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Iduronate-2-sulfatase (IDS) is a lysosomal enzyme involved in the metabolism of the glycosaminoglycans heparan (HS) and dermatan (DS) sulfate. Mutations on IDS gene produce mucopolysaccharidosis II (MPS II), characterized by the lysosomal accumulation of HS and DS, leading to severe damage of the central nervous system (CNS) and other tissues. In this study, we used a neurochemistry and proteomic approaches to identify the brain distribution of IDS and its interacting proteins on wild-type mouse brain. IDS immunoreactivity showed a robust staining throughout the entire brain, suggesting an intracellular reactivity in nerve cells and astrocytes. By using affinity purification and mass spectrometry we identified 187 putative IDS partners-proteins, mainly hydrolases, cytoskeletal proteins, transporters, transferases, oxidoreductases, nucleic acid binding proteins, membrane traffic proteins, chaperons and enzyme modulators, among others. The interactions with some of these proteins were predicted by using bioinformatics tools and confirmed by co-immunoprecipitation analysis and Blue Native PAGE. In addition, we identified cytosolic IDS-complexes containing proteins from predicted highly connected nodes (hubs), with molecular functions including catalytic activity, redox balance, binding, transport, receptor activity and structural molecule activity. The proteins identified in this study would provide new insights about IDS physiological role into the CNS and its potential role in the brain-specific protein networks.

Lysophosphatidic acid (LPA) is an extracellular lipid mediator that regulates nervous system development and functions acting through G protein-coupled receptors (GPCRs). Here we explore the crosstalk between LPA1 receptor and glutamatergic transmission by examining expression of glutaminase (GA) isoforms in different brain areas isolated from wild-type (WT) and KOLPA1 mice. Silencing of LPA1 receptor induced a severe down-regulation of Gls-encoded long glutaminase protein variant (KGA) (glutaminase gene encoding the kidney-type isoforms, GLS) protein expression in several brain regions, particularly in brain cortex and hippocampus. Immunohistochemical assessment of protein levels for the second type of glutaminase (GA) isoform, glutaminase gene encoding the liver-type isoforms (GLS2), did not detect substantial differences with regard to WT animals. The regional mRNA levels of GLS were determined by real time RT-PCR and did not show significant variations, except for prefrontal and motor cortex values which clearly diminished in KO mice. Total GA activity was also significantly reduced in prefrontal and motor cortex, but remained essentially unchanged in the hippocampus and rest of brain regions examined, suggesting activation of genetic compensatory mechanisms and/or post-translational modifications to compensate for KGA protein deficit. Remarkably, Golgi staining of hippocampal regions showed an altered morphology of glutamatergic pyramidal cells dendritic spines towards a less mature filopodia-like phenotype, as compared with WT littermates. This structural change correlated with a strong decrease of active matrix-metalloproteinase (MMP) 9 in cerebral cortex and hippocampus of KOLPA1 mice. Taken together, these results demonstrate that LPA signaling through LPA1 influence expression of the main isoenzyme of glutamate biosynthesis with strong repercussions on dendritic spines maturation, which may partially explain the cognitive and learning defects previously reported for this colony of KOLPA1 mice.

The expression of glutaminase in glial cells has been a controversial issue and matter of debate for many years. Actually, glutaminase is essentially considered as a neuronal marker in brain. Astrocytes are endowed with efficient and high capacity transport systems to recapture synaptic glutamate which seems to be consistent with the absence of glutaminase in these glial cells. In this work, a comprehensive study was devised to elucidate expression of glutaminase in neuroglia and, more concretely, in astrocytes. Immunocytochemistry in rat and human brain tissues employing isoform-specific antibodies revealed expression of both Gls and Gls2 glutaminase isozymes in glutamatergic and GABAergic neuronal populations as well as in astrocytes. Nevertheless, there was a different subcellular distribution: Gls isoform was always present in mitochondria while Gls2 appeared in two different locations, mitochondria and nucleus. Confocal microscopy and double immunofluorescence labeling in cultured astrocytes confirmed the same pattern previously seen in brain tissue samples. Astrocytic glutaminase expression was also assessed at the mRNA level, real-time quantitative RT-PCR detected transcripts of four glutaminase isozymes but with marked differences on their absolute copy number: the predominance of Gls isoforms over Gls2 transcripts was remarkable (ratio of 144:1). Finally, we proved that astrocytic glutaminase proteins possess enzymatic activity by in situ activity staining: concrete populations of astrocytes were labeled in the cortex, cerebellum and hippocampus of rat brain demonstrating functional catalytic activity. These results are relevant for the stoichiometry of the Glu/Gln cycle at the tripartite synapse and suggest novel functions for these classical metabolic enzymes.

Cancer cells develop and succeed by shifting to different metabolic programs compared with their normal cell counterparts. One of the classical hallmarks of cancer cells is their higher glycolysis rate and lactate production even in the presence of abundant O2 (Warburg effect). Another common metabolic feature of cancer cells is a high rate of glutamine (Gln) consumption normally exceeding their biosynthetic and energetic needs. The term Gln addiction is now widely used to reflect the strong dependence shown by most cancer cells for this essential nitrogen substrate after metabolic reprogramming. A Gln/glutamate (Glu) cycle occurs between host tissues and the tumor in order to maximize its growth and proliferation rates. The mechanistic basis for this deregulated tumor metabolism and how these changes are connected to oncogenic and tumor suppressor pathways are becoming increasingly understood. Based on these advances, new avenues of research have been initiated to find novel therapeutic targets and to explore strategies that interfere with glutamine metabolism as anticancer therapies. In this review, we provided an updated overview of glutamine addiction in glioma, the most prevalent type of brain tumor.

Glutamine/glutamate homeostasis must be exquisitely regulated in mammalian brain and glutaminase (GA, E.C. 3.5.1.2) is one of the main enzymes involved. The products of GA reaction, glutamate and ammonia, are essential metabolites for energy and biosynthetic purposes but they are also hazardous compounds at concentrations beyond their normal physiological thresholds. The classical pattern of GA expression in mammals has been recently challenged by the discovery of novel transcript variants and protein isoforms. Furthermore, the interactome of brain GA is also starting to be uncovered adding a new level of regulatory complexity. GA may traffic in brain and unexpected locations, like cytosol and nucleus, have been found for GA isoforms. Finally, the expression of GA in glial cells has been reported and its potential implications in ammonia homeostasis are discussed.

Metabolism is fundamental to cell survival, growth, and behavior. Tumor cells have an enhanced demand for nutrients which provide biosynthetic building blocks and cellular energy to sustain their proliferative status. Altered metabolism is a hallmark of cancer. The term metabolic reprogramming has been coined to describe the whole range of metabolic abnormalities accompanying tumorigenesis and metastasis. Increased glutamine uptake and glutaminolysis are key metabolic traits that have been consistently found on a wide range of human and experimental cancers. Glutaminase proteins control the first step in the glutaminolytic process: the conversion of glutamine to glutamate and ammonium ions. Glutaminase expression has been correlated with malignancy and growth rate on a great variety of tumors. Recent works are now starting to uncover the differential expression of glutaminase isoenzymes and their regulation by oncogenes and tumor suppressor genes. In parallel, glutaminase isoforms are attracting great interest as novel cancer chemotherapeutic targets. The focus of this chapter will highlight the role of glutamine addiction in tumor biology with particular emphasis on the interaction between the host and the tumor.

Glutaminase is expressed in most mammalian tissues and cancer cells, but recent studies are now revealing a considerably degree of complexity in its pattern of expression and functional regulation. Novel transcript variants of the mammalian glutaminase Gls2 gene have been recently found and characterized in brain. Co-expression of different isoforms in the same cell type would allow cells to fine-tune their Gln/Glu levels under a wide range of metabolic states. Moreover, the discovery of protein interacting partners and novel subcellular localizations, for example nucleocytoplasmic in neurons and astrocytes, strongly suggest non-neurotransmission roles for Gls2 isoforms associated with transcriptional regulation and cellular differentiation. Of note, Gls isoforms have been considered as an important trophic factor for neuronal differentiation and postnatal development of brain regions. On the other hand, glutaminases are taking center stage in tumor biology as new therapeutic targets to inhibit metabolic reprogramming of cancer cells. Interestingly, glutaminase isoenzymes play seemingly opposing roles in cancer cell growth and proliferation; this issue will be also succinctly discussed with special emphasis on brain tumors.

Cancer cells require a robust supply of reduced nitrogen to produce nucleotides, non-essential amino acids and a high cellular redox activity. Glutamine provides a major substrate for respiration as well as nitrogen for the production of proteins, hexosamines, and macromolecules. Therefore, glutamine is one of key molecules in cancer metabolism during cell proliferation. The notion of targeting glutamine metabolism in cancer, originally rationalized by the number of pathways fed by this nutrient, has been reinforced by more recent studies demonstrating that its metabolism is regulated by oncogenes. Glutamine can exert its effects by modulating redox homeostasis, bioenergetics, nitrogen balance or other functions, including by being a precursor of glutathione, the major nonenzymatic cellular antioxidant. Glutaminase (GA) is the first enzyme that converts glutamine to glutamate, which is in turn converted to alpha-ketoglutarate for further metabolism in the tricarboxylic acid cycle. Different GA isoforms in mammals are encoded by two genes, Gls and Gls2. As each enzymatic form of GA has distinct kinetic and molecular characteristics, it has been speculated that the differential regulation of GA isoforms may reflect distinct functions or requirements in different tissues or cell states. GA encoded by Gls gene (GLS) has been demonstrated to be regulated by oncogenes and to support tumor cell growth. GA encoded by Gls2 gene (GLS2) reduces cellular sensitivity to reactive oxygen species associated apoptosis possibly through glutathione-dependent antioxidant defense, and therefore to behave more like a tumor suppressor. Thus, modulation of GA function may be a new therapeutic target for cancer treatment.

Cannabinoid CB1 receptors peripherally modulate energy metabolism. Here, we investigated the role of CB1 receptors in the expression of glucose/pyruvate/tricarboxylic acid (TCA) metabolism in rat abdominal muscle. Dihydrolipoamide dehydrogenase (DLD), a flavoprotein component (E3) of alpha-ketoacid dehydrogenase complexes with diaphorase activity in mitochondria, was specifically analyzed. After assessing the effectiveness of the CB1 receptor antagonist AM251 (3 mg kg(-1), 14 days) on food intake and body weight, we could identified seven key enzymes from either glycolytic pathway or TCA cycle-regulated by both diet and CB1 receptor activity-through comprehensive proteomic approaches involving two-dimensional electrophoresis and MALDI-TOF/LC-ESI trap mass spectrometry. These enzymes were glucose 6-phosphate isomerase (GPI), triosephosphate isomerase (TPI), enolase (Eno3), lactate dehydrogenase (LDHa), glyoxalase-1 (Glo1) and the mitochondrial DLD, whose expressions were modified by AM251 in hypercaloric diet-induced obesity. Specifically, AM251 blocked high-carbohydrate diet (HCD)-induced expression of GPI, TPI, Eno3 and LDHa, suggesting a down-regulation of glucose/pyruvate/lactate pathways under glucose availability. AM251 reversed the HCD-inhibited expression of Glo1 and DLD in the muscle, and the DLD and CB1 receptor expression in the mitochondrial fraction. Interestingly, we identified the presence of CB1 receptors at the membrane of striate muscle mitochondria. DLD over-expression was confirmed in muscle of CB1-/- mice. AM251 increased the pyruvate dehydrogenase and glutathione reductase activity in C2C12 myotubes, and the diaphorase/oxidative activity in the mitochondria fraction. These results indicated an up-regulation of methylglyoxal and TCA cycle activity. Findings suggest that CB1 receptors in muscle modulate glucose/pyruvate/lactate pathways and mitochondrial oxidative activity by targeting DLD. This work was supported by CIBERobn (CB06/03/1008), Ministerio de Economia y Competitividad (MINECO) (PG: BFU2012-33334), Instituto de Salud Carlos III (ISCIII), MINECO, cofunded by UE-ERDF program (JS: CP12/03109), Red de Trastornos Adictivos (FRF: RD12/0028/0001, PG: RD12/0028/0004, JM: RD12/0028/0013), The Basque Country Government (PG: BCG IT764-13), Consejeria de Economia, Innovaciin y Ciencia, Junta de Andalucia, UE-ERDF (FRF: CTS-8221, JM: CVI-6656), Consejeria de Salud, Junta de Andalucia, UE-ERDF (FRF: SAS111224), and University of the Basque Country UPV/EHU (PG: UFI11/41). JS, FJP and AS hold "Miguel Servet" research contracts from the National System of Health, ISCIII, UE-ERDF (CP12/03109, CP14/00212, and CP14/00173 respectively).

The synthesis of neurotransmitter glutamate in brain is mainly carried out by glutaminase enzymes. This synthesis must be exquisitely regulated because of its harmful potential giving rise to excitotoxic damage. It is noteworthy that two glutaminase isozymes coded by different genes are expressed in the brain of mammals. The need for two genes and two isozymes to support the single process of glutamate synthesis is unexplained, and identifying the role of each glutaminase is an important factor in understanding glutamate-mediated neurotransmission. Multiple transcripts for glutaminase genes and simultaneous expression of glutaminase isoforms have been reported in mammalian tissues and cells. The recent discovery of protein interacting partners widens the possibilities of regulatory mechanisms controlling these biosynthetic enzymes. The expression of distinct isozymes and binding partners may represent the biochemical and molecular basis to achieve fine-tuning control of glutamate synthesis in different cell types or developmental states. In this review, we will briefly summarize recent works on glutaminase proteins in mammals, with particular emphasis on brain studies. We present convergent evidence supporting the existence of a novel glutaminase isozyme in mammalian tissues.

Glutamine behaves as a key nutrient for tumors and rapidly dividing cells. Glutaminase is the main glutamine-utilizing enzyme in these cells, and its activity correlates with glutamine consumption and growth rate. We have carried out the antisense L-type glutaminase inhibition in human MCF7 breast cancer cells, in order to study its effect on the hexosamine pathway and the pattern of protein O-glycosylation. The antisense mRNA glutaminase expressing cells, named ORF19, presented a 50% lower proliferation rate than parental cells, showing a more differentiated phenotype. ORF19 cells had an 80% reduction in glutamine:fructose-6-P amidotransferase activity, which is the rate-limiting step of the hexosamine pathway. Although the overall cellular protein O-glycosylation did not change, the O-glycosylation status of several key proteins was altered. O-glycosylation of O-GlcNAc transferase (OGT), the enzyme that links N-acetylglucosamine to proteins, was fivefold lower in ORF19 than in wild type cells. Inhibition of glutaminase also provoked a 10-fold increase in Sp1 expression, and a significant decrease in the ratio of O-glycosylated to total protein for both Sp1 and the Rpt2 proteasome component. These changes were accompanied by a higher Sp1 transcriptional activity. Proteome analysis of O-glycosylated proteins permitted the detection of two new OGT target proteins: the chaperonin TCP-1 theta and the oncogene Ets-related protein isoform 7. Taken together, our results support the hexosamine pathway and the O-glycosylation of proteins being a sensor mechanism of the nutritional and energetic states of the cell.

BackgroundGlutaminase is expressed in most mammalian tissues and cancer cells, but the regulation of its expression is poorly understood. An essential step to accomplish this goal is the characterization of its species- and cell-specific isoenzyme pattern of expression. Our aim was to identify and characterize transcript variants of the mammalian glutaminase Gls2 gene.Methodology/principal findingsWe demonstrate for the first time simultaneous expression of two transcript variants from the Gls2 gene in human, rat and mouse. A combination of RT-PCR, primer-extension analysis, bioinformatics, real-time PCR, in vitro transcription and translation and immunoblot analysis was applied to investigate GLS2 transcripts in mammalian tissues. Short (LGA) and long (GAB) transcript forms were isolated in brain and liver tissue of human, rat and mouse. The short LGA transcript arises by a combination of two mechanisms of transcriptional modulation: alternative transcription initiation and alternative promoter. The LGA variant contains both the transcription start site (TSS) and the alternative promoter in the first intron of the Gls2 gene. The full human LGA transcript has two in-frame ATGs in the first exon, which are missing in orthologous rat and mouse transcripts. In vitro transcription and translation of human LGA yielded two polypeptides of the predicted size, but only the canonical full-length protein displayed catalytic activity. Relative abundance of GAB and LGA transcripts showed marked variations depending on species and tissues analyzed.Conclusions/significanceThis is the first report demonstrating expression of alternative transcripts of the mammalian Gls2 gene. Transcriptional mechanisms giving rise to GLS2 variants and isolation of novel GLS2 transcripts in human, rat and mouse are presented. Results were also confirmed at the protein level, where catalytic activity was demonstrated for the human LGA protein. Relative abundance of GAB and LGA transcripts was species- and tissue-specific providing evidence of a differential regulation of GLS2 transcripts in mammals.