Peptide Model of the Mutant Proinsulin Syndrome. I. Design and Clinical Correlation

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.

Peptide Model of the Mutant Proinsulin Syndrome. II. Nascent Structure and Biological Implications

“…The biological importance of these CD-and NMR-derived biophysical parameters is demonstrated by their further correlation with levels of ER stress induced by expression of the corresponding mutant proinsulin (Figures 10C, D) (18). Although each MIDY mutation alters an invariant framework residue-conserved among both vertebrate insulins and vertebrate insulin-like growth factors (23,61,71) -the less severe biophysical consequences of Phe B24 !Ser in consistent with the delayed onset of DM in patients with this mutation (4,33).…”

“…Increasing the net negative charge through these acidic substitutions would also be expected to enhance solubility and retard competing formation of amyloid (69). A one-disulfide model of an initial proinsulin folding intermediate was thus obtained by pairwise substitution of exposed cystine B7-A7 by Ser and internal cystine A6-A11 by Ala (18). The present study builds on our foundational characterization of the 1SS peptide model to interrogate nascent structure by twodimensional 1 H and 1 H-13 C NMR spectroscopy.…”

“…GPRs in MPS may be more straightforward: the extent of β-cell dysfunction and velocity of β-cell loss (together determining age of diabetes onset in a particular patient) may reflect mutation-specific molecular properties, i.e., whether a given amino-acid substitution is associated with a severe (PNDM) or mild (MODY) perturbation to foldability. In our initial study [preceding article in this issue ( 18 )] we designed a 49-residue peptide model of an early on-pathway proinsulin folding intermediate and its application to three representative MIDY mutations. This single-chain model contains only one disulfide bridge and is thus designated 1SS .…”

“…The 49-residue synthetic precursor (designated “DesDi”) exhibits remarkable folding efficiency, enabling preparation of certain insulin analogs refractory to classical chain combination ( 27 ). In the 1SS model cystine B7-A7 (solvent exposed in native insulin) is pairwise substituted by Ser whereas cystine A6-A11 (buried in the core of native insulin) is pairwise substituted by Ala ( 18 ). Segmental α-helical propensity and solubility were augmented by acidic surface substitutions His B10 →Asp ( 28 , 29 ) and Thr A8 →Glu ( 30 ).…”

“…Whereas the side chains of Leu B15 and Leu A16 are buried in the hydrophobic core ( Figures 2B, C, E, F ), the aromatic side chain of Phe B24 packs within a crevice overlying internal cystine B19-A20 such that one side of the aromatic ring is exposed to solvent ( Figures 2B–D ). Initial characterization of these peptides was described in our companion article ( 18 ). Whereas the Pro substitutions introduced marked perturbations in folding efficiency in a mini-proinsulin [“DesDi”, ( 27 )], Ser B24 was well tolerated.…”