Project description:Besides being building blocks for protein synthesis, amino acids serve a wide variety of cellular functions, including acting as metabolic intermediates for ATP generation and for redox homeostasis. Upon amino acid deprivation, free uncharged tRNAs trigger GCN2-ATF4 to mediate the well-characterized transcriptional amino acid response (AAR). However, it is not clear whether the deprivation of different individual amino acids triggers identical or distinct AARs. Here, we characterized the global transcriptional response upon deprivation of one amino acid at a time. With the exception of glycine, which was not required for the proliferation of MCF7 cells, we found that the deprivation of most amino acids triggered a shared transcriptional response that included the activation of ATF4, p53 and TXNIP. However, there was also significant heterogeneity among different individual AARs. The most dramatic transcriptional response was triggered by methionine deprivation, which activated an extensive and unique response in different cell types. We uncovered that the specific methionine-deprived transcriptional response required creatine biosynthesis. This dependency on creatine biosynthesis was caused by the consumption of S-Adenosyl-L-methionine (SAM) during creatine biosynthesis that helps to deplete SAM under methionine deprivation and reduces histone methylations. As such, the simultaneous deprivation of methionine and sources of creatine biosynthesis (either arginine or glycine) abolished the reduction of histone methylation and the methionine-specific transcriptional response. Arginine-derived ornithine was also required for the complete induction of the methionine-deprived specific gene response. Collectively, our data identify a previously unknown set of heterogeneous amino acid responses and reveal a distinct methionine-deprived transcriptional response that results from the crosstalk of arginine, glycine and methionine metabolism via arginine/glycine-dependent creatine biosynthesis. RNA was extracted by RNAeasy kits (Qiagen) from the MCF7 or PC3 cells which exposed to the control full DMEM or deprived one (or all) amino acid media for 24 or 48 hours.

Project description:Protozoa of the genus Leishmania are the causative agents of leishmaniasis in humans. These parasites cycle between promastigotes in the sand fly mid-gut and amastigotes in phagolysosome of mammalian macrophages. During infection, host up-regulate nitric oxide synthase and parasite induce host arginase expression, both of which use arginine as a substrate. These elevated activities deplete macrophage arginine pools, a situation that invading Leishmania must overcome since it is an essential amino acid. Leishmania donovani imports exogenous arginine via a mono-specific amino acid transporter (AAP3) and utilizes it primarily through the polyamine pathway to provide precursors for trypanothione biosynthesis. Here we report the discovery of a pathway whereby promastigote and amastigote forms of the Leishmania sense the lack of environmental arginine and respond with rapid up-regulation in AAP3 expression and activity, as well as several other transporters. Significantly, this arginine deprivation response is also activated in parasites during macrophage infection. Phosphoproteomic analyses of L. donovani promastigotes have implicated a Mitogen-Activated Protein Kinase 2 (MPK2)-mediated signaling cascade in this response and L. mexicana mutants lacking MPK2 are unable to respond to arginine deprivation. In this study, we established that Leishmania cells sense the absence of arginine in their environment; both in culture (axenic promastigotes and amastigotes) and in macrophages during infection (amastigotes). This study describes the first amino acid deprivation sensing mechanism and the pathway that transduce this response, and reveals a novel host-pathogen metabolic interplay. Total RNA from Ten Leishmania donovani samples were analyzed using RNA-Seq. Cells from two life stages (promastigotes and amastigotes) were grown in axenic culture in the presence and absense of arginine. For each condition two biological replicates were grown and analyzed. In addition two macrophage grown amastigotes were analyzed.

Project description:Cancer immunosuppression involves cellular pathways appropriated by tumors to escape immune surveillance but are normally required for tissue homeostasis and repair, self-tolerance, and successful transplantation. Among these pathways, enzymes that degrade tryptophan and arginine are thought to suppress activated T cells in the tumor microenvironment and are therefore attractive drug targets. However, how amino acid metabolism controls the development and progression of cancer has remained elusive, since commonly used implantable tumor models may not faithfully recapitulate the complex cellular and biochemical interactions within tumor microenvironments. To address this, we generated a novel, penetrant autochthonous model of neuroblastoma to accurately quantify tumor growth non-invasively and simultaneously genetically manipulate the enzymes that mediate tryptophan and arginine metabolism, and the cells that express them. We established that tumor development and growth were dependent on infiltrating CCR2+ bone marrow-derived myeloid cells but independent of Stat6-dependent ‘M2’ macrophages. Macrophages can modulate CD4+ T cell activation, proliferation, and differentiation by consuming amino acids and forcing the T cells to remain in the G1 phase of the cell cycle. Indeed, we found that CD4+ T cells deprived of arginine increased Foxp3 expression and adopted a unique regulatory-like phenotype. Notably, depletion of CD4+ T cells like CCR2+ macrophages, virtually eliminated tumor formation. When rare tumors did form in CD4-depleted mice, they contained Foxp3+ CD8 T cells, suggesting regulatory T cell activity is a fundamental requirement for tumor development. When we ablated macrophage arginase 1 (Arg1), an enzyme that locally consumes arginine, in the tumor-prone background, almost no tumors were observed. Therefore, Arg1 and arginine metabolism form a pro-tumor pathway. We extended these findings to two pathways of programmed myeloid tryptophan metabolism (IDO1, IDO2, IL4i1). Like Arg1, expression of these enzymes was confined to myeloid cells in humans and mice, and each enzyme was independently essential for tumor formation and growth. The protective effects of genetic ablation of each myeloid amino acid pathways uniformly occurred early in tumor formation, arguing that a network of immune cells controlled by myeloid amino acid metabolism and CD4+ T cells is important for the origins of a pro-tumor environment. Our results establish an unappreciated potency of macrophage amino acid metabolism in promoting malignancy, and suggest that these pathways can be disabled by targeting of myeloid amino acid metabolism. OVA-specific CD4+ T cells were stimulated with peptide in the presence of APCs (CD3-depleted splenocytes) either in control complete RPMI-1640 (containing 10% dialyzed FBS) or in RPMI-1640 (containing 10% dialyzed FBS) containing 1% of the normal arginine levels (12uM) to characterize the effects of limited arginine availability on gene expression.

Project description:Besides being building blocks for protein synthesis, amino acids serve a wide variety of cellular functions, including acting as metabolic intermediates for ATP generation and for redox homeostasis. Upon amino acid deprivation, free uncharged tRNAs trigger GCN2-ATF4 to mediate the well-characterized transcriptional amino acid response (AAR). However, it is not clear whether the deprivation of different individual amino acids triggers identical or distinct AARs. Here, we characterized the global transcriptional response upon deprivation of one amino acid at a time. With the exception of glycine, which was not required for the proliferation of MCF7 cells, we found that the deprivation of most amino acids triggered a shared transcriptional response that included the activation of ATF4, p53 and TXNIP. However, there was also significant heterogeneity among different individual AARs. The most dramatic transcriptional response was triggered by methionine deprivation, which activated an extensive and unique response in different cell types. We uncovered that the specific methionine-deprived transcriptional response required creatine biosynthesis. This dependency on creatine biosynthesis was caused by the consumption of S-Adenosyl-L-methionine (SAM) during creatine biosynthesis that helps to deplete SAM under methionine deprivation and reduces histone methylations. As such, the simultaneous deprivation of methionine and sources of creatine biosynthesis (either arginine or glycine) abolished the reduction of histone methylation and the methionine-specific transcriptional response. Arginine-derived ornithine was also required for the complete induction of the methionine-deprived specific gene response. Collectively, our data identify a previously unknown set of heterogeneous amino acid responses and reveal a distinct methionine-deprived transcriptional response that results from the crosstalk of arginine, glycine and methionine metabolism via arginine/glycine-dependent creatine biosynthesis.

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A Bioinformatic Analysis of Arginine-Sensitive Regulation of rat Hepatic Gene Expression

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