GLS1 depletion inhibited colorectal cancer proliferation and migration via redox/Nrf2/autophagy-dependent pathway.

Cancer cells can metabolize glutamine to replenish TCA cycle intermediates for cell survival. Glutaminase (GLS1) is over-expressed in multiple cancers, including colorectal cancer (CRC). However, the role of GLS1 in colorectal cancer development has not yet fully elucidated. In this study, we found that GLS1 levels were significantly increased in CRC cells. Knockdown of GLS1 by shRNAs as well as GLS1 inhibitor BPTES decreased DLD1 and SW480 cell proliferation, colony formation and migration. Knockdown of GLS1 as well as BPTES induced reactive oxygen species (ROS) production, down-regulation of GSH/GSSG ratio, an decrease in Nrf2 protein expression and an increase in cytoplasmic Nrf2 protein expression in DLD1 and SW480 cells. Furthermore, Knockdown of GLS1 as well as BPTES inhibited autophagy pathway, antioxidant NAC and Nrf2 activator could reversed inhibition of GLS1-mediated an decrease in autophagic flux in DLD1 and SW480 cells. Depletion of GLS1-induced inhibition of DLD1 and SW480 CRC cell proliferation, colony formation and migration was reversed by autophagy inducer rapamycin. These results suggest that targeting GLS1 might be a new potential therapeutic target for the treatment of CRC.

Bioprospecting of the agaricomycete Ganoderma australe GPC191 as novel source for L-asparaginase production.

L-Asparaginase is a therapeutically and industrially-competent enzyme, acting predominantly as an anti-neoplastic and anti-cancerous agent. The existing formulations of prokaryotic L-asparaginase are often toxic and contain L-glutaminase and urease residues, thereby increasing the purification steps. Production of L-glutaminase and urease free L-asparaginase is thus desired. In this research, bioprospecting of isolates from the less explored class Agaricomycetes was undertaken for L-asparaginase production. Plate assay (using phenol red and bromothymol blue dyes) was performed followed by estimation of L-asparaginase, L-glutaminase and urease activities by Nesslerization reaction for all the isolates. The isolate displaying the desired enzyme production was subjected to morphological, molecular identification, and phylogenetic analysis with statistical validation using Jukes-Cantor by Neighbour-joining tree of Maximum Likelihood statistical method. Among the isolates, Ganoderma australe GPC191 with significantly high zone index value (5.581 ± 0.045 at 120 h) and enzyme activity (1.57 ± 0.006 U/mL), devoid of L-glutaminase and urease activity was selected. The present study for the first-time reported G. australe as the potential source of L-glutaminase and urease-free L-asparaginase and also is one of the few studies contributing to the literature of G. australe in India. Hence, it can be postulated that it may find its future application in pharmaceutical and food industries.

Glutaminase Deficiency Caused by Short Tandem Repeat Expansion in GLS.

We report an inborn error of metabolism caused by an expansion of a GCA-repeat tract in the 5' untranslated region of the gene encoding glutaminase (GLS) that was identified through detailed clinical and biochemical phenotyping, combined with whole-genome sequencing. The expansion was observed in three unrelated patients who presented with an early-onset delay in overall development, progressive ataxia, and elevated levels of glutamine. In addition to ataxia, one patient also showed cerebellar atrophy. The expansion was associated with a relative deficiency of GLS messenger RNA transcribed from the expanded allele, which probably resulted from repeat-mediated chromatin changes upstream of the GLS repeat. Our discovery underscores the importance of careful examination of regions of the genome that are typically excluded from or poorly captured by exome sequencing.

Identification of a Loss-of-Function Mutation in the Context of Glutaminase Deficiency and Neonatal Epileptic Encephalopathy.

Importance: The identification and understanding of the monogenic causes of neurodevelopmental disorders are of high importance for personalized treatment and genetic counseling. Objective: To identify and characterize novel genes for a specific neurodevelopmental disorder characterized by refractory seizures, respiratory failure, brain abnormalities, and death in the neonatal period; describe the outcome of glutaminase deficiency in humans; and understand the underlying pathological mechanisms. Design, Setting, and Participants: We performed exome sequencing of cases of neurodevelopmental disorders without a clear genetic diagnosis, followed by genetic and bioinformatic evaluation of candidate variants and genes. Establishing pathogenicity of the variants was achieved by measuring metabolites in dried blood spots by a hydrophilic interaction liquid chromatography method coupled with tandem mass spectrometry. The participants are 2 families with a total of 4 children who each had lethal, therapy-refractory early neonatal seizures with status epilepticus and suppression bursts, respiratory insufficiency, simplified gyral structures, diffuse volume loss of the brain, and cerebral edema. Data analysis occurred from October 2017 to June 2018. Main Outcomes and Measures: Early neonatal epileptic encephalopathy with glutaminase deficiency and lethal outcome. Results: A total of 4 infants from 2 unrelated families, each of whom died less than 40 days after birth, were included. We identified a homozygous frameshift variant p.(Asp232Glufs*2) in GLS in the first family, as well as compound heterozygous variants p.(Gln81*) and p.(Arg272Lys) in GLS in the second family. The GLS gene encodes glutaminase (Enzyme Commission 3.5.1.2), which plays a major role in the conversion of glutamine into glutamate, the main excitatory neurotransmitter of the central nervous system. All 3 variants probably lead to a loss of function and thus glutaminase deficiency. Indeed, glutamine was increased in affected children (available z scores, 3.2 and 11.7). We theorize that the potential reduction of glutamate and the excess of glutamine were a probable cause of the described physiological and structural abnormalities of the central nervous system. Conclusions and Relevance: We identified a novel autosomal recessive neurometabolic disorder of loss of function of glutaminase that leads to lethal early neonatal encephalopathy. This inborn error of metabolism underlines the importance of GLS for appropriate glutamine homeostasis and respiratory regulation, signal transduction, and survival.

Glutamine fuels proliferation but not migration of endothelial cells.

Endothelial metabolism is a key regulator of angiogenesis. Glutamine metabolism in endothelial cells (ECs) has been poorly studied. We used genetic modifications and 13C tracing approaches to define glutamine metabolism in these cells. Glutamine supplies the majority of carbons in the tricyclic acid (TCA) cycle of ECs and contributes to lipid biosynthesis via reductive carboxylation. EC-specific deletion in mice of glutaminase, the initial enzyme in glutamine catabolism, markedly blunts angiogenesis. In cell culture, glutamine deprivation or inhibition of glutaminase prevents EC proliferation, but does not prevent cell migration, which relies instead on aerobic glycolysis. Without glutamine catabolism, there is near complete loss of TCA intermediates, with no compensation from glucose-derived anaplerosis. Mechanistically, addition of exogenous alpha-ketoglutarate replenishes TCA intermediates and rescues cellular growth, but simultaneously unveils a requirement for Rac1-dependent macropinocytosis to provide non-essential amino acids, including asparagine. Together, these data outline the dependence of ECs on glutamine for cataplerotic processes; the need for glutamine as a nitrogen source for generation of biomass; and the distinct roles of glucose and glutamine in EC biology.

Specific behavioural phenotype and secondary cognitive decline as a result of an 8.6 Mb deletion of 2q32.2q33.1.

Chromosomal abnormalities involving 2q32q33 deletions are very rare and present with a specific phenotype. This case report describes a 37-year-old female patient with 2q32q33 microdeletion syndrome presenting with the characteristic features, but with the addition of secondary cognitive decline. Molecular karyotyping was performed on the patient and her parents. It revealed an 8.6 megabase deletion with the proximal breakpoint in the chromosome band 2q32.2 and the distal breakpoint in 2q33.1. The deletion encompassed 22 known genes, including theGLS,MYO1B,TMEFF2,PGAP1andSATB2genes. The observed deletion was confirmed using a paralogue ratio test. This case report provides further evidence that theSATB2gene, together withGLS,MYO1B,TMEFF2and possiblyPGAP1,is a crucial gene in 2q32q33 microdeletion syndrome. TheSATB2gene seems to be crucial for the behavioural problems noted in our case, but deletion of theGLS,MYO1BandTMEFF2genes presumably contributed to the more complex behavioural characteristics observed. Our patient is also, to our knowledge, the only patient with 2q32q33 microdeletion syndrome with secondary cognitive decline.

Network of brain protein level changes in glutaminase deficient fetal mice.

Glutaminase is a multifunctional enzyme encoded by gene Gls involved in energy metabolism, ammonia trafficking and regeneration of neurotransmitter glutamate. To address the proteomic basis for the neurophenotypes of glutaminase-deficient mice, brain proteins from late gestation wild type, Gls+/- and Gls-/- male mice were subjected to two-dimensional gel electrophoresis, with subsequent identification by mass spectrometry using nano-LC-ESI-MS/MS. Protein spots that showed differential genotypic variation were quantified by immunoblotting. Differentially expressed proteins unambiguously identified by MS/MS included neurocalcin delta, retinol binding protein-1, reticulocalbin-3, cytoskeleton proteins fascin and tropomyosin alpha-4-chain, dihydropyrimidinase-related protein-5, apolipoprotein IV and proteins from protein metabolism proteasome subunits alpha type 2, type 7, heterogeneous nuclear ribonucleoprotein C1/C2 and H, voltage-gated anion-selective channel proteins 1 and 2, ATP synthase subunit ß and transitional endoplasmic reticulum ATPase. An interaction network determined by Ingenuity Pathway Analysis revealed a link between glutaminase and calcium, Akt and retinol signaling, cytoskeletal elements, ATPases, ion channels, protein synthesis and the proteasome system, intermediary, nucleic acid and lipid metabolism, huntingtin, guidance cues, transforming growth factor beta-1 and hepatocyte nuclear factor 4-alpha. The network identified involves (a) cellular assembly and organization and (b) cell signaling and cell cycle, suggesting that Gls is crucial for neuronal maturation.

Glutaminase-deficient mice display hippocampal hypoactivity, insensitivity to pro-psychotic drugs and potentiated latent inhibition: relevance to schizophrenia.

Dysregulated glutamatergic neurotransmission has been strongly implicated in the pathophysiology of schizophrenia (SCZ). Recently, presynaptic modulation of glutamate transmission has been shown to have therapeutic promise. We asked whether genetic knockdown of glutaminase (gene GLS1) to reduce glutamatergic transmission presynaptically by slowing the recycling of glutamine to glutamate, would produce a phenotype relevant to SCZ and its treatment. GLS1 heterozygous (GLS1 het) mice showed about a 50% global reduction in glutaminase activity, and a modest reduction in glutamate levels in brain regions relevant to SCZ pathophysiology, but displayed neither general behavioral abnormalities nor SCZ-associated phenotypes. Functional imaging, measuring regional cerebral blood volume, showed hippocampal hypometabolism mainly in the CA1 subregion and subiculum, the inverse of recent clinical imaging findings in prodromal and SCZ patients. GLS1 het mice were less sensitive to the behavioral stimulating effects of amphetamine, showed a reduction in amphetamine-induced striatal dopamine release and in ketamine-induced frontal cortical activation, suggesting that GLS1 het mice are resistant to the effects of these pro-psychotic challenges. Moreover, GLS1 het mice showed clozapine-like potentiation of latent inhibition, suggesting that reduction in glutaminase has antipsychotic-like properties. These observations provide further support for the pivotal role of altered glutamatergic synaptic transmission in the pathophysiology of SCZ, and suggest that presynaptic modulation of the glutamine-glutamate pathway through glutaminase inhibition may provide a new direction for the pharmacotherapy of SCZ.

Mice lacking brain/kidney phosphate-activated glutaminase have impaired glutamatergic synaptic transmission, altered breathing, disorganized goal-directed behavior and die shortly after birth.

Neurotransmitter glutamate has been thought to derive mainly from glutamine via the action of glutaminase type 1 (GLS1). To address the importance of this pathway in glutamatergic transmission, we knocked out GLS1 in mice. The insertion of a STOP cassette by homologous recombination produced a null allele that blocked transcription, encoded no immunoreactive protein, and abolished GLS1 enzymatic activity. Null mutants were slightly smaller, were deficient in goal-directed behavior, hypoventilated, and died in the first postnatal day. No gross or microscopic defects were detected in peripheral organs or in the CNS. In cultured neurons from the null mutants, miniature EPSC amplitude and duration were normal; however, the amplitude of evoked EPSCs decayed more rapidly with sustained 10 Hz stimulation, consistent with an observed reduction in depolarization-evoked glutamate release. Because of this activity-dependent impairment in glutamatergic transmission, we surmised that respiratory networks, which require temporal summation of synaptic input, would be particularly affected. We found that the amplitude of inspirations was decreased in vivo, chemosensitivity to CO2 was severely altered, and the frequency of pacemaker activity recorded in the respiratory generator in the pre-Bötzinger complex, a glutamatergic brainstem network that can be isolated in vitro, was increased. Our results show that although alternate pathways to GLS1 glutamate synthesis support baseline glutamatergic transmission, the GLS1 pathway is essential for maintaining the function of active synapses, and thus the mutation is associated with impaired respiratory function, abnormal goal-directed behavior, and neonatal demise.

Lack of expression of the liver-type glutaminase (LGA) mRNA in human malignant gliomas.

In the central nervous system (CNS), liver-type glutaminase (LGA) shows a unique nuclear localization suggesting its role in the regulation of transcription rather than in the cellular glutamine metabolism. RT-PCR analysis of RNA derived from postoperative tissue samples revealed the absence or only traces of LGA mRNA in all (9) cases of malignant gliomas (astrocytoma anaplasticum, AA, WHO grade III; glioblastoma multiforme, WHO grade IV) examined. The RNA was strongly expressed in the non-neoplastic tissue derived from the same patients (6 cases), and in most of the brain metastases from different organs (5 out of 7 cases). By contrast, the mRNAs coding for the kidney-type glutaminase (KGA) and its less ubiquitous isoform GAC, which catalyze degradation of the cytoplasmic pool of Gln, were expressed in all the tissues examined. The lack of LGA may be thus considered as a useful negative diagnostic marker of highly malignant gliomas in situ.