Journal: Cell

Article Title: The Parkinson’s disease protein alpha-synuclein is a modulator of Processing-bodies and mRNA stability

doi: 10.1016/j.cell.2022.05.008

Figure Lengend Snippet: Key resources table

Article Snippet: BCA Protein Assay Kit , Pierce , 23225.

Techniques: Western Blot, Virus, Recombinant, Protease Inhibitor, Lysis, Magnetic Beads, Membrane, Transfection, Expressing, Bicinchoninic Acid Protein Assay, Silver Staining, In Situ, Sample Prep, Luciferase, Reporter Assay, Multiplex sample analysis, Biomarker Discovery, Marker, Generated, Software, Mass Spectrometry, Imaging

Journal: Brain

Article Title: Heterozygous PINK1 p.G411S increases risk of Parkinson’s disease via a dominant-negative mechanism

doi: 10.1093/brain/aww261

Figure Lengend Snippet: p.G411S, but not p.Q456X heterozygotes show persistently impaired PINK1 kinase activity under endogenous conditions over time. Primary human skin fibroblasts were left untreated or stressed with 1 µM of the potassium ionophore valinomycin (VM) for the indicated times to depolarize mitochondrial membranes. Two cell lines per genotype were analysed. PINK1 genotypes of individuals are stated as wild-type (WT), heterozygous (HET) or homozygous (HOMO) along with the respective PINK1 mutation. ( A ) Representative western blots with total cell lysates were analysed for levels of PINK1, phosphorylated ubiquitin (p-Ser65-Ub), parkin, and its substrate mitofusin 2 (MFN2). Closed and open triangles indicate unmodified MFN2 and its ubiquitinated forms, respectively. Anti-vinculin was used as loading control. ( B and C ) Quantification of protein levels from western blots of six independent experiments. Shown is the mean per genotype ± SEM. Statistical significance was assessed with two-way ANOVA, Tukey’s post hoc test (* P < 0.05; ** P < 0.005; *** P < 0.0005). ( B ) Shown are levels of PINK1 and p-Ser65-Ub normalized to wild-type at 24 h. Levels of total, unmodified ubiquitin were comparable from all cells and at all times (data not shown). p.Q456X heterozygotes showed significantly reduced levels of PINK1, concomitant with initially decreased levels of p-Ser65-Ub. However, p.G411S showed PINK1 levels similar to wild-type, but significantly reduced levels of p-Ser65-Ub over time. ( C ) Shown are relative levels of parkin and MFN2 normalized to 0 h valinomycin treatment. Absolute levels of parkin were similar in wild-type and all heterozygous PINK1 mutant cells, but were significantly elevated in p.Q456X homozygotes; however, no difference was seen for MFN2 (data not shown). ( D ) Quantitative MSD assay in 96-well plate format of p-Ser65-Ub levels from cell lysates. Cells were treated with 1 µM valinomycin (VM) for the indicated times, lysed and p-S65-Ub was captured with phospho-specific ubiquitin antibodies and detected with total ubiquitin antibodies. Shown is the mean per genotype ± SEM normalized to wild-type at 24 h from three independent experiments. Statistical significance was assessed with two-way ANOVA, Tukey’s post-hoc test (* P < 0.05; *** P < 0.0005). ( E ) Quantitative high content imaging in 96-well plate format of p-Ser65-Ub intensity in primary human fibroblasts before and after valinomycin (VM) treatment. Shown is the mean per genotype ± SEM normalized to wild-type at 0 h and 24 h from three independent experiments. Statistical significance was assessed with two-way ANOVA, Tukey’s post hoc test (* P < 0.05; ** P < 0.005; *** P < 0.0005).

Article Snippet: Detection of biotinylated ubiquitin with streptavidin-HRP (Pierce) served as loading control.

Techniques: Activity Assay, Mutagenesis, Western Blot, Ubiquitin Proteomics, Control, Imaging

Journal: Brain

Article Title: Heterozygous PINK1 p.G411S increases risk of Parkinson’s disease via a dominant-negative mechanism

doi: 10.1093/brain/aww261

Figure Lengend Snippet: Ubiquitin binding to PINK1 and prediction of phosphorylation efficiencies. Ubiquitin was docked as a substrate to each half of the PINK1 kinase wild-type homo- (G411/G411) and mutant (G411/S411) heterodimers. Given is a tabular summary of distances [Å] between the Ser65 oxygen of ubiquitin (Ub-Ser65-O) and the terminal phosphate of the bound ATP molecule as well as key PINK1 atoms ( A ). Relevant PINK1 atoms include the oxygens of Ser228 and Ser402 (Ser228/402-O) as well as the alpha carbons of either G411 or S411 (G411/S411-Cα). Both unmodified and auto-phosphorylated (p-Ser228/402) forms of PINK1 were analysed and compared for each subunit from the respective dimers. Greater distances between Ub-Ser65 and ATP as well as Ser228 and Ser402 of PINK1 in both wild-type and mutant subunits of the heterodimer, compared to wild-type homodimer, likely result in less efficient phosphorylation of the substrate. Interestingly, phosphorylation of PINK1 Ser228 and Ser402 in the wild-type homodimer facilitates an optimal alignment of ATP and Ub-Ser65 in the active site of PINK1. ( B–G ) The corresponding magnifications of the kinase domain (between the N- and C-lobes) of the PINK1 molecules with ubiquitin docked near the active site. Ser228 and Ser402 (or p-Ser228 and p-Ser402) are shown in Van der Waals spheres with the carbons coloured to match the domain colours. ATP is shown with orange carbons and structure in Van der Waals. The ubiquitin molecule is shown in beige ribbons with cyan carbon and the Ser65 residue in Van der Waals. G411 or S411 of PINK1 is shown above the site. Corresponding full-length PINK1 dimer structures in complex with ubiquitin can be found in . A schematic representation of the analysed distances between the respective atoms is depicted in .

Article Snippet: Detection of biotinylated ubiquitin with streptavidin-HRP (Pierce) served as loading control.

Techniques: Ubiquitin Proteomics, Binding Assay, Phospho-proteomics, Mutagenesis, Residue

Journal: Brain

Article Title: Heterozygous PINK1 p.G411S increases risk of Parkinson’s disease via a dominant-negative mechanism

doi: 10.1093/brain/aww261

Figure Lengend Snippet: The PINK1 p.G411S mutation exerts a dominant-negative mechanism. HeLa cells were used to confirm a dominant-negative effect of the p.G411S mutation on kinase activity of PINK1 wild-type that translated into reduced activation of parkin downstream. ( A ) HeLa cells were simultaneously transfected with specific PINK1 siRNA and siRNA-resistant PINK1-V5 wild-type or mutants (p.Q456X or p.G411S). Control cells were transfected with the corresponding empty vector (-) and with scrambled (scr) or PINK1 siRNA. Cells were treated with 15 µM CCCP for the indicated times and levels of phosphorylated ubiquitin were assessed by anti-p-Ser65-Ub. Endogenous and overexpressed PINK1 levels were monitored by anti-PINK1 and anti-V5 antibodies, respectively. Anti-GAPDH served as loading control. ( B ) HeLa cells were co-transfected with the indicated combinations of PINK1-V5 and PINK1-mCherry constructs or respective empty vector controls (-) and treated with 15 µM CCCP for 3 h. PINK1-V5 was immunoprecipitated (IP: V5) and the interaction between wild-type and mutant PINK1 was analysed by western blot. PINK1 wild-type and p.G411S strongly interacted with themselves and with each other to a similar extent. Black and grey triangles indicate full-length (wild-type and p.G411S) and truncated (p.Q456X) PINK1 protein, respectively. ( C ) In vitro ubiquitin phosphorylation assay confirms reduced kinase activity of p.G411S mutant and partial dominant-negative effects on PINK1 wild-type. HeLa cells were transfected with V5-tagged PINK1 wild-type, p.G411S, p.Q456X or a combination of wild-type plus p.G411S or p.Q456X. Cells were then treated with 15 µM CCCP for 3 h and PINK1 was immunoprecipitated with anti-V5. V5 immunoprecipitates were incubated with biotinylated ubiquitin in kinase reaction buffer. Anti-V5 antibody was used to show equal PINK1 levels in the IP. Black and grey triangles indicate full-length (wild-type and p.G411S) and truncated (p.Q456X) PINK1 protein, respectively. Phosphorylation of ubiquitin was determined by anti-p-Ser65-Ub antibody and total ubiquitin was detected by streptavidin-HRP that served as a loading control. Quantification of the p-Ser65-Ub/streptavidin ratio from three independent experiments is provided below. Values represent mean ± SEM, normalized to the average of wild-type values. Statistical significance was assessed by one-way ANOVA with Tukey’s post hoc test (* P < 0.05; ** P < 0.005; *** P < 0.0005). ( D ) High content imaging of HeLa cells stably expressing EGFP–parkin was used to quantify parkin activation and translocation to damaged mitochondria upon overexpression of PINK1 variants. While co-expression of p.G411S with PINK1 wild-type significantly reduced parkin activation, p.Q456X showed no dominant-negative effect. HeLa cells were simultaneously transfected with specific PINK1 siRNA and siRNA-resistant PINK1-V5 wild-type or mutants (p.Q456X or p.G411S) or their combinations as indicated. mCherry was used as a transfection control. Control cells were transfected with empty vector with scrambled (scr) or PINK1 siRNA. Cells were left untreated (0 h) or treated with 10 µM CCCP for 2 h. Parkin translocation to mitochondria was measured in transfected, mCherry co-expressing cells only. Values represents mean ± SEM, n = 3. Statistical significance was assessed by two-way ANOVA with Tukey’s post hoc test (*** P < 0.0005; ns = not significant).

Article Snippet: Detection of biotinylated ubiquitin with streptavidin-HRP (Pierce) served as loading control.

Techniques: Mutagenesis, Dominant Negative Mutation, Activity Assay, Activation Assay, Transfection, Control, Plasmid Preparation, Ubiquitin Proteomics, Construct, Immunoprecipitation, Western Blot, In Vitro, Phospho-proteomics, Incubation, Imaging, Stable Transfection, Expressing, Translocation Assay, Over Expression