A linear pathway for inositol pyrophosphate metabolism revealed by 18O labeling and model reduction

The homeostasis of intracellular inorganic phosphate is essential for eukaryotic metabolism and is regulated by the INPHORS signalling pathway, which employs inositol pyrophosphates (IPPs) as key intermediary messengers. This study investigates the metabolic pathways of inositol pyrophosphates (IPPs) in the yeast cell line ΔSPX and the human tumor cell line HCT116. Utilizing pulse-labelling experiments with 18O water and ordinary differential equation (ODE) models, we explore the synthesis and turnover of the highly phosphorylated IPP, 1,5-InsP8. Our findings challenge the notion that 1,5-InsP8 can be synthesized through distinct routes, revealing a linear reaction sequence in both systems. Employing model reduction via the profile likelihood method, we achieved statistically concise identifiability analysis that led to significant biological insights. In yeast, we determined that 1,5-InsP8 production primarily occurs through the phosphorylation of 5-InsP7, with the pathway involving 1-InsP7 deemed unnecessary as its removal did not compromise model accuracy. Crucially, this prediction of altered IPP concentrations was validated experimentally in vip1Δ and kcs1Δ knockout strains, providing orthogonal biological support for the reduced model. In HCT116 cells, 1,5-InsP8 synthesis is mainly driven by 1-InsP7, with variations observed across different experimental conditions. These results underscore the utility of model reduction in enhancing our understanding of metabolic pathways, coupling predictive modeling with experimental validation, and providing a framework for future investigations into the regulation and implications of linear IPP pathways in eukaryotic cells.Author summary: This research reveals how cells produce 1,5-InsP8, a key molecule in phosphate homeostasis, a vital part of cellular metabolism. Using isotope tracing and mathematical modelling, the study shows that both yeast and human cancer cells follow a linear, rather than branching, pathway to build this molecule. The analysis identified essential steps in the process, challenging previous assumptions about how these molecules are synthesized. By improving our understanding of inositol pyrophosphate metabolism, this work offers new insights into how cells regulate phosphate levels, with implications for cellular energy supply and metabolism in general.