Project description:Metabolic reprogramming is a hallmark of cancer. However, mechanisms underlying metabolic reprogramming and how altered metabolism in turn enhances tumorigenicity are poorly understood. Here, we report that arginine levels are elevated in hepatocellular carcinoma (HCC) despite reduced expression of arginine synthesis genes, in murine and patient tumors. Tumor cells accumulate high levels of arginine due to increased uptake and reduced arginine-to-polyamine conversion. Importantly, the high levels of arginine promote tumor formation via further metabolic reprogramming, including changes in glucose, amino acid, nucleotide, and fatty acid metabolism. Mechanistically, arginine binds RNA-binding motif protein 39 (RBM39) to control expression of metabolic genes. RBM39-mediated upregulation of asparagine synthesis leads to enhanced arginine uptake, creating a positive feedback loop to sustain high arginine levels and oncogenic metabolism. Thus, arginine is a second messenger-like molecule that reprograms metabolism to promote tumor growth.
Project description:The overall goal of this study was to identify methylated arginine residues on the Trypanosoma brucei RNA binding protein, ZFP3, and determine their functions in transcriptome regulation. We expressed Ty-tagged ZFP3 in procyclic form T. brucei, immunoprecipitated triplicate samples with anti-Ty antibodies, and subjected these samples to mass spectrometry to identify posttranslational modifications. We identified a highly methylated RGG domain, abundant R methylation at R109 in an unexpected context, and abundant tyrosine phosphorylation at Y4. To identify which protein arginine methyltransferases catalyzed these R methylation events, we performed in vitro methylation assays with TbPRMT1, TbPRMT6, and TbPRMT7 along with wild type and mutant ZFP3 substrates. These experiments showed that TbPRMT1 and TbPRMT7 methylate the RGG domain, while TbPRMT6 modifies R109. To ask how R methylations within affect ZFP3 transcriptome regulation, we overexpressed WT-ZFP3-Ty, R68,71K-ZFP3-Ty (hypomethylated) or R68,71F-ZFP3-Ty (methylmimic) in procyclic T. brucei. Overerexpression of WT-ZFP3-Ty lead to an increased expression of 749 tranascripts and decreased expression of 169 transcripts (1.5-fold change; padj<0.05). Overexpression of hypomethylated or methylmimic ZFP3 nearly abolished the capacity of ZFP3 to regulate three transcriptome, leading to changes in the abundances of less than 10 transcripts in either cell line. From these data, we conclude that arginine residues in the ZFP3 RGG domain are critical for its gene regulatory function.
Project description:Arginine utilization in Pseudomonas aeruginosa with multiple catabolic pathways represents one of the best examples of metabolic versatility of this organism. To identify genes of this complex arginine network, we employed DNA microarray to analyze the transcriptional profiles of this organism in response to L-arginine. While most genes in arginine uptake, regulation and metabolism have been identified as members of the ArgR regulon in our previous study, eighteen putative transcriptional units of 38 genes including the two known genes of the arginine dehydrogenase (ADH) pathway, kauB and gbuA, were found inducible by exogenous L-arginine but independent of ArgR. We conducted three independent microarray experiments in the presence (experimental) of L-Glutamate or D-Arginine. P. aeruginosa PAO1 was grown aerobically in minimal medium P with 300 rpm shaking at 37°C, in the presence of L-Glu with or without the addition of D-Arg at 20 mM.
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.
Project description:submission for the paper "Systems level profiling of arginine starvation in ASS1 negative sarcomas reveals ERK and MYC adaptive metabolic reprogramming of TCA anaplerosis and lipid metabolism"
Project description:In natural environments, photosynthetic organisms adjust their metabolism to cope with the fluctuating availability of combined nitrogen-sources, a growth limiting factor. For the acclimation, the dynamic degradation/synthesis of tetrapyrrolic pigments as well as of the amino acid arginine is pivotal; however, there was no evidence that these processes could be functionally coupled. Using co-immunopurification and spectral shift assays we found that in the cyanobacterium Synechocystis sp. PCC 6803 the arginine-related ArgD and CphB enzymes form protein complexes with Gun4, an essential factor for chlorophyll biosynthesis. Gun4 binds ArgD with high affinity, and the ArgD-Gun4 complex strongly accumulates in cells supplemented with ornithine, a key intermediate of the arginine pathway. Elevated ornithine levels restricted de novo synthesis of tetrapyrrolic pigments, which arrested the recovery from nitrogen deficiency. Our data reveals a direct cross-talk between tetrapyrrole biosynthesis and arginine metabolism that clarifies the importance of balancing photosynthetic pigment synthesis with nitrogen homeostasis.
Project description:The N6-methyladenosine (m6A) RNA modification serves crucial functions in RNA metabolism; however, the molecular mechanisms underlying the regulation of m6A are not well understood. Here, we establish arginine methylation of METTL14, a component of the m6A methyltransferase complex, as a novel pathway that controls the function of m6A in DNA repair. Specifically, protein arginine methyltransferase 1 (PRMT1) interacts with and methylates the intrinsically disordered C-terminus of METTL14, which promotes its interaction with RNA substrates, enhances its RNA methylation activity, and is crucial for its interaction with RNAPII. Mouse embryonic stem cells (mESCs) expressing arginine methylation-deficient METTL14 exhibit dramatically reduced global m6A levels. Transcriptome-wide m6A analysis reveals that arginine methylation-dependent m6A enhances the translation of genes essential for the repair of DNA interstrand crosslinks; thus, METTL14 arginine methylation-deficient mESCs are hypersensitive to DNA crosslinking agents. Collectively, these findings reveal important aspects of m6A regulation that could have broad implications in normal development and in diseases such as cancer.
Project description:Metabolic reprogramming is a hallmark of cancer. However, mechanisms underlying metabolic reprogramming and how altered metabolism in turn enhances tumorigenicity are poorly understood. Here, we report that arginine levels are elevated in hepatocellular carcinoma (HCC) despite reduced expression of arginine synthesis genes, in murine and patient tumors. Tumor cells accumulate high levels of arginine due to increased uptake and reduced arginine-to-polyamine conversion. Importantly, the high levels of arginine promote tumor formation via further metabolic reprogramming, including changes in glucose, amino acid, nucleotide, and fatty acid metabolism. Mechanistically, arginine binds RNA-binding motif protein 39 (RBM39) to control expression of metabolic genes. RBM39-mediated upregulation of asparagine synthesis leads to enhanced arginine uptake, creating a positive feedback loop to sustain high arginine levels and oncogenic metabolism. Thus, arginine is a second messenger-like molecule that reprograms metabolism to promote tumor growth.
Project description:Arginine utilization in Pseudomonas aeruginosa with multiple catabolic pathways represents one of the best examples of metabolic versatility of this organism. To identify genes of this complex arginine network, we employed DNA microarray to analyze the transcriptional profiles of this organism in response to L-arginine. While most genes in arginine uptake, regulation and metabolism have been identified as members of the ArgR regulon in our previous study, eighteen putative transcriptional units of 38 genes including the two known genes of the arginine dehydrogenase (ADH) pathway, kauB and gbuA, were found inducible by exogenous L-arginine but independent of ArgR.