Cathepsin S (CTSS), a lysosomal cysteine protease, has been reported to be associated with extracellular matrix (ECM) degradation, thus promoting cell migration and invasion, but whether CTSS regulates other intracellular mechanisms during metastasis remains unknown. The expression of CTSS was knocked down using siRNA transfection, and enzymatic activity was inhibited by the highly-selective CTSS inhibitor RJW-58. The results of in vitro functional assays, western blot analysis, and an in vivo colonization model demonstrated that CTSS was positively related to cellular adhesive ability. Moreover, both CTSS knockdown and inhibition significantly decreased Ca2+ influx via store-operated Ca2+ entry (SOCE) without changing STIM1 and Orai1 expression levels, while RJW-58 dose-dependently reduced the activation of the Ca2+-dependent downstream effectors, NFAT1 and Rac1. The results of immunoprecipitation assays demonstrated that CTSS could bind to STIM1, which was reversed by CTSS inhibition. In addition, confocal microscopy and super-resolution imaging showed that CTSS inhibition led to STIM1 puncta accumulation in the endoplasmic reticulum and reduced the interaction between active STIM1 and EB1. In conclusion, we have demonstrated for the first time that the lysosomal cysteine protease, CTSS, plays an important role in mediating Ca2+ homeostasis by regulating STIM1 trafficking, which leads to the suppression of cell migration and invasion.

中文翻译：

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溶酶体半胱氨酸蛋白酶组织蛋白酶S通过调节储存操纵的Ca2 +进入而参与癌细胞的运动。

组织蛋白酶S（CTSS），一种溶酶体半胱氨酸蛋白酶，据报道与细胞外基质（ECM）降解有关，从而促进细胞迁移和侵袭，但尚不清楚CTSS是否在转移过程中调节其他细胞内机制。使用siRNA转染可降低CTSS的表达，并通过高度选择性的CTSS抑制剂RJW-58抑制酶的活性。体外功能测定，蛋白质印迹分析和体内定植模型的结果表明，CTSS与细胞粘附能力呈正相关。此外，CTSS的抑制和抑制都通过存储操作的Ca2 +进入（SOCE）显着减少了Ca2 +的流入，而没有改变STIM1和Orai1的表达水平，而RJW-58剂量依赖性地降低了Ca2 +依赖性下游效应子的激活，NFAT1和Rac1。免疫沉淀分析的结果表明，CTSS可以与STIM1结合，而CTTIM的抑制作用可以逆转STIM1。此外，共聚焦显微镜和超分辨率成像显示，CTSS抑制导致内质网中STIM1点状积累，并减少了活性STIM1和EB1之间的相互作用。总之，我们首次证明了溶酶体半胱氨酸蛋白酶CTSS通过调节STIM1转运在介导Ca2 +稳态中起重要作用，从而抑制了细胞迁移和侵袭。共聚焦显微镜和超分辨率成像显示，CTSS抑制导致内质网中STIM1点状积累，并减少了活性STIM1和EB1之间的相互作用。总之，我们首次证明了溶酶体半胱氨酸蛋白酶CTSS通过调节STIM1转运在介导Ca2 +稳态中起重要作用，从而抑制了细胞迁移和侵袭。共聚焦显微镜和超分辨率成像显示，CTSS抑制导致内质网中STIM1点状积累，并减少了活性STIM1和EB1之间的相互作用。总之，我们首次证明了溶酶体半胱氨酸蛋白酶CTSS通过调节STIM1转运在介导Ca2 +稳态中起重要作用，从而抑制了细胞迁移和侵袭。