Coordinated Regulation of the Orosomucoid-Like Gene Family Expression Controls de Novo Ceramide Synthesis in Mammalian Cells [Molecular Bases of Disease]

December 17th, 2014 by Kiefer, K., Carreras–Sureda, A., Garcia–Lopez, R., Rubio–Moscardo, F., Casas, J., Fabrias, G., Vicente, R.

The orosomucoid-like family of proteins (ORMDLs) is involved in the regulation of de novo sphingolipid synthesis, calcium homeostasis, and unfolded protein response. Single nucleotide polymorphisms (SNP) that increase ORMDL3 expression have been associated with various immune/inflammatory diseases, although the pathophysiological mechanisms underlying this association are poorly understood. ORMDL proteins are claimed to be inhibitors of the serine palmitoyl transferase (SPT). However, it is not clear whether individual ORMDLs expression levels may have an impact on ceramide synthesis. The present study addressed the interaction with and regulation of SPT activity by ORMDLs in order to clarify their pathophysiological relevance. We have measured ceramide production in HEK 293 cells incubated with palmitate as a direct substrate for SPT reaction. Our results showed that a coordinated overexpression of the three isoforms inhibits the enzyme completely, while individual ORMDLs are not as effective. Immunoprecipitation and Fluorescence Resonance Energy Transfer (FRET) studies showed that mammalian ORMDLs form oligomeric complexes that change conformation depending on cellular sphingolipid levels. Finally, using macrophages as a model, we demonstrate that mammalian cells modify ORMDL genes expression levels coordinately in order to regulate de novo ceramide synthesis pathway. In conclusion, we have shown a physiological modulation of SPT activity by the general ORMDL expression levels regulation. Moreover, because single ORMDL3 protein alteration produces an incomplete inhibition of SPT activity, this work argues against the idea that ORMDL3 pathophysiology could be explained by a simple on/off mechanism on SPT activity.
  • Posted in Journal of Biological Chemistry, Publications
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