Amyloidogenicity of recombinant human pro-islet amyloid polypeptide (ProIAPP)

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.     

A Hot‐Segment‐Based Approach for the Design of Cross‐Amyloid Interaction Surface Mimics as Inhibitors of Amyloid Self‐Assembly

The design of inhibitors of protein–protein interactions mediating amyloid self‐assembly is a major challenge mainly due to the dynamic nature of the involved structures and interfaces. Interactions of amyloidogenic polypeptides with other proteins are important modulators of self‐assembly. Here we present a hot‐segment‐linking approach to design a series of mimics of the IAPP cross‐amyloid interaction surface with Aβ (ISMs) as nanomolar inhibitors of amyloidogenesis and cytotoxicity of Aβ, IAPP, or both polypeptides. The nature of the linker determines ISM structure and inhibitory function including both potency and target selectivity. Importantly, ISMs effectively suppress both self‐ and cross‐seeded IAPP self‐assembly. Our results provide a novel class of highly potent peptide leads for targeting protein aggregation in Alzheimer’s disease, type 2 diabetes, or both diseases and a chemical approach to inhibit amyloid self‐assembly and pathogenic interactions of other proteins as well.     

Structural Insight into IAPP-Derived Amyloid Inhibitors and Their Mechanism of Action

Designed peptides derived from the islet amyloid polypeptide (IAPP) cross-amyloid interaction surface with Aβ (termed interaction surface mimics or ISMs) have been shown to be highly potent inhibitors of Aβ amyloid self-assembly. However, the molecular mechanism of their function is not well understood. Using solution-state and solid-state NMR spectroscopy in combination with ensemble-averaged dynamics simulations and other biophysical methods including TEM, fluorescence spectroscopy and microscopy, and DLS, we characterize ISM structural preferences and interactions. We find that the ISM peptide R3-GI is highly dynamic, can adopt a β-like structure, and oligomerizes into colloid-like assemblies in a process that is reminiscent of liquid–liquid phase separation (LLPS). Our results suggest that such assemblies yield multivalent surfaces for interactions with Aβ40. Sequestration of substrates into these colloid-like structures provides a mechanistic basis for ISM function and the design of novel potent anti-amyloid molecules.     

Inhibition of hIAPP amyloid-fibril formation and apoptotic cell death by a designed hIAPP amyloid- core-containing hexapeptide

The pathogenesis of type II diabetes is associated with the aggregation of the 37-residue human islet amyloid polypeptide (hIAPP) into cytotoxic beta sheet aggregates and fibrils. We have recently shown that introduction of two N-methyl rests in the beta sheet- and amyloid-core-containing sequence hIAPP(22-27), or NFGAIL converted this amyloidogenic and cytotoxic sequence into nonamyloidogenic and noncytotoxic NF(N-Me)GA(N-Me)IL. Here, we show that NF(N-Me)GA(N-Me)IL is able to bind with high-affinity full-length hIAPP and to inhibit its fibrillogenesis. NF(N-Me)GA(N-Me)IL also inhibits hIAPP-mediated apoptotic beta cell death. By contrast, unmodified NFGAIL does not inhibit hIAPP amyloidogenesis and cytotoxicity, suggesting that N-methylation conferred on NFGAIL the properties of NF(N-Me)GA(N-Me)IL. These results support the concept that rational N-methylation of hIAPP amyloid-core sequences may be a valuable strategy to design pancreatic-amyloid diagnostics and therapeutics for type II diabetes.