MINT MINT, Dpt of Biology, University of Rome Tor Vergata MINT http://mint.bio.uniroma2.it/mint url>http://mint.bio.uniroma2.it/mint/ https://mint.bio.uniroma2.it MINT-[0-9]+ https://mint.bio.uniroma2.it/index.php/results-interactions/?id=${ac} The Molecular INTeraction database (MINT) is a relational database designed to store interactions between biological molecules. http://mint.bio.uniroma2.it http://mint.bio.uniroma2.it/mint anonymous-2013h-4 Synuclein as an intrinsically disordered monomer: fact or artifact? in vitro In vitro ms interact detect detection by mass spectrometry ms interact detect predetermined predetermined participant predetermined Coelho-Cerqueira E., Carmo-Gonçalves P., Sá Pinheiro A., Cortines J., Follmer C. follmer@iq.ufrj.br FEBS J. (1742-464X) 2013 Only protein-protein interactions imex curation anonymous-2013h-7 Synuclein as an intrinsically disordered monomer: fact or artifact? in vitro In vitro cd circular dichroism CD CD cd predetermined predetermined participant predetermined Coelho-Cerqueira E., Carmo-Gonçalves P., Sá Pinheiro A., Cortines J., Follmer C. follmer@iq.ufrj.br FEBS J. (1742-464X) 2013 Only protein-protein interactions imex curation anonymous-2013h-5 Synuclein as an intrinsically disordered monomer: fact or artifact? in vitro In vitro crosslink cross-linking study crosslink western blot western blot Immuno blot Immuno blot Coelho-Cerqueira E., Carmo-Gonçalves P., Sá Pinheiro A., Cortines J., Follmer C. follmer@iq.ufrj.br FEBS J. (1742-464X) 2013 Only protein-protein interactions imex curation anonymous-2013h-6 Synuclein as an intrinsically disordered monomer: fact or artifact? in vitro In vitro molecular sieving molecular sieving Gel Filtration Size Exclusion Chromatography Size Exclusion Chromatography Gel Filtration Sizing column predetermined predetermined participant predetermined Coelho-Cerqueira E., Carmo-Gonçalves P., Sá Pinheiro A., Cortines J., Follmer C. follmer@iq.ufrj.br FEBS J. (1742-464X) 2013 Only protein-protein interactions imex curation 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 1-131 1430484 n/a aplha-Syn 1430488 unspecified role unspecified role neutral component neutral component purified purified ecoli Escherichia coli (strain K12) Stoichiometry: 2.0 direct interaction direct interaction false false false f2c Mass spectrometry (MALDI-TOF/TOF) was used to confirm the oligomeric state of cross-linked α-syn. A molecular mass of 14,458 Da was obtained from the MALDI-TOF/TOF spectrum of non-cross-linked α-syn purified using the heating protocol (Figure 2C), and no differences were observed in comparison with α- syn purified using the acidification protocol (data not shown). After cross-linking, both heat- and acid-purified α-syn exhibited a minor population of dimers and trimers with the predominant monomer. For the α-syn purified using the heating protocol, a very small population of tetramers (64,269 Da) was also observed. Together, the analysis of crosslinked α-syn by both SDS-PAGE and mass spectroscopy indicated that the protein is mostly monomeric with a small population of oligomers, notably dimers. 123-1 1430484 n/a aplha-Syn 1430488 unspecified role unspecified role neutral component neutral component purified purified ecoli Escherichia coli (strain K12) Stoichiometry: 1.0 n/a aplha-Syn 1430488 unspecified role unspecified role neutral component neutral component purified purified ecoli Escherichia coli (strain K12) Stoichiometry: 1.0 direct interaction direct interaction false false false f2c Mass spectrometry (MALDI-TOF/TOF) was used to confirm the oligomeric state of cross-linked α-syn. A molecular mass of 14,458 Da was obtained from the MALDI-TOF/TOF spectrum of non-cross-linked α-syn purified using the heating protocol (Figure 2C), and no differences were observed in comparison with α- syn purified using the acidification protocol (data not shown). After cross-linking, both heat- and acid-purified α-syn exhibited a minor population of dimers and trimers with the predominant monomer. For the α-syn purified using the heating protocol, a very small population of tetramers (64,269 Da) was also observed. Together, the analysis of crosslinked α-syn by both SDS-PAGE and mass spectroscopy indicated that the protein is mostly monomeric with a small population of oligomers, notably dimers. 1-134 1430485 n/a aplha-Syn 1430488 unspecified role unspecified role neutral component neutral component purified purified ecoli Escherichia coli (strain K12) Stoichiometry: 1.0 n/a aplha-Syn 1430488 unspecified role unspecified role neutral component neutral component purified purified ecoli Escherichia coli (strain K12) Stoichiometry: 1.0 direct interaction direct interaction false false false f1b f1c f4b Both heat- and acid-purified α-syn displayed a CD spectrum with a negative band at 200 nm that is typical of disordered structures, although a slight band at approximately 220 nm was verified for the α-syn obtained using the heating protocol (Figure 1B). Additionally, the α-syn purified by both the acidification and heating methods displayed similar fibrillogenic properties; typical sigmoidal curves for the kinetics of fibrillization were observed, with an initial lag phase, associated with the nucleation process, followed by an exponential growth phase (Figure 1C). 1-132 1430486 n/a aplha-Syn 1430488 unspecified role unspecified role neutral component neutral component purified purified ecoli Escherichia coli (strain K12) Stoichiometry: 2.0 direct interaction direct interaction false false false f2a f2b f3g f4a To determine the oligomeric state of the recombinant α-syn purified with either the heating or the acidification protocol, the protein was cross-linked with GHD and then analyzed by SDS-PAGE (Figure 2). After cross-linking, a smallpopulation of α-syn was found as dimers (3-5% of total α-syn) with an apparent molecular mass of ~34 kDa, as determined using the Totallab software (Figures 2A and 2B). These species were also revealed by western blot using the monoclonal antibody anti-human α-syn 14H2L1. We also observed a very small population of α-syn running at ~60 kDa. Because α-syn monomers and dimers exhibit respective molecular masses of 18 and 34 kDa according to SDS-PAGE, this 60 kDa protein may be either a tetrameric or trimeric form of α-syn. 1-133 1430487 n/a aplha-Syn 1430488 unspecified role unspecified role neutral component neutral component purified purified ecoli Escherichia coli (strain K12) Stoichiometry: 2.0 direct interaction direct interaction false false false f3d f3e f3f Collectively, these data provide convincing evidence that α-syn dimers are highly dynamic and exist in equilibrium with the monomeric species