Ectopic expression of a deletion variant of cystathionine gamma-synthase 2 (GmCGS2) in soybean increases expression of genes encoding sulfur amino acid-rich proteins
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PROVIDER: PRJNA1284234 | ENA |
REPOSITORIES: ENA
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Project description:A deletion variant of GmCGS2 was ectopically expressed in soybean with a diverse range of expression levels and patterns. A combination of a soybean ubiquitin promoter and an in-frame deletion in GmCGS2, from K66 to S143, resulted in increased seed Met and Cys contents without incurring a yield penalty in a ten-location field trial. Transgenic events of this construct had increases of 41 to 114%, 10 to 12%, and 4.7 to 7.3% in free Met, total Cys, and total Met, respectively. RNAseq analysis of developing seed revealed increased transcript for three Cys-rich Bowman-Birk protease inhibitor (BBI) genes, and for two Met-rich 2S albumin genes, with little effect apparent on genes of primary sulfur metabolism. Trypsin-agarose chromatography, protease inhibition assays, and mass spectrometry analysis revealed increased BBI and 2S albumin proteins. The additional BBI protein was determined to be 1.5 to 1.8% of the protein in transgenic seed, sufficient to account for the increased Cys content. The observation that overexpressing an enzyme involved in Met biosynthesis that used Cys as a substrate still increased total Cys more than total Met suggested a lack of native Met-rich soybean proteins capable of incorporating the additional free Met.
2025-08-30 | GSE301182 | GEO
Project description:Cystathionine Beta Synthase (CBS), an enzyme that utilizes a methionine precursor to generate glutathione (GSSH), has been implicated in the mitigation of ROS production in COPD lung. Additionally, post-translational modification analysis on mouse and human cells treated with cigarette smoke showed the unique presence of mono- and di-methylated histone H3 lysine 27 that was not seen in controls, suggesting that cigarette smoke could lead to loss of Polycomb Repressive Complex 2 (PRC2) activity. To elucidate CBS as a potential upstream target for PRC2 inhibition, we overexpressed CBS in Human Bronchial Epithelial Cells (HBECs), grew them as 2D cultures, then conducted bulk RNA seq analysis.
2022-06-30 | GSE183858 | GEO
Project description:Cystathionine beta synthase (CBS) Over expression vs PLenti6 Expression Vector Human Bronchial Epithelial Cells (HBEC)
| PRJNA762118 | ENA
Project description:Ectopic ATP synthase is a functional onco-protein increases cell proliferation when transported to plasma membrane of cancer cells. Our previous study performed large scale gene silencing screening indicated ER and mitochondrial transport pathways may lead to ectopic ATP synthase expression. Silencing dynamin-related protein 1 (Drp1), mitofusin-1 (Mfn1) and Parkin affected ectopic ATP synthases expression. However, the underlying trafficking mechanism is poorly understood. Here, we analyzed our membrane and mitochondrial proteome of lung cancer A549 cells and found that both nuclear-encoded ATP synthase subunits and mitochondrial-encoded components-ATP6 translocated to cell surface, indicating that ATP synthase subunits assembled in mitochondria. Furthermore, serum starvation enhanced ATP synthase translocation to plasma membrane, Mdivi-1, a chemical inhibitor of the mitochondrial fission protein Drp1, rescued the phenomena. Additionally, image quantification of mitochondria, showing that mitochondrial fission preference cells expressed more eATP synthase. Therefore, we proposed that eATP synthase trafficking may be related to mitochondrial dynamics. Additionally, ICC and flow cytometry revealed the expression of a critical transcription factor associated with high-risk neuroblastoma, MYCN, correlated with eATP synthase expression. To better understand whether MYCN mediated mitochondrial fission and affected ATP synthase trafficking, we first analyzed MYCN ChIP-sequencing data and found Drp1, Mfns and Parkin possessed the consensus DNA-binding motif of MYCN. Further high-resolution image analysis showed higher mitochondrial fission and eATP synthase expression in MYCN-amplified neuroblastoma. Last, silencing MYCN reduced the fission level by detecting DRP1. In summary, we suggest that trafficking of ectopic ATP synthase may via mitochondrial dynamics.
2023-05-10 | PXD007036 | Pride
Project description:Abstract Purpose: Cystathionine ?-synthase (CBS), a key enzyme in the transsulfuration metabolic pathway, converts homocysteine to cystathionine, which is converted to cysteine required for the synthesis of major retinal antioxidant glutathione (GSH). Enzyme activity assays suggest that CBS is present in human and pig retina, however recent studies reported that CBS is not expressed in mouse retina. We found this species difference puzzling. Given the plethora of studies using mouse retina as a model system, coupled with the importance of GSH in retina, we investigated CBS expression in mouse retina at the molecular and cell biological level.Wildtype (WT) mice or mice lacking the gene encoding CBS (cbs(-)(/)(-)) were used in these studies. RNA and protein were isolated from retinas and liver (positive control) for the analysis of cbs gene expression by RT-PCR and CBS protein expression by Western blotting, respectively. CBS was analyzed by immunofluorescence in retinal cryosections and primary retinal cells (ganglion, Müller, retinal pigment epithelial). CBS enzyme activity was measured in primary Müller cells.RT-PCR revealed robust cbs expression in WT liver, brain and retina. Western blotting detected CBS in retina, brain and liver of WT mice, but not in cbs(-)(/)(-) mice liver. In immunohistochemical studies, CBS was present abundantly in the ganglion cell layer of retina; it was detected also in primary isolations of Müller, RPE and ganglion cells. CBS activity was detected in Müller cells by fluorescent detection of H2S.We have compelling molecular evidence that CBS is expressed in mouse retina at the gene and protein level. Our immunofluorescence data suggest that it is present in several retinal cell types and the data from the enzyme activity assay suggest activity in Müller cells. These findings set the stage to investigate the role of CBS and the transsulfuration pathway in the generation of GSH in mouse retina.
| S-EPMC3731150 | biostudies-literature
Project description:Ectopic adenosine triphosphate synthase (eATP synthase) was found on the cell surface to generate extracellular ATP in various cancer cells. Recently using proteomics approach, several reports identified ATP synthase in extracellular vesicles (EVs) which are important in cell-cell communications. However, the functions of ATP synthase in EVs are still unclear. Here we increased the expression of eATP synthase on the plasma membrane of lung cancer A549 cells via 0.1% FBS starvation and collected EVs including microvesicles and exosomes from the culture medium using serial centrifugation. Both of them were assuredly isolated and confirmed by transmission electron microscopy, nano tracking analysis, and specific EV markers. We also used western blot to demonstrate the presence of ATP synthase in EVs. Besides, dot blot experiment revealed that the F1 portion faced towards extracellular space using anti-ATP beta subunit antibody. Under starvation, the expression of eATP synthase was enhanced on cell surface and exosomes as well as increased EV secretion. To further investigate the functions, we first elucidated whether eATP synthase on EV membrane produced ATP by ATP luciferase analysis. Our data showed that there was no significant ATP production. Moreover, we performed proteomic analysis using dimethyl labeling and LC-MS/MS to compare the difference between EVs released by A549 control and starvation. We identified a total of 1504 and 869 proteins in microvesicles and exosomes, respectively. Using 1.5- and 0.67-fold change as the thresholds, there were 171 and 46 differential expressed proteins in microvesicles and exosomes, respectively. Seven ATP synthase subunits in microvesicles and five in exosomes were identified. The proteomic data were further analyzed using gene set enrichment analysis (GSEA) within the C2 (curated gene sets) subclasses and setting 0.25 as FDR cutoff to find out biological functions related to eATP synthase. Among these up-regulated terms, metabolism, cell cycle and apoptosis were enriched, which indicating that starvation influences cell behavior. Additionally, the immune response is upregulated strikingly in exosomes within C2 gene set. One of these immune response pathways, MHC-1 antigen presentation, was previously proved to interact with eATP synthase which could be recognized by several immune cells.
2023-05-22 | PXD014995 | Pride
Project description:Cystathionine-β-Synthase Promotes Colon Carcinogenesis
| PRJNA356616 | ENA
Project description:BackgroundCystathionine β-lyase performs an essential role in the transsulfuration pathway by its primary reaction of forming homocysteine from cystathionine. Understanding how the Neurospora crassa met-2⁺ gene, which encodes cystathionine β-lyase, is regulated is important in determining the basis of the cellular control of transsulfuration. The aim of this study was to determine the nature of a potential regulatory connection of met-2⁺ to the Neurospora sulfur regulatory network.FindingsThe cystathionine β-lyase (met-2⁺) gene was cloned by the identification of a cosmid genomic clone capable of transforming a met-2 mutant to methionine prototrophy and subsequently characterized. The gene contains a single intron and encodes a protein of 457 amino acids with conserved residues predicted to be important for catalysis and pyridoxal-5'-phosphate co-factor binding. The expression of met-2⁺ in wild-type N. crassa increased 3.1-fold under sulfur-limiting growth conditions as compared to the transcript levels seen under high sulfur growth conditions (i.e., repressing conditions). In a Δcys-3 strain, met-2⁺ transcript levels were substantially reduced under either low- or high-sulfur growth conditions. In addition, the presence of CYS3 activator binding sites on the met-2⁺ promoter was demonstrated by gel mobility shift assays.ConclusionsIn this report, we demonstrate the sulfur-regulated expression of the met-2⁺ gene and confirm its connection to the N. crassa sulfur regulatory circuit by the reduced expression observed in a Δcys-3 mutant and the in vitro detection of CYS3 binding sites in the met-2⁺ promoter. The data further adds to our understanding of the regulatory dynamics of transsulfuration.
| S-EPMC3716945 | biostudies-literature
Project description:Adenosine triphosphate (ATP) synthase is located on the inner membrane of mitochondria which generate ATP for various cellular processes. Our previous studies showed that ATP synthases existed on plasma membrane of several types of cancer cells, which was defined as ectopic ATP synthase. However, the trafficking pathway of ectopic ATP synthase is still unclear. To investigate how ATP synthase subunits are involved in the trafficking process, we performed shot-gun proteomic analysis to reveal the plasma membrane proteins of A549 lung cancer cells and identified four subunits of ATP synthases, ATP5A1, ATP5B, ATP5H, and MT-ATP6. With the results of gene set enrichment analysis, we inferred that ectopic ATP synthase subunits are assembled as complex consistently and then translocated to cell surface through the trafficking of mitochondria. On the other hand, microtubules are involved in many intracellular transport. During protein trafficking, mitochondria and vesicles are transported to their final destination along microtubules by motor proteins such as kinesins and dyneins. To explore the function of microtubules in the trafficking of ectopic ATP synthase, we treated MCF-7 breast and A549 lung cancer cells with nocodazole, a microtubule depolymerization agent, and found that the expression level of ectopic ATP synthase decreased using both immunocytochemistry and flow cytometry. Besides, since kinesin family member 5B (KIF5B) is an important motor proteins involved in the transport of mitochondria, we further performed the immunoprecipitation of KIF5B followed by liquid chromatography−tandem mass spectrometry (LC−MS/MS). The result revealed that one of the mitochondrial outer membrane proteins, dynamin-1 like proteins (Drp1), was able to associate to KIF5B. From our recent research results and other reports show that Drp1 is a mediator of mitochondrial dynamic through regulation of mitochondrial fission. Taken together, our findings suggested that Drp1-KIF5B complex-mediated mitochondrial trafficking along microtubules may play a critical role in the transport of ectopic ATP synthase
2023-05-10 | PXD006791 | Pride
Project description:UnlabelledCystathionine β-synthase (CBS) catalyzes metabolic reactions that convert homocysteine to cystathionine. To assess the role of CBS in human glioma, cells were stably transfected with lentiviral vectors encoding shRNA targeting CBS or a nontargeting control shRNA, and subclones were injected into immunodeficient mice. Interestingly, decreased CBS expression did not affect proliferation in vitro but decreased the latency period before rapid tumor xenograft growth after subcutaneous injection and increased tumor incidence and volume following orthotopic implantation into the caudate-putamen. In soft-agar colony formation assays, CBS knockdown subclones displayed increased anchorage-independent growth. Molecular analysis revealed that CBS knockdown subclones expressed higher basal levels of the transcriptional activator hypoxia-inducible factor 2α (HIF2α/EPAS1). HIF2α knockdown counteracted the effect of CBS knockdown on anchorage-independent growth. Bioinformatic analysis of mRNA expression data from human glioma specimens revealed a significant association between low expression of CBS mRNA and high expression of angiopoietin-like 4 (ANGPTL4) and VEGF transcripts, which are HIF2 target gene products that were also increased in CBS knockdown subclones. These results suggest that decreased CBS expression in glioma increases HIF2α protein levels and HIF2 target gene expression, which promotes glioma tumor formation.ImplicationsCBS loss-of-function promotes glioma growth.
| S-EPMC4274393 | biostudies-literature