Selection for Nonamyloidogenic Mutants of Islet Amyloid Polypeptide (IAPP) Identifies an Extended Region for Amyloidogenicity

Abstract

The aggregation of the 37-residue polypeptide IAPP, as either insoluble amyloid or as small oligomers, appears to play a direct role in the death of pancreatic β-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 E. 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 non-amyloidogenic 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.The aggregation of misfolded proteins into toxic oligomers and fibers has been linked to a variety of diseases such as type II diabetes, Alzheimer's disease and Parkinson's disease. In type II diabetes the amyloid-forming peptide is islet amyloid polypeptide (IAPP, amylin). This 37 amino acid polypeptide misfolds and forms aggregates within the pancreas. This misfolding into toxic aggregates, such as small soluble oligomers or large fibers, is believed to contribute to the loss of pancreatic β-cells. While the exact role of IAPP in type II diabetes is unclear, it is known that IAPP is found as extracellular deposits of amyloid in approximately 95% of patients afflicted with type II diabetes (1-3). IAPP has also been shown to be a toxic agent in vitro when added to human islet β-cells (4).Many of the therapeutic strategies for preventing or slowing the progression of amyloid diseases such as type II diabetes, involve slowing or preventing the aggregation of the amyloidogenic proteins. To this end, a great deal of effort has been made in identifying the amino acids responsible for the aggregation-prone nature of amyloidogenic peptides such as †

Protein Misfolding and Aggregation in Alzheimer’s Disease and Type 2 Diabetes Mellitus

“…The relevance of this mid region in aggregation is underlined by a single point mutation, a Ser20Gly substitution, which enhances amyloid formation, leads to β-cell death and an early onset familial form of T2DM found in Japan [160]. Of note, mutations outside of this mid region can eliminate amyloid formation, indicating that one cannot rule out contributions from other regions impacting IAPP’s amyloidogenicity [161, 162]. …”

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“…Because of this, it is possible that substances capable of inhibiting the aggregation of one amyloid protein may also be capable of inhibiting others. Because IAPP is considered to be one of the most amyloidogenic proteins known, substances found to inhibit this amyloid protein may be potential therapeutics for other amyloid proteins, such as Aβ42 aggregation in Alzheimer’s disease or α-synuclein in Parkinson’s disease (Fox et al, 2010). …”

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