Schlosser Christine, Sharrouf Kinda, Papadopoulou Alkmini A, Haug-Kröper Martina, Singh Suman, Johler Maximilian, Pettinger Jonas, Horn Henrike, Koch Marco, Hoeppner Sabine, Fluhrer Regina
Biochemistry and Molecular Biology, Institute of Theoretical Medicine, Faculty of Medicine, University of Augsburg, Augsburg, Germany.
Anatomy and Cell Biology, Institute of Theoretical Medicine, Faculty of Medicine, University of Augsburg, Augsburg, Germany.
Commun Biol. 2025 Apr 30;8(1):686. doi: 10.1038/s42003-025-08102-y.
Signal peptide peptidase-like (SPPL) proteases, members of the intramembrane aspartyl protease family, have attracted increased interest due to their involvement in immune cell differentiation and cellular glycan structure regulation. However, the enzymatic domain involved in substrate recognition remains enigmatic. Here we provide evidence that the N-terminal protease-associated (PA) domains of the SPPL2 subfamily are involved in substrate recognition and discrimination of substrates that differ slightly in their luminal/extracellular domain. Presence of the SPPL2c PA domain impairs SPPL2a/b mediated tumor necrosis factor α (TNFα) initial cleavage, kinetics, and processivity in cells and in vitro. In contrast, the SPPL2a PA domain enhances processing by SPPL2b. Additionally, we demonstrate non-canonical shedding activity of SPPL3 on full-length TNFα and that the ability for consecutive cleavage differs within the SPPL-family and is mainly based on the SPPL2a/b membrane spanning body. This provides the basis to finally understand the mechanistic differences of these homologous proteases.
信号肽肽酶样（SPPL）蛋白酶是膜内天冬氨酰蛋白酶家族的成员，由于其参与免疫细胞分化和细胞聚糖结构调节而受到越来越多的关注。然而，参与底物识别的酶结构域仍然是个谜。在这里，我们提供证据表明，SPPL2亚家族的N端蛋白酶相关（PA）结构域参与底物识别以及对管腔/细胞外结构域略有不同的底物的区分。SPPL2c PA结构域的存在会损害SPPL2a/b介导的细胞内和体外肿瘤坏死因子α（TNFα）的初始切割、动力学和持续合成能力。相反，SPPL2a PA结构域会增强SPPL2b的加工能力。此外，我们证明了SPPL3对全长TNFα具有非经典的脱落活性，并且连续切割的能力在SPPL家族中有所不同，主要基于SPPL2a/b跨膜结构域。这为最终理解这些同源蛋白酶的机制差异提供了基础。
