PXD067431

PXD067431 is an original dataset announced via ProteomeXchange.

Title	Mechanism of ORMDL proteins turnover via proteasome and autophagy is cell-type dependent and tied to ceramide homeostasis
Description	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.
HostingRepository	PRIDE
AnnounceDate	2026-03-16
AnnouncementXML	Submission_2026-03-16_04:47:48.805.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Marek Vrbacky
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: NEWT:9606;
ModificationList	iodoacetamide derivatized residue
Instrument	Orbitrap Exploris 480

Revision	Datetime	Status	ChangeLog Entry
0	2025-08-17 23:46:03	ID requested
⏵ 1	2026-03-16 04:47:49	announced

10.1096/fj.202502924rr;

Mrkacek M, Tumova M, Puskasu A, Utekal P, Vrbacky M, Kuchar L, Draber P, Bugajev V, Turnover via Proteasome and Autophagy Is Cell-Type Dependent and Tied to Ceramide Homeostasis. FASEB J, 40(6):e71655(2026) [pubmed]

submitter keyword: autophagy, sphingolipid, LC3, fumonisin B1, proteasome degradation,ORMDL3, myriocin

Viktor Bugajev
contact affiliation	Laboratory of Signal Transduction, Institute of Molecular Genetics, Czech Academy of Sciences, Prague
contact email	viktor.bugajev@img.cas.cz
lab head
Marek Vrbacky
contact affiliation	Czech Academy of Sciences
contact email	proteom.krc@gmail.com
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD067431
     1. Label: PRIDE project
     2. Name: Mechanism of ORMDL proteins turnover via proteasome and autophagy is cell-type dependent and tied to ceramide homeostasis