Selection and Characterization of a Nanobody Biosensor of GTP-Bound RHO Activities
Selection and Characterization of a Nanobody Biosensor of GTP-Bound RHO Activities
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DOI：
10.3390/antib8010008
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发表时间：
2019-01-09
期刊：
影响因子：
4.7
通讯作者：
Olichon, Aurelien
中科院分区：
文献类型：
--
作者：
Keller, Laura;Bery, Nicolas;Olichon, Aurelien
RHO （Ras同源）GTP酶是激活与鸟苷三磷酸（GTP）结合的关键信号通路的分子开关，涉及肌动蛋白细胞骨架动力学。之前，我们从合成的噬菌体展示文库中选择了纳米体RH12，它结合了RHOA （Ras同源家族成员a）的gtp结合的活性构象。然而，当作为细胞内抗体表达时，其对RHO信号的阻断作用导致肌动蛋白纤维的丢失，进而影响细胞形状和细胞存活。在这里，为了设计一个RHOA-GTP激活的细胞内生物传感器，我们筛选了相同的噬菌体纳米体库，并鉴定了另一个RHOA-GTP选择性细胞内纳米体，但没有明显的毒性。重组RH57纳米体在体外对gtp结合的小gtp酶RHOA/B/C亚群具有高亲和力。RH57的细胞内表达允许与内源性RHOA亚家族的gtp结合状态选择性共沉淀。当作为荧光融合蛋白表达时，在内源性RHO激活的刺激下，色体GFP-RH57定位于质膜内。最后，利用RH57纳米体构建了基于bret的RHO激活生物传感器（生物发光共振能量转移）。BRET信号的动态范围可能为开发基于细胞筛选RHOA亚家族激活调节剂提供新的机会。
RHO (Ras HOmologous) GTPases are molecular switches that activate, in their state bound to Guanosine triphosphate (GTP), key signaling pathways, which involve actin cytoskeleton dynamics. Previously, we selected the nanobody RH12, from a synthetic phage display library, which binds the GTP-bound active conformation of RHOA (Ras Homologous family member A). However, when expressed as an intracellular antibody, its blocking effect on RHO signaling led to a loss of actin fibers, which in turn affected cell shape and cell survival. Here, in order to engineer an intracellular biosensor of RHOA-GTP activation, we screened the same phage nanobody library and identified another RHO-GTP selective intracellular nanobody, but with no apparent toxicity. The recombinant RH57 nanobody displays high affinity towards GTP-bound RHOA/B/C subgroup of small GTPases in vitro. Intracellular expression of the RH57 allowed selective co-precipitation with the GTP-bound state of the endogenous RHOA subfamily. When expressed as a fluorescent fusion protein, the chromobody GFP-RH57 was localized to the inner plasma membrane upon stimulation of the activation of endogenous RHO. Finally, the RH57 nanobody was used to establish a BRET-based biosensor (Bioluminescence Resonance Energy Transfer) of RHO activation. The dynamic range of the BRET signal could potentially offer new opportunities to develop cell-based screening of RHOA subfamily activation modulators.