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

Abstract

The ORM/ORMDL family proteins function as regulatory subunits of the serine palmitoyltransferase (SPT) complex, which is the initiating and rate-limiting enzyme in sphingolipid biosynthesis. This complex is tightly regulated by cellular sphingolipid levels, but the sphingolipid sensing mechanism is unknown. Here we show that purified human SPT-ORMDL complexes are inhibited by the central sphingolipid metabolite ceramide. We have solved the cryo-EM structure of the SPT-ORMDL3 complex in a ceramide-bound state. Structure-guided mutational analyses reveal the essential function of this ceramide binding site for the suppression of SPT activity. Structural studies indicate that ceramide can induce and lock the N-terminus of ORMDL3 into an inhibitory conformation. Furthermore, we demonstrate that childhood amyotrophic lateral sclerosis (ALS) variants in the SPTLC1 subunit cause impaired ceramide sensing in the SPT-ORMDL3 mutants. Our work elucidates the molecular basis of ceramide sensing by the SPT-ORMDL complex for establishing sphingolipid homeostasis and indicates an important role of impaired ceramide sensing in disease development.

The structure of the Orm2-containing serine palmitoyltransferase complex reveals distinct inhibitory potentials of yeast Orm proteins

“…3B). Homologs from human and Arabidopsis thaliana SPTs in complex with ceramides share this general binding mode 26,29,30 , indicating a conserved inhibitory function. Interestingly, the ceramide is located near the N-terminal loop, which includes the Ypk1 phosphorylation target sites (Fig.…”

“…Ceramides have been identified as common negative regulators of SPT activity from yeast to man 21,26,29 . Model building of SPOT-Orm2 3A revealed the presence of an elongated non-protein density within the monomeric and dimeric complex.…”

“…S 7A, B). Therefore, further structural differences in the N-terminal parts are possible, and previous studies have already hinted at an important role for the N-terminus in the inhibitory function of Orm proteins in other species 26,29 . To investigate differences in the Orm1- and Orm2-containing complexes, we analyzed the activities of the purified complexes using in vitro activity assays.…”

Simultaneous deletion of ORMDL1 and ORMDL3 proteins disrupts immune cell homeostasis

“…Dysregulation of sphingolipid synthesis leads to many human diseases, where increased, as well as decreased, sphingolipid production is critical for disease development and manifestation ( 15 , 43 – 45 ). ORMDL proteins are essential regulators of sphingolipid production by ceramide-sensitive inhibition of the SPT enzyme ( 3 , 8 , 9 , 14 ). In the current study, we analyzed the immune cells of mice in which one or two Ormdl genes were deleted.…”

“…The importance of ORMDL3 in the control of SPT enzymatic activity was confirmed just recently when the structure of the SPT enzymatic complex with ORMDL3 was solved ( 6 , 7 ). Production of sphingolipids is controlled by a feedback loop, when the ORMDL-SPT inhibitory complex is stabilized by direct binding of ceramides ( 8 , 9 ). Another feedback loop acts through sensing of increased levels of sphingosine-1-phosphate (S1P) by the S1P receptor 1 (S1PR1).…”