Synthetic studies of the mutant proinsulin syndrome demonstrate correlation between folding efficiency and age of diabetes onset.

Purpose: Heterozygous mutations in the insulin gene can give rise to a monogenic diabetes syndrome due to toxic misfolding of the variant proinsulin in the endoplasmic reticulum (ER) of pancreatic β-cells. Clinical mutations are widely distributed in the sequence (86 amino acids). Misfolding induces chronic ER stress and interferes in Methods: To enable efficient preparation of non-foldable variants, we developed a four-segment native chemical-ligation scheme that exploits two native cysteines (Cys Results: Folding of the reduced and unfolded polypeptides was investigated under three conditions: pH 10.6 (which promotes disulfide pairing as in the pharmaceutical manufacture of insulin) and pH 7.4 in the absence or presence of "foldase" protein disulfide isomerase. Whereas wild-type proinsulin efficiently folds to form a single dominant product (in accordance with classical studies), the clinical variants exhibited marked impairment, especially at neutral pH. Conclusion: Among representative clinical variants, relative folding yields correlated with both degree of ER stress in cell culture and ages of clinical diabetes onset (neonatal, adolescence or adulthood). Implications for the native mechanism of nascent protein folding are discussed.