Targeting the Proline-Glutamine-Asparagine-Arginine Metabolic Axis in Amino Acid Starvation Cancer Therapy.
ABSTRACT: Proline, glutamine, asparagine, and arginine are conditionally non-essential amino acids that can be produced in our body. However, they are essential for the growth of highly proliferative cells such as cancers. Many cancers express reduced levels of these amino acids and thus require import from the environment. Meanwhile, the biosynthesis of these amino acids is inter-connected but can be intervened individually through the inhibition of key enzymes of the biosynthesis of these amino acids, resulting in amino acid starvation and cell death. Amino acid starvation strategies have been in various stages of clinical applications. Targeting asparagine using asparaginase has been approved for treating acute lymphoblastic leukemia. Targeting glutamine and arginine starvations are in various stages of clinical trials, and targeting proline starvation is in preclinical development. The most important obstacle of these therapies is drug resistance, which is mostly due to reactivation of the key enzymes involved in biosynthesis of the targeted amino acids and reprogramming of compensatory survival pathways via transcriptional, epigenetic, and post-translational mechanisms. Here, we review the interactive regulatory mechanisms that control cellular levels of these amino acids for amino acid starvation therapy and how drug resistance is evolved underlying treatment failure.
Project description:Several amino acids were found to stimulate glycogen synthesis and lipogenesis, and to inhibit ketogenesis in isolated rat hepatocytes. When hepatocytes were incubated in the presence of 20 mM-glucose, the amino acids could be classified in decreasing order of efficiency as follows: glutamine and proline, alanine, aminoisobutyric acid, asparagine and histidine for stimulation of glycogen synthesis; glutamine, proline and alanine for stimulation of lipogenesis; proline and glutamine for inhibition of ketogenesis. The study of the time course revealed that the rates were not linear and were preceded by a lag period. In all conditions studied, glutamine and proline were found to have similar quantitative effects on glycogen synthesis and lipid metabolism. However, their effects differ qualitatively. Indeed, the effects of proline on glycogen synthesis, lipogenesis and glutamate and aspartate content were faster. Moreover, proline increased the hydroxybutyrate/acetoacetate ratio, whereas glutamine did not change it. Incubation of hepatocytes with aminoisobutyric acid or under hypo-osmotic conditions, which increased cell volume and mimicked the amino acid-induced stimulation of glycogen synthesis, had little effect on lipogenesis. In hepatocytes incubated without glucose, ketogenesis was inhibited, in decreasing order of efficiency, by alanine, asparagine, glutamine and proline. Under these conditions, glutamine increased, alanine decreased and asparagine did not affect the concentration of malonyl-CoA. This indicates that the latter cannot be responsible for the inhibition of ketogenesis by alanine and asparagine.
Project description:Dehalococcoides species are key players in the anaerobic transformation of halogenated solvents at contaminated sites. Here, we analyze isotopologue distributions in amino acid pools from peptides of Dehalococcoides strain CBDB1 after incubation with (13)C-labeled acetate or bicarbonate as a carbon source. The resulting data were interpreted with regard to genome annotations to identify amino acid biosynthesis pathways. In addition to using gas chromatography-mass spectrometry (GC-MS) for analyzing derivatized amino acids after protein hydrolysis, we introduce a second, much milder method, in which we directly analyze peptide masses after tryptic digest and peptide fragments by nano-liquid chromatography-electrospray ionization-tandem mass spectrometry (nano-LC-ESI-MS/MS). With this method, we identify isotope incorporation patterns for 17 proteinaceous amino acids, including proline, cysteine, lysine, and arginine, which escaped previous analyses in Dehalococcoides. Our results confirmed lysine biosynthesis via the ?-aminoadipate pathway, precluding lysine formation from aspartate. Similarly, the isotopologue pattern obtained for arginine provided biochemical evidence of its synthesis from glutamate. Direct peptide MS/MS analysis of the labeling patterns of glutamine and asparagine, which were converted to glutamate and aspartate during protein hydrolysis, gave biochemical evidence of their precursors and confirmed glutamate biosynthesis via a Re-specific citrate synthase. By addition of unlabeled free amino acids to labeled cells, we show that in strain CBDB1 none of the 17 tested amino acids was incorporated into cell mass, indicating that they are all synthesized de novo. Our approach is widely applicable and provides a means to analyze amino acid metabolism by studying specific proteins even in mixed consortia.
Project description:The addition of organic solvents to ?-amino acids in aqueous solution could be an effective method in crystallization. We reviewed the available data on the solubility of ?-amino acids in water, water-ethanol mixtures, and ethanol at 298.15 K and 0.1 MPa. The solubility of l-alanine, l-proline, l-arginine, l-cysteine, and l-lysine in water and ethanol mixtures and the solubility of l-alanine, l-proline, l-arginine, l-cysteine, l-lysine, l-asparagine, l-glutamine, l-histidine, and l-leucine in pure ethanol systems were measured and are published here for the first time. The impact on the solubility of amino acids that can convert in solution, l-glutamic acid and l-cysteine, was studied. At lower concentrations, only the ninhydrin method and the ultraperfomance liquid chromatography (UPLC) method yield reliable results. In the case of ?-amino acids that convert in solution, only the UPLC method was able to discern between the different ?-amino acids and yields reliable results. Our results demonstrate that ?-amino acids with similar physical structures have similar changes in solubility in mixed water/ethanol mixtures. The solubility of l-tryptophan increased at moderate ethanol concentrations.
Project description:The composition of Serra da Estrela PDO cheeses (total fat, total protein, salt and free amino acids) was assessed using NIR spectrophotometry and UPLC-DAD-MS/MS. In total, 24 cheeses were acquired from 6 certified cheesemakers located in 5 different municipalities within the delimited PDO geographical region. Cheeses were produced from raw ewe milk of two autochthonous Portuguese sheep breeds, between November 2017 and March 2018, and were acquired after 45 days of maturation. The data include the mean (and respective standard deviations) levels of moisture (%), total fat (%), total protein (%) and salt (%), obtained by NIR spectrophotometry. As well the mean (and respective standard deviations) of free amino acids contents (mg/100 g of cheese, in wet basis) evaluated using a UPLC-DAD-MS/MS method are shown. The latter data include information regarding 8 essential free amino acids (histidine, leucine-isoleucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine) and 9 non-essential free amino acids (arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, proline, tyrosine and serine). Leucine and isoleucine, being isomers, were quantified together. Leucine-isoleucine, phenylalanine and serine were the most abundant essential free amino acids and cysteine, proline and asparagine were the most abundant non-essential free amino acids. Free amino acids contents depended on the cheese producer as well as on the production time-period.
Project description:Cancer cells reprogram metabolism to coordinate their rapid growth. They addict on glutamine metabolism for adenosine triphosphate generation and macromolecule biosynthesis. In this study, we report that glutamine deprivation retarded cell growth and induced prosurvival autophagy. Autophagy inhibition by chloroquine significantly enhanced glutamine starvation induced growth inhibition and apoptosis activation. Asparagine deprivation by L-asparaginase exacerbated growth inhibition induced by glutamine starvation and autophagy blockage. Similar to glutamine starvation, inhibition of glutamine metabolism with a chemical inhibitor currently under clinical evaluation was synthetically lethal with chloroquine and L-asparaginase, drugs approved for the treatment of malaria and leukemia, respectively. In conclusion, inhibiting glutaminolysis was synthetically lethal with autophagy inhibition and asparagine depletion. Therefore, targeting glutaminolysis could be a promising approach for colorectal cancer treatment.
Project description:The facultative intracellular pathogen Salmonella enterica resides in a specific membrane-bound compartment termed the Salmonella-containing vacuole (SCV). Despite being segregated from access to metabolites in the host cell cytosol, Salmonella is able to efficiently proliferate within the SCV. We set out to unravel the nutritional supply of Salmonella in the SCV with focus on amino acids. We studied the availability of amino acids by the generation of auxotrophic strains for alanine, asparagine, aspartate, glutamine, and proline in a macrophage cell line (RAW264.7) and an epithelial cell line (HeLa) and examined access to extracellular nutrients for nutrition. Auxotrophies for alanine, asparagine, or proline attenuated intracellular replication in HeLa cells, while aspartate, asparagine, or proline auxotrophies attenuated intracellular replication in RAW264.7 macrophages. The different patterns of intracellular attenuation of alanine- or aspartate-auxotrophic strains support distinct nutritional conditions in HeLa cells and RAW264.7 macrophages. Supplementation of medium with individual amino acids restored the intracellular replication of mutant strains auxotrophic for asparagine, proline, or glutamine. Similarly, a mutant strain deficient in succinate dehydrogenase was complemented by the extracellular addition of succinate. Complementation of the intracellular replication of auxotrophic Salmonella by external amino acids was possible if bacteria were proficient in the induction of Salmonella-induced filaments (SIFs) but failed in a SIF-deficient background. We propose that the ability of intracellular Salmonella to redirect host cell vesicular transport provides access of amino acids to auxotrophic strains and, more generally, is essential to continuously supply bacteria within the SCV with nutrients.
Project description:The chemotaxis of Vibrio cholerae, the causative agent of cholera, has been implicated in pathogenicity. The bacterium has more than 40 genes for methyl-accepting chemotaxis protein (MCP)-like proteins (MLPs). In this study, we found that glycine and at least 18 L-amino acids, including serine, arginine, asparagine, and proline, serve as attractants to the classical biotype strain O395N1. Based on the sequence comparison with Vibrio parahaemolyticus, we speculated that at least 17 MLPs of V. cholerae may mediate chemotactic responses. Among them, Mlp24 (previously named McpX) is required for the production of cholera toxin upon mouse infection. mlp24 deletion strains of both classical and El Tor biotypes showed defects in taxis toward several amino acids, which were complemented by the expression of Mlp24. These amino acids enhanced methylation of Mlp24. Serine, arginine, asparagine, and proline were shown to bind directly to the periplasmic fragment of Mlp24. The structural information of its closest homolog, Mlp37, predicts that Mlp24 has two potential ligand-binding pockets per subunit, the membrane distal of which was suggested, by mutational analyses, to be involved in sensing of amino acids. These results suggest that Mlp24 is a chemoreceptor for multiple amino acids, including serine, arginine, and asparagine, which were previously shown to stimulate the expression of several virulence factors, implying that taxis toward a set of amino acids plays critical roles in pathogenicity of V. cholerae.
Project description:Cancer is caused by uncontrollable growth of neoplastic cells, leading to invasion of adjacent and distant tissues resulting in death. Cancer cells have specific nutrient(s) auxotrophy and have a much higher nutrient demand compared to normal tissues. Therefore, different metabolic inhibitors or nutrient-depleting enzymes have been tested for their anti-cancer activities. We review recent available laboratory and clinical data on using various specific amino acid metabolic pathways inhibitors in treating cancers. Our focus is on glutamine, asparagine, and arginine starvation. These three amino acids are chosen due to their better scientific evidence compared to other related approaches in cancer treatment. Amino acid-specific depleting enzymes have been adopted in different standard chemotherapy protocols. Glutamine starvation by glutaminase inhibitior, transporter inhibitor, or glutamine depletion has shown to have significant anti-cancer effect in pre-clinical studies. Currently, glutaminase inhibitor is under clinical trial for testing anti-cancer efficacy. Clinical data suggests that asparagine depletion is effective in treating hematologic malignancies even as a single agent. On the other hand, arginine depletion has lower toxicity profile and can effectively reduce the level of pro-cancer biochemicals in patients as shown by ours and others' data. This supports the clinical use of arginine depletion as anti-cancer therapy but its exact efficacy in various cancers requires further investigation. However, clinical application of these enzymes is usually hindered by common problems including allergy to these foreign proteins, off-target cytotoxicity, short half-life and rapidly emerging chemoresistance. There have been efforts to overcome these problems by modifying the drugs in different ways to circumvent these hindrance such as (1) isolate human native enzymes to reduce allergy, (2) isolate enzyme isoforms with higher specificities and efficiencies, (3) pegylate the enzymes to reduce allergy and prolong the half-lives, and (4) design drug combinations protocols to enhance the efficacy of chemotherapy by drug synergy and minimizing resistance. These improvements can potentially lead to the development of more effective anti-cancer treatment with less adverse effects and higher therapeutic efficacy.
Project description:Albumin synthesis was measured in the isolated perfused rat liver by using the livers of both well-fed and starved rats. Starvation markedly decreased albumin synthesis. The livers from starved rats were unable to increase synthesis rates after the addition to the perfusates of single amino acids or the addition of both glucagon and tryptophan. Arginine, asparagine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan and valine, added together to ten times their normal peripheral blood concentrations, restored synthesis rates to normal. The plasma aminogram (i.e. the relative concentrations, of amino acids) was altered by depriving rats of protein for 48h. The use of blood from the deprived rats as perfusate, instead of normal blood, decreased albumin synthesis rates significantly by livers obtained from well-fed rats. The addition of single amino acids, including the non-metabolizable amino acid, alpha-aminoisobutyric acid, to the above mixture increased albumin synthesis rates to normal values. It is concluded that amino acids play an important role in the control of albumin synthesis and that more than one mechanism is probably involved.
Project description:Multiple myeloma (MM) cells consume huge amounts of glutamine and, as a consequence, the amino acid concentration is lower-than-normal in the bone marrow (BM) of MM patients. Here we show that MM-dependent glutamine depletion induces glutamine synthetase in stromal cells, as demonstrated in BM biopsies of MM patients, and reproduced in vitro by co-culturing human mesenchymal stromal cells (MSCs) with MM cells. Moreover, glutamine depletion hinders osteoblast differentiation of MSCs, which is also severely blunted by the spent, low-glutamine medium of MM cells, and rescued by glutamine restitution. Glutaminase and the concentrative glutamine transporter SNAT2 are induced during osteoblastogenesis in vivo and in vitro, and both needed for MSCs differentiation, pointing to enhanced the requirement for the amino acid. Osteoblastogenesis also triggers the induction of glutamine-dependent asparagine synthetase (ASNS), and, among non-essential amino acids, asparagine rescues differentiation of glutamine-starved MSCs, by restoring the transcriptional profiles of differentiating MSCs altered by glutamine starvation. Thus, reduced asparagine availability provides a mechanistic link between MM-dependent Gln depletion in BM and impairment of osteoblast differentiation. Inhibition of Gln metabolism in MM cells and supplementation of asparagine to stromal cells may, therefore, constitute novel approaches to prevent osteolytic lesions in MM.