Project description:The metabolic adaptation of eukaryotic cells to hypoxia involves increasing dependence upon glycolytic ATP production, an event with consequences for both cell bioenergetics and cell fate. This response is regulated at the transcriptional level by HIF-1-dependent transcriptional upregulation of all ten glycolytic enzymes. However, this response alone does not account for the levels of ATP produced in hypoxia. Here, we investigated additional mechanisms of regulating glycolysis in hypoxia. We found that both intestinal epithelial cells treated with inhibitors of transcription and translation and human platelets (which lack nuclei) maintained the capacity for hypoxia-induced glycolysis, suggesting the involvement of a non-transcriptional component to the hypoxia-induced metabolic switch to a highly glycolytic phenotype. Mass spectrometric analysis of the interactome of immunoprecipitated rate-limiting glycolytic enzymes identified hypoxia-sensitive complexes comprising multiple glycolytic enzymes and glucose transporters in intestinal epithelial cells. Surprisingly, the formation of glycolytic complexes, though not dependent upon transcription, occurs via a HIF-1?-dependent mechanism, suggesting that HIF-1? may play a moonlighting role in the formation / maintenance of glycolytic complexes. Furthermore, we provide evidence for the presence of HIF-1? in cytosolic fractions of hypoxic cells which physically associated with the glucose transporter GLUT1 and the glycolytic enzyme PFKP in a hypoxia-sensitive manner. In conclusion, we hypothesize that HIF-1? plays a role in initiation and/or maintenance of glycolytic complexes in intestinal epithelial cells under hypoxic conditions in a manner which optimizes catalytic efficiency of the pathway by facilitating substrate channeling of glycolytic intermediates between sequential pathway enzymes. In hypoxia, cells undergo a metabolic switch to increased glycolysis. This has important implications for cell behavior, phenotype, and fate in both healthy and cancerous cells. Here we describe a mechanism by which HIF-1, in addition to increasing glycolytic enzyme expression, promotes glycolysis via the formation of a metabolic complex.

Project description:As a result of ancestral whole genome and small-scale duplication events, the genome of Saccharomyces cerevisiaeM-bM-^@M-^Ys, and of many eukaryotes, still contain a substantial fraction of duplicated genes. In all investigated organisms, metabolic pathways, and more particularly glycolysis, are specifically enriched for functionally redundant paralogs. In ancestors of the Saccharomyces lineage, the duplication of glycolytic genes is purported to have played an important role leading to S. cerevisiae current lifestyle favoring fermentative metabolism even in the presence of oxygen and characterized by a high glycolytic capacity. In modern S. cerevisiae, the 12 glycolytic reactions leading to the biochemical conversion from glucose to ethanol are encoded by 27 paralogs. In order to experimentally explore the physiological role of this genetic redundancy, a yeast strain with a minimal set of 14 paralogs was constructed (MG strain). Remarkably, a combination of quantitative, systems approach and of semi-quantitative analysis in a wide array of growth environments revealed the absence of phenotypic response to the cumulative deletion of 13 glycolytic paralogs. This observation indicates that duplication of glycolytic genes is not a prerequisite for achieving the high glycolytic fluxes and fermentative capacities that are characteristic for S. cerevisiae and essential for many of its industrial applications and argues against gene dosage effects as a means for fixing minor glycolytic paralogs in the yeast genome. MG was carefully designed and constructed to provide a robust, prototrophic platform for quantitative studies, and is made available to the scientific community. The goals of the present study are to experimentally explore genetic redundancy in yeast glycolysis by cumulative deletion of minor paralogs and to provide a new experimental platform for fundamental yeast research by constructing a yeast strain with a functional M-bM-^@M-^Xminimal glycolysisM-bM-^@M-^Y. To this end, we deleted 13 minor paralogs, leaving only the 14 major paralogs for the S. cerevisiae glycolytic pathway. The cumulative impact of deleting all minor paralogs was investigated by two complementary approaches. A first, quantitative analysis focused on the impact on glycolytic flux under a number of controlled cultivation conditions that, in wild-type strains, result in different glycolytic fluxes. These quantitative growth studies were combined with transcriptome, enzyme-activity and intracellular metabolite assays to capture potential small phenotypic effects. A second, semi-quantitative characterization explored the phenotype of the M-bM-^@M-^Xminimal glycolysisM-bM-^@M-^Y strain under a wide array of experimental conditions to identify potential context-dependent phenotypes

			Action	DRS
	MODEL2012150003?filename=Ecoli_Growth_Glycolysis.xml	Xml
	MODEL2012150003?filename=all_model_fluxes.csv	Csv
	MODEL2012150003?filename=all_model_init_conds.csv	Csv
	MODEL2012150003?filename=all_model_parameters.csv	Csv
	MODEL2012150003?filename=metabolite_abbrevs.csv	Csv

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Haiman2020 Functional enzyme states in the E.coli glycolytic proteome metabolic model

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