation in different cell lines, including MCF7 breast cancer cells. Taken together, these results demonstrate how the substrate specificity of an E3 ubiquitin ligase can be reprogrammed to generate designer uAbs against difficult-to-drug targets, enabling a modular platform for remodeling the mammalian proteome.

中文翻译：

工程单一泛特异性泛在体用于靶向降解所有形式的内源 ERK 蛋白激酶

Ubiquibodies (uAbs) 是一种可定制的蛋白质组编辑技术，利用与合成结合蛋白基因融合的 E3 泛素连接酶，将稳定的目标蛋白 (POI) 引导至 26S 蛋白酶体进行降解。工程化 uAb 以翻译后方式加速外源或内源 POI 周转的能力，为剖析细胞蛋白的多种功能特性以及扩展可成药蛋白质组以包括尚未发现的致瘤蛋白家族提供了一种简单而强大的工具。 -通过常规抑制剂成功用药。在这里，我们描述了由 Hsc70 相互作用蛋白 (CHIP) 的人羧基末端组成的 uAb 工程，CHIP 是一种高度模块化的人 E3 泛素连接酶，与不同设计的锚蛋白重复蛋白 (DARPins) 结合，与非磷酸化（无活性）和/或双磷酸化（活性）形式的细胞外信号调节激酶 1 和 2 (ERK1/2)。结果发现，其中两种 uAb 是全局 ERK 降解剂，泛特异性捕获所有内源性 ERK1/2 蛋白形式，并将它们重定向至蛋白酶体，以便在不同细胞系（包括 MCF7 乳腺癌细胞）中降解。总而言之，这些结果证明了如何对 E3 泛素连接酶的底物特异性进行重新编程，以生成针对难以药物靶标的设计 uAb，从而实现用于重塑哺乳动物蛋白质组的模块化平台。

更新日期：2021-09-17

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