Lu Huixin, Mazumder Mostafizur, Jaikaran Anna S I, Kumar Anil, Leis Eric K, Xu Xiuling, Altmann Michael, Cochrane Alan, Woolley G Andrew
Department of Chemistry , University of Toronto , 80 St. George Street , Toronto , ON M5S 3H6 , Canada.
Institut für Biochemie und Molekulare Medizin , Universität Bern , Bühlstr. 28 , CH-3012 Bern , Switzerland.
ACS Synth Biol. 2019 Apr 19;8(4):744-757. doi: 10.1021/acssynbio.8b00386. Epub 2019 Apr 4.
The precise spatiotemporal regulation of protein synthesis is essential for many complex biological processes such as memory formation, embryonic development, and tumor formation. Current methods used to study protein synthesis offer only a limited degree of spatiotemporal control. Optogenetic methods, in contrast, offer the prospect of controlling protein synthesis noninvasively within minutes and with a spatial scale as small as a single synapse. Here, we present a hybrid yeast system where growth depends on the activity of human eukaryotic initiation factor 4E (eIF4E) that is suitable for screening optogenetic designs for the down-regulation of protein synthesis. We used this system to screen a diverse initial panel of 15 constructs designed to couple a light switchable domain (PYP, RsLOV, AsLOV, Dronpa) to 4EBP2 (eukaryotic initiation factor 4E binding protein 2), a native inhibitor of translation initiation. We identified cLIPS1 (circularly permuted LOV inhibitor of protein synthesis 1), a fusion of a segment of 4EBP2 and a circularly permuted version of the LOV2 domain from Avena sativa, as a photoactivated inhibitor of translation. Adapting the screen for higher throughput, we tested small libraries of cLIPS1 variants and found cLIPS2, a construct with an improved degree of optical control. We show that these constructs can both inhibit translation in yeast harboring a human eIF4E in vivo, and bind human eIF4E in vitro in a light-dependent manner. This hybrid yeast system thus provides a convenient way for discovering optogenetic constructs that can regulate human eIF4E-dependent translation initiation in a mechanistically defined manner.
蛋白质合成精确的时空调节对于许多复杂的生物学过程至关重要，如记忆形成、胚胎发育和肿瘤形成。目前用于研究蛋白质合成的方法仅提供有限程度的时空控制。相比之下，光遗传学方法提供了在数分钟内以小至单个突触的空间尺度无创控制蛋白质合成的前景。在此，我们展示了一种杂交酵母系统，其生长依赖于人类真核起始因子4E（eIF4E）的活性，该系统适用于筛选用于下调蛋白质合成的光遗传学设计。我们使用该系统筛选了一个多样化的初始构建体库，其中15个构建体旨在将光可切换结构域（PYP、RsLOV、AsLOV、Dronpa）与4EBP2（真核起始因子4E结合蛋白2）偶联，4EBP2是翻译起始的天然抑制剂。我们鉴定出cLIPS1（蛋白质合成的环状排列LOV抑制剂1），它是4EBP2的一段与燕麦LOV2结构域的环状排列版本的融合体，作为一种光激活的翻译抑制剂。为了适应更高通量的筛选，我们测试了cLIPS1变体的小型文库，并发现了cLIPS2，这是一种光学控制程度更高的构建体。我们表明，这些构建体既能在体内含有人类eIF4E的酵母中抑制翻译，又能在体外以光依赖的方式与人eIF4E结合。因此，这种杂交酵母系统为发现能够以机械定义的方式调节人类eIF4E依赖性翻译起始的光遗传学构建体提供了一种便捷方法。
