Shortcut barcoding and early pooling for scalable multiplex single-cell reduced-representation CpG methylation sequencing at single nucleotide resolution.

DNA methylation is essential for a wide variety of biological processes, yet the development of a highly efficient and robust technology remains a challenge for routine single-cell analysis. We developed a multiplex scalable single-cell reduced representation bisulfite sequencing (msRRBS) technology. It allows cell-specific barcoded DNA fragments of individual cells to be pooled before bisulfite conversion, free of enzymatic modification or physical capture of the DNA ends, and achieves read mapping rates of 62.5 ± 3.9%, covering 60.0 ± 1.4% of CpG islands and 71.6 ± 1.6% of promoters in K562 cells. Its reproducibility is shown in duplicates of bulk cells with close to perfect correlation (R = 0.97-0.99). At a low 1 Mb of clean reads, msRRBS provides highly consistent coverage of CpG islands and promoters, outperforming the conventional methods with orders of magnitude reduction in cost. Here, we use this method to characterize the distinct methylation patterns and cellular heterogeneity of six cell lines, plus leukemia and hepatocellular carcinoma models. Taking 4 h of hands-on time, msRRBS offers a unique, highly efficient approach for dissecting methylation heterogeneity in a variety of multicellular systems.

Regulatory role of Chitinase 3-like 1 gene in papillary thyroid carcinoma proved by integration analyses of single-cell sequencing with cohort and experimental validations.

Papillary thyroid carcinoma (PTC) is one of the most common thyroid carcinomas. The gross extrathyroidal extension and extensive metastases of PTC lead to high rates of recurrence and poor clinical outcomes. However, the mechanisms underlying PTC development are poorly understood. In this study, using single-cell RNA sequencing, the transcriptome profiles of two PTC patients were addressed, including PTC1 with low malignancy and good prognosis and PTC2 with high malignancy and poor prognosis. We found that epithelial subcluster Epi02 was the most associated with the malignant development of PTC cells, with which the fold change of Chitinase 3-like 1 (CHI3L1) is on the top of the differentially expressed genes between PTC1 and PTC2 (P < 0.001). However CHI3L1 is rarely investigated in PTC as far. We then studied its role in PTC with a series of experiments. Firstly, qRT-PCR analysis of 14 PTC patients showed that the expression of CHI3L1 was positively correlated with malignancy. In addition, overexpression or silencing of CHI3L1 in TPC-1 cells, a PTC cell line, cultured in vitro showed that the proliferation, invasion, and metastasis of the cells were promoted or alleviated by CHI3L1. Further, immunohistochemistry analysis of 110 PTC cases revealed a significant relationship between CHI3L1 protein expression and PTC progression, especially the T (P < 0.001), N (P < 0.001), M stages (P = 0.007) and gross ETE (P < 0.001). Together, our results prove that CHI3L1 is a positive regulator of malignant development of PTC, and it promotes proliferation, invasion, and metastasis of PTC cells. Our study improves understanding of the molecular mechanisms underlying the progression of PTC and provides new insights for the clinical diagnosis and treatment of PTC.

Glucose promotes cell growth and casein synthesis via ATF4/Nrf2-Sestrin2- AMPK-mTORC1 pathway in dairy cow mammary epithelial cells.

In the dairy industry, glucose (Glu) is used as bioactive substance to increase milk yield. However, the molecular regulation underneath needs further clarification. Here, the regulation and its molecular mechanism of Glu on cell growth and casein synthesis of dairy cow mammary epithelial cells (DCMECs) were investigated. When Glu was added from DCMECs, both cell growth, ß-casein expression and the mechanistic target of rapamycin complex 1 (mTORC1) pathway were increased. Overexpression and silencing of mTOR revealed that Glu promoted cell growth and ß-casein expression through the mTORC1 pathway. When Glu was added from DCMECs, both Adenosine 5'-monophosphate-activated protein kinase α (AMPKα) and Sestrin2 (SESN2) expression were decreased. Overexpression and silencing of AMPKα or SESN2 uncovered that AMPKα suppressed cell growth and ß-casein synthesis through inhibiting mTORC1 pathway, and SESN2 suppressed cell growth and ß-casein synthesis through activating AMPK pathway. When Glu was depleted from DCMECs, both activating transcription factor 4 (ATF4) and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) expression were increased. Overexpression or silencing of ATF4 or Nrf2 demonstrated that Glu depletion promoted SESN2 expression through ATF4 and Nrf2. Together, these results indicate that in DCMECs, Glu promoted cell growth and casein synthesis via ATF4/Nrf2-SESN2-AMPK-mTORC1 pathway.

MustSeq, an alternative approach for multiplexible strand-specific 3' end sequencing of mRNA transcriptome confers high efficiency and practicality.

RNA-seq has been widely used to reveal the molecular mechanism of variants of life process. We have developed an alternative method, MustSeq, which generates multiple second strands along a single 1st strand cDNA by random-priming initiation, immediately after reverse transcription for each RNA extract using sample-barcoded poly-dT primers, then 3' ends-enriching PCR is applied to construct the library. Unlike the conventional RNA seq, MustSeq avoids procedures such as mRNA isolation, fragmentation and RNA 5'-end capture, enables early pooling of multiple samples, and requires only one twentieth of sequencing reads of full-length sequencing. We demonstrate the power and features of MustSeq comparing with TruSeq and NEBNext RNA-seq, two conventional full-length methods and QuantSeq, an industrial 3' end method. In cancer cell lines, the reads distribution of CDS-exon as well as genes, lncRNAs and GO terms detected by MustSeq are closer than QuantSeq to TruSeq. In mouse hepatocarcinoma and healthy livers, MustSeq enriches the same pathways as by NEBNext, and reveals the molecular profile of carcinogenesis. Overall MustSeq is a robust and accurate RNA-seq method allowing efficient library construction, sequencing and analysis, particularly valuable for analysis of differentially expressed genes with a large number of samples. MustSeq will greatly accelerate the application of bulk RNA-seq on different fields, and potentially applicable for single cell RNA-seq.

Methionine stimulates GlyRS phosphorylation via the GPR87-CDC42/Rac1-MAP3K10 signaling pathway.

Glycyl-tRNA synthetase (GlyRS) has non-canonical roles beyond aminoacylation, but the molecular mechanism is largely unknown. We have previously found that GlyRS is phosphorylated in the cytoplasm of bovine mammary epithelial cells (bMECs) in response to amino acid stimulation, and the phosphorylated GlyRS enters nucleus to stimulate gene expression for milk synthesis. In this study, we aim to uncover the upstream kinase of GlyRS and reveal the signaling pathways that methionine (Met) stimulates GlyRS phosphorylation. We show that mitogen-activated protein kinase 10 (MAP3K10) interacts with GlyRS in bMECs by Co-IP, mass spectrometry, and Western blotting analysis. We further identify that MAP3K10 is an upstream kinase of GlyRS by in vitro kinase assay and MAP3K10 stimulates NFκB1 phosphorylation via activating GlyRS. We also uncover that Met stimulates GlyRS phosphorylation via the GPR87-CDC42/Rac1-MAP3K10 signaling pathway. Our findings help to understand the molecular mechanism of GlyRS in cellular signaling transduction.

Amino acids stimulate glycyl-tRNA synthetase nuclear localization for mammalian target of rapamycin expression in bovine mammary epithelial cells.

Amino acids are required for the activation of mammalian target of rapamycin (mTOR) to increase cell growth, protein and lipid synthesis, and inhibit autophagy. However, the mechanism through which amino acids activate the mTOR signaling is still largely unknown. In our previous study, we discovered that glycyl-tRNA synthetase (GlyRS) is a key mediator of amino-acid-induced mTOR expression and activation in bovine mammary epithelial cells (BMECs). Here we show that amino acids stimulate GlyRS nuclear localization for mTOR expression in BMECs. Met stimulates GlyRS nuclear localization, and the nuclear GlyRS is cleaved into a C-terminus-containing truncated form. We prove that GlyRS has a bipartite nuclear leading sequences, and GlyRS is phosphorylated at Thr544 and Ser704 in the cytoplasm under the stimulation of amino acids (Met, Leu, and Lys). The nuclear GlyRS physically binds to nuclear factor kappa B1, triggers its phosphorylation, thereby enhancing mRNA expression of its target genes including mTOR, S6K1, and 4EBP1. We further demonstrate that GlyRS is required for the inhibition of autophagy by Met. Thus our work elucidates that amino acids trigger GlyRS phosphorylation and nuclear localization to enhance the mRNA expression of mTOR.

GlyRS is a new mediator of amino acid-induced milk synthesis in bovine mammary epithelial cells.

Amino acids are required for the mammalian target of rapamycin (mTOR) signaling pathway and milk synthesis in bovine mammary epithelial cells (BMECs). However, the mechanism through which amino acids activate this pathway is largely unknown. Here we show that glycyl-tRNA synthetase (GlyRS) mediates amino acid-induced activation of the mTOR-S6K1/4EBP1 pathway, and milk protein and fat synthesis in BMECs. Among 19 aminoacyl-tRNA synthetases, only the mRNA expression of GlyRS and Leucyl-tRNA synthetase (LeuRS) were significantly increased by several amino acids including Met and Leu. We then observed that GlyRS knockdown abolished the stimulation of Met on milk protein and fat synthesis in BMECs, whereas GlyRS overexpression led to more significantly increased milk synthesis in cells treated with Met. By western blotting and qualitative real time-polymerase chain reaction analysis (qRT-PCR) analysis, we next revealed that GlyRS is required for amino acid-induced activation of the mTOR-S6K1/4EBP1 pathway. Thus, this study establishes that GlyRS mediates amino acid-induced activation of the mTOR pathway, thereby regulating milk protein and fat synthesis.

Transcriptional and Proteomic Analysis Revealed a Synergistic Effect of Aflatoxin M1 and Ochratoxin A Mycotoxins on the Intestinal Epithelial Integrity of Differentiated Human Caco-2 Cells.

Aflatoxin M1 (AFM1) is a common mycotoxin in dairy milk, and it is typically concurrently present with other mycotoxins that may represent a threat to food safety. However, knowledge of how AFM1, alone or in combination with other mycotoxins, may affect human intestinal epithelial integrity remain to be established. We employed transcriptome and proteome analysis integrated with biological validation to reveal the molecular basis underlining the effect of exposure to AFM1, ochratoxin A (OTA), or both on the intestinal epithelial integrity of differentiated Caco-2 cells. Exposure to 4 µg/mL of OTA was found to disrupt human gut epithelial integrity, whereas 4 µg/mL of AFM1 did not. The integrated transcriptome and proteome analysis of AFM1 and OTA, alone or in combination, indicate the synergistic effect of the two mycotoxins in disrupting intestinal integrity. This effect was mechanistically linked to a broad range of pathways related to intestinal integrity enriched by down-regulated genes and proteins, associated with focal adhesion, adheren junction, and gap junction pathways. Furthermore, the cross-omics analysis of mixed AFM1 and OTA compared to OTA alone suggest that kinase family members, including myosin light-chain kinase, mitogen-activated protein kinases, and protein kinase C, are the potential key regulators in modulating intestinal epithelial integrity. These findings provide novel insight into the synergistic detrimental role of multiple mycotoxins in disrupting intestinal integrity and, therefore, identify potential targets to improve milk safety related to human health.

Tudor-SN Regulates Milk Synthesis and Proliferation of Bovine Mammary Epithelial Cells.

Tudor staphylococcal nuclease (Tudor-SN) is a highly conserved and ubiquitously expressed multifunctional protein, related to multiple and diverse cell type- and species-specific cellular processes. Studies have shown that Tudor-SN is mainly expressed in secretory cells, however knowledge of its role is limited. In our previous work, we found that the protein level of Tudor-SN was upregulated in the nucleus of bovine mammary epithelial cells (BMEC). In this study, we assessed the role of Tudor-SN in milk synthesis and cell proliferation of BMEC. We exploited gene overexpression and silencing methods, and found that Tudor-SN positively regulates milk synthesis and proliferation via Stat5a activation. Both amino acids (methionine) and estrogen triggered NFκB1 to bind to the gene promoters of Tudor-SN and Stat5a, and this enhanced the protein level and nuclear localization of Tudor-SN and p-Stat5a. Taken together, these results suggest the key role of Tudor-SN in the transcriptional regulation of milk synthesis and proliferation of BMEC under the stimulation of amino acids and hormones.

Molecular network including eIF1AX, RPS7, and 14-3-3γ regulates protein translation and cell proliferation in bovine mammary epithelial cells.

14-3-3γ, an isoform of the 14-3-3 protein family, was proved to be a positive regulator of mTOR pathway. Here, we analyzed the function of 14-3-3γ in protein synthesis using bovine mammary epithelial cells (BMECs). We found that 14-3-3γ interacted with eIF1AX and RPS7 by 14-3-3γ coimmunoprecipitation (CoIP) and matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight (MALDI-TOF/TOF) peptide mass fingerprinting analysis. These interactions of 14-3-3γ with eIF1AX and RPS7 were further confirmed by colocalization and fluorescence resonance energy transfer (FRET) analysis. We also found that methionine could promote protein synthesis and trigger the protein expression levels of 14-3-3γ, eIF1AX and RPS7. Analysis of overexpression and inhibition of 14-3-3γ confirmed that it positively affected the protein expression levels of eIF1AX, RPS7, Stat5 and mTOR pathway to promote protein synthesis and cell proliferation in BMECs. We further showed that overexpression of eIF1AX and RPS7 also triggered protein translation and cell proliferation. From these results, we conclude that molecular network including eIF1AX, RPS7, and 14-3-3γ regulates protein translation and cell proliferation in BMECs.

GSK3ß regulates milk synthesis in and proliferation of dairy cow mammary epithelial cells via the mTOR/S6K1 signaling pathway.

Glycogen synthase kinase 3 (GSK3) is a serine/threonine kinase, whose activity is inhibited by AKT phosphorylation. This inhibitory phosphorylation of GSK3ß can in turn play a regulatory role through phosphorylation of several proteins (such as mTOR, elF2B) to promote protein synthesis. mTOR is a key regulator in protein synthesis and cell proliferation, and recent studies have shown that both GSK3ß and mTORC1 can regulate SREBP1 to promote fat synthesis. Thus far, however, the cross talk between GSK3ß and the mTOR pathway in the regulation of milk synthesis and associated cell proliferation is not well understood. In this study the interrelationship between GSK3ß and the mTOR/S6K1 signaling pathway leading to milk synthesis and proliferation of dairy cow mammary epithelial cells (DCMECs) was analyzed using techniques including GSK3ß overexpression by transfection, GSK3ß inhibition, mTOR inhibition and methionine stimulation. The analyses revealed that GSK3ß represses the mTOR/S6K1 pathway leading to milk synthesis and cell proliferation of DCMECs, whereas GSK3ß phosphorylation enhances this pathway. Conversely, the activated mTOR/S6K1 signaling pathway downregulates GSK3ß expression but enhances GSK3ß phosphorylation to increase milk synthesis and cell proliferation, whereas inhibition of mTOR leads to upregulation of GSK3ß and repression of GSK3ß phosphorylation, which in turn decreases milk synthesis, and cell proliferation. These findings indicate that GSK3ß and phosphorylated GSK3ß regulate milk synthesis and proliferation of DCMECs via the mTOR/S6K1 signaling pathway. These findings provide new insight into the mechanisms of milk synthesis.

Impact of genomic and epigenomic alterations of multigene on a multicancer pedigree.

BACKGROUND: Germline mutations have been identified in a small number of hereditary cancers, but the genetic predisposition for many familial cancers remains to be elucidated. METHODS: This study identified a Chinese pedigree that presented different cancers (breast cancer, BRCA; adenocarcinoma of the esophagogastric junction, AEG; and B-cell acute lymphoblastic leukemia, B-ALL) in each of the three generations. Whole-genome sequencing and whole-exome sequencing were performed on peripheral blood or bone marrow and cancer biopsy samples. Whole-genome bisulfite sequencing was conducted on the monozygotic twin brothers, one of whom developed B-ALL. RESULTS: According to the ACMG guidelines, bioinformatic analysis of the genome sequencing revealed 20 germline mutations, particularly mutations in the DNAH11 (c.9463G > A) and CFH (c.2314G > A) genes that were documented in the COSMIC database and validated by Sanger sequencing. Forty-one common somatic mutated genes were identified in the cancer samples, displaying the same type of single nucleotide substitution Signature 5. Meanwhile, hypomethylation of PLEK2, MRAS, and RXRA as well as hypermethylation of CpG island associated with WT1 was shown in the twin with B-ALL. CONCLUSIONS: These findings reveal genomic alterations in a pedigree with multiple cancers. Mutations found in the DNAH11, CFH genes, and other genes predispose to malignancies in this family. Dysregulated methylation of WT1, PLEK2, MRAS, and RXRA in the twin with B-ALL increases cancer susceptibility. The similarity of the somatic genetic changes among the three cancers indicates a hereditary impact on the pedigree. These familial cancers with germline and somatic mutations, as well as epigenomic alterations, represent a common molecular basis for many multiple cancer pedigrees.

Goat mammary gland expression of Cecropin B to inhibit bacterial pathogens causing mastitis.

The antibacterial peptide Cecropin B (CB), isolated from the giant silk moth, has been shown to effectively eliminate bacteria. In this study, the effects of transgenic CB on dairy goat mammary epithelial cells (DGMECs) and dairy goat mammary gland were investigated. The DNA of CB from silkworm was amplified by reverse transcription PCR (RT-PCR) and then fused to the eukaryotic expression vector pECFP-C1. The recombinant plasmid pECFP-Cecropin B (pECFP-CB) was used for the transfection of DGMECs, and the expression of transgenic CB and the antibacterial activity of it were confirmed by western blot and agar diffusion reaction respectively. The stable DGMEC line transfected by pECFP-CB was obtained by screening with G418. In vivo experiment, pECFP-CB was injected into dairy goat mammary gland, and also the expression and antibacterial activity of transgenic CB were confirmed. Results of this study: transgenic CB can be expressed in DGMECs and dairy goat mammary gland, and inhibit the mastitis caused by Staphylococcus aureus.

SOCS3-mediated blockade reveals major contribution of JAK2/STAT5 signaling pathway to lactation and proliferation of dairy cow mammary epithelial cells in vitro.

Suppressor of cytokine signaling 3 (SOCS3) is a cytokine-induced negative feedback-loop regulator of cytokine signaling. More and more evidence has proved it to be an inhibitor of signal transducers and activators of transcription 5 (STAT5). Here, we used dairy cow mammary epithelial cells (DCMECs) to analyze the function of SOCS3 and the interaction between SOCS3 and STAT5a. The expression of SOCS3 was found in cytoplasm and nucleus of DCMECs by fluorescent immunostaining. Overexpression and inhibition of SOCS3 brought a remarkable milk protein synthesis change through the regulation of JAK2/STAT5a pathway activity, and SOCS3 expression also decreased SREBP-1c expression and fatty acid synthesis. Inhibited STAT5a activation correlated with reduced SOCS3 expression, which indicated that SOCS3 gene might be one of the targets of STAT5a activation, DCMECs treated with L-methionine (Met) resulted in a decrease of SOCS3 expression. SOCS3 could also decrease cell proliferation and viability by CASY-TT detection. Together, our findings indicate that SOCS3 acts as an inhibitor of JAK2/STAT5a pathway and disturbs fatty acid synthesis by decreasing SREBP-1c expression, which validates its involvement in both milk protein synthesis and fat synthesis. In aggregate, these results reveal that low SOCS3 expression is required for milk synthesis and proliferation of DCMECs in vitro.

Sodium acetate promotes fat synthesis by suppressing TATA element modulatory factor 1 in bovine mammary epithelial cells.

Short-chain fatty acids are important nutrients that regulate milk fat synthesis. They regulate milk synthesis via the sterol regulatory element binding protein 1 (SREBP1) pathway; however, the details are still unknown. Here, the regulation and mechanism of sodium acetate (SA) in milk fat synthesis in bovine mammary epithelial cells (BMECs) were assessed. BMECs were treated with SA supplementation (SA+) or without SA supplementation (SA-), and milk fat synthesis and activation of the SREBP1 pathway were increased (P = 0.0045; P = 0.0042) by SA+ and decreased (P = 0.0068; P = 0.0031) by SA-, respectively. Overexpression or inhibition of SREBP1 demonstrated that SA promoted milk fat synthesis (P = 0.0045) via the SREBP1 pathway. Overexpression or inhibition of TATA element modulatory factor 1 (TMF1) demonstrated that TMF1 suppressed activation of the SREBP1 pathway (P = 0.0001) and milk fat synthesis (P = 0.0022) activated by SA+. Overexpression or inhibition of TMF1 and SREBP1 showed that TMF1 suppressed milk fat synthesis (P = 0.0073) through the SREBP1 pathway. Coimmunoprecipitation analysis revealed that TMF1 interacted with SREBP1 in the cytoplasm and suppressed the nuclear localization of SREBP1 (P = 0.0066). The absence or presence of SA demonstrated that SA inhibited the expression of TMF1 (P = 0.0002) and the interaction between TMF1 and SREBP1 (P = 0.0001). Collectively, our research suggested that TMF1 was a new negative regulator of milk fat synthesis. In BMECs, SA promoted the SREBP1 pathway and milk fat synthesis by suppressing TMF1. This study enhances the current understanding of the regulation of milk fat synthesis and provides new scientific data for the regulation of milk fat synthesis.

Glutamine Regulates Cell Growth and Casein Synthesis through the CYTHs/ARFGAP1-Arf1-mTORC1 Pathway in Bovine Mammary Epithelial Cells.

In the dairy industry, glutamine (Gln) is often used as a feed additive to increase milk yield and quality; however, the molecular regulation underneath needs further clarification. Here, with bovine mammary epithelial cells (BMECs), the effects and mechanisms of Gln on cell growth and casein synthesis were assessed. When Gln was added or depleted from BMECs, both cell growth and ß-casein (CSN2) expression were increased or decreased, respectively. Overexpressing or inhibiting the mechanistic target of rapamycin (mTOR) revealed that Gln regulated cell growth and CSN2 synthesis through the mTORC1 pathway. A similar intervention of ADP-ribosylation factor 1 (Arf1) uncovered that Gln activated the mTORC1 pathway through Arf1. We next observed that both guanine nucleotide exchange factors, Cytohesin-1/2/3 (CYTH1/2/3, CYTHs) and ADP-ribosylation factor GTPase activating protein 1 (ARFGAP1), interacted with Arf1. Inhibiting CYTHs or ARFGAP1 showed that Gln supplement or depletion activated or inactivated Arf1 through CYTHs or ARFGAP1, respectively. Collectively, this study demonstrated that Gln positively regulated cell growth and casein synthesis in BMECs, which works through the CYTHs/ARFGAP1-Arf1-mTORC1 pathway. These results greatly enhanced current understanding regarding the regulation of the mTOR pathway and provided new insights for the processes of cell growth and casein synthesis by amino acids, particularly Gln.

Efficiency of Traditional Chinese medicine targeting the Nrf2/HO-1 signaling pathway.

Cardiovascular disease (CVD) is a significant cause of death worldwide. Because of its major individual differences in genetic background, pathogenesis, and disease progression pattern, the mortality risk rate remains high following conventional Western medicine diagnosis under current guidelines. Traditional Chinese medicine (TCM) has important multi-target, multi-pathway, and multi-layer benefits that can effectively address western medicine deficiencies. It was therefore commonly used in CVD diagnosis. Oxidative stress is also one of the main factors of CVD. Likewise, this main reaction regulator is the nuclear factor erythroid-2-related (Nrf2) factor. When activated, it can be transferred to the nucleus and initiated in the downstream pathway, thus playing an anti-oxidant stress role. As one of the most crucial endogenous protection systems in the body, Nrf2-related / heme oxygenase 1 (Nrf2/HO-1) signaling pathway is Nrf2's most classic approach to playing roles. Recently, various advances have been made to research and explain TCM by manipulating this pathway to treat CVD using modern molecular biology and other approaches. This analysis summarized the relationship between Nrf2/HO-1 signaling route, CVD and TCM. Further, Autodock calculation was also conducted to determine the binding amino acid on this TCM to Nrf2 and HO-1.

Sestrin2 Negatively Regulates Casein Synthesis through the SH3BP4-mTORC1 Pathway in Response to AA Depletion or Supplementation in Cow Mammary Epithelial Cells.

Sestrin2 (SESN2) negatively regulates the mammalian target of rapamycin complex 1 (mTORC1) pathway and casein synthesis in response to amino acid (AA) depletion in cow mammary epithelial cells (CMECs); however, the underlying mechanism is unclear. In the current study, the regulation of SESN2 on AA-mediated ß-casein (CSN2) synthesis in CMECs and its mechanism were investigated. Overexpression and silencing of SESN2 demonstrated that SESN2 negatively regulated AA-mediated expression of CSN2 and mTORC1 pathway. Co-immunoprecipitation analysis showed that SESN2 interacted with SH3 domain-binding protein 4 (SH3BP4). Overexpression and silencing of SH3BP4 demonstrated that SH3BP4 negatively regulated AA-mediated expression of CSN2 and mTORC1 pathway and that SESN2 negatively regulated expression of CSN2 and mTORC1 pathway through the SH3BP4 in the presence and absence of AA. The absence or presence of AA demonstrated that AA negatively regulated expression and nuclear localization of activating transcription factor 4 (ATF4). Overexpression and silencing of ATF4 demonstrated that AA negatively regulated SESN2 expression through ATF4. Together, these results indicate that SESN2 negatively regulates the mTORC1 pathway and subsequent CSN2 synthesis through the SH3BP4 in response to AA absence or presence in CMECs.

Proteomic Analysis of Lysosomal Membrane Proteins in Bovine Mammary Epithelial Cells Illuminates Potential Novel Lysosome Functions in Lactation.

Lactation of bovine mammary epithelial cells (BMEC) is a complex biological process that involves in various organelles. Studies have shown that lysosome and lysosomal membrane proteins (LMP) plays an important role in lactation of BMEC. But the LMP of BMEC remains poorly understood. To obtain a global view of the LMP of BMEC and the affect of lysosome on lactation, the LMP of BMEC was identified using sequential windowed acquisition of all theoretical mass spectra (LC-SWATH/MS). 1214 LMP were identified and 559 were reported to be localized on lysosomal membrane for the first time in BMEC. Gene ontology annotation of these identified proteins showed that both previously reported casein synthesis-related LMP, such as LAMTOR1, 2, 3, and rRagC, and newly identified casein and milk fat synthesis-related LMP, such as EIF4E and ACAA1, were found. KEGG pathway analysis of these identified proteins showed that some pathways involved in lactation, such as PI3K-Akt, mTOR, insulin, PPAR, and JAK-STAT pathway, were found. The lysosomal location of five proteins (PRKCA, EIF4E, ACAA1, HRAS, and THBS1) was analyzed by laser confocal microscopy, and all five were associated with the lysosomal membrane. These findings help to elucidate lysosome functions in the regulation of lactation. The results implicate lysosomes as important organelles in regulation of lactation of BMEC that have been previously undervalued.

Proteomic analyses reveal GNG12 regulates cell growth and casein synthesis by activating the Leu-mediated mTORC1 signaling pathway.

In cow mammary epithelial cells (CMECs), cell growth and casein synthesis are regulated by amino acids (AAs), and lysosomes are important organelles in this regulatory process, but the mechanisms remain unclear. Herein, lysosomal membrane proteins (LMPs) in CMECs in the presence (Leu+) and absence (Leu-) of leucine were quantitatively analysed using Sequential Windowed Acquisition of All Theoretical Fragment Ion (SWATH) mass spectrometry. In identified LMPs, Guanine nucleotide-binding protein subunit gamma-12 (GNG12) was a markedly up-regulated protein in Leu+ group. CMECs were treated with Leu+ or Leu-, expression and lysosomal localization of GNG12 were decreased in response to Leu absence. Overexpressing or inhibiting GNG12 demonstrated that cell growth, casein synthesis and activation of the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway were all up-regulated by GNG12. Cell growth, casein synthesis and mTORC1 signaling pathway were decreased in response to Leu absence, but these decreases were partially restored by GNG12 overexpression, and those effects were partially reversed by inhibiting GNG12. Co-immunoprecipitation analysis showed that GNG12 activates the mTORC1 pathway via interaction with Ragulator. Taken together, these results suggest that GNG12 is a positive regulator of the Leu-mediated mTORC1 signaling pathway in CMECs that promotes cell growth and casein synthesis.