Centre de Recherche en Cancérologie de Toulouse (CRCT), Université de Toulouse, Institut National de la Santé et de la Recherche Médicale (INSERM), Centre National de la Recherche Scientifique (CNRS), Université Toulouse III-Paul Sabatier, Centre de Recherches en Cancérologie de Toulouse (CRCT), Toulouse, France.
Laboratoire de Biologie Médicale Oncologique, IUCT-Oncopôle, Toulouse, France.
Front Immunol. 2022 Aug 18;13:980539. doi: 10.3389/fimmu.2022.980539. eCollection 2022.
Strategies based on intracellular expression of artificial binding domains present several advantages over manipulating nucleic acid expression or the use of small molecule inhibitors. Intracellularly-functional nanobodies can be considered as promising macrodrugs to study key signaling pathways by interfering with protein-protein interactions. With the aim of studying the RAS-related small GTPase RHOA family, we previously isolated, from a synthetic phage display library, nanobodies selective towards the GTP-bound conformation of RHOA subfamily proteins that lack selectivity between the highly conserved RHOA-like and RAC subfamilies of GTPases. To identify RHOA/ROCK pathway inhibitory intracellular nanobodies, we implemented a stringent, subtractive phage display selection towards RHOA-GTP followed by a phenotypic screen based on F-actin fiber loss. Intracellular interaction and intracellular selectivity between RHOA and RAC1 proteins was demonstrated by adapting the sensitive intracellular protein-protein interaction reporter based on the tripartite split-GFP method. This strategy led us to identify a functional intracellular nanobody, hereafter named RH28, that does not cross-react with the close RAC subfamily and blocks/disrupts the RHOA/ROCK signaling pathway in several cell lines without further engineering or functionalization. We confirmed these results by showing, using SPR assays, the high specificity of the RH28 nanobody towards the GTP-bound conformation of RHOA subfamily GTPases. In the metastatic melanoma cell line WM266-4, RH28 expression triggered an elongated cellular phenotype associated with a loss of cellular contraction properties, demonstrating the efficient intracellular blocking of RHOA/B/C proteins downstream interactions without the need of manipulating endogenous gene expression. This work paves the way for future therapeutic strategies based on protein-protein interaction disruption with intracellular antibodies.
基于细胞内表达人工结合域的策略与操纵核酸表达或使用小分子抑制剂相比具有几个优势。细胞内功能化的纳米抗体可被视为研究关键信号通路的有前途的大分子药物，通过干扰蛋白质-蛋白质相互作用。为了研究与 RAS 相关的小 GTPase RHOA 家族，我们之前从合成噬菌体展示文库中分离出了对 RHOA 亚家族蛋白的 GTP 结合构象具有选择性的纳米抗体，这些纳米抗体对高度保守的 RHOA 样和 RAC 亚家族 GTPases之间缺乏选择性。为了鉴定 RHOA/ROCK 通路抑制性细胞内纳米抗体，我们对 RHOA-GTP 进行了严格的、减法噬菌体展示选择，然后基于 F-肌动蛋白纤维损失进行表型筛选。通过适应基于三部分分裂 GFP 方法的敏感细胞内蛋白质-蛋白质相互作用报告器，证明了 RHOA 和 RAC1 蛋白之间的细胞内相互作用和细胞内选择性。这种策略使我们能够鉴定出一种功能性的细胞内纳米抗体，以下称为 RH28，它与密切相关的 RAC 亚家族没有交叉反应，并在没有进一步工程化或功能化的情况下阻断/破坏几种细胞系中的 RHOA/ROCK 信号通路。我们使用 SPR 测定证实了这些结果，表明 RH28 纳米抗体对 RHOA 亚家族 GTPases 的 GTP 结合构象具有高特异性。在转移性黑色素瘤细胞系 WM266-4 中，RH28 表达触发了与细胞收缩特性丧失相关的拉长细胞表型，证明了有效阻断 RHOA/B/C 蛋白下游相互作用的细胞内阻断，而无需操纵内源性基因表达。这项工作为基于蛋白质-蛋白质相互作用破坏的细胞内抗体的未来治疗策略铺平了道路。
