Rosier Bas J H M, Markvoort Albert J, Gumí Audenis Berta, Roodhuizen Job A L, den Hamer Anniek, Brunsveld Luc, de Greef Tom F A
Laboratory of Chemical Biology, Department of Biomedical Engineering, Eindhoven University of Technology, The Netherlands.
Institute for Complex Molecular Systems, Eindhoven University of Technology, The Netherlands.
Nat Catal. 2020 Mar;3(3):295-306. doi: 10.1038/s41929-019-0403-7. Epub 2020 Jan 6.
Living cells regulate key cellular processes by spatial organisation of catalytically active proteins in higher-order signalling complexes. These act as organising centres to facilitate proximity-induced activation and inhibition of multiple intrinsically weakly associating signalling components, which makes elucidation of the underlying protein-protein interactions challenging. Here we show that DNA origami nanostructures provide a programmable molecular platform for the systematic analysis of signalling proteins by engineering a synthetic DNA origami-based version of the apoptosome, a multi-protein complex that regulates apoptosis by co-localizing multiple caspase-9 monomers. Tethering of both wildtype and inactive caspase-9 variants to a DNA origami platform demonstrates that enzymatic activity is induced by proximity-driven dimerization with half-of-sites reactivity, and additionally, reveals a multivalent activity enhancement in oligomers of three and four enzymes. Our results offer fundamental insights in caspase-9 activity regulation and demonstrate that DNA origami-based protein assembly platforms have the potential to inform the function of other multi-enzyme complexes involved in inflammation, innate immunity and cell death.
活细胞通过在高阶信号复合物中对具有催化活性的蛋白质进行空间组织来调节关键的细胞过程。这些复合物作为组织中心，促进多个内在弱关联信号成分的邻近诱导激活和抑制，这使得阐明潜在的蛋白质-蛋白质相互作用具有挑战性。在这里，我们表明，DNA折纸纳米结构通过构建基于合成DNA折纸的凋亡小体（一种通过共定位多个半胱天冬酶-9单体来调节细胞凋亡的多蛋白复合物）版本，为信号蛋白的系统分析提供了一个可编程的分子平台。将野生型和无活性的半胱天冬酶-9变体连接到DNA折纸平台上表明，酶活性是由邻近驱动的二聚化以半位点反应性诱导的，此外，还揭示了三聚体和四聚体酶中多价活性增强。我们的结果为半胱天冬酶-9活性调节提供了基本见解，并表明基于DNA折纸的蛋白质组装平台有可能为参与炎症、先天免疫和细胞死亡的其他多酶复合物的功能提供信息。
