Diabetes mellitus due to the toxic misfolding of proinsulin variants

Abstract

Dominant mutations in the human insulin gene can lead to pancreatic β-cell dysfunction and diabetes mellitus due to toxic folding of a mutant proinsulin. Analogous to a classical mouse model (the Akita mouse), this monogenic syndrome highlights the susceptibility of human β-cells to endoreticular stress due to protein misfolding and aberrant aggregation. The clinical mutations directly or indirectly perturb native disulfide pairing. Whereas the majority of mutations introduce or remove a cysteine (leading in either case to an unpaired residue), non-cysteine-related mutations identify key determinants of folding efficiency. Studies of such mutations suggest that the evolution of insulin has been constrained not only by its structure and function, but also by the susceptibility of its single-chain precursor to impaired foldability.

Figure 1. Biosynthesis of proinsulin

(A) Pathway begins with preproinsulin (top): signal peptide (gray), B-domain (blue), dibasic BC junction (green), C-domain (red), dibasic CA junction (green), and A-domain (red). Specific disulfide pairing in the ER yields native proinsulin (middle panels). BC- and CA cleavage (mediated by prohormone convertases PC1 and PC2) releases insulin and C-peptide (bottom). (B) Solution structure of proinsulin: insulin-like moiety and disordered connecting peptide (dashed line). A- and B-domains are shown in red and blue, respectively; C-domain contains a nascent α-helical turn near the CA junction [49]. Cystines are labeled in yellow boxes. The solution structure exploited an engineered monomer (DKP-proinsulin) as characterized by multi-dimensional ¹H-¹³C-¹⁵N NMR. Panel A is adapted from Ref panel B depicts a representative member of an ensemble of solution structures (Protein Databank entry 2KQP).

Figure 2. Energy landscape paradigm

(A) Successive disulfide pairing enables a sequence of folding trajectories on ever-steeper funnel-shaped free-energy landscapes. (B) Preferred pathway of disulfide pairing. Initial formation of cystine A20-B19 (left) is directed by a nascent hydrophobic core comprising the central B-domain α-helix (residues B9-B19), part of the C-terminal B-chain β-strand (B24-B26), and part of the C-terminal A-domain α-helix (A16-A20). Alternative pathways mediate successive disulfide pairing (middle panel) leading in turn to the native state (right). The mechanism of disulfide pairing is perturbed by clinical mutations associated with a monogenic syndrome of DM due to toxic misfolding of the variant proinsulin in the ER. Figure is adapted from Ref ; panel A is adapted from an image kindly provided by J. Williamson.

Figure 3. Critical sites governing the foldability of proinsulin are widely distributed in insulin

Asterisk indicates N-terminal segment of the B chain, which promotes foldability but is dispensable in the mature hormone [100]. Conserved side chains in or adjoining the C-terminal α-helix of the A chain (Leu^A16 and Tyr^A19; C_α purple spheres) and at multiple sites in the B chain (C_α red spheres) impair insulin chain combination in accord with studies of mutant proinsulins in mammalian cell lines and the distribution of clinical non-cysteine mutations in the insulin gene. Contacts between the side chains Phe^B1 and Ile^A13 (C_α blue spheres), although not well ordered in the native state, contribute to the cellular foldability of proinsulin. Residues Ile^A2, Tyr^B26, and Pro^B28 (C_α green spheres) contribute to the structure and stability of the native state but are not required for efficient disulfide pairing in chain combination. Disulfide bridges are as indicated (orange). Coordinates were obtained from Protein Databank file 4INS and correspond to molecule 1 of the classical 2-Zn insulin hexamer [123].

Figure 4. Proposed binding surfaces of insulin

(A) Front view and (B) back view. Whereas the classical receptor-binding surface of insulin engages IR Site 1 (blue; Ref ,, its Site 2-related surface includes hexamer contacts His^B10, Val^B18, Ser^A12, Leu^A13 and Glu^A17 (red). The A- and B chains are otherwise shown in light gray and dark gray, respectively. The structures shown are based on an R crystallographic protomer, which contains a receptor-active positive phi angle at Gly^B24 (in accordance with the results of Ref 76) but is otherwise unlikely to represent the receptor-bound conformation of insulin [128].