Allele-Specific Reprogramming of Cancer Metabolism by the Long Non-coding RNA CCAT2.

Altered energy metabolism is a cancer hallmark as malignant cells tailor their metabolic pathways to meet their energy requirements. Glucose and glutamine are the major nutrients that fuel cellular metabolism, and the pathways utilizing these nutrients are often altered in cancer. Here, we show that the long ncRNA CCAT2, located at the 8q24 amplicon on cancer risk-associated rs6983267 SNP, regulates cancer metabolism in vitro and in vivo in an allele-specific manner by binding the Cleavage Factor I (CFIm) complex with distinct affinities for the two subunits (CFIm25 and CFIm68). The CCAT2 interaction with the CFIm complex fine-tunes the alternative splicing of Glutaminase (GLS) by selecting the poly(A) site in intron 14 of the precursor mRNA. These findings uncover a complex, allele-specific regulatory mechanism of cancer metabolism orchestrated by the two alleles of a long ncRNA.

Pediatric-Like Brain Tumors in Adults.

Pediatric brain tumors are different to those found in adults in pathological type, anatomical site, molecular signature, and probable tumor drivers. Although these tumors usually occur in childhood, they also rarely present in adult patients, either as a de novo diagnosis or as a delayed recurrence of a pediatric tumor in the setting of a patient that has transitioned into adult services.Due to the rarity of pediatric-like tumors in adults, the literature on these tumor types in adults is often limited to small case series, and treatment decisions are often based on the management plans taken from pediatric studies. However, the biology of these tumors is often different from the same tumors found in children. Likewise, adult patients are often unable to tolerate the side effects of the aggressive treatments used in children-for which there is little or no evidence of efficacy in adults. In this chapter, we review the literature and summarize the clinical, pathological, molecular profile, and response to treatment for the following pediatric tumor types-medulloblastoma, ependymoma, craniopharyngioma, pilocytic astrocytoma, subependymal giant cell astrocytoma, germ cell tumors, choroid plexus tumors, midline glioma, and pleomorphic xanthoastrocytoma-with emphasis on the differences to the adult population.

Toward the "Perfect" Shunt: Historical Vignette, Current Efforts, and Future Directions.

As a concept, drainage of excess fluid volume in the cranium has been around for more than 1000 years. Starting with the original decompression-trepanation of Abulcasis to modern programmable shunt systems, to other nonshunt-based treatments such as endoscopic third ventriculostomy and choroid plexus cauterization, we have come far as a field. However, there are still fundamental limitations that shunts have yet to overcome: namely posture-induced over- and underdrainage, the continual need for valve opening pressure especially in pediatric cases, and the failure to reinstall physiologic intracranial pressure dynamics. However, there are groups worldwide, in the clinic, in industry, and in academia, that are trying to ameliorate the current state of the technology within hydrocephalus treatment. This chapter aims to provide a historical overview of hydrocephalus, current challenges in shunt design, what members of the community have done and continue to do to address these challenges, and finally, a definition of the "perfect" shunt is provided and how the authors are working toward it.

Dipeptidyl peptidase 4 (DPP4) in fecal samples: validation of the extraction methodology and stability in short-term storage conditions.

OBJECTIVES: This study assesses the clinical relevance of dipeptidyl peptidase 4 (DPP4) membrane exopeptidase as a biomarker of inflammatory bowel disease (IBD). A spike-and-recovery approach of DPP4 in fecal samples was used to compare two different methods for protein extraction, followed by a stability assessment. METHODS: Fecal samples of healthy volunteers spiked with known concentrations of recombinant DPP4 were processed using a standard manual extraction protocol and the CALEX® protocol. The two methods were compared by quantification of fecal DPP4 by ELISA, followed by Bland-Altman analysis. For the stability assays DPP4 was extracted from fecal samples and stored under different conditions of temperature and time after collection. RESULTS: In general, the levels of spiked DPP4 in stool samples were lower with the manual protocol than in those obtained with the CALEX® method; this trend was corroborated by Bland-Altman analysis. Nonetheless, variability was within the acceptable limits for both protocols. In the stability assessment, no statistically significant differences were found between the results obtained under the different storage conditions. CONCLUSIONS: Both manual and CALEX® protocols provided equal extraction ability of DPP4 from stool samples. In addition, DPP4 provided flexibility in terms of sample storage enabling the accurate assessment of samples delivered up to a week before analysis.

Subclinical Persistent Inflammation as Risk Factor for Crohn's Disease Progression: Findings From a Prospective Real-World Study of 2 Years.

BACKGROUND AND AIMS: Subclinical intestinal inflammation is common in Crohn's disease (CD). We aimed to explore its impact in the disease progression of infliximab-treated patients and the usefulness of fecal calprotectin (FC) and C-reactive protein (CRP) as surrogate minimally invasive biomarkers. METHODS: The registry-based, prospective, observational, multicenter DIRECT (study to investigate the correlation of fecal calprotectin with serum Drug levels and development of an antI-dRug antibodiEs among adult patients with inflammatory bowel disease reCeiving anti-TNF-alfa treatment or vedoluzimab treatment) study followed infliximab-treated CD patients for 2 years in a tertiary care setting. Persistent inflammation definition was based on FC (>150 µg/g, >250 µg/g, or >350 µg/g) or serum CRP (>3 µg/mL) concentrations over 2 consecutive or at least 3 visits. Patients were categorized according to a composite outcome reflecting disease progression that incorporated surgery; hospitalizations; new fistulae, abscess, or stricture; and treatment escalation. RESULTS: Of 322 DIRECT study patients, 180 asymptomatic, infliximab treated on maintenance regimen were included in the analysis. Patients developing the composite endpoint (n = 96) presented higher median levels of FC (205 [interquartile range, 98-515] µg/g; P = .045) but not of CRP (2.50 [interquartile range, 0.80-6.00] µg/mL; P = .895). Biomarker-defined persistent subclinical inflammation prevalence ranged from 24% to 81%. Considering FC >250 µg/g in 2 consecutive visits, prevalence was 50%, odds of achieving the endpoint were increased 3-fold (odds ratio, 2.996 [95% confidence interval, 1.557-5.776]), and time-to-outcome occurrence was significantly lower among subjects with persistent inflammation (median time: 11 months). Both clinical-related and treatment-related components were significantly associated with persistent inflammation. Definitions based on CRP >3 µg/mL, FC >150 µg/g, FC >350 µg/g, double biomarkers (FC >250 µg/g and/or CRP >3 µg/mL), or more visits did not improve predictive ability. CONCLUSIONS: Persistent inflammation, defined simply and readily by FC >250 µg/g over 2 consecutive visits, was associated with a significantly higher risk and shorter time to occurrence of a composite outcome reflecting disease progression in asymptomatic infliximab-treated CD patients.

Ventriculomegaly in children: nocturnal ICP dynamics identify pressure-compensated but active paediatric hydrocephalus.

INTRODUCTION: Paediatric ventriculomegaly without obvious signs or symptoms of raised intracranial pressure (ICP) is often interpreted as resulting from either relative brain atrophy, arrested "benign" hydrocephalus, or "successful" endoscopic third ventriculostomy (ETV). We hypothesise that the typical ICP "signature" found in symptomatic hydrocephalus can be present in asymptomatic or oligosymptomatic children, indicating pressure-compensated, but active hydrocephalus. METHODS: A total of 37 children fulfilling the mentioned criteria underwent computerised ICP overnight monitoring (ONM). Fifteen children had previous hydrocephalus treatment. ICP was analysed for nocturnal dynamics of ICP, ICP amplitudes (AMP), magnitude of slow waves (SLOW), and ICP/AMP correlation index RAP. Depending on the ONM results, children were either treated or observed. The ventricular width was determined at the time of ONM and at 1-year follow-up. RESULTS: The recordings of 14 children (group A) were considered normal. In the 23 children with pathologic recordings (group B), all ICP values and dependent variables (AMP, SLOW) were significantly higher, except for RAP. In group B, 12 of 15 children had received a pre-treatment and 11 of 22 without previous treatment. All group B children received treatment for hydrocephalus and showed a significant reduction of frontal-occipital horn ratio at 1 year. During follow-up, a positive neurological development was seen in 74% of children of group A and 100% of group B. CONCLUSION: Ventriculomegaly in the absence of signs and symptoms of raised ICP was associated in 62% of cases to pathological ICP dynamics. In 80% of pre-treated cases, ETV or shunt failure was found. Treating children with abnormal ICP dynamics resulted in an outcome at least as favourable as in the group with normal ICP dynamics. Thus, asymptomatic ventriculomegaly in children deserves further investigation and, if associated with abnormal ICP dynamics, should be treated in order to provide a normalised intracranial physiology as basis for best possible long-term outcome.

Dual inhibition of glutaminase and carnitine palmitoyltransferase decreases growth and migration of glutaminase inhibition-resistant triple-negative breast cancer cells.

Triple-negative breast cancers (TNBCs) lack progesterone and estrogen receptors and do not have amplified human epidermal growth factor receptor 2, the main therapeutic targets for managing breast cancer. TNBCs have an altered metabolism, including an increased Warburg effect and glutamine dependence, making the glutaminase inhibitor CB-839 therapeutically promising for this tumor type. Accordingly, CB-839 is currently in phase I/II clinical trials. However, not all TNBCs respond to CB-839 treatment, and the tumor resistance mechanism is not yet fully understood. Here we classified cell lines as CB-839-sensitive or -resistant according to their growth responses to CB-839. Compared with sensitive cells, resistant cells were less glutaminolytic and, upon CB-839 treatment, exhibited a smaller decrease in ATP content and less mitochondrial fragmentation, an indicator of poor mitochondrial health. Transcriptional analyses revealed that the expression levels of genes linked to lipid metabolism were altered between sensitive and resistant cells and between breast cancer tissues (available from The Cancer Genome Atlas project) with low versus high glutaminase (GLS) gene expression. Of note, CB-839-resistant TNBC cells had increased carnitine palmitoyltransferase 2 (CPT2) protein and CPT1 activity levels. In agreement, CB-839-resistant TNBC cells mobilized more fatty acids into mitochondria for oxidation, which responded to AMP-activated protein kinase and acetyl-CoA carboxylase signaling. Moreover, chemical inhibition of both glutaminase and CPT1 decreased cell proliferation and migration of CB-839-resistant cells compared with single inhibition of each enzyme. We propose that dual targeting of glutaminase and CPT1 activities may have therapeutic relevance for managing CB-839-resistant tumors.

Value of computerized shunt infusion study in assessment of pediatric hydrocephalus shunt function-a two center cross-sectional study.

BACKGROUND: Hydrocephalus shunt malfunction can-also in children-occur insidiously without clear symptoms of raised intracranial pressure (ICP) or changes in ventricular size, imposing a diagnostic challenge. Computerized shunt infusion studies enable quantitative shunt function assessment. We report on feasibility and results of this technique in children in a two center cross-sectional study. MATERIAL AND METHODS: Shunt infusion study (SIS) is performed with two needles inserted into a pre-chamber for ICP recording and CSF infusion. After baseline ICP recording, constant rate infusion is started until a new ICP plateau (ICPpl) is reached. Dedicated software containing the shunt's resistance characteristics calculates ICP and its amplitude outflow resistance and critical shunt pressure (CSP). Overall, 203 SIS were performed in 166 children. Shunts were defined as functional if ICPpl was 5 mmHg above CSP and borderline in between. RESULTS: Forty-one shunts (20.2%) were found obstructed, 26 (12.8%) had borderline characteristics, and 136 (67%) were functional. Baseline ICP in obstructed shunts was significantly above shunt operating pressure. CSF outflow resistance (Rout) and ∆ICP plateau were significantly elevated in obstructed shunts, with cut-off thresholds of 8.07 mmHg min/ml and 11.74 mmHg respectively. Subgroup analysis showed smaller ventricles in 69% of revised cases. CONCLUSION: SIS is a feasible, reliable, and radiation-free technique for quantitative shunt assessment to rule out or prove shunt malfunction. Dedicated software containing shunt hydrodynamic characteristics is necessary and small children may need short-term sedation. Due to the clinical and inherent economic advantages, SIS should be more frequently used in pediatric neurosurgery.

Application of Natural Pigments in Ordinary Cooked Ham.

The possibility of obtaining a carmine or pink color on ordinary cooked ham by applying natural dyes from three plant species, namely red radish (Raphanus sativus L.), hibiscus (Roselle sabdariffa L.) and red beetroot (Beta vulgaris L.), was investigated. The extracts were evaluated for the stability at physical-chemical parameters and subjected to cytotoxicity assays in the gastric cell line AGS Encapsulation of the extracts in soybean lecithin liposomes and maltodextrin microcapsules was performed. Lyophilized extracts before and after encapsulation in maltodextrin were applied in the formulation of ordinary cooked ham and used in a pilot scale of production. The color of cooked ham samples from different assays was evaluated visually and by colorimetry. The results suggest that the coloration of ordinary cooked ham obtained with extracts of red beetroot is very promising for future applications in this type of meat product.

Patterns of care: burr-hole cover application for chronic subdural hematoma trepanation.

OBJECTIVE: Skin depressions may appear as undesired effects after burr-hole trepanation for the evacuation of chronic subdural hematomas (cSDH). Placement of burr-hole covers to reconstruct skull defects can prevent skin depressions, with the potential to improve the aesthetic result and patient satisfaction. The perception of the relevance of this practice, however, appears to vary substantially among neurosurgeons. The authors aimed to identify current practice variations with regard to the application of burr-hole covers after trepanation for cSDH. METHODS: An electronic survey containing 12 questions was sent to resident and faculty neurosurgeons practicing in different parts of the world, as identified by an Internet search. All responses completed between September 2018 and December 2018 were considered. Descriptive statistics and logistic regression were used to analyze the data. RESULTS: A total of 604 responses were obtained, of which 576 (95.4%) provided complete data. The respondents' mean age was 42.4 years (SD 10.5), and 86.5% were male. The sample consisted of residents, fellows, junior/senior consultants, and department chairs from 79 countries (77.4% Europe, 11.8% Asia, 5.4% America, 3.5% Africa, and 1.9% Australasia). Skin depressions were considered a relevant issue by 31.6%, and 76.0% indicated that patients complain about skin depressions more or less frequently. Burr-hole covers are placed by 28.1% in the context of cSDH evacuation more or less frequently. The most frequent reasons for not placing a burr-hole cover were the lack of proven benefit (34.8%), followed by additional costs (21.9%), technical difficulty (19.9%), and fear of increased complications (4.9%). Most respondents (77.5%) stated that they would consider placing burr-hole covers in the future if there was evidence for superiority of the practice. The use of burr-hole covers varied substantially across countries, but a country's gross domestic product per capita was not associated with their placement. CONCLUSIONS: Only a minority of neurosurgeons place burr-hole covers after trepanation for cSDH on a regular basis, even though the majority of participants reported complaints from patients regarding postoperative skin depressions. There are significant differences in the patterns of care among countries. Class I evidence with regard to patient satisfaction and safety of burr-hole cover placement is likely to have an impact on future cSDH management.

Glutaminase Affects the Transcriptional Activity of Peroxisome Proliferator-Activated Receptor γ (PPARγ) via Direct Interaction.

Phosphate-activated glutaminases catalyze the deamidation of glutamine to glutamate and play key roles in several physiological and pathological processes. In humans, GLS encodes two multidomain splicing isoforms: KGA and GAC. In both isoforms, the canonical glutaminase domain is flanked by an N-terminal region that is folded into an EF-hand-like four-helix bundle. However, the splicing event replaces a well-structured three-repeat ankyrin domain in KGA with a shorter, unordered C-terminal stretch in GAC. The multidomain architecture, which contains putative protein-protein binding motifs, has led to speculation that glutaminases are involved in cellular processes other than glutamine metabolism; in fact, some proteins have been identified as binding partners of KGA and the isoforms of its paralogue gene, GLS2. Here, a yeast two-hybrid assay identified nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) as a new binding partner of the glutaminase. We show that KGA and GAC directly bind PPARγ with a low-micromolar dissociation constant; the interaction involves the N-terminal and catalytic domains of glutaminases as well as the ligand-binding domain of the nuclear receptor. The interaction occurs within the nucleus, and by sequestering PPARγ from its responsive element DR1, the glutaminases decreased nuclear receptor activity as assessed by a luciferase reporter assay. Altogether, our findings reveal an unexpected glutaminase-binding partner and, for the first time, directly link mitochondrial glutaminases to an unanticipated role in gene regulation.

The origin and evolution of human glutaminases and their atypical C-terminal ankyrin repeats.

On the basis of tissue-specific enzyme activity and inhibition by catalytic products, Hans Krebs first demonstrated the existence of multiple glutaminases in mammals. Currently, two human genes are known to encode at least four glutaminase isoforms. However, the phylogeny of these medically relevant enzymes remains unclear, prompting us to investigate their origin and evolution. Using prokaryotic and eukaryotic glutaminase sequences, we built a phylogenetic tree whose topology suggested that the multidomain architecture was inherited from bacterial ancestors, probably simultaneously with the hosting of the proto-mitochondrion endosymbiont. We propose an evolutionary model wherein the appearance of the most active enzyme isoform, glutaminase C (GAC), which is expressed in many cancers, was a late retrotransposition event that occurred in fishes from the Chondrichthyes class. The ankyrin (ANK) repeats in the glutaminases were acquired early in their evolution. To obtain information on ANK folding, we solved two high-resolution structures of the ANK repeat-containing C termini of both kidney-type glutaminase (KGA) and GLS2 isoforms (glutaminase B and liver-type glutaminase). We found that the glutaminase ANK repeats form unique intramolecular contacts through two highly conserved motifs; curiously, this arrangement occludes a region usually involved in ANK-mediated protein-protein interactions. We also solved the crystal structure of full-length KGA and present a small-angle X-ray scattering model for full-length GLS2. These structures explain these proteins' compromised ability to assemble into catalytically active supra-tetrameric filaments, as previously shown for GAC. Collectively, these results provide information about glutaminases that may aid in the design of isoform-specific glutaminase inhibitors.

Role of Indocyanine Green Videoangiography in Identification of Donor and Recipient Arteries in Cerebral Bypass Surgery.

The identification and preparation of a very good quality donor artery is a crucial step in every superficial temporal artery to middle cerebral artery (STA-MCA) bypass.For flow-preservation bypass performed for trapping of complex MCA aneurysms, the key element is the correct target of the recipient artery. When a cortical recipient artery (M4 segment of the MCA) is selected, this vessel must be a terminal branch of the artery whose sacrifice is necessary for definitive aneurysmal treatment.In this chapter we report on two techniques for (1) intraoperative mapping and preparation of good quality STA branch as the donor artery for STA-MCA bypass (mostly in the case the frontal branch of the STA needs to be used) and (2) selective identification of the correct superficial (M4 cortical) "recipient" artery in flow-preservation STA-MCA bypass performed for managing complex MCA aneurysms.Both techniques are based on the use of microscope-integrated indocyanine green videoangiography (ICG-VA), an intraoperative tool allowing observation and real-time assessment of blood flow in large and small vessels, with distinct evaluation of arterial, capillary, and venous phases.The two techniques contribute, respectively, to (1) reduce the risk of erroneous identification or injury of the donor artery in STA-MCA bypass procedures and (2) eliminate the risk of erroneous revascularization of a non-involved arterial territory in flow-preservation bypass surgery for managing complex MCA aneurysms.

Using microRNA Networks to Understand Cancer.

Human cancers are characterized by deregulated expression of multiple microRNAs (miRNAs), involved in essential pathways that confer the malignant cells their tumorigenic potential. Each miRNA can regulate hundreds of messenger RNAs (mRNAs), while various miRNAs can control the same mRNA. Additionally, many miRNAs regulate and are regulated by other species of non-coding RNAs, such as circular RNAs (circRNAs) and long non-coding RNAs (lncRNAs). For this reason, it is extremely difficult to predict, study, and analyze the precise role of a single miRNA involved in human cancer, considering the complexity of its connections. Focusing on a single miRNA molecule represents a limited approach. Additional information could come from network analysis, which has become a common tool in the biological field to better understand molecular interactions. In this review, we focus on the main types of networks (monopartite, association networks and bipartite) used for analyzing biological data related to miRNA function. We briefly present the important steps to take when generating networks, illustrating the theory with published examples and with future perspectives of how this approach can help to better select miRNAs that can be therapeutically targeted in cancer.

Guanylate-binding protein-1 is a potential new therapeutic target for triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) is characterized by a lack of estrogen and progesterone receptor expression (ESR and PGR, respectively) and an absence of human epithelial growth factor receptor (ERBB2) amplification. Approximately 15-20% of breast malignancies are TNBC. Patients with TNBC often have an unfavorable prognosis. In addition, TNBC represents an important clinical challenge since it does not respond to hormone therapy. METHODS: In this work, we integrated high-throughput mRNA sequencing (RNA-Seq) data from normal and tumor tissues (obtained from The Cancer Genome Atlas, TCGA) and cell lines obtained through in-house sequencing or available from the Gene Expression Omnibus (GEO) to generate a unified list of differentially expressed (DE) genes. Methylome and proteomic data were integrated to our analysis to give further support to our findings. Genes that were overexpressed in TNBC were then curated to retain new potentially druggable targets based on in silico analysis. Knocking-down was used to assess gene importance for TNBC cell proliferation. RESULTS: Our pipeline analysis generated a list of 243 potential new targets for treating TNBC. We finally demonstrated that knock-down of Guanylate-Binding Protein 1 (GBP1 ), one of the candidate genes, selectively affected the growth of TNBC cell lines. Moreover, we showed that GBP1 expression was controlled by epidermal growth factor receptor (EGFR) in breast cancer cell lines. CONCLUSIONS: We propose that GBP1 is a new potential druggable therapeutic target for treating TNBC with enhanced EGFR expression.

Solvothermal Synthesis of Cu2SnS3 Quantum Dots and Their Application in Near-Infrared Photodetectors.

Cu2SnS3 (CTS) quantum dots were synthesized by solvothermal technique with poly(vinylpyrrolidone) (PVP) as a surfactant. The structural and optical properties were studied using X-ray diffraction, transmission electron microscopy, X-ray photoelectron spectroscopy, and UV-vis spectroscopy. The electronic band gap was measured using cyclic voltammetry. The infrared photoresponse of the CTS quantum dots-incorporated device was measured under different illumination intensities of the infrared lamp, 1550 and 1064 nm lasers. The characteristics of the photodetector device, that is, responsivity, external quantum efficiency, and specific detectivity were calculated. This study proves the potential use of CTS quantum dots in infrared photodetectors.

Effect of Illumination Intensities on the Visible and Infrared Photoresponse of Cu2SnS3 Nanostructures.

We report the synthesis of Cu(2)SnS(3) nanostructures using solvothermal technique and the study of its visible and infrared (IR) photoresponse under different illumination intensities. The CTS nano-crystals were found to have tetragonal crystal structure using X-ray Diffraction (XRD). Both flower and sphere shaped structures of around 1.5 µm were obtained as seen using scanning electron microscopy (SEM). Transmission electron microscopy (TEM) was used to study the crystalline nature as well as the different planes present in the crystal. The band gap of the obtained crystals was found to be 1.4 eV using optical studies. The visible photocurrent increased from 0.25 µA at dark to 0.42 µA at 1.05 suns and 1 V applied bias. The sensitivity increased from 1.25 at 0.88 suns to 1.68 at 1.05 suns. The IR photocurrent increased from 0.13 µA at dark to 0.66 µA at 477.7 mWcm(−2). The device exhibited an increase in the sensitivity, responsivity, external quantum efficiency and specific detectivity from 1.23, 0.10 mA/W, 0.016% and 5.02 × 10(8) Jones respectively at 127.4 mWcm(−2 ) to 4.95, 0.46 mA/W, 0.071% and 2.22×10(9) Jones respectively at 477.7 mWcm(−2). The time response of the photocurrent was measured over different ON-OFF cycles and the cyclic stability of the device was verified.

Mitochondrial localization and structure-based phosphate activation mechanism of Glutaminase C with implications for cancer metabolism.

Glutamine is an essential nutrient for cancer cell proliferation, especially in the context of citric acid cycle anaplerosis. In this manuscript we present results that collectively demonstrate that, of the three major mammalian glutaminases identified to date, the lesser studied splice variant of the gene gls, known as Glutaminase C (GAC), is important for tumor metabolism. We show that, although levels of both the kidney-type isoforms are elevated in tumor vs. normal tissues, GAC is distinctly mitochondrial. GAC is also most responsive to the activator inorganic phosphate, the content of which is supposedly higher in mitochondria subject to hypoxia. Analysis of X-ray crystal structures of GAC in different bound states suggests a mechanism that introduces the tetramerization-induced lifting of a "gating loop" as essential for the phosphate-dependent activation process. Surprisingly, phosphate binds inside the catalytic pocket rather than at the oligomerization interface. Phosphate also mediates substrate entry by competing with glutamate. A greater tendency to oligomerize differentiates GAC from its alternatively spliced isoform and the cycling of phosphate in and out of the active site distinguishes it from the liver-type isozyme, which is known to be less dependent on this ion.

Active glutaminase C self-assembles into a supratetrameric oligomer that can be disrupted by an allosteric inhibitor.

The phosphate-dependent transition between enzymatically inert dimers into catalytically capable tetramers has long been the accepted mechanism for the glutaminase activation. Here, we demonstrate that activated glutaminase C (GAC) self-assembles into a helical, fiber-like double-stranded oligomer and propose a molecular model consisting of seven tetramer copies per turn per strand interacting via the N-terminal domains. The loop (321)LRFNKL(326) is projected as the major regulating element for self-assembly and enzyme activation. Furthermore, the previously identified in vivo lysine acetylation (Lys(311) in humans, Lys(316) in mouse) is here proposed as an important down-regulator of superoligomer assembly and protein activation. Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide, a known glutaminase inhibitor, completely disrupted the higher order oligomer, explaining its allosteric mechanism of inhibition via tetramer stabilization. A direct correlation between the tendency to self-assemble and the activity levels of the three mammalian glutaminase isozymes was established, with GAC being the most active enzyme while forming the longest structures. Lastly, the ectopic expression of a fiber-prone superactive GAC mutant in MDA-MB 231 cancer cells provided considerable proliferative advantages to transformed cells. These findings yield unique implications for the development of GAC-oriented therapeutics targeting tumor metabolism.