Design of a mimic of nonamyloidogenic and bioactive human islet amyloid polypeptide (IAPP) as nanomolar affinity inhibitor of IAPP cytotoxic fibrillogenesis

Yan et al. 10.1073/pnas.0507471103.

Supporting Information

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Supporting Figure 7
Supporting Text

Suporting Figure 7

Fig. 7. Self-assembly states of [(N-Me)G24, N-Me)I26]-human islet amyloid polypeptide (IAPP-GI) (A) or IAPP (B) (6.25 mM), as determined by size exclusion chromatography (SEC) at different time points. Results are from two independent experiments.

Supporting Text

Peptide Synthesis

₄₃^α^α^αN^α^αeeN-e

Sedimentation Assays

μM assay] were performed at room temperature. At various time points, aliquots were centrifuged (20,200 ´g

Size Exclusion Chromatography (SEC)

The elution buffer was 10 mM sodium phosphate buffer, pH 7.4, containing 100 mM NaCl; the flow rate was 0.25 ml/min, and detection was at 214 nm. The column was calibrated with proteins and peptides of known molecular weights. Incubations of IAPP or IAPP-GI (6.25 μM) were made in elution buffer, and 800 μl (20 μg) aliquots were applied onto the column at various time points. All samples were centrifuged (17,000 ´ g, 10 min) before application to the column.

Thioflavin T (ThT)-Binding Assays

ed on aliquots of incubations of IAPP, IAPP-GI, or mixtures of both peptides (625 nM, 6.25 μM, and 62.5 μM) made in 10 mM Tris buffer, pH 7.4, containing 2% HFIP (assay buffer 1) or in 50 mM sodium phosphate buffer, pH 7.4, containing 100 mM NaCl and 0.5%»s of an IAPP incubation (16.5 μM in assay buffer 2) were withdrawn at various time points of its fibrillization process and added to an Eppendorf tube containing an equimolar amount of IAPP-GI (in dry form). At the indicated time points, aliquots of the slutions were mixed with a ThT solution (5 μM ThT for peptide concentrations of 6.25-62.5 μM or 200 μM ThT for the 625 nM peptide concentration in 0.1 M (0.05 M for assays performed with assay buffer 2) glycine/NaOH, pH 8.5), and ThT binding was determined²

Far-UV CD Spectroscopy

Adenylate Cyclase Activation Assays

´⁵cells/ml (100 μl/well). After incubation for 24 h (37°₂dded to the cells (200 μl) and incubations were performed for 15 min at 37°nces). Results are expressed as percent of maximum adenylate cyclase activation above the basal level, and maximum stimulation was the stimulation caused by 1 μM IAPP (9).

Receptor-Binding Assays

´⁶¹²⁵specific binding (1 μM IAPP), which was between 13% and 24% of total binding throughout all assays.

Pull-Down Assays

⁰]IAPP-GI (Dig-IAPP-GI) (2.5 μM) was incubated (1 h) alone or in a mixture with N^α⁰.5 μM) in 1 ml of 10 mM sodium phosphate buffer, pH 7.4, (assay buffer) at room temperature. Streptavidin-coupled magnetic beads (Dynabeads M-280 Streptavidin, Dynal, Oslo) were washed with PBSμ

Fluorescence Spectroscopic Titration Studies

FF_minF_maxF_min´K_dK_d²´^1/2FF_minF_maxK_d+

Assessment of Cytotoxicity via 3-[4,5-Dimethylthiazol-2-yl]-2,5-Diphenyltetrazolium Bromide (MTT) Reduction Assays

IAPP incubations in the presence or absence of IAPP-GI were performed at 5 μM (each peptide) in 10 mM sodium phosphate buffer, pH 7.4, containing 1% HFIP or at 16.5 μM in 50 mM sodium phosphate, pH 7.4, containing 100 mM NaCl and 0.5% HFBalone were found. For the studies to determine the inhibitory potency of IAPP-GI, incubations of 5 μM IAPP in the absence or presence of various amounts of IAPP-GI (in 10 mM sodium phosphate buffer, pH 7.4, 1% HFIP) were performed for 24 h. Solutions wer

Assessment of Apoptosis via ELISA

After incubation of the cells for 24 h (37°C, humidified atmosphere with 5% CO₂), an aliquot of an IAPP solution (5 μM, in 10 mM sodium phosphate buffer, pH 7.4) or a solution containing a mixture of IAPP (5 μM) with IAPP-GI (5 μM) or vehicle alone were applied to the cells at peptide final concentrations of 500 nM. After incubation for 20 h, cells were detached from the wells by short treatment with a trypsin/EDTA solution, counted, and apoptosis was assessed by the Cell Death Detection ELISA kit which quantitates the generated nucleosomes (Roche Diagnostics, Mannheim, Germany), as described (2).

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