Ceramide sensing by human SPT-ORMDL complex for establishing sphingolipid homeostasis

Area of Science:

• Biochemistry and Molecular Biology
• Cellular Metabolism
• Neuroscience

Background:

• The serine palmitoyltransferase (SPT) complex initiates sphingolipid biosynthesis, a crucial cellular process.
• ORM/ORMDL proteins are regulatory subunits of SPT, but the mechanism of sphingolipid sensing remains elusive.
• Sphingolipid homeostasis is vital for cellular function, and its dysregulation is implicated in diseases.

Purpose of the Study:

• To elucidate the molecular mechanism by which the SPT-ORMDL complex senses cellular sphingolipid levels.
• To determine the structural basis of ceramide inhibition of the SPT-ORMDL complex.
• To investigate the role of ceramide sensing defects in the pathogenesis of childhood amyotrophic lateral sclerosis (ALS).

Main Methods:

• Purification and structural analysis (cryo-electron microscopy) of human SPT-ORMDL3 complexes.
• Biochemical assays to assess SPT activity and inhibition by ceramide.
• Structure-guided mutagenesis to identify key residues in ceramide binding and regulation.
• Analysis of patient-derived variants in SPTLC1 associated with childhood ALS.

Main Results:

• Purified SPT-ORMDL complexes are directly inhibited by ceramide, a central sphingolipid metabolite.
• The cryo-EM structure reveals ceramide binding to the SPT-ORMDL3 complex, inducing an inhibitory conformation.
• Mutational analysis confirms the ceramide binding site is essential for SPT activity suppression.
• Childhood ALS variants in SPTLC1 lead to impaired ceramide sensing in SPT-ORMDL3 mutants.

Conclusions:

• The study reveals the molecular basis of ceramide sensing by the SPT-ORMDL complex, explaining sphingolipid homeostasis.
• Ceramide binding to SPT-ORMDL proteins acts as a feedback mechanism to regulate sphingolipid biosynthesis.
• Impaired ceramide sensing due to genetic variants is implicated in the development of childhood ALS.