Distribution, regulation and function of a novel lysine PTM in metabolic disease

新型赖氨酸 PTM 在代谢疾病中的分布、调控和功能

• 批准号: 9057774
• 负责人: Raymond E Moellering
• 金额: $ 24.9万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-07 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9057774
• 关键词: Active Sites; Allosteric Regulation; Anabolism; Antibodies; Biochemical; Biological Markers; Biology; Cancer Biology; Cancer cell line; Cancerous; Carbon; Catalysis; Cell Line; Cell Proliferation; Cell Survival; Cells; Critiques; Data; Data Set; Detection; Development; Disease; Enzymes; Feedback; Glioblastoma; Glucose; Glucose Transporter; Glycolysis; Goals; Health; Human; In Vitro; Individual; Intervention; Label; Link; Lysine; Malignant Neoplasms; Maps; Metabolic Control; Metabolic Diseases; Metabolic Pathway; Metabolism; Methods; Mitochondria; Modification; Mus; Normal Cell; Normal tissue morphology; Output; Pathologic; Pathway interactions; Peptides; Phenotype; Phosphoric Monoester Hydrolases; Post-Translational Protein Processing; Process; Production; Protein Dephosphorylation; Proteins; Proteomics; Reaction; Regulation; Reporting; Research; Respiration; Role; Sirtuins; Site; Technology; Therapeutic; Therapeutic Intervention; Tissues; Translations; Tumor Tissue; Warburg Effect; Western Blotting; Writing; aerobic glycolysis; base; cancer cell; cancer therapy; deacylation; enzyme activity; glucose metabolism; glycerate 1,3-biphosphate; human disease; human tissue; meetings; mouse model; novel; protein structure function; response; tool; tumor progression

DESCRIPTION (provided by applicant): The post-translational modification (PTM) of proteins and their allosteric regulation by endogenous metabolites represent conserved regulatory mechanisms in biology. At the confluence of these two processes, we report here that the primary glycolytic intermediate 1,3-bisphosphoglycerate reacts with select lysine residues in proteins to form the novel PTM 3-phosphoglyceryl-lysine (pgK). This reaction, which does not require enzyme catalysis, but rather exploits the electrophilicity of 1,3-bisphosphoglycerate, was found by proteomic profiling to be enriched on select classes of proteins, most prominently in or around the active sites of glycolytic enzymes themselves. This distribution was consistent with the spatial localization of target proteins to GAPDH and 1,3- BPG biosynthesis, which is additionally supported by the pgK-labeling of proteins outside of glycolysis known to associate with GAPDH. On glycolytic enzymes in both cancer cell lines and mouse tissues, higher glucose exposure was correlated with accumulation of pgK-modifications on functional lysines. Several pgK- modification sites in glycolytic enzymes were found to inhibit enzyme activity in response to increased glucose exposure, thus creating an intrinsic feedback mechanism that decreases carbon flux through glycolysis and leads to build up and redirection of central metabolites into biosynthetic pathways shown to be essential for cancer cell proliferation. Increased glucose metabolism is both pathologic and ubiquitous in cancer cells, and is irrevocably linked to the altered expression or activity of glucose transporters, glycolytic enzymes and a rewiring of metabolism that leads to a reliance on aerobic glycolysis. These phenotypes are collectively known as the Warburg Effect, and have been mechanistically attributed to the redirection of glucose-derived carbon away from ATP production by mitochondrial respiration and toward the synthesis of anabolic metabolites necessary for cancer cell survival, proliferation and aggressiveness. Our preliminary data presented herein are consistent with increased pgK modification being both a cause and a consequence of the altered glucose metabolism observed in cancer cells. This resubmission application aims to construct a comprehensive understanding of the distribution, regulation and biologic consequences of pgK-modifications in normal mammalian biology and cancer. Tools and methods will be developed to permit the enhanced detection and quantification of pgK-modification sites in cell lines, tissues and tumors. These tools will then be applied to characterize the enzyme(s) responsible for metabolic control of pgK formation as well as pgK turnover observed in human cancer cell lines. These datasets will be integrated to permit targeted modulation of pgK- levels in aggressive, glycolytic cancer cell lines, which will be assessed for functional changes in central carbon metabolism and aggressive phenotypes associated with the Warburg Effect. Finally, I plan to quantitatively map pgK-modification status during tumor progression in both an orthotopic mouse model as well as in primary human glioblastoma cells. Together these studies will establish the comprehensive landscape of this novel, metabolically-encoded PTM in both cancerous and normal cells. These data will be extremely valuable to further our understanding of altered metabolism in cancer cells, aid in the development disease biomarkers related to these changes in metabolism and ultimately highlight potential points of therapeutic intervention for the treatment of cancer.

描述（由申请方提供）：蛋白质的翻译后修饰（PTM）及其内源性代谢物的变构调节代表了生物学中保守的调节机制。在这两个过程的汇合处，我们在这里报告的主要糖酵解中间体1，3-bisphosphoglycerate反应与选择的赖氨酸残基在蛋白质中形成新的PTM 3-磷酸甘油基-赖氨酸（pgK）。该反应不需要酶催化，而是利用了1，3-二磷酸甘油酸的亲电性，通过蛋白质组学分析发现，该反应在选择的蛋白质类别上富集，最突出的是在糖酵解酶本身的活性位点中或周围。这种分布与靶蛋白对GAPDH和1，3- BPG生物合成的空间定位一致，这另外得到已知与GAPDH相关的糖酵解之外的蛋白质的pgK标记的支持。在癌细胞系和小鼠组织中的糖酵解酶上，较高的葡萄糖暴露与功能性赖氨酸上pgK修饰的积累相关。发现糖酵解酶中的几个pgK修饰位点响应于增加的葡萄糖暴露而抑制酶活性，从而产生内在反馈机制，其减少通过糖酵解的碳通量，并导致中心代谢物的积累和重定向到生物合成途径中，所述生物合成途径显示为癌细胞增殖所必需的。葡萄糖代谢增加是病理性的，并且在癌细胞中普遍存在，并且与葡萄糖转运蛋白、糖酵解酶的表达或活性的改变以及导致对有氧糖酵解的依赖的代谢的重新布线（rewiring）不可分割地相关。这些表型统称为瓦尔堡效应，并且在机械上归因于葡萄糖衍生的碳从线粒体呼吸产生的ATP重定向到合成癌细胞存活、增殖和侵袭性所必需的合成代谢物。我们在此提供的初步数据与增加的pgK修饰一致，这既是在癌细胞中观察到的葡萄糖代谢改变的原因，也是其结果。该重新提交申请旨在全面了解正常哺乳动物生物学和癌症中pgK修饰的分布，调节和生物学后果。将开发工具和方法，以增强对细胞系、组织和肿瘤中pgK修饰位点的检测和定量。然后将这些工具应用于表征负责pgK形成的代谢控制以及在人癌细胞系中观察到的pgK周转的酶。将整合这些数据集以允许靶向调节侵袭性糖酵解癌细胞系中的pgK水平，将评估与瓦尔堡效应相关的中心碳代谢和侵袭性表型的功能变化。最后，我计划在原位小鼠模型和原代人胶质母细胞瘤细胞中定量绘制肿瘤进展过程中的pgK修饰状态。这些研究将共同建立这种新的代谢编码PTM在癌细胞和正常细胞中的全面景观。这些数据将是非常有价值的，以进一步了解癌细胞中的代谢改变，帮助开发与这些代谢变化相关的疾病生物标志物，并最终突出治疗癌症的治疗干预的潜在点。