Selection for nonamyloidogenic mutants of islet amyloid polypeptide (IAPP) identifies an extended region for amyloidogenicity.
The aggregation of the 37-residue protein, islet amyloid polypeptide (IAPP), as either insoluble amyloid or as small oligomers, appears to play a direct role in the death of pancreatic beta-islet cells in type II diabetes. While IAPP has been known to be the primary component of type II diabetes amyloid, the molecular interactions responsible for this aggregation have not been identified. To identify the aggregation-prone region(s), we constructed a library of randomly generated point mutants of IAPP. This mutant IAPP library was expressed in Escherichia coli as genetic fusions to the reporter protein enhanced green fluorescent protein (EGFP). Because IAPP aggregates rapidly, both independently and when fused to EGFP, the fusion protein does not yield a functional, fluorescent EGFP. However, mutations of IAPP that result in nonamyloidogenic sequences remain soluble and allow EGFP to fold and fluoresce. Using this screen, we identified 22 single mutations, 4 double mutations, and 2 triple mutations of IAPP that appear to be less amyloidogenic than wild-type human IAPP. A comparison of these sequences suggests residues 13 and 15-17 comprise an additional aggregation-prone region outside of the main amyloidogenic region of IAPP.
Fox
Ayano
A
Department of Chemistry and Biochemistry, Loyola Marymount University, 1 LMU Drive, Los Angeles, California 90045, USA.
Snollaerts
Thibaut
T
Errecart Casanova
Camille
C
Calciano
Anastasia
A
Nogaj
Luiza A
LA
Moffet
David A
DA
D000595:Amino Acid Sequence; D000682:Amyloid / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D058228:Islet Amyloid Polypeptide; D018625:Microscopy, Atomic Force; D008969:Molecular Sequence Data; D009154:Mutation; D017510:Protein Folding; D017433:Protein Structure, Secondary
D000682:Amyloid; D058228:Islet Amyloid Polypeptide; D049452:Green Fluorescent Proteins