Changes in the nuclear proteomic profiling of human glioblastoma cells after glutaminase overexpression

A proteomics study of the nuclear protein profiling of human glioblastoma cells with targeted glutaminase expression. The study was performed with protein microarrays and SELDI-TOF technology.Human glutaminase (GA) enzymes are the products of two genes, GLS and GLS2. GA isozymes play markedly different roles in tumour biology: GLS isoforms (KGA and GAC) are related to cell growth and proliferation, whereas GLS2 isoforms (GAB and LGA) are associated with low proliferation rates and are characteristics for resting or quiescent cells. Furthermore, the GAB isoform has been recently involved in transcriptional regulation. Due to the proposed roles of GAB isoform in cellular differentiation and transcriptional modulation, the aim of the present study is to profile differentially expressed nuclear proteins in order to discover putative biomarkers associated with GAB overexpression in glioma cells. Nuclear cell proteins were incubated with strong anion exchange (Q10) and weak cation exchange (CM10) ProteinChip arrays and analyzed using surface-enhanced laser desorption/ionization time-of- flight mass spectrometry (SELDI-TOF/MS) proteomics technology. T98G-GAB nuclear protein expression profiles were compared with those of T98G-WT and empty vector transfected T98G-pcDNA3 with the ProteinChip Data Manager Client 4.0 software. Eighteen proteins were found to be differentially expressed between control cells and GAB-transfected T98G cells. Nine proteins with m/z between 5.5-29.7 were identified to be highly expressed in T98G-GAB (P≤ 0.01), while the expression of two proteins with m/z values of 7.8 and 8.5 were higher in control cells (P≤ 0.01). Our study shows the potential of proteomics profiling to get a deep insight into the role of nuclear GAB in brain tumors in order to assess its suitability as a novel anti-cancer therapeutic target. References Pérez-Gómez C, et al. Co-expression of glutaminase K and L isoenzymes in human tumour cells. Biochem. J. 2005; 386, 535-42. Szeliga M, et al. Transfection with liver-type glutaminase (LGA) cDNA alteres gene expression and reduces viability, migration and proliferation of T98G glioma cells. Glia 2009; 57, 1014-23.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.This work was supported by Grant SAF2010-17573 from the Ministry of Science and Innovation of Spain, Excellence Grant CVI-6656 from the regional Andalusian government (Junta de Andalucia), and Grant RD06/1012 of the RTA RETICS network from the Spanish Health Institute Carlos III

Glutaminase and MMP-9 downregulation in cortex and hippocampus of LPA1 receptor null mice correlate with altered dendritic spine plasticity

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

Ratones knock-out del receptor lpa1 de ácido lisofosfatídico presentan un acusado déficit de la isoenzima glutaminasa KGA (GLS) y una morfología alterada en las espinas dendríticas de hipocampo y corteza

Objectives: The objective of the present study was to utilize mice with knocked-down lysophosphatidic acid 1 (LPA1) receptor to ascertain changes in glutamatergic transmission that may help to explain part of the cognitive and memory deficits shown by these KO-LPA1 mice. Material & methods: A well characterized KO-LPA1 mouse strain was used as animal model and compared with wild-type (WT) and heterozygous animals. Expression studies were implemented by immunohistochemistry and Western analysis of mouse brain regions, real-time quantitative RT-PCR of GA isoforms, enzymatic analysis of regional GA activity and Golgi staining to assess dendritic spine morphology and density. Results: A strong reduction of KGA immunoreactivity was mostly revealed in cerebral cortex and hippocampus of KO-LPA1 mice versus WT and heterozygous animals. In contrast, neither mRNA levels nor enzyme activity were significantly altered in KO mice suggesting compensatory mechanisms for neurotransmitter Glu synthesis. Interestingly, Golgi staining of hippocampal and cortical neurons revealed a clear morphology change toward a less-mature undifferentiated spine phenotype, without changes in the total number of spines. Conclusions: The molecular mechanisms underlying KGA downregulation in null LPA1 mutant mice are unknown. However, LPA increases neuronal differentiation, arborization and neurite outgrowth of developing neurons, while Gln-derived Glu, through GA reaction, has been also involved in neuronal growth and differentiation. It is tempting to speculate that downregulation of KGA protein in KO-LPA1 mice induce morphological changes in dendritic spines of cortical and hippocampal neurons which, in turn, may account for memory and cognitive deficits shown by KO-LPA1 mice.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Acknowledgements: Red de Trastornos Adictivos, RTA, (RD12/0028/0013/) RETICS, ISCIII, y Consejería Innovación, Ciencia y Empresa, Junta de Andalucía (Proyecto de Excelencia CVI-6656)

Pharmacological Blockade of Cannabinoid CB1 Receptors in Diet-Induced Obesity Regulates Mitochondrial Dihydrolipoamide Dehydrogenase in Muscle

Funding: This work was supported by CIBERobn (CB06/03/1008), Ministerio de Economía y Competitividad (MINECO) (PG: BFU2012-33334), Instituto de Salud Carlos III (ISCIII), MINECO, co-funded 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), Consejería de Economía, Innovación y Ciencia, Junta de Andalucía, UE-ERDF (FRF: CTS-8221, JM: CVI-6656), Consejería de Salud, Junta de Andalucía, 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)Peer reviewedPublisher PD

Nuclear translocation of glutaminase GLS2 in human cancer cells associates with proliferation arrest and differentiation

Glutaminase (GA) catalyzes the first step in mitochondrial glutaminolysis playing a key role in cancer metabolic reprogramming. Humans express two types of GA isoforms: GLS and GLS2. GLS isozymes have been consistently related to cell proliferation, but the role of GLS2 in cancer remains poorly understood. GLS2 is repressed in many tumor cells and a better understanding of its function in tumorigenesis may further the development of new therapeutic approaches. We analyzed GLS2 expression in HCC, GBM and neuroblastoma cells, as well as in monkey COS-7 cells. We studied GLS2 expression after induction of differentiation with phorbol ester (PMA) and transduction with the full-length cDNA of GLS2. In parallel, we investigated cell cycle progression and levels of p53, p21 and c-Myc proteins. Using the baculovirus system, human GLS2 protein was overexpressed, purified and analyzed for posttranslational modifications employing a proteomics LC-MS/MS platform. We have demonstrated a dual targeting of GLS2 in human cancer cells. Immunocytochemistry and subcellular fractionation gave consistent results demonstrating nuclear and mitochondrial locations, with the latter being predominant. Nuclear targeting was confirmed in cancer cells overexpressing c-Myc- and GFP-tagged GLS2 proteins. We assessed the subnuclear location finding a widespread distribution of GLS2 in the nucleoplasm without clear overlapping with specific nuclear substructures. GLS2 expression and nuclear accrual notably increased by treatment of SH-SY5Y cells with PMA and it correlated with cell cycle arrest at G2/M, upregulation of tumor suppressor p53 and p21 protein. A similar response was obtained by overexpression of GLS2 in T98G glioma cells, including downregulation of oncogene c-Myc. Furthermore, human GLS2 was identified as being hypusinated by MS analysis, a posttranslational modification which may be relevant for its nuclear targeting and/or function. Our studies provide evidence for a tumor suppressor role of GLS2 in certain types of cancer. The data imply that GLS2 can be regarded as a highly mobile and multilocalizing protein translocated to both mitochondria and nuclei. Upregulation of GLS2 in cancer cells induced an antiproliferative response with cell cycle arrest at the G2/M phase

Detection of kinase domain mutations in BCR::ABL1 leukemia by ultra-deep sequencing of genomic DNA

The screening of the BCR::ABL1 kinase domain (KD) mutation has become a routine analysis in case of warning/failure for chronic myeloid leukemia (CML) and B-cell precursor acute lymphoblastic leukemia (ALL) Philadelphia (Ph)-positive patients. In this study, we present a novel DNA-based next-generation sequencing (NGS) methodology for KD ABL1 mutation detection and monitoring with a 1.0E-4 sensitivity. This approach was validated with a well-stablished RNA-based nested NGS method. The correlation of both techniques for the quantification of ABL1 mutations was high (Pearson r = 0.858, p < 0.001), offering DNA-DeepNGS a sensitivity of 92% and specificity of 82%. The clinical impact was studied in a cohort of 129 patients (n = 67 for CML and n = 62 for B-ALL patients). A total of 162 samples (n = 86 CML and n = 76 B-ALL) were studied. Of them, 27 out of 86 harbored mutations (6 in warning and 21 in failure) for CML, and 13 out of 76 (2 diagnostic and 11 relapse samples) did in B-ALL patients. In addition, in four cases were detected mutation despite BCR::ABL1 < 1%. In conclusion, we were able to detect KD ABL1 mutations with a 1.0E-4 sensitivity by NGS using DNA as starting material even in patients with low levels of disease

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

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.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.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

Design and generation of a glutaminase GLS2 conditional knockout mice

Mammalian glutaminase (GA; EC 3.5.1.2) is the main enzyme involved in brain generation of glutamate (Glu). This amino acid acts as an excitatory neurotransmitter within the CNS, and it is also implicated in behavioral sensitization through the mesolimbic pathway. Two different GA genes have been described: Gls that encodes the isozymes KGA and GAC, and Gls2, which encodes GAB and LGA isozymes. Gls and Gls2 isoforms are co-expressed in different brain regions and cells. Of note, location of Gls2-encoded isoforms in neuronal nuclei suggests a novel role in the regulation of gene expression. The co-expression of different GA isoforms in mammalian brain is so far unexplained. Our objective is to study the cerebral function of Gls2; for this purpose, we develop a conditional knockout (KO) mouse model to silence GAB and LGA expression in brain. A vector carrying the Gls2 gene from exon 1 to 12 (obtained from the EUCOMM consortium) was transfected by electroporation into B6D2F1 murine embryonic stem cells (ES). These ES were selected by geneticin and PCR-genotyped before their microinjection in 8-cell stage embryos (Swiss strain). Embryo implantation was performed in pseudopregnant state mice, which leads to chimeric pups. This vector targets chromosome 10 and will yield a conditional KO mouse model, since exons 2 to 7 are included between loxP sites. The chimeric pups carrying this modification within their germ line were used to generate the homozygous Gls2 (-/-) mice. After integration of the vector in both alleles, the mice will be mated with mutant Cre mice, which express this recombinase enzyme under control of the synapsin specific promoter. This will result in a deletion of the exons 2 to 7 giving rise to null Gls2 mutants mainly in the following brain areas: cortex, hippocampus, amygdala and cerebellum, which are essential for glutamatergic transmission and related to the mesolimbic pathway.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Evaluación de un programa de atención sanitaria con enfermera tutora en cirugía de traumatología en un hospital de Granada 2004

Fundamento: La tendencia actual en la atención al paciente es hacia un aumento de la calidad en los servicios sanitarios. El objetivo de este trabajo fue evaluar los resultados de la aplicación de un programa de atención sanitaria a través de una enfermera de referencia que acogía al paciente al ingreso hospitalario, le visitaba regularmente durante su estancia y le resolvía dudas y problemas. Métodos: Estudio cuasi-experimental realizado en pacientes (149 casos y 454 controles) ingresados para cirugía traumatológica en el Hospital Virgen de las Nieves de Granada. Al ingreso se recogieron variables sociodemográficas y clínicas. A los 15 días tras el alta se les administró la ¿Escala heteroevaluada de ansiedad de Zung¿ y un cuestionario ad hoc sobre la satisfacción con diferentes aspectos de la atención sanitaria durante la estancia en el hospital y la comprensión de la información recibida. Se realizó análisis bivariante y análisis de regresión lineal y regresión logística multivariante con estudio de variables confundentes. Resultados: Para la variable nivel de ansiedad los pacientes no tutorizados en el análisis de regresión lineal multivariante tenían un coeficiente B=2,64 (p<0,01), controlando por la otras variables en el modelo final: sexo, presencia de cuidador informal actividad profesional, días de estancia, y comprensión de la información sanitaria. Para la variable de resultado insuficiente comprensión de la información los pacientes no tutorizados tenían una Odds Ratio de 3,48 en el análisis de regresión logística multivariante controlando por educación y presencia de cuidador informal. La satisfacción con la amabilidad del personal y con el medio hospitalario no se modificó significativamente aunque el porcentaje de insatisfacción fue mayor en el grupo no tutorizado, 15% frente a 11% (p= 0,34), y 18% frente a 12% (p=0,11) respectivamente. Conclusiones: El programa de atención continuada aplicado se ha manifestado eficaz para los pacientes, aumentando la comprensión de la información y disminuyendo los niveles de ansiedad

Immunoblot analysis of human SHSY-5Y neuroblastoma cells and rat liver and brain mitochondria.

<p>Cell extracts of human neuroblastoma SHSY-5Y cells (left and center panel) and mitochondria isolated from rat liver and brain (right panel) were analyzed by SDS-PAGE and Western blotting. Blots were revealed by chemiluminescence using polyclonal antibodies raised against the exclusive first exon of human GAB protein (left panel) or against the whole human GAB protein which recognize both GAB and LGA proteins (center and right panel). M, lanes containing the molecular mass markers indicated on the left margin; SHSY-5Y, lanes loaded with protein extracts isolated from human neuroblastoma cells; Liver 1 and 2: lanes containing total liver protein extracts and mitochondrial protein extracts, respectively; Brain 3 and 4: lanes containing total brain protein extracts and mitochondrial protein extracts, respectively.</p