Mutant INS-Gene Induced Diabetes of Youth: Proinsulin Cysteine Residues Impose Dominant-Negative Inhibition on Wild-Type Proinsulin Transport

Abstract

Recently, a syndrome of Mutant I NS-gene-induced Diabetes of Youth (MIDY, derived from one of 26 distinct mutations) has been identified as a cause of insulin-deficient diabetes, resulting from expression of a misfolded mutant proinsulin protein in the endoplasmic reticulum (ER) of insulin-producing pancreatic beta cells. Genetic deletion of one, two, or even three alleles encoding insulin in mice does not necessarily lead to diabetes. Yet MIDY patients are INS-gene heterozygotes; inheritance of even one MIDY allele, causes diabetes. Although a favored explanation for the onset of diabetes is that insurmountable ER stress and ER stress response from the mutant proinsulin causes a net loss of beta cells, in this report we present three surprising and interlinked discoveries. First, in the presence of MIDY mutants, an increased fraction of wild-type proinsulin becomes recruited into nonnative disulfide-linked protein complexes. Second, regardless of whether MIDY mutations result in the loss, or creation, of an extra unpaired cysteine within proinsulin, Cys residues in the mutant protein are nevertheless essential in causing intracellular entrapment of co-expressed wild-type proinsulin, blocking insulin production. Third, while each of the MIDY mutants induces ER stress and ER stress response; ER stress and ER stress response alone appear insufficient to account for blockade of wild-type proinsulin. While there is general agreement that ultimately, as diabetes progresses, a significant loss of beta cell mass occurs, the early events described herein precede cell death and loss of beta cell mass. We conclude that the molecular pathogenesis of MIDY is initiated by perturbation of the disulfide-coupled folding pathway of wild-type proinsulin.

Autophagy in adipose tissue and the beta cell: implications for obesity and diabetes

“…This mutant proinsulin is trapped in the ER, where it generates ER stress and marked reduction of insulin secretion, resulting in diabetes [91,92]. Similar mutations were found in a rare form of human congenital diabetes, MIDY (mutant INS gene-induced diabetes of youth syndrome) [93]. Intriguingly, in Akita islets autophagy was not reduced; still, stimulation of autophagy by the mTORC1 inhibitors rapamycin or Torin1 (an mTOR kinase inhibitor) alleviated stress and prevented beta cell apoptosis, while inhibition of autophagy severely augmented cellular stress.…”

Protective hinge in insulin opens to enable its receptor engagement

“…In both the free and μIR-bound hormone, the side chain of Phe B24 abuts cystine B19-A20. Although secretion of Ser B24 -insulin was observed in a patient (46), Ser B24 was found in a β-cell line to perturb disulfide pairing (52). Ser B24 -associated DM may thus have dual origins-impaired receptor binding by the mutant insulin (46) and β-cell dysfunction resulting from chronic endoplasmic reticular (ER) stress (52).…”

“…Although secretion of Ser B24 -insulin was observed in a patient (46), Ser B24 was found in a β-cell line to perturb disulfide pairing (52). Ser B24 -associated DM may thus have dual origins-impaired receptor binding by the mutant insulin (46) and β-cell dysfunction resulting from chronic endoplasmic reticular (ER) stress (52). The extent of impaired binding (14-fold) is less marked than that of insulins Chicago or Wakayama: Phe B25 →Leu (50-fold) or Val A3 →Leu (1000-fold) (31,46).…”

“…The degu B chain contains multiple anomalous substitutions predicted to impair dimerization (Tyr B26 →Arg, Thr B27 →Pro, and Pro B28 →His) and the zinc-stabilized hexamer assembly (His B10 →Asn). The marked susceptibility of such rodents to β-cell dysfunction when fed a diet high in carbohydrates (especially free sugars, which are almost absent in its natural plant-based diet) may reflect both extracellular-and intracellular proteotoxicity, respectively, due to amyloidogenesis and impaired folding of the divergent proinsulin-the latter leading to ER stress as in human neonatal DM (52). The anomalous molecular features of degu insulin may thus honor in the breach our suggestion that the closed conformation of insulin and its stable self-assembly in secretory vesicles (2,39) evolved to protect the β cell from proteotoxicity.…”

Insulin analog with additional disulfide bond has increased stability and preserved activity

“…Introduction of a single Cys leads to reduced stability due to an increased likelihood of disulfide scrambling. 30 Also, introduction of a single Cys has been associated with mutant INS gene-induced diabetes of youth 39 underlining the negative evolutionary selection for single Cys mutations. Consequently, introduction of the two Cys would need to occur in a single evolutionary step-a very improbable event-to avoid a deleterious single Cys substitution mutation.…”