Elucidating the regulation of mitochondrial glutaminase in cancer progression

阐明线粒体谷氨酰胺酶在癌症进展中的调节

• 批准号: 8456503
• 负责人: Kelly E Sullivan
• 金额: $ 3.87万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-01-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8456503
• 关键词: Acetyl Coenzyme A; Adverse effects; Affect; Alkaline Phosphatase; Alternative Splicing; Am 80; Amino Acids; Ammonia; Anchorage-Independent Growth; Binding; Breast Cancer Cell; Cancer Cell Growth; Carbon; Cell Proliferation; Cells; Citric Acid Cycle; Data; Enzymes; Epithelial Cells; Event; Exhibits; Fellowship; Fibroblasts; GLS2 gene; Genes; Glucose; Glutamate Dehydrogenase; Glutamates; Glutaminase; Glutamine; Glycolysis; Goals; Growth; Human; Hydrolysis; Kidney; Knowledge; Laboratories; Lead; Link; Lipids; Liver; Lysine; MDA MB 231; Malignant Neoplasms; Mammary gland; Mass Spectrum Analysis; Messenger RNA; Metabolic; Metabolic Activation; Metabolic Pathway; Metabolism; Mitochondria; Mutation; Normal Cell; Nucleic Acids; Oncogenic; Outcome; Pathway interactions; Pharmaceutical Preparations; Phosphorylation; Phosphorylation Site; Play; Post-Translational Protein Processing; Production; Proliferating; Protein Biosynthesis; Protein Isoforms; Proteins; Pyruvate; RNA Splicing; Regulation; Role; SKBR3; Signal Transduction; Source; Testing; Therapeutic Intervention; United States National Institutes of Health; Up-Regulation; Variant; Warburg Effect; base; cancer cell; cell transformation; chemotherapeutic agent; chemotherapy; citrate carrier; design; enzyme activity; inhibitor/antagonist; metaplastic cell transformation; mutant; novel; programs; protein expression; public health relevance; research study; rho GTP-Binding Proteins; small molecule; transcription factor; tumor progression

DESCRIPTION (provided by applicant): Rapidly proliferating cancer cells are highly dependent on glutamine metabolism for the biosynthesis of protein, lipid, and nucleic acid building blocks, which are required for growth and proliferation. Many intermediates of these critical biosynthetic pathways are derived from the TCA cycle. Given that glucose-derived carbon is largely excreted as lactate rather than entering the TCA cycle (i.e., the "Warburg effect"), cancer cells adapt by upregulating glutaminolysis, which involves the 2-step conversion of glutamine to ?-ketoglutarate, a TCA cycle intermediate. As such, activation of the metabolic enzyme glutaminase C (GAC), which catalyzes the first step of glutaminolysis, namely the hydrolysis of glutamine to glutamate, is vital for the growth and proliferation of these cells. Indeed, a small molecule GAC inhibitor, referred to as 968, significantly reduces the growth of breast cancer cells. Furthermore, 968 does not affect the survival of nontransformed mammary epithelial cells, indicating that GAC participates in an alternative metabolic pathway that is dispensable for the growth of quiescent cells. Understanding the regulation of GAC is thus key for the design of novel chemotherapeutic agents that specifically target cancer cells without inducing the side effects often observed with classical chemotherapy. GAC activation has previously been linked to the signaling of hyper-activated Rho GTPases and the transcription factor NF-?B, but very little is known regarding the regulatory events controlling GAC activity. Thus, the specific aims of this proposal will identify the mechanisms leading to the upregulation of GAC activity in cancer cells. Preliminary data indicate that post-translational modifications (PTMs), particularly phosphorylation and the recently discovered lysine succinylation/malonylation, may be critical for the activation of GAC. Specifically, alkaline phosphatase treatment of GAC immunoprecipitated from transformed cells drastically reduces the basal activity of this enzyme, indicating that phosphorylation of GAC or a GAC-binding partner is required for high activity. On the other hand, knockdown of Sirtuin 5, a novel desuccinylase/demalonylase, reduces GAC basal activity by ~50%, suggesting that succinylation/ malonylation suppresses GAC activity. As such, mass spectrometry analysis will be used to identify GAC PTMs, followed by a mutation-based analysis in which phosphomimetic and phosphorylation- and succinylation/malonylation-defective mutants will be generated. These mutants will be examined for their ability to contribute to cellular transformation and to rescue the growth of GAC-depleted breast cancer cells. Additionally, preliminary results indicate that the splice-variant of GAC, termed kidney-type glutaminase (KGA), plays a role in regulating GAC activity through protein expression levels. As a long-term goal, the ability of KGA to contribute to transformation and regulate GAC activity will be investigated.

描述（由申请人提供）：快速增殖的癌细胞高度依赖于谷氨酰胺代谢，用于蛋白质，脂质和核酸构建块的生物合成，这是生长和增殖所必需的。这些关键生物合成途径的许多中间体源自TCA循环。鉴于葡萄糖衍生的碳在很大程度上被排泄为乳酸，而不是进入TCA循环（即“沃伯格效应”），癌细胞通过上调谷氨酰胺溶解来适应，谷氨酰胺溶解涉及谷氨酰胺至吗？谷氨酰胺至？ - 酮甲酸酯，TCA周期，TCA周期。因此，代谢酶C（GAC）的激活催化了谷氨酰胺溶解的第一步，即谷氨酰胺向谷氨酸的水解，对这些细胞的生长和增殖至关重要。实际上，称为968的小分子GAC抑制剂显着降低了乳腺癌细胞的生长。此外，968不会影响非转化的乳腺上皮细胞的存活，这表明GAC参与了一种替代代谢途径，该途径可用于静态细胞的生长。因此，了解GAC的调节是设计新型化学治疗剂的关键，这些化学治疗剂专门针对癌细胞而不诱导经典化学疗法经常观察到的副作用。 GAC激活先前已与过度激活的Rho GTPase和转录因子NF-？B的信号联系在一起，但是关于控制GAC活性的调节事件知之甚少。因此，该提案的具体目的将确定导致癌细胞中GAC活性上调的机制。初步数据表明，翻译后修饰（PTM），尤其是磷酸化和最近发现的赖氨酸琥珀酰化/误导性，可能对GAC的激活至关重要。具体而言，从转化细胞中对GAC免疫沉淀的碱性磷酸酶处理大幅度降低了该酶的基础活性，表明GAC或GAC结合伴侣的磷酸化需要高活性。另一方面，一种新型的desuccinylase/ demalonylase Sirtuin 5敲低可将GAC基础活性降低约50％，这表明琥珀酰化/误导化抑制了GAC活性。因此，质谱分析将用于鉴定GAC PTM，然后进行基于突变的分析，在该分析中，将产生磷酸化和磷酸化和磷酸化和琥珀酸化/丙二酰化缺陷突变体。将检查这些突变体的能力，有助于细胞转化并挽救贫血乳腺癌细胞的生长。此外，初步结果表明，GAC的剪接变体称为肾型谷氨酰胺酶（KGA），在通过蛋白质表达水平调节GAC活性中起作用。作为一个长期目标，将研究KGA有助于转化和调节GAC活动的能力。