(241h) Modeling Sphingolipid Metabolism and Prediction of Orosomucoid (ORM) Proteins’ Regulatory Roles: 15 N Stable Isotope Labeling Studies

Sphingolipids are a diverse and essential class of lipids found in eukaryotic cells, ranging from human plasma membranes to plant endomembranes, where they play crucial roles in growth regulation and stress response. These complex lipids are synthesized through a tightly regulated biosynthetic pathway, and their metabolism is finely tuned to maintain cellular homeostasis. Despite their importance, the intricate interplay between sphingolipid biosynthesis and catabolism is not yet fully understood. To investigate the regulatory mechanisms governing sphingolipid metabolism in Arabidopsis thaliana, we combined targeted ¹⁵N isotope labeling experiments with high-throughput metabolomics and in silico dynamic modeling. Our approach involved using a kinetic model of the sphingolipid pathway to explore the complex regulatory interactions between orosomucoid protein (ORM), a subunit of the pathway limiting enzyme, and key sphingolipid metabolites under steady-state conditions. Our study also identified a mid-stream enzyme, sphingoid-base hydroxylase (SBH), which catalyzes the hydroxylation of sphinganine to phytosphingosine, as essential for Arabidopsis cell maintenance. Loss of SBH resulted in aberrant phenotypes. Dynamic flux analysis revealed the crucial regulatory role of the long-chain-base kinase (LCBK) in maintaining homeostasis by mediating SBH activity. Furthermore, our research showed the synergism between fatty acid 2-hydroxylase (FA2H), class II ceramide synthase (CS2), and glucosylceramide synthase (GCS), which plays a role in maintaining cellular homeostasis. These findings not only shed light on the intricate regulation of sphingolipid metabolism in plants but also have practical applications in crop engineering. By better understanding the balance between sphingolipid biosynthesis and catabolism, we can engineer crops with improved yield and stress resistance. Moreover, our modeling framework is a powerful tool for generating testable hypotheses and minimizing the need for exhaustive experimental effort in future studies. Overall, this research showcases the value of systems biology approaches in elucidating metabolic pathway regulation and its potential for practical applications.