Understanding the diauxic shift in Saccharomyces cerevisiae and its evolutionarily conserved mechanisms of regulation

The diauxic shift is a change in metabolism in Saccharomyces cerevisiae whereby glucose consumption fuels glycolytic fermentation but then shifts to respiration by ethanol import upon glucose exhaustion. In comparison, cancer cells have increased aerobic glycolysis and lactic acid excretion compared to noncancerous cells. Cancer cells undergo glycolysis upon glucose induction, much like yeast cells do. One protein that regulates the diauxic shift is the glucose response transcription factor, GCR1. After a high growth state and as glucose becomes limited, Gcr1 is deactivated as the cell switches to respiration. By studying the mechanisms and metabolism of the diauxic shift, much can be learned about diseases in other organisms that have deregulated glycolytic metabolism, such as cancer in humans. First, to model metabolism and to be able to characterize the phenotype of a biological system based off of transcriptomic, proteomic, or genomic datasets, condition-specific metabolic modeling software was developed. The Algorithm for Simplified Metabolic ANalysIs by Altering Networks and Deducing flux Estimates for VIsuaLization (TASMANIAN DEVIL) is comprised of four independently functional modules: gene activity determination, genome-scale metabolic model importation and simplification to reduce network complexity, robust heuristic model building and metabolic flux prediction using steady-state flux balance analysis, and flux visualization from a reference network topology. Publically available transcriptomic and measured flux datasets for yeast were used to validate the software and assumptions used for simplifying networks. TASMANIAN DEVIL is easily installable and can be utilized by researchers in many fields. After TASMANIAN DEVIL was developed, it was used to character the metabolism of GCR1 mutant transcriptomic studies before and after the diauxic shift. It was modeled that prior to the diauxic shift, there is a high rate of metabolic flux through glycolysis, the pentose phosphate pathway, and biosynthesis, in part generated by anaplerosis. Upon ethanol import, there is a decrease in these pathways and an increase in the citric acid cycle, oxidative phosphorylation, and arginine metabolism. The modeling predicted that the upregulation of the glycine cleavage complex (GCV1, GCV2, and GCV3 in the mitochondria) by GCR1 creates an efficient way to generate one carbon building blocks for biosynthesis for adenine production along with other purines. Gcr1 is deactivated by inositol pyrophosphorylation from 5-diphosphoinositol pentakisphosphate (5PP-IP5), which is the addition of a phosphate group to a prephosphorylated serine residue through a nonenzymatic cleavage. The process is highly endergonic, requiring a near physiological level Michaelis constant for ATP. This predicts that GCR1 is therefore a regulator of its own deactivation, and it becomes downregulated after a previous high growth state. Finally, it was investigated how pyrophosphorylation may be conserved in humans to regulate homeostasis. A conserved multi-domain was identified over the residues where pyrophosphorylation occurs on GCR1, which may enable pyrophosphorylation to take place. In humans, several inositol polyphosphatases contain this domain near their phosphatases, perhaps indicating a positive feedback loop for the continued formation of inositol pyrophosphates. GCR1 and its transcriptional partners were also found to be cyclically expressed. The levels of 5PP-IP5 are periodically modulated with the cell cycle in mammals, influencing the activities of the proteins it pyrophosphorylates and binds to, thus governing cell cycle checkpoints. It has been shown that 5PP-IP5 regulates p53-mediated apoptosis upon DNA damage and that it inactivates AKT to downregulate glycolytic metabolism. In cancer cells, there are several ways that the inositol pyrophosphate pathway can become deregulated. However, focusing on reactivating these mechanisms of regulation may provide an effective way to target cancer cells