IntAct European Bioinformatics Institute IntAct intact http://www.ebi.ac.uk/ http://www.ebi.ac.uk/intact/query/${ac} EBI-[0-9]+|IA:[0-9]+ INTerAction database (IntAct) provides an open source database and toolkit for the storage, presentation and analysis of molecular interactions. http://www.ebi.ac.uk/intact European Bioinformatics Institute; Wellcome Trust Genome Campus; Hinxton, Cambridge; CB10 1SD; United Kingdom http://www.ebi.ac.uk/intact/ http://www.ebi.ac.uk/ rosalind-2015-4 in vitro In vitro comigration in sds comigration in sds page comigration in sds western blot western blot Immuno blot Immuno blot Rosalind F. Roberts , Richard Wade-Martins , Javier Alegre-Abarrategui Neurodegeneration - Publications depicting interactions involved in neurodegenerative diseases. SNCA and amyloid -beta oligomers were produced by incubating the recombinant human proteins with the lipid peroxidation byproduct 4-hydroxy-2-nonenal. (see pmid:22867050). SNCA fibrils were produced by shaking the recombinant human protein for 5 days. javier.alegre@dpag.ox.ac.uk Brain; A Journal of Neurology 2015 imex curation Rosalind F. Roberts , Richard Wade-Martins , Javier Alegre-Abarrategui, Rosalind F. Roberts , Richard Wade-Martins , Javier Alegre-Abarrategui, Rosalind F. Roberts , Richard Wade-Martins , Javier Alegre-Abarrategui, Rosalind F. Roberts , Richard Wade-Martins , Javier Alegre-Abarrategui Parkinsons - Interactions investigated in the context of Parkinsons disease Affinomics - Interactions curated for the Affinomics consortium. Outeiro et al., 2008 describes SNCA constructs of 140 amino acids; this corresponds to UniProt Isoform 1. The exact amyloid-beta product (from Tocris Bioscience) is not specified; hence the longer peptide sequences have been blasted and matches found. The sequence of all Tocris products is within UniProt P37840 aas 672-714. The protein "Beta-amyloid protein 42" has been used here (aas 672-713). Only protein-protein interactions imex curation rosalind-2015-5 in vitro In vitro electron microscopy electron microscopy Electron crystallography Electron cryomicroscopy Electron crystallography Electron cryomicroscopy predetermined predetermined participant predetermined Rosalind F. Roberts , Richard Wade-Martins , Javier Alegre-Abarrategui Neurodegeneration - Publications depicting interactions involved in neurodegenerative diseases. SNCA and amyloid -beta oligomers were produced by incubating the recombinant human proteins with the lipid peroxidation byproduct 4-hydroxy-2-nonenal. (see pmid:22867050). SNCA fibrils were produced by shaking the recombinant human protein for 5 days. javier.alegre@dpag.ox.ac.uk Brain; A Journal of Neurology 2015 imex curation Rosalind F. Roberts , Richard Wade-Martins , Javier Alegre-Abarrategui, Rosalind F. Roberts , Richard Wade-Martins , Javier Alegre-Abarrategui, Rosalind F. Roberts , Richard Wade-Martins , Javier Alegre-Abarrategui, Rosalind F. Roberts , Richard Wade-Martins , Javier Alegre-Abarrategui Parkinsons - Interactions investigated in the context of Parkinsons disease Affinomics - Interactions curated for the Affinomics consortium. Outeiro et al., 2008 describes SNCA constructs of 140 amino acids; this corresponds to UniProt Isoform 1. The exact amyloid-beta product (from Tocris Bioscience) is not specified; hence the longer peptide sequences have been blasted and matches found. The sequence of all Tocris products is within UniProt P37840 aas 672-714. The protein "Beta-amyloid protein 42" has been used here (aas 672-713). Only protein-protein interactions imex curation rosalind-2015-1 human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. bifc bimolecular fluorescence complementation bifc tag fluorescence tag visualisation by fluorescence tag fluorescence Rosalind F. Roberts , Richard Wade-Martins , Javier Alegre-Abarrategui Neurodegeneration - Publications depicting interactions involved in neurodegenerative diseases. javier.alegre@dpag.ox.ac.uk Brain; A Journal of Neurology 2015 imex curation Rosalind F. Roberts , Richard Wade-Martins , Javier Alegre-Abarrategui, Rosalind F. Roberts , Richard Wade-Martins , Javier Alegre-Abarrategui, Rosalind F. Roberts , Richard Wade-Martins , Javier Alegre-Abarrategui, Rosalind F. Roberts , Richard Wade-Martins , Javier Alegre-Abarrategui Parkinsons - Interactions investigated in the context of Parkinsons disease Affinomics - Interactions curated for the Affinomics consortium. Outeiro et al., 2008 describes SNCA constructs of 140 amino acids; this corresponds to UniProt Isoform 1. The exact amyloid-beta product (from Tocris Bioscience) is not specified; hence the longer peptide sequences have been blasted and matches found. The sequence of all Tocris products is within UniProt P37840 aas 672-714. The protein "Beta-amyloid protein 42" has been used here (aas 672-713). Only protein-protein interactions imex curation rosalind-2015-2 human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. pla proximity ligation assay pELISA pELISA PLA antibody detection identification by antibody antibody detection Rosalind F. Roberts , Richard Wade-Martins , Javier Alegre-Abarrategui Neurodegeneration - Publications depicting interactions involved in neurodegenerative diseases. As stated in Methods section:- "Alpha-synuclein bimolecular fluorescence complementation assay" -cells were imaged, fixed for immunofluorescence AND AS-PLA. "AS-PLA":-the same alpha-synuclein antibody (syn211)was used to make both probes. This antibody has previously been shown to display blocking activity on the epitope (El-Agnaf et al., 2006) and thus should prevent more than one probe binding per alpha-synuclein molecule, allowing AS-PLA to recognize only alpha-synuclein molecules that are interacting (i.e. oligomers) and not monomers. javier.alegre@dpag.ox.ac.uk Brain; A Journal of Neurology 2015 imex curation Rosalind F. Roberts , Richard Wade-Martins , Javier Alegre-Abarrategui, Rosalind F. Roberts , Richard Wade-Martins , Javier Alegre-Abarrategui, Rosalind F. Roberts , Richard Wade-Martins , Javier Alegre-Abarrategui, Rosalind F. Roberts , Richard Wade-Martins , Javier Alegre-Abarrategui Parkinsons - Interactions investigated in the context of Parkinsons disease Affinomics - Interactions curated for the Affinomics consortium. Outeiro et al., 2008 describes SNCA constructs of 140 amino acids; this corresponds to UniProt Isoform 1. The exact amyloid-beta product (from Tocris Bioscience) is not specified; hence the longer peptide sequences have been blasted and matches found. The sequence of all Tocris products is within UniProt P37840 aas 672-714. The protein "Beta-amyloid protein 42" has been used here (aas 672-713). Only protein-protein interactions imex curation rosalind-2015-3 human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. pla proximity ligation assay pELISA pELISA PLA antibody detection identification by antibody antibody detection Rosalind F. Roberts , Richard Wade-Martins , Javier Alegre-Abarrategui Neurodegeneration - Publications depicting interactions involved in neurodegenerative diseases. FKBP-FRB-rapamycin assay:-Rapamycin can bind simultaneously to two different proteins, FKBP and mTOR, forming a ternary complex. FKBP-alpha-synuclein and FRB-alpha-synuclein constructs were transfected into HEK293 cells which were then fixed for AS-PLA analysis. Interactions between alpha-synuclein monomers are conditionally induced in the presence of rapamycin. "AS-PLA":-the same alpha-synuclein antibody (syn211)was used to make both probes. This antibody has previously been shown to display blocking activity on the epitope (El-Agnaf et al., 2006) and thus should prevent more than one probe binding per alpha-synuclein molecule, allowing AS-PLA to recognize only alpha-synuclein molecules that are interacting (i.e. oligomers) and not monomers. Details of the FKBP and FRB constructs were taken from the Gruber et al., 2006 reference javier.alegre@dpag.ox.ac.uk Brain; A Journal of Neurology 2015 imex curation Rosalind F. Roberts , Richard Wade-Martins , Javier Alegre-Abarrategui, Rosalind F. Roberts , Richard Wade-Martins , Javier Alegre-Abarrategui, Rosalind F. Roberts , Richard Wade-Martins , Javier Alegre-Abarrategui, Rosalind F. Roberts , Richard Wade-Martins , Javier Alegre-Abarrategui Parkinsons - Interactions investigated in the context of Parkinsons disease Affinomics - Interactions curated for the Affinomics consortium. Outeiro et al., 2008 describes SNCA constructs of 140 amino acids; this corresponds to UniProt Isoform 1. The exact amyloid-beta product (from Tocris Bioscience) is not specified; hence the longer peptide sequences have been blasted and matches found. The sequence of all Tocris products is within UniProt P37840 aas 672-714. The protein "Beta-amyloid protein 42" has been used here (aas 672-713). Only protein-protein interactions imex curation rosalind-2015-6 in vitro In vitro pla proximity ligation assay pELISA pELISA PLA antibody detection identification by antibody antibody detection Rosalind F. Roberts , Richard Wade-Martins , Javier Alegre-Abarrategui, Rosalind F. Roberts , Richard Wade-Martins , Javier Alegre-Abarrategui, Rosalind F. Roberts , Richard Wade-Martins , Javier Alegre-Abarrategui, Rosalind F. Roberts , Richard Wade-Martins , Javier Alegre-Abarrategui Neurodegeneration - Publications depicting interactions involved in neurodegenerative diseases. SNCA and amyloid -beta oligomers were produced by incubating the recombinant human proteins with the lipid peroxidation byproduct 4-hydroxy-2-nonenal. (see pmid:22867050). SNCA fibrils were produced by shaking the recombinant human protein for 5 days. Outeiro et al., 2008 describes SNCA constructs of 140 amino acids; this corresponds to UniProt Isoform 1. The exact amyloid-beta product (from Tocris Bioscience) is not specified; hence the longer peptide sequences have been blasted and matches found. The sequence of all Tocris products is within UniProt P37840 aas 672-714. The protein "Beta-amyloid protein 42" has been used here (aas 672-713). javier.alegre@dpag.ox.ac.uk Brain; A Journal of Neurology 2015 Only protein-protein interactions imex curation imex curation Parkinsons - Interactions investigated in the context of Parkinsons disease Affinomics - Interactions curated for the Affinomics consortium. rosalind-2015-7 human-brain Homo sapiens brain brain Brain [cerebrum] pla proximity ligation assay pELISA pELISA PLA antibody detection identification by antibody antibody detection Rosalind F. Roberts , Richard Wade-Martins , Javier Alegre-Abarrategui, Rosalind F. Roberts , Richard Wade-Martins , Javier Alegre-Abarrategui Neurodegeneration - Publications depicting interactions involved in neurodegenerative diseases. Post-mortem tissue from eight patients with Parkinson’s disease;medulla, midbrain and cingulate cortex . Cingulate cortex from patients with dementia with Lewy bodies, substantia nigra from patients with multiple system atrophy. javier.alegre@dpag.ox.ac.uk Brain; A Journal of Neurology 2015 imex curation Rosalind F. Roberts , Richard Wade-Martins , Javier Alegre-Abarrategui, Rosalind F. Roberts , Richard Wade-Martins , Javier Alegre-Abarrategui, Rosalind F. Roberts , Richard Wade-Martins , Javier Alegre-Abarrategui, Rosalind F. Roberts , Richard Wade-Martins , Javier Alegre-Abarrategui Parkinsons - Interactions investigated in the context of Parkinsons disease Affinomics - Interactions curated for the Affinomics consortium. Outeiro et al., 2008 describes SNCA constructs of 140 amino acids; this corresponds to UniProt Isoform 1. The exact amyloid-beta product (from Tocris Bioscience) is not specified; hence the longer peptide sequences have been blasted and matches found. The sequence of all Tocris products is within UniProt P37840 aas 672-714. The protein "Beta-amyloid protein 42" has been used here (aas 672-713). Only protein-protein interactions imex curation p37840-1 Alpha-synuclein NACP140 SNCA NACP PARK1 Non-A beta component of AD amyloid Non-A4 component of amyloid precursor protein protein human Homo sapiens Human MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA p05067-pro_0000000092 Amyloid-beta protein 42 Alzheimer disease amyloid protein Cerebral vascular amyloid peptide APP A4 AD1 ABPP PreA4 Protease nexin-II APPI Amyloid precursor protein Amyloid-beta A4 protein Alzheimer disease amyloid A4 protein homolog Amyloid-beta (A4) precursor protein protein protein human Homo sapiens Human DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA 672 713 syua_human Alpha-synuclein Non-A beta component of AD amyloid Non-A4 component of amyloid precursor SNCA NACP PARK1 protein protein human Homo sapiens Human MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA snca-snca-9 1788160 N/A 1788167 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) N/A 1788167 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) direct interaction direct interaction false false false 3Ai Oligomers and fibrils were formed. app-app-9 1788160 N/A 1788168 unspecified role unspecified role neutral component neutral component N/A 1788168 unspecified role unspecified role neutral component neutral component direct interaction direct interaction false false false 3Aii Oligomers formed. snca-snca-10 1788161 N/A 1788167 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) N/A 1788167 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) direct interaction direct interaction false false false 3Bi Oligomers and fibrils were formed. Oligomers were seen as small round structures arranged in chains of variable lengths;fibrils showed a fibrillar helical ultrastructure. app-app-66 1788161 N/A 1788168 unspecified role unspecified role neutral component neutral component N/A 1788168 unspecified role unspecified role neutral component neutral component direct interaction direct interaction false false false 3Bi Oligomers formed. 3qewr-1 1788162 N/A 1788167 unspecified role unspecified role neutral component neutral component c_terminus (aas 156-238) cgfp cGFP c-term range C-terminal range c-term range c-term range C-terminal range c-term range false N/A 1788167 unspecified role unspecified role neutral component neutral component n_terminus (aas1-155) ngfp nGFP n-term range n-terminal range n-term range n-term range n-terminal range n-term range false physical association physical association false false false 1Aii,1Ci snca-snca-6 1788163 N/A 1788167 unspecified role unspecified role neutral component neutral component c_terminus (aas 156-238) cgfp cGFP c-term range C-terminal range c-term range c-term range C-terminal range c-term range false N/A 1788167 unspecified role unspecified role neutral component neutral component n_terminus (aas1-155) ngfp nGFP n-term range n-terminal range n-term range n-term range n-terminal range n-term range false proximity proximity close-contact colocalization neighbourhood interaction false false false 1Cii snca-snca-5 1788164 N/A 1788167 unspecified role unspecified role neutral component neutral component Rapamycin-binding domain-MTOR (I2021-K2113) Rapamycin-binding domain of MTOR fusion protein fusion protein undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false N/A 1788167 unspecified role unspecified role neutral component neutral component FKBP1A (G2-E108) fusion protein fusion protein undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false proximity proximity close-contact colocalization neighbourhood interaction false false false 2, Supp fig 2. A 4-fold increase in AS-PLA signal was observed in cells that had been exposed to rapamycin for 1 h post-transfection. snca-snca-11 1788165 N/A 1788167 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) N/A 1788167 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) direct interaction direct interaction false false false 3Bi, Supp Fig 2. Oligomers and fibrils were formed. snca-snca-12 1788166 N/A 1788169 unspecified role unspecified role neutral component neutral component N/A 1788169 unspecified role unspecified role neutral component neutral component proximity proximity close-contact colocalization neighbourhood interaction false false false 4,5,6. Supp fig 3 In tissue from post-mortem Parkinsons patients, Pale bodies, extrasomal Lewy bodies and other early lesions in the brainstem displayed prominent AS-PLA staining (medulla and midbrain). AS-PLA reveals diffuse cytoplasmic staining in neurons that contained neither pale bodies nor Lewy bodies [Fig. 4A(iv)]. This suggests that AS-PLA is able to detect alpha-synuclein oligomerization in cells very early in the aggregation process. In tissue from patients with dementia with Lewy bodies, cortical Lewy bodies were detected by AS-PLA; in multiple system atrophy cases, diseased glial cells harbouring glial cytoplasmic inclusions showed cytoplasmic labelling with AS-PLA.