Leucyl-tRNA Synthetase Regulates Casein Synthesis in Dairy Cows via the mTORC1-LAT1 Pathway

Leucyl-tRNA synthetase (LARS) is an essential multifunctional enzyme in mammals, pivotal in maintaining cellular protein and amino acid equilibrium. It facilitates tRNA aminoacylation, initiating intracellular protein synthesis, and serves as an intracellular leucine sensor. This sensor function enables LARS to activate the mTORC1 pathway via Rag GTPase binding, playing a critical role in protein synthesis regulation. Despite its significance, the precise mechanisms of these functions are yet to be fully delineated. This study focuses on LARS, specifically its role in modulating milk protein synthesis. In this study,the bovine mammary epithelial cells were selected as the research subject, and stable LARS-OE and LARS-KD cell lines were constructed, which were verified by Cell Counting Kit-8, Click-iT EdU , Western blot, RT-qPCR, and immunoconfocal techniques. Our findings show that LARS overexpression in bovine mammary epithelial cells (MAC-T) not only enhances cell proliferation but also mediates intracellular leucine levels, thereby increasing casein production through the mTORC1 pathway. LARS further boosts casein expression via the mTORC1-LAT1(mechanistic Target of Rapamycin Complex 1, L-type Amino Transporters 1) pathway. Remarkably, 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 its augmented 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. s: In summary, by detecting intracellular leucine levels, LARS regulates LAT1 expression and membrane positioning through the mTORC1 pathway, ultimately influencing casein synthesis. These insights lay a theoretical groundwork for advancing milk protein production and offer novel strategies for improving dairy product quality.