Mechanism of ORMDL proteins turnover via proteasome and autophagy is cell-type dependent and tied to ceramide homeostasis

ABSTRACT: ORMDL proteins are essential negative regulators of the serine palmitoyltransferase (SPT) complex, thereby controlling the rate of de novo sphingolipid synthesis. Although mammalian ORMDL proteins undergo rapid turnover, the mechanisms regulating their stability are not completely understood, with conflicting observations reported across several studies. Here, we combined lipidomics, proteomics, and biochemical assays to investigate the regulation of ORMDL proteins in HEK293 cells and primary mouse bone marrow–derived mast cells (BMMCs). Inhibition of the SPT complex by myriocin or of ceramide synthases by fumonisin B1 (FB1) profoundly altered sphingolipid composition but induced minimal global proteomic changes, while consistently reducing amounts of ORMDL proteins. In contrast, overexpression of a single-chain SPT increased ORMDL proteins levels alongside elevated sphingolipids, an effect abolished by myriocin or FB1 treatment. Loss of ORMDL proteins closely correlated with ceramide depletion and, in HEK293 cells, could be prevented by proteasome inhibition, whereas autophagy had no effect. In BMMCs, both pathways contributed to the regulation of ORMDL proteins, consistent with high basal autophagic activity reflected by elevated LC3-II levels. Mutation of conserved asparagines (N11/N13) in ORMDL3, which mediate ceramide binding and stabilization of the inhibitory conformation, disrupted association with SPTLC1 and SPTLC2, mimicking myriocin-induced complex dissociation, while FB1 had a weaker effect. These findings identify ceramide depletion as the primary trigger for ORMDL proteins degradation in BMMCs and HEK293 cells, and reveal a proteasome-dependent pathway that can be supplemented by autophagy in cell types with high basal autophagic activity.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Marek Vrbacky  

LAB HEAD: Viktor Bugajev

PROVIDER: PXD067431 | Pride | 2026-03-16

REPOSITORIES: Pride