Betti Camilla, Schymkowitz Joost, Rousseau Frederic, Russinova Eugenia
Department of Plant Biotechnology and Bioinformatics, Ghent University, Technologiepark 927, 9052, Gent, Belgium.
Center for Plant Systems Biology, VIB, Technologiepark 927, 9052, Gent, Belgium.
Methods Mol Biol. 2018;1676:109-127. doi: 10.1007/978-1-4939-7315-6_6.
Protein aggregation is determined by 5-15 amino acids peptides of the target protein sequence, so-called aggregation-prone regions (APRs) that specifically self-associate to form β-structured inclusions. The presence of APRs in a target protein can be predicted by a dedicated algorithm, such as TANGO. Synthetic aggregation-prone proteins are designed by expressing specific APRs fused to a fluorescent carrier for stability and visualization. Previously, the stable expression of these proteins in Zea mays (maize) has been demonstrated to induce aggregation of target proteins with specific localization, such as the starch-degrading enzyme α-glucan water dikinase, giving rise to plants displaying knockdown phenotypes. Here, we describe how to design synthetic aggregation-prone proteins to harness the sequence specificity of APRs to generate aggregation-associated phenotypes in a targeted manner and in different subcellular compartments. This method points toward the application of induced targeted aggregation as a useful tool to knock down protein functions in maize and to generate crops with improved traits.
蛋白质聚集由靶蛋白序列中的5至15个氨基酸肽段决定，这些肽段即所谓的易聚集区域（APR），它们会特异性地自我缔合形成β结构内含物。靶蛋白中APR的存在可通过专用算法（如TANGO）进行预测。通过表达与荧光载体融合的特定APR来设计合成易聚集蛋白，以实现稳定性和可视化。此前已证明，这些蛋白在玉米中稳定表达可诱导具有特定定位的靶蛋白聚集，如淀粉降解酶α-葡聚糖水二激酶，从而使植物表现出基因敲低表型。在此，我们描述了如何设计合成易聚集蛋白，以利用APR的序列特异性，在不同亚细胞区室中以靶向方式产生聚集相关表型。该方法表明诱导靶向聚集可作为一种有用工具，用于在玉米中敲低蛋白功能并培育具有改良性状的作物。
