Abstract
The mutant proinsulin syndrome is a monogenic cause of diabetes mellitus due to toxic misfolding of insulin’s biosynthetic precursor. Also designated mutant INS-gene induced diabetes of the young (MIDY), this syndrome defines molecular determinants of foldability in the endoplasmic reticulum (ER) of β-cells. Here, we describe a peptide model of a key proinsulin folding intermediate and variants containing representative clinical mutations; the latter perturb invariant core sites in native proinsulin (LeuB15→Pro, LeuA16→Pro, and PheB24→Ser). The studies exploited a 49-residue single-chain synthetic precursor (designated DesDi), previously shown to optimize in vitro efficiency of disulfide pairing. Parent and variant peptides contain a single disulfide bridge (cystine B19-A20) to provide a model of proinsulin’s first oxidative folding intermediate. The peptides were characterized by circular dichroism and redox stability in relation to effects of the mutations on (a) in vitro foldability of the corresponding insulin analogs and (b) ER stress induced in cell culture on expression of the corresponding variant proinsulins. Striking correlations were observed between peptide biophysical properties, degree of ER stress and age of diabetes onset (neonatal or adolescent). Our findings suggest that age of onset reflects the extent to which nascent structure is destabilized in proinsulin’s putative folding nucleus. We envisage that such peptide models will enable high-resolution structural studies of key folding determinants and in turn permit molecular dissection of phenotype-genotype relationships in this monogenic diabetes syndrome. Our companion study (next article in this issue) employs two-dimensional heteronuclear NMR spectroscopy to define site-specific perturbations in the variant peptides.
Highlights
The mutant proinsulin syndrome (MPS) is a monogenic cause of diabetes mellitus (DM) presenting at a broad range of ages: onset can occur either in the neonatal period, childhood, adolescence or early adulthood [1–4]
Whereas the majority of mutant INS-gene induced diabetes of the young (MIDY) mutations introduce or remove a Cys residue—in either case leading to an odd number of thiol groups and risk of aberrant intermolecular disulfide pairing [19]—Permanent Neonatal Diabetes Mellitus (PNDM)- and Maturity Onset Diabetes of the Young (MODY) phenotypes are associated with non-cysteinerelated mutations [20]
The latter amino-acid substitutions generally occur at sites conserved among vertebrate insulins [and in most cases shared by vertebrate insulin-like growth factors (IGFs) [21]]
Summary
The mutant proinsulin syndrome (MPS) is a monogenic cause of diabetes mellitus (DM) presenting at a broad range of ages: onset can occur either in the neonatal period, childhood, adolescence or early adulthood [1–4]. Designated Mutant INS-gene Induced Diabetes of the Young (MIDY) [11], MPS pertains to patients traditionally classified, on the basis of age at presentation, as either Permanent Neonatal Diabetes Mellitus (PNDM) or Maturity Onset Diabetes of the Young (MODY) [12] This phenotypic spectrum may reflect polygenic differences in b-cell biology [13] or intrinsic mutation-dependent biophysical properties of the variant proinsulins [11, 14, 15]. Whereas the majority of MIDY mutations introduce or remove a Cys residue—in either case leading to an odd number of thiol groups and risk of aberrant intermolecular disulfide pairing [19]—PNDM- and MODY phenotypes are associated with non-cysteinerelated mutations [20] The latter amino-acid substitutions generally occur at sites conserved among vertebrate insulins [and in most cases shared by vertebrate insulin-like growth factors (IGFs) [21]]. First introduced in studies of bovine pancreatic trypsin inhibitor [25], peptide models of protein-folding intermediates have provided a general approach toward dissecting critical molecular interactions guiding the conformational search of a nascent polypeptide (see Box 1 and Figure 2) [31, 32]
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