Department of Genomic Medicine and Environmental Toxicology, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico City, México.
Cognitive Neuroscience, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Mexico City, México.
J Mol Endocrinol. 2020 Oct;65(3):45-57. doi: 10.1530/JME-19-0253.
The calpain-10 (CAPN10) protease is implicated in the translocation of the glucose transporter 4 (GLUT4), which is retained in the Golgi matrix via the Tether containing a UBX domain for GLUT4 (TUG) protein. Insulin stimulation induces the proteolytic processing of TUG, which leads to the translocation of GLUT4 to the cell membrane. We tested whether TUG is a CAPN10 substrate. Proteolysis of TUG by calpains was assessed using a cell-free system containing calpain-1 and TUG. In situ proteolysis of TUG by calpains was demonstrated in 3T3-L1 adipocytes in the presence of insulin or calpain inhibitors to modulate calpain activity. Proteolysis of TUG by CAPN10 was confirmed using transient or stable silencing of CAPN10 in 3T3-L1 adipocytes. Calpains proteolyzed the C-terminus of TUG in vitro. In adipocytes, insulin-induced cleavage of TUG was correlated with the activation of calpains. Treatment with calpain inhibitors reduced TUG cleavage, resulting in impaired GLUT4 translocation without altering Akt phosphorylation. Furthermore, CAPN10 but not calpain-1 or calpain-2 colocalized with GLUT4 in the absence of insulin, and their colocalization was reduced after stimulation with insulin. Finally, we demonstrated that CAPN10 knockdown reduced the proteolysis of TUG without altering the phosphorylation of Akt or the expression of the Usp25m protease. Thus, our results provide evidence that the TUG protein is cleaved by CAPN10 to regulate GLUT4 translocation.
钙蛋白酶-10（CAPN10）蛋白酶参与葡萄糖转运体 4（GLUT4）的易位，GLUT4 通过含有 GLUT4 的 UBX 结构域的连接蛋白（TUG）蛋白保留在高尔基体基质中。胰岛素刺激诱导 TUG 的蛋白水解处理，导致 GLUT4 易位到细胞膜。我们测试了 TUG 是否是 CAPN10 的底物。使用含有钙蛋白酶-1 和 TUG 的无细胞系统评估 TUG 被钙蛋白酶的蛋白水解。在存在胰岛素或钙蛋白酶抑制剂以调节钙蛋白酶活性的情况下，在 3T3-L1 脂肪细胞中证明了 TUG 的原位钙蛋白酶蛋白水解。使用 3T3-L1 脂肪细胞中 CAPN10 的瞬时或稳定沉默来证实 TUG 被 CAPN10 蛋白水解。钙蛋白酶在体外蛋白水解 TUG 的 C 末端。在脂肪细胞中，胰岛素诱导的 TUG 切割与钙蛋白酶的激活相关。用钙蛋白酶抑制剂处理可减少 TUG 切割，导致 GLUT4 易位受损，而不改变 Akt 磷酸化。此外，在没有胰岛素的情况下，CAPN10 而不是钙蛋白酶-1 或钙蛋白酶-2 与 GLUT4 共定位，并且在用胰岛素刺激后其共定位减少。最后，我们证明 CAPN10 敲低减少了 TUG 的蛋白水解，而不改变 Akt 的磷酸化或 Usp25m 蛋白酶的表达。因此，我们的结果提供了证据，表明 TUG 蛋白被 CAPN10 切割以调节 GLUT4 易位。
