Leucyl-tRNA synthetase regulates casein synthesis in dairy cows via the mTORC1-LAT1 pathway

Objective Leucyl-tRNA synthetase (LARS) is an essential multifunctional enzyme in mammals, pivotal in maintaining cellular protein and amino acid balance. It facilitates tRNA aminoacylation, initiating intracellular protein synthesis, and serves as an intracellular leucine sensor. The sensor function enables LARS to activate the mTORC1 pathway via Rag GTPase binding, playing a critical role in the regulation of protein synthesis. Despite its significance, the precise mechanisms of these functions are yet to be fully delineated. This study examines LARS and its role in modulating milk protein synthesis. Methods This study utilizes stable bovine mammary epithelial cell lines, LARS overexpression and LARS knockdown, validated by using Cell Counting Kit-8, Click-iT EdU, Western blot, real-time quantitative polymerase chain reaction, and immunoconfocal techniques. Results Our findings show that LARS overexpression in bovine mammary epithelial cells (MAC-T) enhances cell proliferation and resultes intracellular leucine levels, thereby increasing casein production through the mTORC1 pathway. LARS enhances casein expression via the mechanistic Target of Rapamycin Complex 1, L-type Amino Transporters 1 (mTORC1-LAT1) pathway. This interaction is supported by a positive feedback mechanism from LAT1, enhancing the activation of the mTORC1 pathway. Additionally, LARS overexpression leads to increased LAT1 expression, improved LAT1 stability, and augmented its localization at the membrane. Our research indicates that LARS’s enhancement of LAT1 expression is contingent on its dual roles in translation and leucine sensing, whereas its impact on LAT1 localization is exclusively dependent on its leucine sensing function. Conclusion LARS regulates LAT1 expression and membrane positioning through the mTORC1 pathway by detecting intracellular leucine levels, thereby influencing casein synthesis. These insights lay a theoretical groundwork for enhancing milk protein production and offer novel strategies for improving the quality of dairy products.