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 Blockade of Islet Amyloid Polypeptide Fibrillation and Cytotoxicity by the Secretory Chaperones 7B2 and proSAAS Blockade of Islet Amyloid Polypeptide Fibrillation and Cytotoxicity by the Secretory Chaperones 7B2 and proSAAS FEBS Lett. (0014-5793) 2013 imex curation Peinado J.R., Sami F., Rajpurohit N., Lindberg I. ilind001@umaryland.edu 30-Sept-2013: Contacted by IntAct-Help. Only protein-protein interactions in vitro In vitro fluorescence fluorescence technology fluorescence predetermined predetermined participant predetermined Peinado J.R., Sami F., Rajpurohit N., Lindberg I. ilind001@umaryland.edu Accepted 2013-SEP-06 by LPERFETTO 30-Sept-2013: Contacted by IntAct-Help. FEBS Lett. (0014-5793) 2013 Only protein-protein interactions imex curation iapp_human Islet amyloid polypeptide IAPP Amylin Diabetes-associated peptide Insulinoma amyloid peptide protein protein human Homo sapiens Human MGILKLQVFLIVLSVALNHLKATPIESHQVEKRKCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTYGKRNAVEVLKREPLNYLPL DIP protein P10997 original sequence version: 138 not sure about the protein origin DIP protein P10997 original sequence version: 92 DIP protein P10997 original sequence version: 88 mint AA8B1F7FD9FCB4BD iapp-iapp 1 hIAPP 2 unspecified role unspecified role unspecified role unspecified role hIAPP 2 unspecified role unspecified role unspecified role unspecified role purified purified direct interaction direct interaction f1 f2 f3 We investigated hIAPP fibrillation using an in vitro fluorescence assay using a ThT assay. In our hands, concentrations ranging from 5 to 20 μM of hIAPP exhibited complete fibrillation within an hour at 25 C and 10 μM was chosen for further assays (Fig. 1A). It has been shown that fibrillation of hIAPP is inhibited by insulin [25]. In our assays 5 μM insulin blocked 80% of hIAPP fibrillation, while an irrelevant control protein such as carbonic anhydrase did not block fibrillation (Fig. 1B). We found 90% inhibition of hIAPP fibrillation with 10 μM 7B2, while 1 μM resulted in 50% inhibition (Fig. 1C). ProSAAS is also abundant in the pancreas [27]; in order to evaluate whether proSAAS also possesses anti-fibrillation ability, we carried out an assay using proSAAS concentrations between 0.1 and 10 μM. Under these conditions 10 μM 21 kDa proSAAS blocked hIAPP fibrillation completely and 0.1 μM still retained some inhibitory activity on fibril formation (Fig. 1D). To elucidate the region within 7B2 responsible for the anti-fibrillation effect, we performed in vitro fibrillation assays with N-terminally truncated proteins. Structure-function analysis using truncated forms of 7B2 revealed that the anti-aggregation effect was greatest using 21-kDa 7B2 (Fig. 2A), although 10 μM 7B230-150 and 7B268-150 partially inhibited hIAPP fibrillation by 30 and 20% respectively (Fig. 2B). two truncated peptides (Fig. 3A) were synthesized (proSAAS97-137 and proSAAS138-180, see Materials and Methods) to test whether α-helices II and III play a role in proSAAS-induced inhibition of fibrillation. However, neither of these two peptides was able to inhibit fibrillation in vitro (Fig. 3B). We conclude that residues 1-97 in the N-terminal domain contain key determinants for the anti-fibrillation ability of proSAAS on hIAPP