Sensitivity of amyloid formation by human islet amyloid polypeptide to mutations at residue 20.
Islet amyloid polypeptide (IAPP, amylin) is responsible for amyloid formation in type 2 diabetes and in islet cell transplants. The only known natural mutation found in mature human IAPP is a Ser20-to-Gly missense mutation, found with small frequency in Chinese and Japanese populations. The mutation appears to be associated with increased risk of early-onset type 2 diabetes. Early measurements in the presence of organic co-solvents showed that S20G-IAPP formed amyloid more quickly than the wild type. We confirm that the mutant accelerates amyloid formation under a range of conditions including in the absence of co-solvents. Ser20 adopts a normal backbone geometry, and the side chain makes no steric clashes in models of IAPP amyloid fibers, suggesting that the increased rate of amyloid formation by the mutant does not result from the relief of steric incompatibility in the fiber state. Transmission electronic microscopy, circular dichroism, and seeding studies were used to probe the structure of the resulting fibers. The S20G-IAPP peptide is toxic to cultured rat INS-1 (transformed rat insulinoma-1) β-cells. The sensitivity of amyloid formation to the identity of residue 20 was exploited to design a variant that is much slower to aggregate and that inhibits amyloid formation by wild-type IAPP. An S20K mutant forms amyloid with an 18-fold longer lag phase in homogeneous solution. Thioflavin T binding assays, together with experiments using a p-cyanophenylalanine (p-cyanoPhe) variant of human IAPP, show that the designed S20K mutant inhibits amyloid formation by human IAPP. The experiments illustrate how p-cyanoPhe can be exploited to monitor amyloid formation even in the presence of other amyloidogenic proteins.
Department of Chemistry, Stony Brook University, Stony Brook, NY 11794-3400, USA.
Ping
P
Cao
Ling-Hsien
LH
Tu
Andisheh
A
Abedini
Olesya
O
Levsh
Rehana
R
Akter
Vadim
V
Patsalo
Ann Marie
AM
Schmidt
Daniel P
DP
Raleigh
D000595:Amino Acid Sequence; D002478:Cells, Cultured; D002942:Circular Dichroism; D006801:Humans; D058228:Islet Amyloid Polypeptide / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D011487:Protein Conformation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D058228:Islet Amyloid Polypeptide