An Achilles' Heel in an Amyloidogenic Protein and Its Repair

Abstract

Insulin fibrillation provides a model for a broad class of amyloidogenic diseases. Conformational distortion of the native monomer leads to aggregation-coupled misfolding. Whereas ␤-cells are protected from proteotoxicity by hexamer assembly, fibrillation limits the storage and use of insulin at elevated temperatures. Here, we have investigated conformational distortions of an engineered insulin monomer in relation to the structure of an insulin fibril. Anomalous 13 C NMR chemical shifts and rapid 15 N-detected 1 H-2 H amide-proton exchange were observed in one of the three classical ␣-helices (residues A1-A8) of the hormone, suggesting a conformational equilibrium between locally folded and unfolded A-chain segments. Whereas hexamer assembly resolves these anomalies in accordance with its protective role, solid-state 13 C NMR studies suggest that the A-chain segment participates in a fibril-specific ␤-sheet. Accordingly, we investigated whether helicogenic substitutions in the A1-A8 segment might delay fibrillation. Simultaneous substitution of three ␤-branched residues (Ile A2 3 Leu, Val A3 3 Leu, and Thr A8 3 His) yielded an analog with reduced thermodynamic stability but marked resistance to fibrillation. Whereas amide-proton exchange in the A1-A8 segment remained rapid, 13 C␣ chemical shifts exhibited a more helical pattern. This analog is essentially without activity, however, as Ile A2 and Val A3 define conserved receptor contacts. To obtain active analogs, substitutions were restricted to A8. These analogs exhibit high receptor-binding affinity; representative potency in a rodent model of diabetes mellitus was similar to wild-type insulin. Although 13 C␣ chemical shifts remain anomalous, significant protection from fibrillation is retained. Together, our studies define an "Achilles' heel" in a globular protein whose repair may enhance the stability of pharmaceutical formulations and broaden their therapeutic deployment in the developing world.Insulin, a hormone central to the regulation of metabolism ( Fig. 1), is a prototypical amyloidogenic protein. In pathological amyloid deposits, such proteins undergo non-native cross-␤-assembly to form linear polymers (fibrils) (1), a hallmark of diseases of toxic misfolding (2). Fibrillation of insulin when stored above room temperature has long complicated its use in the treatment of diabetes mellitus (DM) 4 (3). Why is insulin susceptible to fibrillation, and how may such degradation be prevented? These questions are of both basic and applied interest. In this study, we employ 13 C and 15 N NMR spectroscopy to identify a dynamic anomaly in an engineered insulin monomer (4). Molecular repair of an unstable polypeptide segment (its "Achilles' heel") provides evidence of a link to the mechanism of fibrillation and yields novel analogs that may enhance the stability, efficacy, and safety of insulin replacement therapy in the treatment of DM in the developed and developing world.The mechanism of insulin fibrillation is outlined in Fig. 2. Of signal importance is...

Protective hinge in insulin opens to enable its receptor engagement

“…5). The activity of analog 1 as an engineered monomer, evaluated by receptor-binding affinity in vitro and glycemic potency in a rat model of DM (22), was at least as high as that of WT insulin (SI Appendix, Figs. S4B and 7).…”

“…Lack of correlation between fibrillation lag times of insulin analogs and native state stability (22,27) reflects a key aspect of fibrillation: the unfolded-state ensemble is off-pathway (Fig. 9, Top).…”

“…Insulin-stimulated receptor autophosphorylation and protein kinase B phosphorylation were assessed in an IGF-1R-deficient mouse embryo fibroblast cell line expressing the human insulin receptor (isoform B) (61). Potency of insulin analogs was tested in male Sprague-Dawley rats rendered diabetic by streptozotocin (22). Statistical significance was assessed using a Student t test.…”

“…Fibrils were induced at 37°C by gentle agitation of a solution of insulin analog at 60 μM in PBS (pH 7.4) and monitored by thioflavin T fluorescence and transmission electron microscopy (EM) (22).…”

“…CD spectra acquisition, assays of guanidine denaturation, and thermodynamic modeling were undertaken as previously described (22).…”