A precondition for efficient proinsulin export from the endoplasmic reticulum (ER) is that proinsulin meets ER quality control folding requirements, including formation of the Cys(B19)–Cys(A20) “interchain” disulfide bond, facilitating formation of the Cys(B7)–Cys(A7) bridge. The third proinsulin disulfide, Cys(A6)–Cys(A11), is not required for anterograde trafficking, i.e., a “lose-A6/A11” mutant [Cys(A6), Cys(A11) both converted to Ser] is well secreted. Nevertheless, an unpaired Cys(A11) can participate in disulfide mispairings, causing ER retention of proinsulin. Among the many missense mutations causing the syndrome of Mutant INS gene-induced Diabetes of Youth (MIDY), all seem to exhibit perturbed proinsulin disulfide bond formation. Here, we have examined a series of seven MIDY mutants [including G(B8)V, Y(B26)C, L(A16)P, H(B5)D, V(B18)A, R(Cpep + 2)C, E(A4)K], six of which are essentially completely blocked in export from the ER in pancreatic β-cells. Three of these mutants, however, must disrupt the Cys(A6)–Cys(A11) pairing to expose a critical unpaired cysteine thiol perturbation of proinsulin folding and ER export, because when introduced into the proinsulin lose-A6/A11 background, these mutants exhibit native-like disulfide bonding and improved trafficking. This maneuver also ameliorates dominant-negative blockade of export of co-expressed wild-type proinsulin. A growing molecular understanding of proinsulin misfolding may permit allele-specific pharmacological targeting for some MIDY mutants.
A precondition for efficient proinsulin export from the endoplasmic reticulum (ER) is that proinsulin meets ER quality control folding requirements, including formation of the Cys(B19)-Cys(A20) interchain disulfide bond, facilitating formation of the Cys(B7)-Cys(A7) bridge. The third proinsulin disulfide, Cys(A6)-Cys(A11), is not required for anterograde trafficking, i.e., a lose-A6/A11 mutant [Cys(A6), Cys(A11) both converted to Ser] is well secreted. Nevertheless, an unpaired Cys(A11) can participate in disulfide mispairings, causing ER retention of proinsulin. Among the many missense mutations causing the syndrome of Mutant INS gene-induced Diabetes of Youth (MIDY), all seem to exhibit perturbed proinsulin disulfide bond formation. Here we have examined a series of seven MIDY mutants [including G(B8)V, Y(B26)C, L(A16)P, H(B5)D, V(B18)A, R(Cpep+2)C, E(A4)K], six of which are essentially completely blocked in export from the ER in pancreatic β-cells. Three of these mutants, however, must disrupt the Cys(A6) Cys(A11) pairing to expose a critical unpaired cysteine thiol perturbation of proinsulin folding and ER export, because when introduced into the proinsulin lose-A6/A11 background, these mutants exhibit native-like disulfide bonding and improved trafficking. This maneuver also ameliorates dominant-negative blockade of export of co-expressed wild-type proinsulin. A growing molecular understanding of proinsulin misfolding may permit allele-specific pharmacological targeting for some MIDY mutants.
A striking number of SNPs and rare mutations have been identified in PCSK1, the gene that codes for the enzyme proprotein convertase 1/3 (PC1/3) which proteolytically activates prohormones within the secretory pathway. All infants bearing two copies of catalytically inactivating mutations, including G209R, exhibit severe neonatal malabsorption requiring parenteral nutrition for months and subsequently develop additional endocrinopathies, often including diabetes and obesity. In order to create a mouse model to explore the underlying mechanism of the malabsorption phenomenon and the endocrinopathies, a G209R point mutation was introduced into exon 6 of mouse Pcsk1 using CRISPR-Cas9 genome editing. Fifty-six live pups were collected at postnatal days one or two; however, most homozygous G209R mutant pups succumbed by day 2, and surviving pups were severely dwarfed. In homozygous, but not heterozygous pups, blood glucose levels were significantly lower with elevated plasma insulin-like immunoreactivity and accumulation of unprocessed proinsulin in G209R pancreas compared to the wild type pups from the same litters. The POMC product α-MSH (produced by PC2 from PC1/3-generated ACTH) has been strongly implicated in obesity mechanisms. We found pituitary POMC processing to ACTH was also affected by the G209R mutation in combined anterior and intermediate pituitary lobes. ACTH was markedly reduced in homozygote pituitary, with significant accumulation of POMC. Using Western blotting, we observed a significant reduction in PC1/3 protein in homozygote brains, while PC2 protein levels remained unaffected. Most likely due to the continued presence of PC2, pituitary and brain levels of α-MSH were not impaired, suggesting that α-MSH itself is not involved in the phenotype. Prior studies have shown that G209R PC1/3 is not efficiently trafficked out of the ER; further studies will examine the contribution of misfolded G209R PC1/3 to possible cellular ER stress, as well as determine peptide hormone levels in brain and peripheral tissues.
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