José A. Campos-Sandoval

Glutamine homeostasis and mitochondrial dynamics

Glutamine is a multifaceted amino acid that plays key roles in many metabolic pathways and also fulfils essential signaling functions. Although classified as non-essential, recent evidence suggests that glutamine is a conditionally essential amino acid in several physiological situations. Glutamine homeostasis must therefore be exquisitely regulated and mitochondria represent a major site of glutamine metabolism in numerous cell types. Glutaminolysis is mostly a mitochondrial process with repercussions in organelle structure and dynamics suggesting a tight and mutual control between mitochondrial form and cell bioenergetics. In this review we describe an updated account focused on the critical involvement of glutamine in oxidative stress, mitochondrial dysfunction and tumour cell proliferation, with special emphasis in the initial steps of mitochondrial glutamine pathways: transport into the organelle and hydrolytic deamidation through glutaminase enzymes. Some controversial issues about glutamine catabolism within mitochondria are also reviewed.

Co-expression of glutaminase K and L isoenzymes in human tumour cells

The pattern of expression of glutaminase isoenzymes in tumour cells has been investigated to clarify its role in the malignant transformation and the prospect of its use as a clinically relevant factor. Using leukaemia cells from medullar blood of human patients and several established human cancer cell lines, we have developed a competitive RT (reverse transcriptase)-PCR assay to quantify simultaneously K-type (kidney-type) and L-type (liver-type) glutaminase mRNAs. Co-expression of both transcripts and higher amounts of L-type mRNA were always found in all cancer cell types analysed. However, mature lymphocytes from the medullar blood of a patient suffering aplasia did not express the K-type transcript and showed a 15-fold increase of L-type transcript. Co-expression was also confirmed at the protein level using isoform-specific antibodies; nevertheless, it did not correlate with the relative abundance of glutaminase transcripts and strong K-type protein signals were detected. On the other hand, marked differences were found with regard to glutamate inhibition and phosphate activation of tumour glutaminase activity. Taken together, the protein data suggest that K isoform would account for the majority of glutaminase activity in these human tumour cells. The results confirm that simultaneous expression of both isoenzymes in human cancer cells is a more frequent event than previously thought. Furthermore, the present work and other previous data suggest that K isoform is up-regulated with increased rates of proliferation, whereas prevalence of the L isoform seems to be related with resting or quiescent cell states.

Glutaminase isoenzymes as key regulators in metabolic and oxidative stress against cancer.

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.

A novel glutaminase isoform in mammalian tissues

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.

Antisense glutaminase inhibition modifies the O-GlcNAc pattern and flux through the hexosamine pathway in breast cancer cells†

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 θ 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. J. Cell. Biochem. 103: 800–811, 2008.

Expression of the scaffolding PDZ protein glutaminase-interacting protein in mammalian brain

A human brain cDNA clone coding for a novel PDZ‐domain protein of 124 amino acids was previously isolated in our laboratory. The protein was termed glutaminase‐interacting protein (GIP), because it interacts with the C‐terminal region of the human L‐type glutaminase (LGA). The pattern of expression and functions of GIP in brain are completely unknown, so its significance remains undefined. Here we describe the expression of GIP mRNA and protein in mammalian brain. Northern blot analysis revealed that GIP mRNA was ubiquitous in most regions of human brain but was particularly abundant in spinal cord. The presence of the protein in rat and monkey brain was studied at the regional, cellular, and subcellular level by immunocytochemistry. The protein was found to be present in both neurons and astrocytes, with a cytosolic and mitochondrial subcellular localization. Double immunofluorescence labeling with anti‐GIP and anti‐LGA antibodies using confocal microscopy revealed colocalization of both proteins in astrocyte cell processes and their perivascular end feet. Electron microscopy of rat brain neurons revealed GIP immunoreactivity concentrated also in the nuclear envelope and the plasma membrane. The multiple locations for GIP in mammalian brain are in agreement with known protein interaction partners reported for this PDZ protein. The findings presented here support a role of GIP as an important scaffold in both astrocytes and neurons and point toward astrocytic processes and perivascular end feet as plausible anatomical substrates for interaction with glutaminase.

Attenuation of cocaine-induced conditioned locomotion is associated with altered expression of hippocampal glutamate receptors in mice lacking LPA1 receptors

RationaleLysophosphatidic acid is a phospholipid mediator that modulates neurodevelopment and neurogenesis in the hippocampus through its actions on LPA1 receptors. Emerging evidences support LPA1 as a mediator of learning and emotional behaviour. There are no studies addressing its role on behaviours associated to drug abuse.ObjectivesWe examined whether genetic deletion of LPA1 receptor in maLPA1-null mice affected either cocaine-induced conditioned locomotion (CL) or behavioural sensitization (BS) induced by repeated cocaine exposure. We also analysed whether cocaine induced changes in the expression of functional markers of both dopamine- and glutamate-related genes in the striatum and the dorsal hippocampus.MethodsWe monitored cocaine-induced CL and BS in both genotypes of mice. Striatal dopamine and hippocampal glutamate-related genes were measured by real-time quantitative PCR, Western blot, and immunohistochemistry.ResultsmaLPA1-null mice exhibit an attenuated CL response after cocaine conditioning but a normal BS after repeated cocaine exposure. These behavioural changes were associated to alterations on the expression of metabotropic mGLUR3 glutamate receptors and on the actions of cocaine on the GLUR1 subunit of AMPA glutamate receptors in the hippocampus of maLPA1 animals. Striatal dopaminergic markers (tyrosine hydroxylase, dopamine D1 receptor, and dopamine transporter DAT), were similar in both genotypes and were equally affected by cocaine exposure.ConclusionThe present results indicate that the lack of LPA1 receptor affect cocaine-induced conditioned locomotion but not behavioural sensitization. The findings suggest that LPA1 receptor may be necessary for a normal associative contextual learning associated to cocaine, probably through the modulation of hippocampal glutamatergic circuits.

Mammalian Glutaminase Gls2 Gene Encodes Two Functional Alternative Transcripts by a Surrogate Promoter Usage Mechanism

Background Glutaminase 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 Findings We 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/Significance This 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.

Both GLS silencing and GLS2 overexpression synergize with oxidative stress against proliferation of glioma cells.

Mitochondrial glutaminase (GA) plays an essential role in cancer cell metabolism, contributing to biosynthesis, bioenergetics, and redox balance. Humans contain several GA isozymes encoded by the GLS and GLS2 genes, but the specific roles of each in cancer metabolism are still unclear. In this study, glioma SFxL and LN229 cells with silenced isoenzyme glutaminase KGA (encoded by GLS) showed lower survival ratios and a reduced GSH-dependent antioxidant capacity. These GLS-silenced cells also demonstrated induction of apoptosis indicated by enhanced annexin V binding capacity and caspase 3 activity. GLS silencing was associated with decreased mitochondrial membrane potential (ΔΨm) (JC-1 dye test), indicating that apoptosis was mediated by mitochondrial dysfunction. Similar observations were made in T98 glioma cells overexpressing glutaminase isoenzyme GAB, encoded by GLS2, though some characteristics (GSH/GSSG ratio) were different in the differently treated cell lines. Thus, control of GA isoenzyme expression may prove to be a key tool to alter both metabolic and oxidative stress in cancer therapy. Interestingly, reactive oxygen species (ROS) generation by treatment with oxidizing agents: arsenic trioxide or hydrogen peroxide, synergizes with either KGA silencing or GAB overexpression to suppress malignant properties of glioma cells, including the reduction of cellular motility. Of note, negative modulation of GLS isoforms or GAB overexpression evoked lower c-myc and bcl-2 expression, as well as higher pro-apoptotic bid expression. Combination of modulation of GA expression and treatment with oxidizing agents may become a therapeutic strategy for intractable cancers and provides a multi-angle evaluation system for anti-glioma pre-clinical investigations.Key messageSilencing GLS or overexpressing GLS2 induces growth inhibition in glioma cell lines.Inhibition is synergistically enhanced after arsenic trioxide (ATO) or H2O2 treatment.Glutatione levels decrease in GLS-silenced cells but augment if GLS2 is overexpressed.ROS synergistically inhibit cell migration by GLS silencing or GLS2 overexpression.c-myc, bid, and bcl-2 mediate apoptosis resulting from GLS silencing or GLS2 overexpression.

New insights into brain glutaminases: Beyond their role on glutamatergic transmission

The synthesis of glutamate in brain must be exquisitely regulated because of its harmful potential giving rise to excitotoxic damage. In this sense, a stringent control based on multiple regulatory mechanisms should be expected to be exhibited by the biosynthetic enzymes responsible of glutamate generation, to assure that glutamate is only synthesized at the right place and at the right time. Glutaminase is considered as the main glutamate-producer enzyme in brain. Recently, novel glutaminase isoforms and extramitochondrial locations for these proteins have been discovered in the brain of mammals: identifying the function of each isozyme is essential for understanding the role of glutaminases in cerebral function. In addition, the interactome of glutaminases is starting to be uncovered adding a new level of regulatory complexity with important functional consequences, including selective and regulated targeting to concrete cellular locations. Finally, recent progress has identified glutaminase to be also present in astrocytes which precludes its classical consideration as a neuron-specific enzyme.