Proinsulin Is Refractory to Protein Fibrillation

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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...

supporting

confidence: 78%

mentioning

confidence: 99%

An Achilles' Heel in an Amyloidogenic Protein and Its Repair

Yang

Petkova

Huang

et al. 2010

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“…We suggest instead that an evolutionary constraint is imposed by requirements of protein folding and trafficking in the ␤-cell. Interchain interactions by His B5 in a nascent proinsulin polypeptide may enhance its folding efficiency in the endoplasmic reticulum (as it does in chain combination in vitro), in turn protecting the ␤-cell from toxic protein misfolding (64,65). Importantly, the genetic link between misfolding of proinsulin and human ␤-cell dysfunction (described above in relation to neonatal diabetes) (61) suggests that the foldability of proinsulin has imposed a powerful constraint on the evolution of allowed insulin sequences.…”

Section: B5mentioning

confidence: 99%

The Structure of a Mutant Insulin Uncouples Receptor Binding from Protein Allostery

Wan

Huang

Whittaker

et al. 2008

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The zinc insulin hexamer undergoes allosteric reorganization among three conformational states, designated T 6 , T 3 R 3 f , and R 6 . Although the free monomer in solution (the active species) resembles the classical T-state, an R-like conformational change is proposed to occur upon receptor binding. Here, we distinguish between the conformational requirements of receptor binding and the crystallographic TR transition by design of an active variant refractory to such reorganization. Our strategy exploits the contrasting environments of His B5 in wild-type structures: on the T 6 surface but within an intersubunit crevice in R-containing hexamers. The TR transition is associated with a marked reduction in His B5 pK a , in turn predicting that a positive charge at this site would destabilize the R-specific crevice. Remarkably, substitution of His B5 (conserved among eutherian mammals) by Arg (occasionally observed among other vertebrates) blocks the TR transition, as probed in solution by optical spectroscopy. Similarly, crystallization of Arg B5 -insulin in the presence of phenol (ordinarily a potent inducer of the TR transition) yields T 6 hexamers rather than R 6 as obtained in control studies of wild-type insulin. The variant structure, determined at a resolution of 1.3 Å , closely resembles the wild-type T 6 hexamer. Whereas Arg B5 is exposed on the protein surface, its side chain participates in a solvent-stabilized network of contacts similar to those involving His B5 in wild-type T-states. The substantial receptor-binding activity of Arg B5 -insulin (40% relative to wild type) demonstrates that the function of an insulin monomer can be uncoupled from its allosteric reorganization within zinc-stabilized hexamers.Insulin is a small globular protein containing two chains, A (21 residues) and B (30 residues). In pancreatic ␤-cells, the hormone is stored as Zn 2ϩ -stabilized hexamers, which form microcrystalline arrays within specialized secretory granules (1). The hexamers dissociate upon secretion into the portal circulation, enabling the hormone to function as a zinc-free monomer. Structure-function relationships have been inferred from patterns of sequence conservation (2) and extensively probed by mutagenesis (2-10). A variety of evidence suggests that insulin undergoes a change in conformation on binding to the insulin receptor (IR) 2 (11). A model for induced fit is provided by the TR transition, a long range allosteric reorganization of zinc insulin hexamers (12). The structural basis of the TR transition has been extensively investigated by x-ray crystallography (13-15). In this paper, we investigate the relationship between such allosteric reorganization and biological activity. Experimental design exploits the contrasting structures of classical hexamers to introduce an electrostatic block to the TR transition.The TR transition encompasses three families of zinc insulin hexamers, designated T 6 , T 3 R 3 f , and R 6 (Fig. 1). Interconversion among these families is regulated by ionic strength (13, 16...

“…Such pathological processes are exemplified by the toxic misfolding of insulin and proinsulin (31). Due to the presence of the C domain, proinsulin is markedly less susceptible to fibrillation than is insulin (35). We suggest that despite its flexibility, the C-domain sequence has evolved to minimize its propensity to fold or misfold.…”

mentioning

confidence: 99%

“…We suggest that despite its flexibility, the C-domain sequence has evolved to minimize its propensity to fold or misfold. The extreme resistance of the isolated C-peptide to fibrillation (35), which is otherwise a universal property of polypeptides, highlights the special nature of such a nonfolding sequence.…”

mentioning

confidence: 99%

Solution Structure of Proinsulin

Yang

Hua

Liu

et al. 2010

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The folding of proinsulin, the single-chain precursor of insulin, ensures native disulfide pairing in pancreatic ␤-cells. Mutations that impair folding cause neonatal diabetes mellitus. Although the classical structure of insulin is well established, proinsulin is refractory to crystallization. Here, we employ heteronuclear NMR spectroscopy to characterize a monomeric analogue. Proinsulin contains a native-like insulin moiety (Aand B-domains); the tethered connecting (C) domain (as probed by { 1 H}-15 N nuclear Overhauser enhancements) is progressively less ordered. Although the BC junction is flexible, residues near the CA junction exhibit ␣-helical-like features. Relative to canonical ␣-helices, however, segmental 13 C ␣/␤ chemical shifts are attenuated, suggesting that this junction and contiguous A-chain residues are molten. We propose that flexibility at each C-domain junction facilitates prohormone processing. Studies of protease SPC3 (PC1/3) suggest that C-domain sequences contribute to cleavage site selection. The structure of proinsulin provides a foundation for studies of insulin biosynthesis and its impairment in monogenic forms of diabetes mellitus.