Structural basis of tRNA recognition by the m3C RNA methyltransferase METTL6 in complex with SerRS seryl-tRNA synthetase.

Methylation of cytosine 32 in the anticodon loop of tRNAs to 3-methylcytosine (m3C) is crucial for cellular translation fidelity. Misregulation of the RNA methyltransferases setting this modification can cause aggressive cancers and metabolic disturbances. Here, we report the cryo-electron microscopy structure of the human m3C tRNA methyltransferase METTL6 in complex with seryl-tRNA synthetase (SerRS) and their common substrate tRNASer. Through the complex structure, we identify the tRNA-binding domain of METTL6. We show that SerRS acts as the tRNASer substrate selection factor for METTL6. We demonstrate that SerRS augments the methylation activity of METTL6 and that direct contacts between METTL6 and SerRS are necessary for efficient tRNASer methylation. Finally, on the basis of the structure of METTL6 in complex with SerRS and tRNASer, we postulate a universal tRNA-binding mode for m3C RNA methyltransferases, including METTL2 and METTL8, suggesting that these mammalian paralogs use similar ways to engage their respective tRNA substrates and cofactors.

Domain collapse and active site ablation generate a widespread animal mitochondrial seryl-tRNA synthetase.

Through their aminoacylation reactions, aminoacyl tRNA-synthetases (aaRS) establish the rules of the genetic code throughout all of nature. During their long evolution in eukaryotes, additional domains and splice variants were added to what is commonly a homodimeric or monomeric structure. These changes confer orthogonal functions in cellular activities that have recently been uncovered. An unusual exception to the familiar architecture of aaRSs is the heterodimeric metazoan mitochondrial SerRS. In contrast to domain additions or alternative splicing, here we show that heterodimeric metazoan mitochondrial SerRS arose from its homodimeric ancestor not by domain additions, but rather by collapse of an entire domain (in one subunit) and an active site ablation (in the other). The collapse/ablation retains aminoacylation activity while creating a new surface, which is necessary for its orthogonal function. The results highlight a new paradigm for repurposing a member of the ancient tRNA synthetase family.

Seryl-tRNA Synthetase Shows a Noncanonical Activity of Upregulating Laccase Transcription in Trametes hirsuta AH28-2 Exposed to Copper Ion.

The function of Seryl-tRNA synthetase in fungi during gene transcription regulation beyond translation has not been reported. Here, we report a seryl-tRNA synthetase, ThserRS, which can negatively regulate laccase lacA transcription in Trametes hirsuta AH28-2 under exposure to copper ion. ThserRS was obtained through yeast one-hybrid screening using a bait sequence of lacA promoter (-502 to -372 bp). ThserRS decreased while lacA increased at the transcription level in T. hirsuta AH28-2 in the first 36 h upon CuSO4 induction. Then, ThserRS was upregulated, and lacA was downregulated. ThserRS overexpression in T. hirsuta AH28-2 resulted in a decrement in lacA transcription and LacA activity. By comparison, ThserRS silencing led to increased LacA transcripts and activity. A minimum of a 32-bp DNA fragment containing two putative xenobiotic response elements could interact with ThserRS, with a dissociation constant of 919.9 nM. ThserRS localized in the cell cytoplasm and nucleus in T. hirsuta AH28-2 and was heterologously expressed in yeast. ThserRS overexpression also enhanced mycelial growth and oxidative stress resistance. The transcriptional level of several intracellular antioxidative enzymes in T. hirsuta AH28-2 was upregulated. Our results demonstrate a noncanonical activity of SerRS that acts as a transcriptional regulation factor to upregulate laccase expression at an early stage after exposure to copper ions. IMPORTANCE Seryl-tRNA synthetase is well known for the attachment of serine to the corresponding cognate tRNA during protein translation. In contrast, its functions beyond translation in microorganisms are underexplored. We performed in vitro and cell experiments to show that the seryl-tRNA synthetase in fungi with no UNE-S domain at the carboxyl terminus can enter the nucleus, directly interact with the promoter of the laccase gene, and negatively regulate the fungal laccase transcription early upon copper ion induction. Our study deepens our understanding of the Seryl-tRNA synthetase noncanonical activities in microorganisms. It also demonstrates a new transcription factor for fungal laccase transcription.

Exploration of seryl tRNA synthetase to identify potent inhibitors against leishmanial parasites.

Aminoacyl-tRNA synthetases are crucial enzymes for cellular protein metabolism and have been considered as an attractive target for development of new antimicrobials. In the current study, seryl tRNA synthetase of Leishmania donovani (LdSerRS) and its mutants were purified and characterized through biochemical and structural methods. Purified LdSerRS was found to be enzymatically active and exhibited more alpha helices in secondary structure. The enzymatic activity of purified protein was observed as highest near physiological temperature and pH. Mutation in ATP binding residues (R295 and E297) demonstrated reduction in the affinity for cofactor with no significant deviation in secondary structure. In vitro inhibition studies with ureidosulfocoumarin derivatives helped to identify Comp 5l as a specific inhibitor for leishmanial SerRS that showed lesser potency towards purified HsSerRS. The identified compound presented competitive mode of inhibition for LdSerRS and also revealed druglikeness along with very low toxicity for human macrophages. Structural analysis of protein and ligand complex depicted the binding of Comp 5l into the cofactor binding site of LdSerRS with high affinity succeeded by validation employing molecular dynamics simulations. Altogether, our study presents a promising scaffold to explore small molecules to target the enzymatic activity of leishmanial SerRS to develop the specific therapeutics.

Selective degradation of tRNASer(AGY) is the primary driver for mitochondrial seryl-tRNA synthetase-related disease.

Mitochondrial translation is of high significance for cellular energy homeostasis. Aminoacyl-tRNA synthetases (aaRSs) are crucial translational components. Mitochondrial aaRS variants cause various human diseases. However, the pathogenesis of the vast majority of these diseases remains unknown. Here, we identified two novel SARS2 (encoding mitochondrial seryl-tRNA synthetase) variants that cause a multisystem disorder. c.654-14T > A mutation induced mRNA mis-splicing, generating a peptide insertion in the active site; c.1519dupC swapped a critical tRNA-binding motif in the C-terminus due to stop codon readthrough. Both mutants exhibited severely diminished tRNA binding and aminoacylation capacities. A marked reduction in mitochondrial tRNASer(AGY) was observed due to RNA degradation in patient-derived induced pluripotent stem cells (iPSCs), causing impaired translation and comprehensive mitochondrial function deficiencies. These impairments were efficiently rescued by wild-type SARS2 overexpression. Either mutation caused early embryonic fatality in mice. Heterozygous mice displayed reduced muscle tissue-specific levels of tRNASers. Our findings elucidated the biochemical and cellular consequences of impaired translation mediated by SARS2, suggesting that reduced abundance of tRNASer(AGY) is a key determinant for development of SARS2-related diseases.

The mitochondrial seryl-tRNA synthetase SARS2 modifies onset in spastic paraplegia type 4.

PURPOSE: Hereditary spastic paraplegia type 4 is extremely variable in age at onset; the same variant can cause onset at birth or in the eighth decade. We recently discovered that missense variants in SPAST, which influences microtubule dynamics, are associated with earlier onset and more severe disease than truncating variants, but even within the early and late-onset groups there remained significant differences in onset. Given the rarity of the condition, we adapted an extreme phenotype approach to identify genetic modifiers of onset. METHODS: We performed a genome-wide association study on 134 patients bearing truncating pathogenic variants in SPAST, divided into early- and late-onset groups (aged ≤15 and ≥45 years, respectively). A replication cohort of 419 included patients carrying either truncating or missense variants. Finally, age at onset was analyzed in the merged cohort (N = 553). RESULTS: We found 1 signal associated with earlier age at onset (rs10775533, P = 8.73E-6) in 2 independent cohorts and in the merged cohort (N = 553, Mantel-Cox test, P < .0001). Western blotting in lymphocytes of 20 patients showed that this locus tends to upregulate SARS2 expression in earlier-onset patients. CONCLUSION: SARS2 overexpression lowers the age of onset in hereditary spastic paraplegia type 4. Lowering SARS2 or improving mitochondrial function could thus present viable approaches to therapy.

Bladder Cancer-Derived Small Extracellular Vesicles Promote Tumor Angiogenesis by Inducing HBP-Related Metabolic Reprogramming and SerRS O-GlcNAcylation in Endothelial Cells.

A malformed tumour vascular network provokes the nutrient-deprived tumour microenvironment (TME), which conversely activates endothelial cell (EC) functions and stimulates neovascularization. Emerging evidence suggests that the flexible metabolic adaptability of tumour cells helps to establish a metabolic symbiosis among various cell subpopulations in the fluctuating TME. In this study, the authors propose a novel metabolic link between bladder cancer (BCa) cells and ECs in the nutrient-scarce TME, in which BCa-secreted glutamine-fructose-6-phosphate aminotransferase 1 (GFAT1) via small extracellular vesicles (sEVs) reprograms glucose metabolism by increasing hexosamine biosynthesis pathway flux in ECs and thus enhances O-GlcNAcylation. Moreover, seryl-tRNA synthetase (SerRS) O-GlcNAcylation at serine 101 in ECs promotes its degradation by ubiquitination and impeded importin α5-mediated nuclear translocation. Intranuclear SerRS attenuates vascular endothelial growth factor transcription by competitively binding to the GC-rich region of the proximal promotor. Additionally, GFAT1 knockout in tumour cells blocks SerRS O-GlcNAcylation in ECs and attenuates angiogenesis both in vitro and in vivo. However, administration of GFAT1-overexpressing BCa cells-derived sEVs increase the angiogenetic activity in the ECs of GFAT1-knockout mice. In summary, this study suggests that inhibiting sEV-mediated GFAT1 secretion from BCa cells and targeting SerRS O-GlcNAcylation in ECs may serve as novel strategies for BCa antiangiogenetic therapy.

Loss of seryl-tRNA synthetase (SARS1) causes complex spastic paraplegia and cellular senescence.

BACKGROUND: Aminoacyl-tRNA synthetases (ARS) are key enzymes catalysing the first reactions in protein synthesis, with increasingly recognised pleiotropic roles in tumourgenesis, angiogenesis, immune response and lifespan. Germline mutations in several ARS genes have been associated with both recessive and dominant neurological diseases. Recently, patients affected with microcephaly, intellectual disability and ataxia harbouring biallelic variants in the seryl-tRNA synthetase encoded by seryl-tRNA synthetase 1 (SARS1) were reported. METHODS: We used exome sequencing to identify the causal variant in a patient affected by complex spastic paraplegia with ataxia, intellectual disability, developmental delay and seizures, but without microcephaly. Complementation and serylation assays using patient's fibroblasts and an Saccharomyces cerevisiae model were performed to examine this variant's pathogenicity. RESULTS: A de novo splice site deletion in SARS1 was identified in our patient, resulting in a 5-amino acid in-frame insertion near its active site. Complementation assays in S. cerevisiae and serylation assays in both yeast strains and patient fibroblasts proved a loss-of-function, dominant negative effect. Fibroblasts showed an abnormal cell shape, arrested division and increased beta-galactosidase staining along with a senescence-associated secretory phenotype (raised interleukin-6, p21, p16 and p53 levels). CONCLUSION: We refine the phenotypic spectrum and modes of inheritance of a newly described, ultrarare neurodevelopmental disorder, while unveiling the role of SARS1 as a regulator of cell growth, division and senescence.

The Escherichia coli serS gene promoter region overlaps with the rarA gene.

Deletion of the entire gene encoding the RarA protein of Escherichia coli results in a growth defect and additional deficiencies that were initially ascribed to a lack of RarA function. Further work revealed that most of the effects reflected the presence of sequences in the rarA gene that affect expression of the downstream gene, serS. The serS gene encodes the seryl aminoacyl-tRNA synthetase. Decreases in the expression of serS can trigger the stringent response. The sequences that affect serS expression are located in the last 15 nucleotides of the rarA gene.

Glucose-sensitive acetylation of Seryl tRNA synthetase regulates lipid synthesis in breast cancer.

Abnormally enhanced de novo lipid biosynthesis has been increasingly realized to play crucial roles in the initiation and progression of varieties of cancers including breast cancer. However, the mechanisms underlying the dysregulation of lipid biosynthesis in breast cancer remain largely unknown. Here, we reported that seryl tRNA synthetase (SerRS), a key enzyme for protein biosynthesis, could translocate into the nucleus in a glucose-dependent manner to suppress key genes involved in the de novo lipid biosynthesis. In normal mammary gland epithelial cells glucose can promote the nuclear translocation of SerRS by increasing the acetylation of SerRS at lysine 323. In SerRS knock-in mice bearing acetylation-defective lysine to arginine mutation, we observed increased body weight and adipose tissue mass. In breast cancer cells the acetylation and nuclear translocation of SerRS are greatly inhibited. Overexpression of SerRS, in particularly the acetylation-mimetic lysine to glutamine mutant, dramatically inhibits the de novo lipid synthesis and hence greatly suppresses the proliferation of breast cancer cells and the growth of breast cancer xenografts in mice. We further identified that HDAC4 and HDAC5 regulated the acetylation and nuclear translocation of SerRS. Thus, we identified a SerRS-meditated inhibitory pathway in glucose-induced lipid biosynthesis, which is dysregulated in breast cancer.

Novel SARS2 variants identified in a Chinese girl with HUPRA syndrome.

BACKGROUND: Hyperuricemia, pulmonary hypertension, renal failure, and alkaline intoxication syndrome (HUPRA syndrome) is a rare autosomal recessive mitochondrial disease. SARS2 gene encoding seryl-tRNA synthetase is the only pathogenic gene of HUPRA syndrome. All the previously reported cases with HUPRA syndrome were detected for homozygous mutation. METHODS: We identified compound heterozygous mutations causing HUPRA syndrome using whole-exome sequencing, and verifed pathogenicity with ACMG standards. All the previously published cases with SARS2 mutations were reviewed. RESULTS: SARS2 gene compound heterozygotes variants were detected in this Chinese patient (c.667G>A/c.1205G>A). Bioinformatics studies and protein models predict that a new variant (c.667G>A) is likely to be pathogenic. A total of six patients, five of whom were previously reported with HUPRA syndrome, were analyzed. All of the six had typical clinical manifestations of HUPRA syndrome, except the Chinese girl who had no pulmonary hypertension or alkaline intoxication. The shrunken kidney was more prominent in our proband. The average survival time for previously reported patients was 17 months, and the Chinese girl was 70 months. Three mutation variants were found, including five homozygous mutants, three of which were Palestinian (c.1169A > G), two of which were from a Spanish family (c.1205G> A), and one was a new variant (c.667G>A/c.1205G>A). CONCLUSION: We found a new pathogenic form (compound heterozygous mutation) causing HUPRA syndrome, and identified a novel pathogenic site (c.667G>A) of the SARS2 gene, expanding the spectrum of SARS2 pathogenic variants. The mild phenotype in complex heterozygous mutations is described.

Phosphorylation of seryl-tRNA synthetase by ATM/ATR is essential for hypoxia-induced angiogenesis.

Hypoxia-induced angiogenesis maintains tissue oxygen supply and protects against ischemia but also enhances tumor progression and malignancy. This is mediated through activation of transcription factors like hypoxia-inducible factor 1 (HIF-1) and c-Myc, yet the impact of hypoxia on negative regulators of angiogenesis is unknown. During vascular development, seryl-tRNA synthetase (SerRS) regulates angiogenesis through a novel mechanism by counteracting c-Myc and transcriptionally repressing vascular endothelial growth factor A (VEGFA) expression. Here, we reveal that the transcriptional repressor role of SerRS is inactivated under hypoxia through phosphorylation by ataxia telangiectasia mutated (ATM) and ataxia telangiectasia mutated and RAD3-related (ATR) at Ser101 and Ser241 to attenuate its DNA binding capacity. In zebrafish, SerRSS101D/S241D, a phosphorylation-mimicry mutant, cannot suppress VEGFA expression to support normal vascular development. Moreover, expression of SerRSS101A/S241A, a phosphorylation-deficient and constitutively active mutant, prevents hypoxia-induced binding of c-Myc and HIF-1 to the VEGFA promoter, and activation of VEGFA expression. Consistently, SerRSS101A/S241A strongly inhibits normal and tumor-derived angiogenesis in mice. Therefore, we reveal a key step regulating hypoxic angiogenesis and highlight the importance of nuclear SerRS in post-developmental angiogenesis regulation in addition to vascular development. The role of nuclear SerRS in inhibiting both c-Myc and HIF-1 may provide therapeutic opportunities to correct dysregulation of angiogenesis in pathological settings.

Rational Design of Aptamer-Tagged tRNAs.

Reprogramming of the genetic code system is limited by the difficulty in creating new tRNA structures. Here, I developed translationally active tRNA variants tagged with a small hairpin RNA aptamer, using Escherichia coli reporter assay systems. As the tRNA chassis for engineering, I employed amber suppressor variants of allo-tRNAs having the 9/3 composition of the 12-base pair amino-acid acceptor branch as well as a long variable arm (V-arm). Although their V-arm is a strong binding site for seryl-tRNA synthetase (SerRS), insertion of a bulge nucleotide in the V-arm stem region prevented allo-tRNA molecules from being charged by SerRS with serine. The SerRS-rejecting allo-tRNA chassis were engineered to have another amino-acid identity of either alanine, tyrosine, or histidine. The tip of the V-arms was replaced with diverse hairpin RNA aptamers, which were recognized by their cognate proteins expressed in E. coli. A high-affinity interaction led to the sequestration of allo-tRNA molecules, while a moderate-affinity aptamer moiety recruited histidyl-tRNA synthetase variants fused with the cognate protein domain. The new design principle for tRNA-aptamer fusions will enhance radical and dynamic manipulation of the genetic code.

An orthogonal seryl-tRNA synthetase/tRNA pair for noncanonical amino acid mutagenesis in Escherichia coli.

We report the development of the orthogonal amber-suppressor pair Archaeoglobus fulgidus seryl-tRNA (Af-tRNASer)/Methanosarcina mazei seryl-tRNA synthetase (MmSerRS) in Escherichia coli. Furthermore, the crystal structure of MmSerRS was solved at 1.45 Å resolution, which should enable structure-guided engineering of its active site to genetically encode small, polar noncanonical amino acids (ncAAs).

An isoflavone derivative potently inhibits the angiogenesis and progression of triple-negative breast cancer by targeting the MTA2/SerRS/VEGFA pathway.

Objective: Angiogenesis plays a vital role in tumor growth and metastasis. Here, we aimed to find novel efficient antiangiogenic molecules targeting vascular endothelial growth factor A (VEGFA ) at the transcriptional level to treat triple-negative breast cancer (TNBC). Methods: We used a cell-based seryl tRNA synthetase (SerRS) promoter-driven dual-luciferase reporter system to screen an in-house library of 384 naturally occurring small molecules and their derivatives to find candidate molecules that could upregulate the expression of SerRS, a potent transcriptional repressor of VEGFA. The levels of SerRS and VEGFA were examined by quantitative RT-PCR (qRT-PCR), western blotting, and/or ELISAs in TNBC cells after candidate molecule administration. Zebrafish, the Matrigel plug angiogenesis assay in mice, the TNBC allograft, and xenograft mouse models were used to evaluate the in vivo anti-angiogenic and anti-cancer activities. Furthermore, the potential direct targets of the candidates were identified by proteomics and biochemical studies. Results: We found the most active compound was 3-(4-methoxyphenyl) quinolin-4(1H)-one (MEQ), an isoflavone derivative. In TNBC cells, MEQ treatment resulted in increased SerRS mRNA (P < 0.001) and protein levels and downregulated VEGFA production. Both the vascular development of zebrafish and Matrigel plug angiogenesis in mice were inhibited by MEQ. MEQ also suppressed the angiogenesis in TNBC allografts and xenografts in mice, resulting in inhibited tumor growth and prolonged overall survival (P < 0.05). Finally, we found that MEQ regulated SerRS transcription by interacting with MTA2 (Metastasis Associated 1 Family Member 2). Conclusions: Our findings suggested that the MTA2/SerRS/VEGFA axis is a drug-treatable anti-angiogenic target, and MEQ is a promising anti-tumor molecule that merits further investigation for clinical applications.

Synthesis and structural insights into the binding mode of the albomycin δ1 core and its analogues in complex with their target aminoacyl-tRNA synthetase.

Despite of proven efficacy and well tolerability, albomycin is not used clinically due to scarcity of material. Several attempts have been made to increase the production of albomycin by chemical or biochemical methods. In the current study, we have synthesized the active moiety of albomycin δ1 and investigated its binding mode to its molecular target seryl-trna synthetase (SerRS). In addition, isoleucyl and aspartyl congeners were prepared to investigate whether the albomycin scaffold can be extrapolated to target other aminoacyl-tRNA synthetases (aaRSs) from both class I and class II aaRSs, respectively. The synthesized analogues were evaluated for their ability to inhibit the corresponding aaRSs by an in vitro aminoacylation experiment using purified enzymes. It was observed that the diastereomer having the 5'S, 6'R-configuration (nucleoside numbering) as observed in the crystal structure, exhibits excellent inhibitory activity in contrast to poor activity of its companion 5'R,6'S-diasteromer obtained as byproduct during synthesis. Moreover, the albomycin core scaffold seems well tolerated for class II aaRSs inhibition compared with class I aaRSs. To understand this bias, we studied X-ray crystal structures of SerRS in complex with the albomycin δ1 core structure 14a, and AspRS in complex with compound 16a. Structural analysis clearly showed that diastereomer selectivity is attributed to the steric restraints of the active site of SerRS and AspRS.

Herb-sourced emodin inhibits angiogenesis of breast cancer by targeting VEGFA transcription.

Anti-angiogenesis is an important and promising strategy in cancer therapy. However, the current methods using anti-vascular endothelial growth factor A (VEGFA) antibodies or inhibitors targeting VEGFA receptors are not as efficient as expected partly due to their low efficiencies in blocking VEGFA signaling in vivo. Until now, there is still no method to effectively block VEGFA production in cancer cells from the very beginning, i.e., from the transcriptional level. Here, we aimed to find bioactive small molecules to block VEGFA transcription. Methods: We screened our natural compound pool containing 330 small molecules derived from Chinese traditional herbs for small molecules activating the expression of seryl-tRNA synthetase (SerRS), which is a newly identified potent transcriptional repressor of VEGFA, by a cell-based screening system in MDA-MB-231 cell line. The activities of the candidate molecules on regulating SerRS and VEGFA expression were first tested in breast cancer cells. We next investigated the antiangiogenic activity in vivo by testing the effects of candidate drugs on the vascular development in zebrafish and by matrigel plug angiogenesis assay in mice. We further examined the antitumor activities of candidate drugs in two triple-negative breast cancer (TNBC)-bearing mouse models. Furthermore, streptavidin-biotin affinity pull-down assay, coimmunoprecipitation assays, docking analysis and chromatin immunoprecipitation were performed to identify the direct targets of candidate drugs. Results: We identified emodin that could greatly increase SerRS expression in TNBC cells, consequently reducing VEGFA transcription. Emodin potently inhibited vascular development of zebrafish and blocked tumor angiogenesis in TNBC-bearing mice, greatly improving the survival. We also identified nuclear receptor corepressor 2 (NCOR2) to be the direct target of emodin. Once bound by emodin, NCOR2 got released from SerRS promoter, resulting in the activation of SerRS expression and eventually the suppression of VEGFA transcription. Conclusion: We discovered a herb-sourced small molecule emodin with the potential for the therapy of TNBC by targeting transcriptional regulators NCOR2 and SerRS to suppress VEGFA transcription and tumor angiogenesis.

Seryl-tRNA synthetase specificity for tRNASec in Bacterial Sec biosynthesis.

tRNA synthetases are responsible for decoding the molecular information, from codons to amino acids. Seryl-tRNA synthetase (SerRS), besides the five isoacceptors of tRNASer, recognizes tRNA[Ser]Sec for the incorporation of selenocysteine (Sec, U) into selenoproteins. The selenocysteine synthesis pathway is known and is dependent on several protein-protein and protein-RNA interactions. Those interactions are not fully described, in particular, involving tRNA[Ser]Sec and SerRS. Here we describe the molecular interactions between the Escherichia coli Seryl-tRNA synthetase (EcSerRS) and tRNA[Ser]Sec in order to determine their specificity, selectivity and binding order, leading to tRNA aminoacylation. The dissociation constant of EcSerRS and tRNA[Ser]Sec was determined as (126 ± 20) nM. We also demonstrate that EcSerRS binds initially to tRNA[Ser]Sec in the presence of ATP for further recognition by E. coli selenocysteine synthetase (EcSelA) for Ser to Sec conversion. The proposed studies clarify the mechanism of tRNA[Ser]Sec incorporation in Bacteria as well as of other domains of life.

ASCT2 Is Involved in SARS-Mediated ß-Casein Synthesis of Bovine Mammary Epithelial Cells with Methionine Supply.

The methionine (Met) uptake into mammary cells depends upon the corresponding amino acid (AA) transporters, which play a regulatory role in the mammary protein production beyond transport. Our previous studies have identified that seryl-tRNA synthetase (SARS) could be a novel mediator to regulate essential AA-stimulated casein synthesis in primary bovine mammary epithelial cells (BMECs). However, the regulatory mechanisms of Met in milk protein production in dairy cows remain further clarified. Here, we aimed to investigate the effects of Met on milk protein synthesis in BMECs and explore the underlying mechanism. The effects of Met on the AA transporter, casein synthesis, and the related signaling pathway were evaluated in the BMECs treated with 0.6 mM Met for 6 h combined with or without the inhibition of AA transporter (ASCT2, a neutral AA transporter) activity by the corresponding inhibitor (GPNA). Besides, the effects of SARS on the cells were mainly evaluated in the BMECs treated with 0.6 mM Met for 6 h together with or without SARS knockdown by RNAi interference. The gene expression of AA transporters and pathway-related genes were analyzed by the real-time quantitative polymerase chain reaction method, and the protein expression of related proteins were determined by the western blot assay. Results showed that 0.6 mM Met remarkably enhanced cell growth and ß-casein synthesis compared to the supply of other Met concentrations. Among 13 amino acid transporters, 0.6 mM Met highly increased ASCT2 expression. This Met-stimulated ASCT2 expression and the enhanced mammary intracellular Met uptake were both decreased by the addition of 500 µM GPNA, an inhibitor of ASCT2. In the presence of 0.6 mM Met, the inhibition of ASCT2 activity (by GPNA) and SARS expression (by RNAi) both reduced ß-casein synthesis. Additionally, 0.6 mM Met increased the gene expression of mTOR, S6K1, 4EBP1, and Akt; in contrast, the inhibition of ASCT2 by GPNA lowered the gene expression of these four genes. Collectively, this work suggests that ASCT2 is involved in the SARS-mediated Met stimulation of ß-casein synthesis through enhancing mammary Met uptake and activating the mTOR signaling pathway in BMECs.