Plasma Membrane Repair Is Regulated Extracellularly by Proteases Released from Lysosomes

Eukaryotic cells rapidly repair wounds on their plasma membrane. Resealing is Ca2+-dependent, and involves exocytosis of lysosomes followed by massive endocytosis. Extracellular activity of the lysosomal enzyme acid sphingomyelinase was previously shown to promote endocytosis and wound removal. However, whether lysosomal proteases released during cell injury participate in resealing is unknown. Here we show that lysosomal proteases regulate plasma membrane repair. Extracellular proteolysis is detected shortly after cell wounding, and inhibition of this process blocks repair. Conversely, surface protein degradation facilitates plasma membrane resealing. The abundant lysosomal cysteine proteases cathepsin B and L, known to proteolytically remodel the extracellular matrix, are rapidly released upon cell injury and are required for efficient plasma membrane repair. In contrast, inhibition of aspartyl proteases or RNAi-mediated silencing of the lysosomal aspartyl protease cathepsin D enhances resealing, an effect associated with the accumulation of active acid sphingomyelinase on the cell surface. Thus, secreted lysosomal cysteine proteases may promote repair by facilitating membrane access of lysosomal acid sphingomyelinase, which promotes wound removal and is subsequently downregulated extracellularly by a process involving cathepsin D.

真核细胞可快速修复其质膜上的破损。质膜重封过程依赖钙离子，且先后涉及溶酶体胞吐与大规模内吞作用。此前已有研究表明，溶酶体酶酸性鞘磷脂酶（acid sphingomyelinase）的胞外活性可促进内吞作用与膜破损清除。然而，细胞损伤过程中释放的溶酶体蛋白酶是否参与质膜重封过程，目前仍不明确。本研究证实，溶酶体蛋白酶可调控质膜修复过程。细胞受损伤后可快速检测到胞外蛋白水解活动，抑制该过程则会阻断质膜修复。反之，细胞膜表面蛋白的降解则可促进质膜重封。丰度较高的溶酶体半胱氨酸蛋白酶——组织蛋白酶B（cathepsin B）与组织蛋白酶L（cathepsin L），已知可通过蛋白水解作用重塑细胞外基质，在细胞损伤后会快速释放至胞外，且是高效完成质膜修复所必需的物质。与之相反，抑制天冬氨酸蛋白酶活性，或是通过RNA干扰（RNAi）介导溶酶体天冬氨酸蛋白酶组织蛋白酶D（cathepsin D）的基因沉默，均可增强质膜重封效率；该效应与活性酸性鞘磷脂酶在细胞表面的积累相关。综上，分泌至胞外的溶酶体半胱氨酸蛋白酶，或可通过促进溶酶体酸性鞘磷脂酶抵达细胞膜的方式来推动修复：该酶可促进膜破损清除，随后通过涉及组织蛋白酶D的胞外过程被下调。