Faculty of Medicine, University of Paris Sud, Kremlin-Bicêtre, France.
Cell Biology and Metabolomics Platforms, Gustave Roussy Cancer Campus, Villejuif, France.
Sci Rep. 2018 Oct 8;8(1):14966. doi: 10.1038/s41598-018-33378-y.
The retention using selective hooks (RUSH) system allows to withhold a fluorescent biosensor such as green fluorescent protein (GFP) fused to a streptavidin-binding peptide (SBP) by an excess of streptavidin molecules that are addressed to different subcellular localizations. Addition of biotin competitively disrupts this interaction, liberating the biosensor from its hook. We constructed a human cell line co-expressing soluble secretory-SBP-GFP (ss-SBP-GFP) and streptavidin within the endoplasmic reticulum (ER) lumen and then used this system to screen a compound library for inhibitors of the biotin-induced release of ss-SBP-GFP via the conventional Golgi-dependent protein secretion pathway into the culture supernatant. We identified and validated a series of molecularly unrelated drugs including antianginal, antidepressant, anthelmintic, antipsychotic, antiprotozoal and immunosuppressive agents that inhibit protein secretion. These compounds vary in their capacity to suppress protein synthesis and to compromise ER morphology and Golgi integrity, as well as in the degree of reversibility of such effects. In sum, we demonstrate the feasibility and utility of a novel RUSH-based phenotypic screening assay.
采用选择性钩子（RUSH）系统可以通过过量的链霉亲和素分子将与链霉亲和素结合肽（SBP）融合的荧光生物传感器（如绿色荧光蛋白（GFP））保留下来，这些分子被定位到不同的亚细胞定位。生物素的添加会竞争性地破坏这种相互作用，从而将生物传感器从其钩子上释放出来。我们构建了一种共表达可溶性分泌型 SBP-GFP（ss-SBP-GFP）和链霉亲和素的人细胞系，这些链霉亲和素位于内质网（ER）腔中，然后使用该系统通过传统的高尔基体依赖的蛋白分泌途径筛选化合物文库，以寻找抑制生物素诱导的 ss-SBP-GFP 释放到培养上清液中的抑制剂。我们鉴定并验证了一系列分子上无关的药物，包括抗心绞痛、抗抑郁、驱虫、抗精神病、抗原生动物和免疫抑制药物，它们能抑制蛋白分泌。这些化合物在抑制蛋白合成和破坏 ER 形态和高尔基体完整性的能力，以及这些作用的可逆程度上存在差异。总之，我们证明了一种基于新型 RUSH 的表型筛选测定法的可行性和实用性。
