Stigmasterol (ST), a phytosterol found in food, has various biological activities. However, the effect of ST on milk synthesis in dairy cows remains unclear. Therefore, bovine primary mammary epithelial cells (BMECs) were isolated, cultured, and treated with ST to determine the effect of ST on milk synthesis. The study revealed that 10 μM ST significantly increased milk synthesis in BMECs by activating the mammalian target of rapamycin (mTOR) signaling pathway. Further investigation revealed that this activation depends on the regulatory role of oxysterol binding protein 5 (ORP5). ST induces the translocation of ORP5 from the cytoplasm to the lysosome, interacts with the mTOR, recruits mTOR to target the lysosomal surface, and promotes the activation of the mTOR signaling pathway. Moreover, ST was found to increase ORP5 protein levels by inhibiting its degradation via the ubiquitin-proteasome pathway. Specifically, the E3 ubiquitin ligase membrane-associated cycle-CH-type finger 4 (MARCH4) promotes the ubiquitination and subsequent degradation of ORP5. ST mitigates the interaction between MARCH4 and ORP5, thereby enhancing the structural stability of ORP5 and reducing its ubiquitination. In summary, ST stabilizes ORP5 by inhibiting the interaction between MARCH4 and ORP5, thereby activating mTOR signaling pathway and enhancing milk synthesis.

中文翻译：

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豆甾醇通过抑制 ORP5 泛素化激活 mTOR 信号通路促进牛乳腺上皮细胞乳汁合成


豆甾醇 (ST) 是一种存在于食品中的植物甾醇，具有多种生物活性。然而，ST 对奶牛牛奶合成的影响仍不清楚。因此，我们对牛原代乳腺上皮细胞（BMEC）进行了分离、培养和ST处理，以确定ST对乳汁合成的影响。研究表明，10 μM ST 通过激活哺乳动物雷帕霉素靶点 (mTOR) 信号通路，显着增加 BMEC 的乳汁合成。进一步的研究表明，这种激活取决于氧甾醇结合蛋白 5 (ORP5) 的调节作用。 ST诱导ORP5从细胞质易位至溶酶体，与mTOR相互作用，招募mTOR靶向溶酶体表面，促进mTOR信号通路的激活。此外，ST 通过泛素-蛋白酶体途径抑制 ORP5 蛋白的降解，从而提高 ORP5 蛋白水平。具体而言，E3 泛素连接酶膜相关循环 CH 型指 4 (MARCH4) 促进 ORP5 的泛素化和随后的降解。 ST 减轻了 MARCH4 和 ORP5 之间的相互作用，从而增强了 ORP5 的结构稳定性并减少了其泛素化。综上所述，ST通过抑制MARCH4和ORP5之间的相互作用来稳定ORP5，从而激活mTOR信号通路并增强乳汁合成。