The mitochondrial enzyme glutaminase: Its role in cancer cell metabolism

线粒体谷氨酰胺酶：其在癌细胞代谢中的作用

• 批准号: 8912267
• 负责人: Clint A Stalnecker
• 金额: $ 3.74万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8912267
• 关键词: Attention; Biological; Biological Assay; Biological Models; Cancer Cell Growth; Cancerous; Cell Line; Cell model; Cells; Characteristics; Citric Acid Cycle; Complement; Coupled; DNA; Development; Embryo; Engineering; Enzymes; Fibroblasts; Fluorescence Resonance Energy Transfer; Fluorescent Probes; Glucose; Glutamates; Glutaminase; Glutamine; Glycolysis; Growth; Guanine Nucleotide Exchange Factors; Health; In Vitro; Label; Laboratories; Lead; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Metabolic; Metabolism; Methods; Mitochondria; Modeling; Monitor; Motivation; Mus; Mutate; NMR Spectroscopy; Neoplasms; Normal Cell; Nuclear Magnetic Resonance; Nucleotide Biosynthesis; Nutritional Requirements; Oncogenic; Outcome; Pathway interactions; Phenotype; Play; Portraits; Proliferating; Property; Protein Isoforms; Proteins; Pyruvate; Recombinants; Roentgen Rays; Role; Signal Pathway; Structure; Study models; System; Tetracycline Control; Tetracyclines; Therapeutic; Up-Regulation; alpha ketoglutarate; anticancer research; c-myc Genes; cancer cell; cancer therapy; dimer; enzyme activity; expectation; fatty acid biosynthesis; feeding; glucose metabolism; in vivo; inhibitor/antagonist; inorganic phosphate; interest; lipid biosynthesis; metabolic abnormality assessment; mutant; neoplastic cell; new therapeutic target; novel; novel therapeutics; respiratory; rho GTP-Binding Proteins; small molecule; therapeutic target; transcription factor; treatment strategy; tumor; uptake

DESCRIPTION (provided by applicant): Among the several hallmarks of cancer cells currently gaining attention is the remodeling of cellular metabolism, where glucose and glutamine are imported at high rates and metabolized to provide building blocks for the biosynthesis of lipids, proteins, and DNA required for growth and proliferation. Transformation of normal cells is therefore often accompanied by a dramatic increase in the glycolytic flux although the end product of glycolysis, pyruvate, is diverted from entering the respiratory TCA cycle and instead secreted from the cell as lactate. In order to compensate for the decrease in glucose-derived pyruvate entering the TCA cycle, glutamine is taken up and converted to glutamate by the mitochondrial enzyme glutaminase, where glutamate can then feed an entry point of the TCA cycle upon conversion to α-ketoglutarate. The increased flux of glutamine and its role in providing biosynthetic precursors for proliferation represents a critical step in the metabolic remodeling of cancer cells. In particular, a specific isoform of glutaminase, glutaminase C (GAC), has been shown to be up regulated and activated in Mycand NF-κB-dependent transformation, and as such may prove to be an important therapeutic target. However, the mechanism of activation of GAC is poorly understood, as well as its role in supporting neoplastic growth. Therefore, the aim of this proposal is to investigate the activation of GAC in vitro using a novel fluorescence resonance energy transfer (FRET) assay, complemented by an in vivo metabolic study of model cell systems engineered to express in a tetracycline-dependent manner, the oncogenesMyc and Dbl, as well as constitutively active GAC mutant constructs. The in vitro FRET assay will provide important information regarding how the ability of GAC to undergo a dimer-to-tetramer transition is coupled to the activation of its enzymatic activity, as well as the mode of inhibition of GAC by small molecule inhibitors. Furthermore, recent X-ray crystallographic structures solved by our laboratory will be used to create constitutively active mutated forms of GAC to complement the in vitro FRET assay. These mutants will be incorporated into the engineered inducible cell system to specifically investigate the impact of hyperactive glutaminase activity on the growth properties of the induced cells. This induced "glutamine addicted" cell line, as well as the inducible cell lines capable of expressing the oncogenic transcriptional regulator Myc, and the Rho-GTPase guanine nucleotide exchange factor (GEF) Dbl, will ultimately be characterized using state of the art 13C-labeled glucose and glutamine metabolic tracing methods, including mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. The expectation is that these studies will ultimately provide a detailed portrait of metabolic transformation and a deeper understanding of the role glutaminase plays in supporting cancer cell growth.

描述（由适用提供）：在当前引起关注的癌细胞的几个标志中，是细胞代谢的重塑，在该重塑中，葡萄糖和谷氨酰胺以高速进口，并代谢以提供脂质，蛋白质，蛋白质和蛋白质和DNA的生物合成的基础。因此，正常细胞的转化通常是通过糖酵解通量的急剧增加来实现的，尽管糖酵解的最终产物，丙酮酸，转移到进入呼吸道TCA循环中，而是从细胞中分泌为裂缝。为了补偿进入TCA循环进入谷氨酸衍生的丙酮酸的减少，将谷氨酸接收并通过线粒体酶谷氨酸转化为谷氨酸，然后将谷氨酸在转换为α-酮酸盐酸盐后将TCA循环的入口点馈入TCA循环。谷氨酸的通量增加及其在提供生物合成前体进行增殖的作用是癌细胞代谢重塑的关键步骤。特别是，谷氨酰胺酶，谷氨酰胺酶C（GAC）的特异性同工型已被证明在Mycand NF-κB依赖性转化中受到调节和激活，因此可能被证明是重要的治疗靶标。然而，GAC激活的机制尚不清楚，及其在支持肿瘤生长中的作用。因此，该提案的目的是使用新型的荧光共振能量转移（FRET）测定法研究GAC在体外的激活，该测定是通过体内代谢性研究的模型细胞系统来完成的，该模型细胞系统以四环素依赖性方式，依赖于TetracyClinecogencogenesmyc和ncogenesmyc和dbl以及组成型活跃的GAC突变构建体。体外FRET分析将提供有关GAC经历二聚体到四聚体过渡的能力的重要信息，并与其酶活性的激活以及小分子抑制剂抑制GAC的模式。此外，我们实验室求解的最近X射线晶体学结构将用于创建组成型活跃的突变形式的GAC以完成体外FRET分析。这些突变体将被纳入工程诱导的细胞系统中，以专门研究多活性谷氨酰胺活性对诱导细胞生长特性的影响。这种诱导的“谷氨酰胺上瘾”的细胞系以及能够表达致癌转录的诱导细胞系 调节器MYC和Rho-GTPase Guanine核交换因子（GEF）DBL最终将使用最先进的13C标记的葡萄糖和谷氨酰胺代谢跟踪方法来表征，包括质谱法（MS）和核磁共振（NMR）光谱。期望这些研究最终将提供代谢转化的详细肖像，并对谷氨酰胺在支持癌细胞生长中的作用有更深入的了解。