title: Highly amyloidogenic two-chain peptide fragments are released upon partial digestion of insulin with pepsin.
abstract: Proteases play a well recognized role in the emergence of highly aggregation-prone protein fragments in vivo, whereas in vitro limited proteolysis is often employed to probe different phases of amyloidogenic pathways. Here, we show that addition of moderate amounts of pepsin to acidified bovine insulin at close to physiological temperature results in an abrupt self-assembly of amyloid-like fibrils from partially digested insulin fragments. Biochemical analysis of the pepsin-induced fibrils implicates peptide fragments (named H) consisting of the 13 or 15 N-terminal residues of the A-chain and 11 or 13 N-terminal residues of the B-chain linked by the disulfide bond between Cys-7A-Cys-7B as the main constituents. There are up to eight pepsin-cleavage sites remaining within the double chain peptide, which become protected upon fast fibrillation unless concentration of the enzyme is increased resulting in complete digestion of insulin. Controlled re-association of H-peptides leads to "explosive" fibrillation only under nonreducing conditions implying the key role of the disulfide bond in their amyloidogenicity. Such re-assembled amyloid is similar in terms of morphology and infrared features to typical bovine insulin fibrils, although it lacks the ability to seed the intact protein.
authors.affiliation: From the Protein Biophysics Group, Institute of High Pressure Physics, Polish Academy of Sciences, 01-142 Warsaw, the Department of Analytical Biochemistry.
authors.forename: Marcin
authors.initials: M
authors.lastname: Piejko
authors.affiliation: the Department of Chemistry, Biological and Chemical Research Centre, University of Warsaw, 02-093 Warsaw, Poland.
authors.forename: Robert
authors.initials: R
authors.lastname: Dec
authors.affiliation: From the Protein Biophysics Group, Institute of High Pressure Physics, Polish Academy of Sciences, 01-142 Warsaw, the Department of Chemistry, Biological and Chemical Research Centre, University of Warsaw, 02-093 Warsaw, Poland.
authors.forename: Viktoria
authors.initials: V
authors.lastname: Babenko
authors.affiliation: From the Protein Biophysics Group, Institute of High Pressure Physics, Polish Academy of Sciences, 01-142 Warsaw, Division of Cell Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Hoang
authors.affiliation: the Department of Chemistry, Biological and Chemical Research Centre, University of Warsaw, 02-093 Warsaw, Poland.
authors.forename: Monika
authors.initials: M
authors.lastname: Szewczyk
authors.affiliation: the Department of Analytical Biochemistry, Malopolska Centre of Biotechnology, Jagiellonian University, 30-387 Krakow, and.
authors.forename: Paweł
authors.initials: P
authors.lastname: Mak
authors.affiliation: From the Protein Biophysics Group, Institute of High Pressure Physics, Polish Academy of Sciences, 01-142 Warsaw, the Department of Chemistry, Biological and Chemical Research Centre, University of Warsaw, 02-093 Warsaw, Poland wdzwolak@chem.uw.edu.pl.
authors.forename: Wojciech
authors.initials: W
authors.lastname: Dzwolak
mesh_terms: D058227:Amyloidogenic Proteins / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D002417:Cattle; D018360:Crystallography, X-Ray; D004063:Digestion; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D007328:Insulin / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D010434:Pepsin A / Q000737:chemistry; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D066329:Protein Aggregates; D059748:Proteolysis
chemical_list: D058227:Amyloidogenic Proteins; D004220:Disulfides; D007328:Insulin; D010455:Peptides; D066329:Protein Aggregates; D010434:Pepsin A
keywords: Amyloid; Disulfide; Insulin; Kinetics; Pepsin; Proteolysis