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 sant'anna-2016 in vitro In vitro fluorescence spectr classical fluorescence spectroscopy fluorescence spectr predetermined predetermined participant predetermined Sant'Anna R., Navarro S., Ventura S., Paraoan L., Foguel D. foguel@bioqmed.ufrj.br; salvador.ventura@uab.es; lparaoan@liverpool.ac.uk FEBS letters 2016 Only protein-protein interactions imex curation imex curation sant'anna-2016-1 in vitro In vitro tem transmission electron microscopy tem predetermined predetermined participant predetermined Sant'Anna R., Navarro S., Ventura S., Paraoan L., Foguel D. foguel@bioqmed.ufrj.br; salvador.ventura@uab.es; lparaoan@liverpool.ac.uk FEBS letters 2016 Only protein-protein interactions imex curation imex curation sant'anna-2016-2 in vitro In vitro ir spectrometry infrared spectroscopy ir spectrometry predetermined predetermined participant predetermined Sant'Anna R., Navarro S., Ventura S., Paraoan L., Foguel D. foguel@bioqmed.ufrj.br; salvador.ventura@uab.es; lparaoan@liverpool.ac.uk FEBS letters 2016 Only protein-protein interactions imex curation imex curation sant'anna-2016-3 in vitro In vitro dls dynamic light scattering dls predetermined predetermined participant predetermined Sant'Anna R., Navarro S., Ventura S., Paraoan L., Foguel D. foguel@bioqmed.ufrj.br; salvador.ventura@uab.es; lparaoan@liverpool.ac.uk FEBS letters 2016 Only protein-protein interactions imex curation imex curation cytc_human Cystatin-C Cystatin-3 Neuroendocrine basic polypeptide Gamma-trace Post-gamma-globulin CST3 protein protein human Homo sapiens Human MAGPLRAPLLLLAILAVALAVSPAAGSSPGKPPRLVGGPMDASVEEEGVRRALDFAVGEYNKASNDMYHSRALQVVRARKQIVAGVNYFLDVELGRTTCTKTQPNLDNCPFHDQPHLKRKAFCSFQIYAVPWQGTMTLSKSTCQDA cst3-cst3-1 1931628 N/A 1931632 unspecified role unspecified role neutral component neutral component purified purified N/A 1931632 unspecified role unspecified role neutral component neutral component purified purified direct interaction direct interaction false false false As shown in Fig. 2A, there was a proportional increase in Thioflavin-T (Th-T) emission at increasing concentrations of the peptide, suggesting formation of amyloid-like aggregates in solution. Congo red (CR) binding assays were also performed to confirm the presence of amyloid-like aggregates (Fig. 2A, inset). In order to follow the aggregation kinetics of the signal peptide, increasing concentrations of the peptide were incubated with Th-T and its fluorescence emission was collected over time (Fig. 2B). Notably, even at very low peptide concentration such as 5 μm, maximum emission of Th-T was attained in the first acquisition points, with no further change in the fluorescence signal of the probe even after 3 days under aggregation conditions. This suggests that aggregation of the signal peptide of cystatin C is very fast. f2 cst3-cst3-2 1931629 N/A 1931632 unspecified role unspecified role neutral component neutral component purified purified N/A 1931632 unspecified role unspecified role neutral component neutral component purified purified direct interaction direct interaction false false false Next, we used TEM to study the morphology of the species present at different times of aggregation (Fig. 3A). Interestingly, although Th-T binding has already leveled-off at 1 h under aggregation conditions, the great majority of the aggregated material presented an amorphous appearance and only few amyloid fibrils were observed at this time (Fig. 3A, left). The binding of Th-T to these amorphous species suggests that they already present cross-β fold able to accommodate this probe. These species are on-pathway to fibril formation since after 72 h under aggregation condition only mature fibrils were observed by TEM (Fig. 3A, right). At 24 h, it was already possible to see the presence of fibrils but small amorphous aggregates still remained (Fig. 3A, middle). f3a cst3-cst3-6 1931630 N/A 1931632 unspecified role unspecified role neutral component neutral component purified purified N/A 1931632 unspecified role unspecified role neutral component neutral component purified purified direct interaction direct interaction false false false In order to get insights into the secondary structural changes that take place with the signal peptide upon aggregation, we took advantage of ATR-FTIR (Fig. 3 B). Left panel shows the FTIR spectra of the peptide dried from a stock solution with 100% DMSO (continuous line) and those collected during the first hour of aggregation. As seen, in DMSO the peptide presented a unique and broad peak centered at 1660 cm−1, which corresponds to disordered, random-coiled structures, which is a strong evidence that the peptide is monomeric in DMSO [48-50]. At the initial times of aggregation (up to 1 h), the spectra changed and a new peak at 1628 cm−1 appeared which was assigned to β-sheet structures. At the same time, the peak related to random-coil structures (1660 cm−1) decreased but was still present. The presence of β-sheet structures in these initial aggregates explains why these species bind Th-T (as shown in Fig. 2), but they are not fully organized, as seen by TEM (Fig. 3A, left). As aggregation proceeded for longer times (16, 24 and 72 h), the peak of β-sheet (1627 cm−1) increased even further, with a concomitant decrease of the peak of random coil at 1660 cm−1. Table 1 summarizes the secondary structural changes that take place upon aggregation of the signal peptide of cystatin C. f3b t1 cst3-cst3-7 1931631 N/A 1931632 unspecified role unspecified role neutral component neutral component purified purified N/A 1931632 unspecified role unspecified role neutral component neutral component purified purified direct interaction direct interaction false false false Next, to further confirm the difference in sizes of the species formed at 1 and 24 h, DLS measurements were performed (Fig. 3C). The main sizes of the two more prevalent species present at 1 h were 0.194 nm ± 0.06 nm and 904 ± 32.14 nm. After 24 h under aggregation conditions, there were two species present, one with 947 ± 134 nm and an extreme large species with 3008 ± 500 nm. Interestingly and corroborating the FTIR data, the species with around 900 nm were still present at 24 h. f3c