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

基本信息

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
项目类别：
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

项目摘要

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）及其内源代谢物的变构调节代表了生物学中的保守调节机制。在这两个过程的交汇处，我们在此报告主要糖酵解中间体 1,3-二磷酸甘油酸与蛋白质中选定的赖氨酸残基反应形成新型 PTM 3-磷酸甘油基赖氨酸 (pgK)。该反应不需要酶催化，而是利用 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定类别的蛋白质上富集，最显着的是在糖酵解酶本身的活性位点内或周围。这种分布与目标蛋白在 GAPDH 和 1,3-BPG 生物合成中的空间定位一致，这还得到了已知与 GAPDH 相关的糖酵解之外的蛋白的 pgK 标记的支持。对于癌细胞系和小鼠组织中的糖酵解酶，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 修饰位点被发现会抑制酶活性，以响应葡萄糖暴露的增加，从而产生一种内在的反馈机制，减少糖酵解过程中的碳通量，并导致中心代谢物的积累和重定向到生物合成途径，这被证明是至关重要的用于癌细胞增殖。葡萄糖代谢增加在癌细胞中既是病理性的，也是普遍存在的，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重新布线有着不可逆转的联系。这些表型统称为 Warburg 效应，从机制上来说，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、增殖和侵袭性所需的合成代谢物。我们在此提供的初步数据与在癌细胞中观察到的葡萄糖代谢改变的原因和结果一致，即增加的pgK修饰既是原因也是结果。此次重新提交申请旨在全面了解 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。将开发工具和方法来增强细胞系、组织和肿瘤中 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 周转的酶。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，这些细胞系将被评估中央碳代谢的功能变化和与 Warburg 效应相关的侵袭性表型。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的 pgK 修饰状态。这些研究将共同建立这种新型代谢编码 PTM 在癌细胞和正常细胞中的全面概况。这些数据对于进一步了解癌细胞代谢变化、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癌症治疗干预的潜在要点非常有价值。