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(由申请人提供)：蛋白质的翻译后修饰(PTM)及其由内源代谢物的变构调节在生物学中代表保守的调节机制。在这两个过程的交汇处，我们在这里报道了初级糖酵解中间体1，3-二磷酸甘油酸酯与蛋白质中特定的赖氨酸残基反应生成新型的PTM 3-磷酸甘油-赖氨酸(PGK)。这种反应不需要酶催化，而是利用了1，3-二磷酸甘油酸酯的亲电性，通过蛋白质组学分析发现，这种反应富含在特定类别的蛋白质上，最突出的是糖酵解酶本身的活性部位或周围。这一分布与目标蛋白在GAPDH和1，3-BPG生物合成中的空间定位是一致的，这一分布还得到了糖酵解外已知与GAPDH相关的蛋白质的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。糖酵解酶中的几个PGK修饰位点被发现抑制了酶的活性，以响应增加的葡萄糖暴露，从而创建了一种内在的反馈机制，通过糖酵解减少了碳流，并导致中心代谢物的建立和重定向到对癌细胞增殖至关重要的生物合成途径。在肿瘤细胞中，葡萄糖代谢增强既是一种病理现象，也是一种普遍存在的现象，它与葡萄糖转运体、糖酵解酶的表达或活性改变以及导致依赖有氧糖酵解的代谢重排有着不可挽回的联系。这些表型统称为Warburg效应，机制上归因于葡萄糖衍生的碳从线粒体呼吸产生的ATP转向合成癌细胞生存、增殖和侵袭所需的合成代谢产物。我们在此提供的初步数据与PGK修饰增加是观察到的癌细胞葡萄糖代谢改变的原因和结果是一致的。这项重新提交的申请旨在全面了解PGK修饰在正常哺乳动物生物学和癌症中的分布、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修饰位点的检测和量化。然后，这些工具将被应用于表征负责代谢控制PGK形成的酶(S)以及在人类癌细胞系中观察到的PGK周转。这些数据集将被整合，以允许对侵袭性、糖酵解癌细胞系中的PGK水平进行有针对性的调节，这将被评估中心碳代谢的功能变化和与华宝效应相关的侵袭性表型。最后，我计划在原位小鼠模型和原代人类胶质母细胞瘤细胞中定量定位肿瘤进展过程中的PGK修饰状态。总之，这些研究将建立这种新奇的、代谢编码的PTM在癌细胞和正常细胞中的全面图景。这些数据将极有价值地加深我们对癌细胞代谢变化的了解，有助于开发与这些代谢变化相关的疾病生物标记物，并最终突出潜在的癌症治疗干预点。