Targeting endogenous proteins for degradation through the affinity-directed protein missile system.
Targeting endogenous proteins for degradation through the affinity-directed protein missile system.
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DOI：
10.1098/rsob.170066
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发表时间：
2017-05
期刊：
影响因子：
5.8
通讯作者：
Sapkota GP
中科院分区：
文献类型：
--
作者：
Fulcher LJ;Hutchinson LD;Macartney TJ;Turnbull C;Sapkota GP
内源性蛋白质的靶向蛋白水解作为研究工具包和治疗剂是理想的。CRISPR/Cas9介导的基因敲除是不可逆的，并且通常对许多基因不可行。类似地，RNA干扰方法需要延长治疗时间，可能导致不完全敲除，并且通常与脱靶效应相关。靶向蛋白水解可以克服这些限制。在这份报告中，我们描述了一个亲和导向的蛋白导弹（AdPROM）系统，窝藏冯希佩尔-林道（VHL）蛋白，底物受体的Cullin 2（CUL 2）E3连接酶复合物，拴在多肽粘合剂，选择性地结合和招募内源性靶蛋白的CUL 2-E3连接酶复合物的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SHP 2的合成单体和选择性结合人ASC蛋白的骆驼衍生的VHH纳米抗体，我们证明了人细胞系中内源性SHP 2和ASC的高效AdPROM介导的降解。我们发现，AdPROM介导的细胞中SHP 2的丢失会影响SHP 2的生物学。这项研究首次表明，选择性地识别内源性靶蛋白的小多肽结合剂可以用于AdPROM介导的靶蛋白的破坏。
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 recognize endogenous target proteins can be exploited for AdPROM-mediated destruction of the target proteins.