Real-time monitoring of apoptosis by caspase-3-like protease induced FRET reduction triggered by amyloid aggregation.
Amyloid formation is cytotoxic and can activate the caspase cascade. Here, we monitor caspase-3-like activity as reduction of fluorescence resonance energy transfer (FRET) using the contstruct pFRET2-DEVD containing enhanced cyan fluorescent protin (EYFP) linked by the caspase-3 specific cleavage site residues DEVD. Beta-TC-6 cells were transfected, and the fluoorescence was measured at 440 nm excitation and 535 nm (EYFP) and 480 nm (ECFP) emission wavelength. Cells were incubated with recombinant pro lset Amyloid Polypeptide (rec prolAPP) or the processing metabolites of prolAPP; the N-terminal flanking peptide withIAPP (recN+IAPP); IAPP with the C-terminal flanking peptied (recIAPP+C) and lslet Amyloid Polypeptide (recIAPP) . Peptides were added in solubilized from (50 microM) or as performed amyloid-like fibrils, or as a combination of these. FRET was measured and incubation with a mixture of solubilized peptide and performed fibrils resulted in loss of FRET and apoptosis was determined to occure in cells incubated with recproIAPP (49%), recN+IAPP (46%), recIAPP (72%) and recIAPP+C (59%). These results show that proIAPP and the processing intermediates reside the same cell toxic capacity as IAPP, and they can all have a central role in the reduction of beta-cell number in type 2 diabetes.
Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Johan F
JF
Paulsson
Sebastian W
SW
Schultz
Martin
M
Köhler
Ingo
I
Leibiger
Per-Olof
PO
Berggren
Gunilla T
GT
Westermark
D000595:Amino Acid Sequence; D000682:Amyloid / Q000378:metabolism*; D000818:Animals; D017209:Apoptosis; D053148:Caspase 3 / Q000378:metabolism*; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D003924:Diabetes Mellitus, Type 2 / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D007328:Insulin / Q000378:metabolism; D050417:Insulin-Secreting Cells / Q000378:metabolism / Q000473:pathology; D007340:Insulinoma / Q000378:metabolism* / Q000473:pathology*; D058228:Islet Amyloid Polypeptide; D051379:Mice; D008822:Mice, Transgenic; D010190:Pancreatic Neoplasms / Q000378:metabolism* / Q000473:pathology*; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
D000682:Amyloid; D007328:Insulin; D058228:Islet Amyloid Polypeptide; D011994:Recombinant Proteins; C422904:pro-islet amyloid polypeptide; C505409:Casp3 protein, mouse; D053148:Caspase 3