Spitz Charlotte, Schlosser Christine, Guschtschin-Schmidt Nadja, Stelzer Walter, Menig Simon, Götz Alexander, Haug-Kröper Martina, Scharnagl Christina, Langosch Dieter, Muhle-Goll Claudia, Fluhrer Regina
Biochemistry and Molecular Biology, Institute of Theoretical Medicine, Medical Faculty, University of Augsburg, Universitätsstrasse 2, 86159 Augsburg, Germany.
Karlsruhe Institute of Technology, Institute for Biological Interfaces 4, 76344 Eggenstein- Leopoldshafen, Germany and Karlsruhe Institute of Technology, Institute of Organic Chemistry, 76131 Karlsruhe, Germany.
iScience. 2020 Nov 5;23(12):101775. doi: 10.1016/j.isci.2020.101775. eCollection 2020 Dec 18.
Ectodomain (EC) shedding defines the proteolytic removal of a membrane protein EC and acts as an important molecular switch in signaling and other cellular processes. Using tumor necrosis factor (TNF)α as a model substrate, we identify a non-canonical shedding activity of SPPL2a, an intramembrane cleaving aspartyl protease of the GxGD type. Proline insertions in the TNFα transmembrane (TM) helix strongly increased SPPL2a non-canonical shedding, while leucine mutations decreased this cleavage. Using biophysical and structural analysis, as well as molecular dynamic simulations, we identified a flexible region in the center of the TNFα wildtype TM domain, which plays an important role in the processing of TNFα by SPPL2a. This study combines molecular biology, biochemistry, and biophysics to provide insights into the dynamic architecture of a substrate's TM helix and its impact on non-canonical shedding. Thus, these data will provide the basis to identify further physiological substrates of non-canonical shedding in the future.
胞外域（EC）脱落是指膜蛋白胞外域的蛋白水解去除过程，在信号传导和其他细胞过程中作为重要的分子开关。以肿瘤坏死因子（TNF）α为模型底物，我们鉴定出SPPL2a的一种非经典脱落活性，它是一种GxGD型跨膜天冬氨酸蛋白酶。TNFα跨膜（TM）螺旋中的脯氨酸插入显著增加了SPPL2a的非经典脱落，而亮氨酸突变则降低了这种切割。通过生物物理和结构分析以及分子动力学模拟，我们在TNFα野生型TM结构域中心鉴定出一个柔性区域，它在SPPL2a对TNFα的加工过程中起重要作用。本研究结合分子生物学、生物化学和生物物理学，深入了解底物TM螺旋的动态结构及其对非经典脱落的影响。因此，这些数据将为未来鉴定非经典脱落的更多生理底物提供基础。
