Department of Genetics, Stanford University, Stanford, United States.
Department of Molecular and Cellular Biology, Northwest Labs, Harvard University, Cambridge, United States.
Elife. 2019 Nov 1;8:e48434. doi: 10.7554/eLife.48434.
The small molecule Retro-2 prevents ricin toxicity through a poorly-defined mechanism of action (MOA), which involves halting retrograde vesicle transport to the endoplasmic reticulum (ER). CRISPRi genetic interaction analysis revealed Retro-2 activity resembles disruption of the transmembrane domain recognition complex (TRC) pathway, which mediates post-translational ER-targeting and insertion of tail-anchored (TA) proteins, including SNAREs required for retrograde transport. Cell-based and in vitro assays show that Retro-2 blocks delivery of newly-synthesized TA-proteins to the ER-targeting factor ASNA1 (TRC40). An ASNA1 point mutant identified using CRISPR-mediated mutagenesis abolishes both the cytoprotective effect of Retro-2 against ricin and its inhibitory effect on ASNA1-mediated ER-targeting. Together, our work explains how Retro-2 prevents retrograde trafficking of toxins by inhibiting TA-protein targeting, describes a general CRISPR strategy for predicting the MOA of small molecules, and paves the way for drugging the TRC pathway to treat broad classes of viruses known to be inhibited by Retro-2.
小分子 Retro-2 通过一种作用机制（MOA）来阻止蓖麻毒素的毒性，该机制涉及阻止逆行囊泡运输到内质网（ER）。CRISPRi 遗传相互作用分析显示，Retro-2 的活性类似于破坏跨膜结构域识别复合物（TRC）途径，该途径介导翻译后 ER 靶向和尾部锚定（TA）蛋白的插入，包括逆行运输所需的 SNARE 蛋白。基于细胞和体外的测定表明 Retro-2 阻止了新合成的 TA 蛋白向 ER 靶向因子 ASNA1（TRC40）的输送。使用 CRISPR 介导的诱变鉴定出的 ASNA1 点突变体既消除了 Retro-2 对蓖麻毒素的细胞保护作用，也消除了其对 ASNA1 介导的 ER 靶向的抑制作用。总的来说，我们的工作解释了 Retro-2 如何通过抑制 TA 蛋白靶向来阻止毒素的逆行运输，描述了一种用于预测小分子 MOA 的通用 CRISPR 策略，并为靶向 TRC 途径治疗已知被 Retro-2 抑制的广泛类别的病毒铺平了道路。
