Gabrielli Jacopo, Di Blasi Roberto, Kontoravdi Cleo, Ceroni Francesca
Department of Chemical Engineering, Imperial College London, London, UK.
Imperial College Centre for Synthetic Biology, Imperial College London, London, UK.
Nat Commun. 2025 Jan 2;16(1):328. doi: 10.1038/s41467-024-55311-w.
Degradation tags, otherwise known as degrons, are portable sequences that can be used to alter protein stability. Here, we report that degron-tagged proteins compete for cellular degradation resources in engineered mammalian cells leading to coupling of the degradation rates of otherwise independently expressed proteins when constitutively targeted human degrons are adopted. We show the effect of this competition to be dependent on the context of the degrons. By considering different proteins, degron position and cellular hosts, we highlight how the impact of the degron on both degradation strength and resource coupling changes, with identification of orthogonal combinations. By adopting inducible bacterial and plant degrons we also highlight how controlled uncoupling of synthetic construct degradation from the native machinery can be achieved. We then build a genomically integrated capacity monitor tagged with different degrons and confirm resource competition between genomic and transiently expressed DNA constructs. This work expands the characterisation of resource competition in engineered mammalian cells to protein degradation also including integrated systems, providing a framework for the optimisation of heterologous expression systems to advance applications in fundamental and applied biological research.
降解标签，也称为降解结构域，是可用于改变蛋白质稳定性的可转移序列。在此，我们报告称，当采用组成型靶向的人类降解结构域时，带有降解结构域标签的蛋白质在工程化哺乳动物细胞中竞争细胞降解资源，导致原本独立表达的蛋白质的降解速率相互耦合。我们表明这种竞争的影响取决于降解结构域的背景。通过考虑不同的蛋白质、降解结构域位置和细胞宿主，我们突出了降解结构域对降解强度和资源耦合的影响如何变化，并鉴定出正交组合。通过采用可诱导的细菌和植物降解结构域，我们还突出了如何实现合成构建体降解与天然机制的可控解耦。然后，我们构建了一个用不同降解结构域标记的基因组整合能力监测器，并证实了基因组和瞬时表达的DNA构建体之间的资源竞争。这项工作将工程化哺乳动物细胞中资源竞争的表征扩展到蛋白质降解，还包括整合系统，为优化异源表达系统以推进基础和应用生物学研究中的应用提供了一个框架。
