title: Amyloidogenicity of recombinant human pro-islet amyloid polypeptide (ProIAPP).
abstract: BACKGROUND
  Pancreatic amyloid has been associated with type II diabetes. The major constituent of pancreatic amyloid is the 37-residue peptide islet amyloid polypeptide (IAPP). IAPP is expressed as a 67-residue pro-peptide called ProIAPP which is processed to IAPP following stimulation. While the molecular events underlying IAPP amyloid formation in vitro have been studied, little is known about the role of ProIAPP in the formation of pancreatic amyloid. This has been due in part to the limited availability of purified ProIAPP for conformational and biochemical studies.
  
  
  RESULTS
  We present a method for efficient recombinant expression and purification of ProIAPP and a processing site mutant, mutProIAPP, as thioredoxin (Trx) fusion proteins. Conformation and amyloidogenicity of cleaved ProIAPP and mutProIAPP and the fusion proteins were assessed by circular dichroism, electron microscopy and Congo red staining. We find that ProIAPP and mutProIAPP exhibit strong self-association potentials and are capable of forming amyloid. However, the conformational transitions of ProIAPP and mutProIAPP during aging and amyloidogenesis are distinct from the random coil-to-beta-sheet transition of IAPP. Both proteins are found to be less amyloidogenic than IAPP and besides fibrils a number of non-fibrillar but ordered aggregates form during aging of ProIAPP. ProIAPP aggregates are cytotoxic on pancreatic cells but less cytotoxic than IAPP while mutProIAPP aggregates essentially lack cytotoxicity. The Trx fusion proteins are neither amyloidogenic nor cytotoxic.
  
  
  CONCLUSIONS
  Our studies suggest that ProIAPP has typical properties of an amyloidogenic polypeptide but also indicate that the pro-region suppresses the amyloidogenic and cytotoxic potentials of IAPP.
authors.affiliation: Physiological-Chemical Institute, University of Tübingen, Tübingen, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Krampert
authors.forename: J
authors.initials: J
authors.lastname: Bernhagen
authors.forename: J
authors.initials: J
authors.lastname: Schmucker
authors.forename: A
authors.initials: A
authors.lastname: Horn
authors.forename: A
authors.initials: A
authors.lastname: Schmauder
authors.forename: H
authors.initials: H
authors.lastname: Brunner
authors.forename: W
authors.initials: W
authors.lastname: Voelter
authors.forename: A
authors.initials: A
authors.lastname: Kapurniotu
mesh_terms: D000595:Amino Acid Sequence; D000682:Amyloid / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D001483:Base Sequence; D002470:Cell Survival; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004396:Coloring Agents / Q000737:chemistry; D003224:Congo Red / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D058228:Islet Amyloid Polypeptide; D007515:Islets of Langerhans / Q000166:cytology* / Q000187:drug effects; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011498:Protein Precursors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012996:Solutions; D014407:Tumor Cells, Cultured
chemical_list: D000682:Amyloid; D004396:Coloring Agents; D058228:Islet Amyloid Polypeptide; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D012996:Solutions; D003224:Congo Red