Journal: bioRxiv

Article Title: GHB confers neuroprotection by stabilizing the CaMKIIα hub domain

doi: 10.1101/2020.09.28.310474

Figure Lengend Snippet: (A) Approach for target identification using a combination of competitive photoaffinity labeling (PAL) and affinity purification in rat hippocampal homogenate followed by quantitative proteomics. (B) Representative anti-biotin Western blot of hippocampal homogenate after PAL and competition with 2 . (C) Identification of CaMKIIα from LC-MS/MS data as the best hit from non-linear regression analysis for all proteins, and (D) concentration-dependent competition of individual proteins by 2 during PAL. (E) Target validation by [ 3 H]- 1 autoradiography using brain slices from Camk2a and Camk2b wildtype (+/+) and knockout (-/-) mice (cresyl violet staining for tissue visualization). (F-H) Target validation by [ 3 H]- 1 binding to whole cell homogenate from transfected HEK293T cells. (F) [ 3 H]- 1 saturation binding to CaMKIIα ( n = 5), shown is one representative curve; means ± SD). (G) CaMKIIα competition with GHB ( n = 3), 1 ( n = 5) and 2 ( n = 3), pooled data (means ± SEM). (H) Subtype selectivity of [ 3 H]- 1 for CaMKIIα cf. CaMKIIβ/γ/δ. Data are pooled ( n = 3) for each subtype and depicted as specific binding (% of total).

Article Snippet: 6x Hub human protein, WT hub or full-length human recombinant CaMKIIα (#02-109, Carna Biosciences) were immobilized on to a biosensor surfaces by amine coupling using a 20 mM NaAc pH 5 immobilization buffer.

Techniques: Labeling, Affinity Purification, Western Blot, Liquid Chromatography with Mass Spectroscopy, Concentration Assay, Autoradiography, Knock-Out, Staining, Binding Assay, Transfection

Journal: bioRxiv

Article Title: GHB confers neuroprotection by stabilizing the CaMKIIα hub domain

doi: 10.1101/2020.09.28.310474

Figure Lengend Snippet: (A) Schematic of a single CaMKIIα subunit comprised of a kinase domain (gray), regulatory segment (green), linker (yellow), and hub domain (lilac). 12-14 hub domains oligomerize into the holoenzyme, shown here in an activated form with kinase domains displaced from the hub. (B) Concentration-dependent binding of 2 to immobilized CaMKIIα 6x Hub measured by SPR (top), and Langmuir binding isotherm (bottom), representative data. (C) Ball and stick model of key binding residues (bold), nearby residues, and hydrogen bonds in green-dashed lines. (D) X-ray crystal structure of 2 bound to the CaMKIIα 6x (14-mer) Hub. (E) Close-up view of a single hub subunit showing the key molecular interactions, displacement (flip) of Trp403 with ligand bound highlighted. (F) Quenching of intrinsic fluorescence caused by Trp403 flip (6x Hub) with increasing concentrations of 2 ( n = 8), pooled data (means ± SEM). (G) Mutational analysis of key residues in the pocket using [ 3 H]- 1 equilibrium binding to CaMKIIα-HEK293T overexpressing cells. Compared to wildtype (WT), binding is completely obliterated in a construct lacking the hub as well as in R433Q, R453Q, R469Q, triple mutant R433/453/468/Q (RRR→QQQ) and H395A mutants ( n = 3).

Article Snippet: 6x Hub human protein, WT hub or full-length human recombinant CaMKIIα (#02-109, Carna Biosciences) were immobilized on to a biosensor surfaces by amine coupling using a 20 mM NaAc pH 5 immobilization buffer.

Techniques: Concentration Assay, Binding Assay, Fluorescence, Construct, Mutagenesis

Journal: bioRxiv

Article Title: GHB confers neuroprotection by stabilizing the CaMKIIα hub domain

doi: 10.1101/2020.09.28.310474

Figure Lengend Snippet: (A) Right-shifted thermal shift assay melting curves of CaMKIIα WT hub upon binding of GHB, 1 and 2 ( left ) and 1 concentration-dependence (right), representative data. (B) No effect of 1 on Ca 2+ -stimulated Thr286 phosphorylation. Shown is quantification of mean band intensities of Ca 2+ -stimulated pThr286 levels ( left ) normalized to total CaMKIIα expression of cultured cortical neurons (DIV 18-20) incubated with 50-100 μM Ca 2+ alone or together with 3 mM of 1 for 1 h and representative Western blots ( right ). GAPDH was used as loading control (C) Time-( left ) and concentration-dependent effects of 1 ( right ) on cell survival at 24 h in cultured cortical neurons (DIV 16-18) stimulated with 100-200/20 μM Glu/Gly for 1 h (tat- 4 as control). Cell death was normalized to maximum cell death as measured by LDH release. (One-way ANOVA, post-hoc Dunnett’s test). (D) Quantification of GluN2B-CaMKIIα co-localization in hippocampal neurons (DIV 14-19) exposed to Glu 400 μM for 2 min and immediately fixed ( left ) and representative immunostained images ( righ t). For B-D: Number in bar diagrams indicates number of experiments/individual cultures. Box plots (boxes, 25–75%; whiskers, minimum and maximum; lines, median). (One-way ANOVA, post-hoc Dunnett’s test).

Article Snippet: 6x Hub human protein, WT hub or full-length human recombinant CaMKIIα (#02-109, Carna Biosciences) were immobilized on to a biosensor surfaces by amine coupling using a 20 mM NaAc pH 5 immobilization buffer.

Techniques: Thermal Shift Assay, Binding Assay, Concentration Assay, Expressing, Cell Culture, Incubation, Western Blot