Figure 5.

CZ-48 and NMN Induce Allosteric Conformational Changes of SARM1 Leading to its Activation

(A–H) (A) Diagram of tagged full-length SARM1 and various truncates (for B–F) and the fusion proteins with the luciferase fragments (last three constructs, for G and H). For the sake of brevity the tags FLAG or HA are omitted in this figure unless otherwise specified. (B) HEK-293 cells stably expressing SARM1 and truncates were constructed. SARM1 and TIR were in constitutive expression cassettes, whereas SAM-TIR was in an inducible expression cassette to prevent cell death caused by overexpression of SAM-TIR. The expression levels of the constitutive SARM1 and TIR, and SAM-TIR induced by 0.5 μg/mL Dox for 20 h, were tested by western blots. Asterisks point to the specific bands. (C) Cells from (B) were treated with 100 μM CZ-48 for 8 h, and the cADPR contents were measured. (D and E) HEK-293 cells carrying an inducible expression cassette of SAM-TIR were treated with 100 μM CZ-48 or 0.5 μg/mL Dox, and protein levels were measured by western blots (D) or cADPR levels (E). (F) Proteins were immunoprecipitated and the cyclase activity in vitro was tested by reverse cycling assay with or without the presence of 100 μM NMN. (G and H) HEK-293 cells, co-transfected with the vectors encoding SARM1-LucN/SARM1-LucC or SARM1-LucN/GFPNb-LucC, as a negative control, were treated with 100 μM CZ-48 for 12 h. The expression of fusion proteins (G) and reconstituted luciferase activities (H) were measured by western blots or luciferin incubation reaction, respectively.

All the above experiments were repeated at least three times (means ± SDs; n = 3; Student's t test, **p < 0.01, ****p < 0.0001).