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 Intracellular seeded aggregation of mutant Cu,Zn-superoxide dismutase associated with amyotrophic lateral sclerosis. Intracellular seeded aggregation of mutant Cu,Zn-superoxide dismutase associated with amyotrophic lateral sclerosis. FEBS Lett. (0014-5793) 2013 Furukawa Y., Kaneko K., Watanabe S., Yamanaka K., Nukina N. furukawa@chem.keio.ac.jp. in vitro In vitro biophysical biophysical predetermined predetermined participant predetermined Furukawa Y., Kaneko K., Watanabe S., Yamanaka K., Nukina N. furukawa@chem.keio.ac.jp. FEBS Lett. (0014-5793) 2013 Intracellular seeded aggregation of mutant Cu,Zn-superoxide dismutase associated with amyotrophic lateral sclerosis. Intracellular seeded aggregation of mutant Cu,Zn-superoxide dismutase associated with amyotrophic lateral sclerosis. FEBS Lett. (0014-5793) 2013 Furukawa Y., Kaneko K., Watanabe S., Yamanaka K., Nukina N. furukawa@chem.keio.ac.jp. in vitro In vitro solution sedimentati cosedimentation in solution solution sedimentati western blot western blot Immuno blot Immuno blot Furukawa Y., Kaneko K., Watanabe S., Yamanaka K., Nukina N. furukawa@chem.keio.ac.jp. FEBS Lett. (0014-5793) 2013 Intracellular seeded aggregation of mutant Cu,Zn-superoxide dismutase associated with amyotrophic lateral sclerosis. Intracellular seeded aggregation of mutant Cu,Zn-superoxide dismutase associated with amyotrophic lateral sclerosis. FEBS Lett. (0014-5793) 2013 Furukawa Y., Kaneko K., Watanabe S., Yamanaka K., Nukina N. furukawa@chem.keio.ac.jp. in vitro In vitro tem transmission electron microscopy tem predetermined predetermined participant predetermined Furukawa Y., Kaneko K., Watanabe S., Yamanaka K., Nukina N. furukawa@chem.keio.ac.jp. FEBS Lett. (0014-5793) 2013 sodc_human Superoxide dismutase [Cu-Zn] Superoxide dismutase 1 SOD1 protein protein human Homo sapiens Human MATKAVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVIGIAQ DIP protein P00441 original sequence version: 194 DIP protein P00441 original sequence version: 189 DIP protein P00441 original sequence version: 186 DIP protein P00441 original sequence version: 147 DIP protein P00441 original sequence version: 148 DIP protein P00441 original sequence version: 145 DIP protein P00441 original sequence version: 169 25CA38DA8D564483 sod1-sod1 3 SOD1 4 predetermined predetermined participant predetermined experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component purified purified SOD1 4 predetermined predetermined participant predetermined experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component purified purified direct interaction direct interaction f1d homomint mint Furthermore, fibrillar morphologies of the seeded SOD1I149T aggregates were confirmed by a transmission electron microscopy (Fig. 1D). sod1-sod1-1 1 SOD1 4 predetermined predetermined participant predetermined experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component purified purified SOD1 4 predetermined predetermined participant predetermined experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component purified purified direct interaction direct interaction f1a homomint mint To prepare SOD1 fibrils in vitro, SOD1 with a fALS-causing mutation, I149T, (SOD1I149T) was first demetallated by precipitation with trichloroacetic acid and re-solubilized with a buffer containing a metal chelator, EDTA. Also, a reductant, DTT, was added to keep the disulfide reduced. Constant agitation (shaking at 1,200 rpm) of apo-SOD1SH with I149T mutation increases the solution turbidity, showing aggregation (Fig. 1A). sod1-sod1-2 2 SOD1 4 western blot western blot Immuno blot Immuno blot experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component purified purified SOD1 4 western blot western blot Immuno blot Immuno blot experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component purified purified direct interaction direct interaction f1b f1c homomint mint To test if SOD1 fibrillation can be triggered by a seeding reaction, the SOD1I149T seeds were prepared by shearing spontaneous SOD1I149T aggregates with ultrasonication. We expected that relatively low melting temperature (~42 oC) of SOD1I149T fibrillar aggregates (14) indicate their fragility and thus facilitate shearing of the aggregates. The sheared SOD1I149T aggregates were added as seeds to the solution containing a soluble form of apo and disulfide-reduced SOD1I149T, and the incubation was performed in the absence of any agitation. As revealed by SDS-PAGE (Fig. 1B and 1C), amounts of SOD1I149T in insoluble pellets increased after addition of SOD1I149T seeds, while no insolubilization of SOD1I149T was observed in the absence of seeds.