Journal: Nature Communications

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

doi: 10.1038/s41467-023-39274-y

Figure Lengend Snippet: a Overview of the de novo sphingolipid biosynthetic pathway and the homeostatic regulation of SPT activity by ceramide in an ORMDL-dependent manner. b , c Coomassie blue-stained SDS-PAGE gel ( b ) and the SPT activity ( c ) of purified SPT and various SPT-ORMDL3 complexes. ORMDL3* stands for the ORMDL3 construct from our previous study (ref. ), which appears to express a mixture of minor full-length ORMDL3 protein (ORMDL3-FL) and dominant N-terminus truncated ORMDL3 protein. The newly made SPT-ORMDL3 (ORMDL3-FL) complex could be inhibited by C6-ceramide, whereas the SPT, SPT-ORMDL3*, and SPT-ORMDL3 (ORMDL3-ΔN17) complexes had no response to C6-ceramide. Data in ( c ) are presented as mean values ± SEM of three independent experiments. d SPT activity versus L-serine concentration measured using SPT or SPT-ORMDL3 complex. Data are presented as mean values ± SEM of three independent experiments. The data points were fitted with a Michaelis-Menten equation. e SPT activity versus palmitoyl-CoA concentration for SPT and SPT-ORMDL3 complex. Data are presented as mean values ± SEM of three independent experiments. The data points were fitted with an allosteric sigmoidal equation. f The inhibition curves of human SPT and SPT-ORMDL complexes by C6-ceramide in the in vitro SPT activity assay. Data are presented as mean values ± SEM of three independent experiments. All the estimated IC 50 values were summarized in Supplementary Table . g Selective inhibition of SPT-ORMDL3 by C6-ceramide among various sphingolipid species or analogs. Data are presented as mean values ± SEM of three independent experiments. The different sphingolipid species or analogs were applied at 10 μM in each assay. Source data are provided as a Source Data file.

Article Snippet: To measure the SPT activity inhibition curves of SPT or SPT-ORMDL complexes by C6-ceramide (Avanti, 860506 P), each protein was incubated with various concentrations of C6-ceramide on ice for 1 hr before reaction initiation.

Techniques: Activity Assay, Staining, SDS Page, Purification, Construct, Concentration Assay, Inhibition, In Vitro

Journal: Nature Communications

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

doi: 10.1038/s41467-023-39274-y

Figure Lengend Snippet: a A close-up view of the density map for the ceramide binding site contoured at 6σ. b Detailed views of the ceramide binding site. Residues involved in the interactions are shown as sticks. All potential polar interactions are indicated by red dashed lines. c Functional characterization of the key residues involved in ceramide binding by SPT activity assay. Data are presented as mean values ± SEM of three independent experiments. d , e Inhibition curves of the SPT-ORMDL3 ceramide-binding variants by C6-ceramide. Curves for the variants mediating the polar interactions and hydrophobic interactions were presented in ( d ) and ( e ), respectively. Data are presented as mean values ± SEM of three independent experiments. Source data are provided as a Source Data file.

Article Snippet: To measure the SPT activity inhibition curves of SPT or SPT-ORMDL complexes by C6-ceramide (Avanti, 860506 P), each protein was incubated with various concentrations of C6-ceramide on ice for 1 hr before reaction initiation.

Techniques: Binding Assay, Functional Assay, Activity Assay, Inhibition

Journal: Nature Communications

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

doi: 10.1038/s41467-023-39274-y

Figure Lengend Snippet: a The resolved N-terminus of ORMDL3 in the C6-ceramide-bound SPT-ORMDL3 map. The ORMDL3-N11 (the 11 residues at the N-terminus) are highlighted in magenta; all the other parts are shown in the same coloring scheme as above. The maps are shown as semi-transparent. b Close-up views of the density map and coordination of ORMDL3-N11. The electron densities for ORMDL3-N11, shown as blue mesh, were contoured at 6σ. The residues that form polar interactions with ORMDL3-N11 in SPTLC1, SPTLC2, and ORMDL3 are shown as sticks, and the potential polar interactions are indicated by red dashed lines. c Functional characterization of ORMDL3 N-terminal deletion variants and N2A variant by SPT activity assay. Data are presented as mean values ± SEM of three independent experiments. d The loss of C6-ceramide-mediated inhibition of SPT activity for the ORMDL3 N-terminal deletion variants and N2A variant. Data are presented as mean values ± SEM of three independent experiments. e Comparison of the EM maps for ORMDL3-N11 (upper) and ceramide (lower) in the apo wild-type (WT) SPT-ORMDL3 complex and the ORMDL3-ΔN2 mutant. The ORMDL3-N11 is no longer visible in the ORMDL3-ΔN2 mutant. The ceramide-like density largely disappeared in the EM map of the ORMDL3-ΔN2 mutant. f Release of the N-terminus of ORMDL3 for substrate binding in the ORMDL3-ΔN2 mutant. The ORMDL3-ΔN2 mutant structure was superimposed with the C6-ceramide-bound WT SPT-ORMDL3 structure and S-CoA-bound SPT-ORMDL3* structure (PDB 7CQK). S-CoA, short for S-(2-oxoheptadecyl)-CoA and a nonreactive analog of palmitoyl-CoA, is displayed as black spheres. The C6-ceramide-bound SPT-ORMDL3 structure is colored gray, and the ORMDL3-ΔN2 mutant structure is colored based on the subunits as above. Asn11, the resolved N-terminus of ORMDL3-ΔN2, is shown as a yellow sphere. g Conformational changes of the ceramide-binding site in the ORMDL3-ΔN2 mutant. The ORMDL3-ΔN2 mutant structure (colored based on the subunits) was superimposed with the C6-ceramide-bound WT SPT-ORMDL3 structure (gray). Source data are provided as a Source Data file.

Article Snippet: To measure the SPT activity inhibition curves of SPT or SPT-ORMDL complexes by C6-ceramide (Avanti, 860506 P), each protein was incubated with various concentrations of C6-ceramide on ice for 1 hr before reaction initiation.

Techniques: Functional Assay, Variant Assay, Activity Assay, Inhibition, Comparison, Mutagenesis, Binding Assay

Journal: Nature Communications

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

doi: 10.1038/s41467-023-39274-y

Figure Lengend Snippet: a A close-up view of the ALS-associated SPTLC1 variants. b The SPTLC1 ALS variants exhibit little effect on ORMDL3 binding. The SPT-ORMDL3 complexes bearing ALS variants, purified by size exclusion chromatography, were visualized by Coomassie-blue stained SDS-PAGE gel. c Functional characterization of the ALS variants by SPT activity assay. Data are presented as mean values ± SEM of three independent experiments. d The impaired C6-ceramide-mediated SPT-ORMDL3 activity inhibition for the ALS variants. Data are presented as mean values ± SEM of three independent experiments. Source data are provided as a Source Data file.

Article Snippet: To measure the SPT activity inhibition curves of SPT or SPT-ORMDL complexes by C6-ceramide (Avanti, 860506 P), each protein was incubated with various concentrations of C6-ceramide on ice for 1 hr before reaction initiation.

Techniques: Binding Assay, Purification, Size-exclusion Chromatography, Staining, SDS Page, Functional Assay, Activity Assay, Inhibition

Journal: Nature Communications

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

doi: 10.1038/s41467-023-39274-y

Figure Lengend Snippet: At low ceramide levels, the ORMDL N-terminus remains flexible, thus allowing the entry of substrate for SPT catalysis. The constant reaction of SPT-ORMDL would lead to the accumulation of ceramide, which would inhibit SPT-ORMDL by locking the N-terminus of ORMDL3 into an inhibition conformation. The fixed ORMDL N-terminus would block the entry of substrate for further catalysis. The Asn2 in ORMDL is important for maintaining the inactive conformation of the SPT-ORMDL complex. The Asn13 in ORMDL is critical for ceramide binding. The ALS-associated SPTLC1 variants, located near the transmembrane interface between SPTLC1 and ORMDL, impair the ceramide sensing of the SPT-ORMDL mutants. The consequently unregulated SPT activity of ALS variants would result in unrestrained sphingolipid biosynthesis.

Article Snippet: To measure the SPT activity inhibition curves of SPT or SPT-ORMDL complexes by C6-ceramide (Avanti, 860506 P), each protein was incubated with various concentrations of C6-ceramide on ice for 1 hr before reaction initiation.

Techniques: Inhibition, Blocking Assay, Binding Assay, Activity Assay