Loss of ALK4 Function Promotes Cancer Progression
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Fusion Lumos
ORGANISM(S): Bos Taurus (ncbitaxon:9913) Homo Sapiens (ncbitaxon:9606)
SUBMITTER: 
Gerard Blobe
PROVIDER: MSV000091598 | MassIVE | Thu Mar 30 14:50:00 BST 2023
REPOSITORIES: MassIVE
Similar Datasets
Project description:Most available knowledge on fungal arginine metabolism is derived from studies on Saccharomyces cerevisiae, in which arginine catabolism is initiated by releasing urea via the arginase reaction. Orthologs of the S. cerevisiae genes encoding the first three enzymes in the arginase pathway were cloned from Kluyveromyces lactis and shown to functionally complement the corresponding deletion in S. cerevisiae. Surprisingly, deletion of the single K. lactis arginase gene KlCAR1 did not completely abolish growth on arginine as nitrogen source. Growth rate of mutant strongly increased during serial transfer in shake-flask cultures. A combination of RNAseq-based transcriptome analysis and 13C-15N-based flux analysis was used to elucidate the arginase-independent pathway. Isotopic 13C15N-enrichment in ?-aminobutyrate revealed succinate as the entry point in the TCA cycle of the alternative pathway. Transcript analysis combined with enzyme activity measurements indicated increased expression in the Klcar1? mutant of a guanidinobutyrase (EC.3.5.3.7), an enzyme not previously demonstrated in fungi. Expression of the K. lactis KLLA0F27995g (renamed KlGBU1) encoding guanidinobutyrase enabled S. cerevisiae to use guanidinobutyrate as sole nitrogen source and its deletion in K. lactis almost completely abolish growth on this nitrogen source. Phylogenetic analysis suggests that this enzyme activity is widespread in fungi. The goal of the present study was to characterize arginine catabolism in K. lactis. To this end, CAR1, CAR2 and PRO3 orthologs in K. lactis were identified and functionally analysed by deletion, expression in S. cerevisiae and enzyme activity assays. Since deletion of the arginase gene in K. lactis was found not to completely abolish growth on arginine as a sole nitrogen source, the alternative pathway for arginine catabolism operating in this yeast was studied by a combination of transcriptome analysis, 13C and 15N isotope-based flux analysis and enzyme activity assays in cell extracts. To investigate arginine metabolism in the arginase-negative K. lactis strain, strains GG1632 (Klku80? KlCAR1 reference strain) and IMS0367 (Klcar1? Arg+) were grown in aerobic bioreactor batch cultures on glucose chemically defined medium with arginine as sole nitrogen source. RNA sequencing of samples taken during the exponential phase of growth on glucose-arginine media of the reference strain G1631 and the arginase less strain IMS0367 were compared resulting in the characterization of a new function.
2014-07-16 | E-GEOD-56060 | biostudies-arrayexpress
Project description:Rabbit haemorrhagic disease virus (RHDV) belongs to the family Caliciviridae, genus Lagovirus and is used in Australia as a biocontrol tool to keep the population of european rabbits low. This virus has a positive-sense single-stranded RNA genome that encodes structural (capsid) and non-structural proteins. Due to the lack of an established cell culture system for this virus, some of the non-structural proteins are yet awaiting characterisation and their function is unknown. This work was aimed at the identification of cellular interactors of RHDV proteins p23 and RNA-dependent RNA polymerase (RdRp). p23 has an unknown function and RdRp is the main enzyme that replicates viral genome. SILAC-labeled rabbit kidney (RK13) cells were transfected with the FLAG-tagged viral proteins and GFP-transfected cells served as an internal control. For example, two replicates of heavy Arg and Lys labeled cells were transfected with protein p23 and two replicates of light Arg and Lys labeled cells were transfected with GFP. For the third replicate this was swapped: 'heavy' cells were transfected with GFP and 'light' cells were transfected with p23. After that, cells were harvested, lysed in mild conditions and applied onto anti-FLAG antibody resin for immunoprecipitation. Eluates from the resin were processed for mass spectrometry processing.
2022-10-14 | PXD031619 | Pride
Project description:Today, the most widely used bottom-up proteomics studies necessitate a proteolysis step prior to MS analysis. Trypsin is often the best protease in choice due to its high specificity and MS-favored proteolytic products. Trypsin has been challenged for its low cleavage efficiency at Lys-X bonds, and tandem Lys-C/trypsin digestion approach has been developed to alleviate the problem. However, the identification capacity of the two digestions often disagrees among different research groups. Herein, we report a new tandem digestion using Lys-C and Arg-C (Lys-C/Arg-C), which we have proven to be more efficient and specific than trypsin and Lys-C/trypsin approaches. Analysis of our previous data (Anal. Chem. 2018, 90, 1554-1559) revealed that both Lys-C and Arg-C perform better in trypsin-mode digestion. In particular for Arg-C, the identification capacity is increased to 2.6 times and even comparable with trypsin. The good complementarity, high digestion efficiency and high specificity of Lys-C and Arg-C prompt a tandem Lys-C/Arg-C digestion. We systematically evaluated the Lys-C/Arg-C digestion using qualitative and quantitative proteomics approaches and confirmed its superior performance in digestion efficiency, specificity and identification capacity to the currently widely used trypsin and Lys-C/trypsin digestions. As a result, we concluded that Lys-C/Arg-C digestion approach would be the choice of next-generation digestion approach for better results in both qualitative and quantitative proteomics studies.
2018-07-24 | PXD009797 | Pride
Project description:A long-standing question in developmental and reproductive biology is when the mammalian embryo becomes sufficiently distinct from its oocyte precursor. Myriads of studies examined the messenger RNAs that change during the oocyte-to-embryo transition, whereas proteins have been much less studied, in spite of their greater vicinity to phenotype. In the present study we modified the widely used embryo culture medium KSOM (PMID 12470333, PMID 10859270) to make it apt for our application. We replaced the serum albumin with polyvinylpyrrolidone and also replaced the natural Arginine and Lysine with their “heavy” isotopic variants Arginine 13C 15N and Lysine 13C 15N. Fertilized oocytes were retrieved from oviducts of gonadotropin-primed B6C3F1 females mated to CD1 males, and cultured at 37 degrees Celsius under 5% CO2 in KSOM containing 0.3 mM Arginine 13C 15N and 0.2 mM Lysine 13C 15N, which are the regular concentrations of these two amino acids in KSOM medium (PMID 12470333; PMID 10859270). After 4 days of culture, the embryos of the isotopic group had undergone blastocyst formation just like the control embryos cultured in normal medium. Samples of approx. 500 “heavy”-labeled blastocysts were collected zona-free and subjected to mass spectrometric analysis. The median labeling rate was 83%, ranging from 0% in proteins that did not incorporate any Arginine 13C 15N and Lysine 13C 15N, to 100% in proteins that were completely labeled. Our study demonstrates that a commonly used, chemically defined medium can be adapted for Stable Isotope Labeling by/with Amino acids in Cell culture (SILAC) and combined with high-resolution mass spectrometry, in a preimplantation embryo setting. This allows to tackle long-standing questions in developmental and reproductive biology, such as the identification of putative maternal (0% labeled), putative embryonic (100% labeled) or shared proteins in live mammalian embryos.
2023-10-19 | PXD035570 | Pride
Project description:Neurospora crassa 3838 T(I II)OY338 arg/lys Resequencing
| PRJNA346017 | ENA
Project description:Development of an improved workflow for newly synthesized proteome analysis, based on combined pulsed metabolic labelling with L-azidohomoalanine (AHA) and semi-automated click chemistry-based enrichments with magnetic alkyne agarose beads.
2023-10-19 | PXD036886 | Pride
Project description:Bottom-up proteomics approach has become an important strategy in diverse areas of biological research, and the enzymatic digestion is essential for this technology. Endopeptidase Arg-C catalyzing the hydrolytic cleavage of peptide bonds C-terminal to arginine could be an important protease in bottom-up proteomics. However, it has been seldom applied due to its low specificity and high cost. Reversible chemical derivatization of amine allows a real Arg-C digestion using trypsin. In this paper, a reversible amine derivatization method, citraconylation and decitraconylation were optimized and evaluated for large-scale proteomics studies. Combination of the reversible derivatization and trypsin digestion (termed Arg-C* digestion to distinguish from the conventional Arg-C digestion) resulted in 64.2% more peptide identification (11,925 ± 199 vs 7,262 ± 59) and significantly higher cleavage specificity (95.6% vs 73.6%) than the conventional Arg-C digestion. Comparison of Arg-C* digestion with the widely used trypsin and Lys-C digestion revealed that Arg-C* performed equally or slightly better than Lys-C although not comparable to trypsin. Therefore, the well-established Arg-C* digestion method is a promising approach for proteomics studies and could be used as the prior alternative digestion method to trypsin digestion in order to achieve higher proteome coverage.
2018-01-04 | PXD007994 | Pride
Project description:U2OS cells were split into two aliquots and cultured in one of
the following media (DMEM + 10 % dialysed FCS):
i) light medium: contains Arg-0 and Lys-0
ii) heavy medium: contains Arg-10 and Lys-8
Cells were passaged at least three times in the respective medium to ensure complete
labelling of proteins. SILAC experiments were performed in a forward and a reverse
reaction: for the forward experiment, heavy-labelled cells were irradiated with 20 Gy and
harvested 45 minutes after irradiation, while light-labelled cells were left untreated. In the
reverse experiment, labels were swapped and light-labelled cells were irradiated, while
heavy labelled cells were left untreated.
| MSV000082975 | MassIVE
Project description:We describe the preparation, evaluation, and application of an S100A12 protein-conjugated solid support, hereafter the “A12-resin,” that can remove 99% of Zn(II) from complex biological solutions without significantly perturbing the concentrations of other metal ions. The A12-resin can be applied to selectively deplete Zn(II) from diverse tissue culture media and from other biological fluids including human sera. To further demonstrate the utility of this approach, we investigated metabolic, transcriptomic, and metallomic responses of HEK293T cells cultured in medium depleted of Zn(II) using S100A12. Our data indicate that dividing cells can maintain a constant pool of free Zn(II), even under conditions of severe Zn(II) deprivation. We expect that the A12-resin will facilitate interrogation of disrupted Zn(II) homeostasis in biological settings, uncovering novel roles for Zn(II) in biology.
2018-01-18 | GSE108923 | GEO
Project description:We examine how SUMO post-translational modification of the yeast transcription factor Sko1 affects its binding site selection and affinity. Chromatin immunoprecipitation followed by next-generation sequencing was performed in strains expressing wild-type Sko1 (Sko1-WT) or Sko1 that harbors an Arg-to-Lys mutation at Lys 567 (Sko1-MT), that impairs its sumoylation. We find that, compared with Sko1-WT, SUMO-deficient Sko1 binds numerous additional sites that are near promoters of non-Sko1 target genes. Furthermore, Sko1-MT shows higher occupancy levels than Sko1-WT on most genes that are occupied by both forms. Osmotic stress causes a general rearrangement of Sko1 positions across the genome, but increased numbers of binding sites and occupancy levels are still observed for Sko1-MT after treatment with NaCl. This study indicates that sumoylation attenuates Sko1 association with chromatin and increases its binding specificity.
2019-02-04 | GSE118655 | GEO