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.
Understanding the regulation of metabolic pathways in human diseases is necessary to identify points of intervention and ultimately develop therapeutic strategies. We have identified a novel regulatory mechanism, a post-translation modification of lysines by a reactive metabolite, which directly links glucose metabolism to altered protein structure and function. The goal of the proposed research is to construct a comprehensive understanding of the distribution, regulation and biologic consequences of pgK-modifications in normal mammalian biology and cancer. The written critiques of individual reviewers are provided in essentially unedited form in this section. Please note that these critiques and criteria scores were prepared prior to the meeting and may not have been revised subsequent to any discussions at the review meeting. The Resume and Summary of Discussion section above summarizes the final opinions of the committee.