(158ab) Predicting the Regulatory Role of ORM Proteins in the Sphingolipid Biosynthesis Pathway Using Ensemble Kinetic Modeling

Sphingolipids are an essential component of many eukaryotic cells including human and plant cell’s plasma membrane and endomembranes. In plants, this class of lipids plays several functional roles including providing structural integrity to the membrane, golgi trafficking, and protein organizational domains. In addition, sphingolipids have been implicated in physiological processes such as the signaling of Programmed Cell Death (PCD) and the hypersensitive response associated with plant resistance to bacterial and fungal pathogens. The metabolic pathways associated with sphingolipid biosynthesis are tightly controlled to ensure sufficient sphingolipid availability for normal cell growth. Simultaneously, metabolic controls constrain the accumulation of sphingolipid building blocks responsible for the induction of PCD until this process is required (e.g. during the pathogen triggered hypersensitive response). Recent work has shown that the first committed step in Sphingolipid metabolism catalyzed by serine palmitoyltransferase (SPT) is the primary control point for regulation of this pathway. Furthermore, it has been found that orosomucoid-like (ORM) proteins are involved in the regulation of multiple enzymes in the pathway. Therefore, in this work, a combined computational and experimental approach was taken to mechanistically decipher the regulation of sphingolipid biosynthesis. A compartmentalized metabolic network of sphingolipid biosynthesis in Arabidopsis thaliana comprised of 399 metabolites and 739 reactions has been reconstructed from available literature and the KEGG database. Flux Balance Analysis (FBA) has been used to simulate the steady-state flux distribution of the network at the measured uptake rates of the starting material Sphingosine. An Ensemble Modeling (EM) framework has then been implemented to construct a kinetic model of the metabolic and regulatory network of Sphingolipid biosynthesis. The constructed model was subsequently used to predict which enzymes were regulated by ORMs as well as the metabolites regulating ORM expression. These predictions will be used to experimentally probe the role of ORMs in altering phenotypic characteristics in plants.