Center for Integrated Protein Science Munich (CIPSM) at the Department of Chemistry, Technical University of Munich, Lichtenbergstrasse 4, 85748, Garching, Germany.
Current address: Institute of Molecular Biology and Biophysics, ETH Zürich, 8093, Zürich, Switzerland.
Chembiochem. 2020 Jul 1;21(13):1861-1867. doi: 10.1002/cbic.201900651. Epub 2020 Mar 9.
Proteins that terminally fail to acquire their native structure are detected and degraded by cellular quality control systems. Insights into cellular protein quality control are key to a better understanding of how cells establish and maintain the integrity of their proteome and of how failures in these processes cause human disease. Here we have used genetic code expansion and fast bio-orthogonal reactions to monitor protein turnover in mammalian cells through a fluorescence-based assay. We have used immune signaling molecules (interleukins) as model substrates and shown that our approach preserves normal cellular quality control, assembly processes, and protein functionality and works for different proteins and fluorophores. We have further extended our approach to a pulse-chase type of assay that can provide kinetic insights into cellular protein behavior. Taken together, this study establishes a minimally invasive method to investigate protein turnover in cells as a key determinant of cellular homeostasis.
细胞质量控制系统可以检测和降解那些最终无法获得其天然结构的蛋白质。深入了解细胞的蛋白质质量控制对于更好地理解细胞如何建立和维持其蛋白质组的完整性，以及这些过程的失败如何导致人类疾病至关重要。在这里，我们使用遗传密码扩展和快速生物正交反应，通过基于荧光的测定法在哺乳动物细胞中监测蛋白质周转。我们使用免疫信号分子（白细胞介素）作为模型底物，并表明我们的方法保留了正常的细胞质量控制、组装过程和蛋白质功能，并且适用于不同的蛋白质和荧光团。我们还进一步扩展了我们的方法，使其成为一种可以提供细胞内蛋白质行为动力学见解的脉冲追踪类型的测定法。总之，这项研究建立了一种微创方法来研究细胞内蛋白质周转，这是细胞内稳态的关键决定因素。
