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: 9310455
• 负责人: Raymond E Moellering
• 金额: $ 22.81万
• 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/9310455
• 关键词: Active Sites; Allosteric Regulation; Anabolism; Antibodies; Biochemical; Biological Markers; Biology; Cancer cell line; Cancerous; Carbon; Catalysis; Cell Line; Cell Proliferation; Cell Survival; Cells; Data; Data Set; Detection; Development; Disease; Enzymes; Feedback; Glioblastoma; Glucose; Glucose Transporter; Glycolysis; Goals; Human; In Vitro; Intervention; Isotope Labeling; Label; Link; Lysine; Malignant Neoplasms; Maps; Metabolic; Metabolic Control; Metabolic Diseases; Metabolic Pathway; Metabolism; Methods; Mitochondria; Modification; Mus; Normal Cell; Normal tissue morphology; Output; Pathogenicity; 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; Warburg Effect; Western Blotting; aerobic glycolysis; base; biomarker development; cancer cell; cancer therapy; deacylation; enzyme activity; glucose metabolism; glycerate 1,3-biphosphate; human disease; human tissue; mouse model; novel; protein function; protein structure; public health relevance; response; targeted biomarker; tool; tumor; 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）及其通过内源代谢产物的变构调节代表生物学中保守的调节机制。在这两个过程的汇合期间，我们在这里报告说，主要的糖酵解中间体1,3-双磷酸甘油酸与蛋白质中精选的赖氨酸残基形成新型PTM 3-磷酸甘油赖氨酸（PGK）。这种反应不需要酶催化，而是通过蛋白质组学分析富集在精选的蛋白质上，发现了1,3-双磷酸甘油酸酯的亲电性，最突出的是在糖酵解酶本身的活性位点上。该分布与靶蛋白在GAPDH和1,3-BPG生物合成中的空间定位是一致的，这是由已知与GAPDH相关的糖溶解外的蛋白质的PGK标记来支持的。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赖氨酸的PGK修饰的积累相关。发现糖酵解酶中的几个PGK-修饰位点可抑制酶的活性，以响应增加的葡萄糖暴露，从而产生一种固有的反馈机制，从而通过糖酵解降低碳通量，并导致将中央代谢物累积并重定向到生物合成途径中，这表现为癌细胞的生物体细胞泛滥。在癌细胞中，葡萄糖代谢增加既是病理性的，又是普遍存在的，并且与葡萄糖转运蛋白，糖酵解酶的表达改变或活性的不可撤销相关，以及代谢的重新布线，从而导致对有氧糖酵解的依赖。这些表型统称为沃堡效应，并从机械上归因于线粒体呼吸从ATP产生的葡萄糖衍生碳的重定向，并通过线粒体呼吸和合成癌细胞存活，增殖，增殖，增殖和攻击所必需的零代谢代谢物的合成。本文介绍的我们的初步数据与PGK修饰的增加既是癌细胞中观察到的葡萄糖代谢改变的原因，也是结果。该重新提出的申请旨在构建对正常哺乳动物生物学和癌症中PGK修饰的分布，调节和生物学后果的全面理解。将开发工具和方法，以允许在细胞系，组织和肿瘤中增强PGK修饰位点的检测和定量。然后，这些工具将用于表征负责PGK形成的代谢控制以及在人类癌细胞系中观察到的PGK周转率的酶。这些数据集将集​​成以允许在侵略性，糖酵解癌细胞系中针对PGK-水平的有针对性调节，该癌细胞系将评估中央碳代谢的功能变化和与Warburg效应相关的侵略性表型。最后，我计划在原位小鼠模型以及原代人胶质母细胞瘤细胞中定量绘制肿瘤进展过程中PGK修饰状态。这些研究将共同​​建立癌细胞和正常细胞中这种新型，代谢上的PTM的全面景观。这些数据对于进一步了解癌细胞的代谢改变，有助于与新陈代谢变化有关的发育疾病生物标志物，最终突出治疗癌症治疗的治疗干预潜在点。