Journal: ACS Chemical Neuroscience

Article Title: SARS-CoV-2 Spike Protein Downregulates Cell Surface α7nAChR through a Helical Motif in the Spike Neck

doi: 10.1021/acschemneuro.2c00610

Figure Lengend Snippet: A helical segment of S12 (1145L-1152L) homologous to the intracellular chaperone-binding motif of α7nAChR (L411-V418) is primarily responsible for the S12-induced downregulation of surface α7nAChR. (a) Structures of α7nAChR transmembrane and intracellular domains (PDB code 7RPM ) and S12 (PDB code 7VXF ) showing the helical motif known for binding α7nAChR chaperones. Sequence alignments of the helical segments highlight three hydrophobic residues (blue) critical for binding α7nAChR chaperones and their mutations in S12 AAA . (b) Relative mean intensities of anti-α7nAChR immunofluorescent staining of nonpermeabilized HEK293 cells showing that relative to the control group (α7nAChR/ZsGreen/RIC3/NACHO), coexpressing S12 AAA (+S12 AAA ) caused only ∼5% reduction of surface α7nAChR in contrast to ∼35% reduction by S12 coexpression (+S12, adapted from Figure ). Data for each group were from two independent experiments with a total number of cells n = 8594 (control) and n = 4903 (+S12 AAA ). (c) Relative mean intensities of anti-α7nAChR immunofluorescent staining of nonpermeabilized PC12 cells transiently transfected with cDNA of mVenus alone (control) or +S12 AAA . In contrast to ∼37% reduction by S12 coexpression (red, adapted from Figure ), +S12 AAA coexpression (blue) produced no significant difference in the staining intensity compared to the control. (d) RIC3 pulldown by S12 or S12 AAA from lysates of HEK293 cells coexpressing RIC3 and S12 or S12 AAA . The His-tags on the S12 constructs were used for pulldowns using NiNTA resin. The bound proteins were probed by anti-RIC3 (1:100) and anti-SARS-CoV-2 spike protein (1:250). RIC3 binding to S12 AAA was much reduced compared to S12. RIC3 was not detected in the absence of S12. Data collection was performed ∼36 h after transfections. Data for each group in (b) and (c) were from three independent experiments with a total number of cells n = 7853 (control) and n = 5614 (+S12 AAA ). Data are the mean ± SEM; p values are from one-way ANOVA with Dunnett’s multiple comparisons; p > 0.05 (ns), p ≤ 0.01 (**), p ≤ 0.0001 (****).

Article Snippet: 50 μL of a 50% slurry of NiNTA resin (ProteinArk catalog no. Super-NiNTA10) was added to each group and incubated 2 h with inversion.

Techniques: Binding Assay, Sequencing, Staining, Control, Transfection, Produced, Construct