Journal: Human Molecular Genetics

Article Title: Human R1441C LRRK2 regulates the synaptic vesicle proteome and phosphoproteome in a Drosophila model of Parkinson’s disease

doi: 10.1093/hmg/ddw352

Figure Lengend Snippet: Phosphoproteomics reveals increased phosphorylation of synaptic vesicle proteins. (A) Hierarchical clustering of the correlation matrix determined by Pearson correlation of triplicates indicates differences between R1441C hLRRK2 and WT hLRRK2. Note that these values are calculated based on the direct ratio and not the raw SILAC ratio. (B) Volcano plot of R1441C hLRRK2 versus WT hLRRK2 fly strain. Significance was considered for p-values below 0.05 and fold changes greater than 1.5. (C) Scatter plot of log2 transformed direct ratios between R1441C hLRRK2 and WT hLRRK2 versus control flies. Phosphorylation sites of ankyrin 2, synaptojanin and futsch are colour coded and show strong upregulation of phosphorylation sites in flies overexpressing R1441C hLRRK2. (D) Selected list of significantly regulated phosphopeptides with their modified sequence and protein quantification. P-values are colour coded from a two-sided t-test. (E) Sequence alignment of regulated phosphopeptides of Drosophila synaptojanin and ankyrin 2 to the human and mouse protein sequences.

Article Snippet: The FLAG-/GFP-tagged human synaptojanin-1 (145 kDa isoform) expression plasmid was a kind gift from Pietro De Camilli (Addgene plasmid # 22293), whereas FLAG-tagged human LRRK2 (WT, R1441C, G2019S and D1994A) expression plasmids have been reported previously ( 26 , 28 ).

Techniques: Transformation Assay, Modification, Sequencing

Journal: Human Molecular Genetics

Article Title: Human R1441C LRRK2 regulates the synaptic vesicle proteome and phosphoproteome in a Drosophila model of Parkinson’s disease

doi: 10.1093/hmg/ddw352

Figure Lengend Snippet: hLRRK2 phosphorylates Synaptojanin-1. (A) Human synaptojanin-1 phosphorylation by full-length human LRRK2 (WT, R1441C, G2019S and D1994A) by in vitro kinase assay with [33P]-γ-ATP. Indicated are autoradiographs and Coomassie-stained SDS-PAGE gels to indicate equivalent protein loading. (B) Quantification (n = 3) of synaptojanin-1 phosphorylation signal normalized to total synaptojanin-1 levels. Bars indicate mean ± SEM. ***P < 0.0001 by one-way ANOVA with Dunnett's multiple comparison test. (C) GST-tagged hLRRK2 (residues 970-2527; WT, R1441C or D1994A) recombinant proteins were incubated separately with human SYNJ1 for 60 min using cold ATP. Mixtures were subjected to MS analysis to identify phospho-sites and phosphorylation of synaptojanin-1 at T1173 (Uniprot: J3KPK1) was found to be increased by LRRK2 (WT and R1441C) relative to D1994A LRRK2. (D) MS/MS spectra for the identified phosphorylated peptide of human synaptojanin-1 at T1173 which correlates to pT1131 in Drosophila melanogaster. (E) Interactive proteomics for hLRRK2 in Drosophila brain. hLRRK2 was immunoprecipitated from the fly brain using anti-FLAG antibody and subjected to LC-MS/MS analysis. Logarithmic protein ratios (hLRRK2/control flies) from two biological replicates were plotted against the -log10 p value from a two-sided t-test. FDR cutoff was set to 0.05 and missing quantitative data were replaced by a downshifted Gaussian distribution to mimic the detection limit of the mass spectrometer. (F) A model of how synaptic vesicle trafficking is altered by human LRRK2 expression in the Drosophila brain.

Article Snippet: The FLAG-/GFP-tagged human synaptojanin-1 (145 kDa isoform) expression plasmid was a kind gift from Pietro De Camilli (Addgene plasmid # 22293), whereas FLAG-tagged human LRRK2 (WT, R1441C, G2019S and D1994A) expression plasmids have been reported previously ( 26 , 28 ).

Techniques: In Vitro, Kinase Assay, Staining, SDS Page, Recombinant, Incubation, Tandem Mass Spectroscopy, Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Expressing

Journal: Nature cell biology

Article Title: VAMP2 regulates phase separation of α-synuclein.

doi: 10.1038/s41556-024-01451-6

Figure Lengend Snippet: Fig. 2 | VAMP2 enables αSYN condensate formation in cells. a, Screening of disease-relevant synaptic proteins on αSYN–YFP distribution upon co-expression in HeLa cells. Scale bar, 20 µm. SPH1, synphilin-1; Rab-3A, Ras-related protein Rab-3A; RPH3A, rabphilin-3A; VPS35, vacuolar protein sorting-associated protein 35; Endo-A1, endophilin-A1; HSC70, heat shock cognate 71 kDa protein; auxilin, putative tyrosine-protein phosphatase auxilin; SJ145 and SJ170, synaptojanin-1 isoforms 1-145 and 1-170. b, Cytosolic–nuclear distribution of αSYN–YFP upon ectopic expression in HeLa cells, condensate formation upon co-expression of αSYN–YFP and VAMP2, co-expression of YFP and VAMP2 shows no condensate formation. c, Zoomed-in regions and fluorescence intensity distribution for cells with αSYN–YFP only, αSYN with VAMP2 and YFP with VAMP2. FL, full-length. d, Quantification of cells forming condensates. Data are derived from Incucyte screening, with 16 images per well, three wells per biological repeat and four biological repeats. n indicates biological repeats. Data are mean ± s.d. One-way ANOVA with Dunnett’s multiple comparison test. e, αSYN–YFP condensates

Article Snippet: Synaptojanin 145 and 170, endophilin-A1 and HSC70 constructs were purchased from Addgene (22291, 22292, 47403 and 86031), pcDNA3-Flag-synaptojanin 1-145 and synaptojanin 1-170 were a gift from P. De Camilli (Addgene plasmid 22291; RRID: Addgene_22291; Addgene plasmid 22292; RRID: Addgene_22292)125, full-length endophilin was a gift from P. McPherson (Addgene plasmid 47403; RRID: Addgene_47403)126, pCDNAZeo(-)HSC73 AS was a gift from J. Blum (Addgene plasmid 86031; RRID: Addgene_86031)127.

Techniques: Expressing, Fluorescence, Derivative Assay, Comparison

Journal: Human Molecular Genetics

Article Title: Human R1441C LRRK2 regulates the synaptic vesicle proteome and phosphoproteome in a Drosophila model of Parkinson’s disease

doi: 10.1093/hmg/ddw352

Figure Lengend Snippet: Phosphoproteomics reveals increased phosphorylation of synaptic vesicle proteins. (A) Hierarchical clustering of the correlation matrix determined by Pearson correlation of triplicates indicates differences between R1441C hLRRK2 and WT hLRRK2. Note that these values are calculated based on the direct ratio and not the raw SILAC ratio. (B) Volcano plot of R1441C hLRRK2 versus WT hLRRK2 fly strain. Significance was considered for p-values below 0.05 and fold changes greater than 1.5. (C) Scatter plot of log2 transformed direct ratios between R1441C hLRRK2 and WT hLRRK2 versus control flies. Phosphorylation sites of ankyrin 2, synaptojanin and futsch are colour coded and show strong upregulation of phosphorylation sites in flies overexpressing R1441C hLRRK2. (D) Selected list of significantly regulated phosphopeptides with their modified sequence and protein quantification. P-values are colour coded from a two-sided t-test. (E) Sequence alignment of regulated phosphopeptides of Drosophila synaptojanin and ankyrin 2 to the human and mouse protein sequences.

Article Snippet: In vitro kinase assay In total, 300 ng of purified recombinant human synaptojanin-1 (Origene) was incubated with 1 nM recombinant GST-tagged human LRRK2 (970-2527; WT, R1441C or D1994A; Invitrogen) in 5 µl 10x kinase buffer (Cell Signalling Technology), 1 µl [γ- 33 P]-ATP (1 µCi) in a final volume of 15 µl without incubation and incubated for 60 mins at 30 °C or quenched directly.

Techniques: Transformation Assay, Modification, Sequencing

Journal: Human Molecular Genetics

Article Title: Human R1441C LRRK2 regulates the synaptic vesicle proteome and phosphoproteome in a Drosophila model of Parkinson’s disease

doi: 10.1093/hmg/ddw352

Figure Lengend Snippet: hLRRK2 phosphorylates Synaptojanin-1. (A) Human synaptojanin-1 phosphorylation by full-length human LRRK2 (WT, R1441C, G2019S and D1994A) by in vitro kinase assay with [33P]-γ-ATP. Indicated are autoradiographs and Coomassie-stained SDS-PAGE gels to indicate equivalent protein loading. (B) Quantification (n = 3) of synaptojanin-1 phosphorylation signal normalized to total synaptojanin-1 levels. Bars indicate mean ± SEM. ***P < 0.0001 by one-way ANOVA with Dunnett's multiple comparison test. (C) GST-tagged hLRRK2 (residues 970-2527; WT, R1441C or D1994A) recombinant proteins were incubated separately with human SYNJ1 for 60 min using cold ATP. Mixtures were subjected to MS analysis to identify phospho-sites and phosphorylation of synaptojanin-1 at T1173 (Uniprot: J3KPK1) was found to be increased by LRRK2 (WT and R1441C) relative to D1994A LRRK2. (D) MS/MS spectra for the identified phosphorylated peptide of human synaptojanin-1 at T1173 which correlates to pT1131 in Drosophila melanogaster. (E) Interactive proteomics for hLRRK2 in Drosophila brain. hLRRK2 was immunoprecipitated from the fly brain using anti-FLAG antibody and subjected to LC-MS/MS analysis. Logarithmic protein ratios (hLRRK2/control flies) from two biological replicates were plotted against the -log10 p value from a two-sided t-test. FDR cutoff was set to 0.05 and missing quantitative data were replaced by a downshifted Gaussian distribution to mimic the detection limit of the mass spectrometer. (F) A model of how synaptic vesicle trafficking is altered by human LRRK2 expression in the Drosophila brain.

Article Snippet: In vitro kinase assay In total, 300 ng of purified recombinant human synaptojanin-1 (Origene) was incubated with 1 nM recombinant GST-tagged human LRRK2 (970-2527; WT, R1441C or D1994A; Invitrogen) in 5 µl 10x kinase buffer (Cell Signalling Technology), 1 µl [γ- 33 P]-ATP (1 µCi) in a final volume of 15 µl without incubation and incubated for 60 mins at 30 °C or quenched directly.

Techniques: In Vitro, Kinase Assay, Staining, SDS Page, Recombinant, Incubation, Tandem Mass Spectroscopy, Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Expressing