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Targeting endogenous proteins for degradation through the affinity-directed protein missile system

机译:通过亲和力定向蛋白导弹系统靶向内源蛋白进行降解

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摘要

Targeted proteolysis of endogenous proteins is desirable as a research toolkit and in therapeutics. CRISPR/Cas9-mediated gene knockouts are irreversible and often not feasible for many genes. Similarly, RNA interference approaches necessitate prolonged treatments, can lead to incomplete knockdowns, and are often associated with off-target effects. Targeted proteolysis can overcome these limitations. In this report, we describe an Affinity-directed PROtein Missile (AdPROM) system that harbours the von Hippel-Lindau (VHL) protein, the substrate receptor of the Cullin2 (CUL2) E3 ligase complex, tethered to polypeptide binders that selectively bind and recruit endogenous target proteins to the CUL2-E3 ligase complex for ubiquitination and proteasomal degradation. By using synthetic monobodies that selectively bind the protein tyrosine phosphatase SHP2 and a camelid-derived VHH nanobody that selectively binds the human ASC protein, we demonstrate highly efficient AdPROM-mediated degradation of endogenous SHP2 and ASC in human cell lines. We show that AdPROM-mediated loss of SHP2 in cells impacts SHP2 biology. This study demonstrates for the first time that small polypeptide binders that selectively recognise endogenous target proteins can be exploited for AdPROM-mediated destruction of the target proteins.
机译:内源性蛋白质的靶向蛋白水解是理想的研究工具包和治疗方法。 CRISPR / Cas9介导的基因敲除是不可逆的,对于许多基因而言通常不可行。同样,RNA干扰方法需要延长治疗时间,可能导致基因敲除不完全,并且经常与脱靶作用相关。靶向蛋白水解可以克服这些限制。在本报告中,我们描述了一种亲和力导向的蛋白导弹(AdPROM)系统,该系统包含von Hippel-Lindau(VHL)蛋白,Cullin2(CUL2)E3连接酶复合物的底物受体,与选择性结合和募集多肽结合剂结合在一起CUL2-E3连接酶复合物的内源性靶蛋白,用于泛素化和蛋白酶体降解。通过使用选择性结合蛋白酪氨酸磷酸酶SHP2和选择性结合人ASC蛋白的骆驼来源的VHH纳米抗体的合成单体,我们证明了高效AdPROM介导的人细胞系中内源性SHP2和ASC的降解。我们显示,AdPROM介导的细胞中SHP2的损失影响SHP2生物学。这项研究首次证明,选择性识别内源性靶蛋白的小多肽结合剂可用于AdPROM介导的靶蛋白破坏。

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