Abstract
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.
Highlights
Patients bearing autosomal dominant diabetogenic mutations in the INS gene [1, 2] develop a syndrome referred to as Mutant INS gene-induced Diabetes of Youth {MIDY [3,4,5]; Pancreatic β-cells can synthesize > 6000 new proinsulin molecules/second [16]
We recognize that MIDY mutant mice bearing the heterozygous Ins2-Munich allele—encoding proinsulin–C(A6)S—develop severe insulin-deficient diabetes [20], indicating that an unpaired proinsulin–Cys(A11) creates an impediment to proinsulin trafficking through disulfide mispairings [21] that can result in catastrophic proinsulin misfolding [19]
Each MIDY mutant was engineered into the hProCpepMyc cDNA encoding human proinsulin bearing myctagged C-peptide [12]
Summary
Patients bearing autosomal dominant diabetogenic mutations in the INS gene [1, 2] develop a syndrome referred to as Mutant INS gene-induced Diabetes of Youth {MIDY [3,4,5]; Pancreatic β-cells can synthesize > 6000 new proinsulin molecules/second [16]. P are each shown in red font within white circles, signifying resistance to secretion rescue in the lose-A6/A11 background. The remaining studied mutations are shown as yellow font in black circles, signify MIDY mutants that exhibit significant secretion rescue within the lose-A6/A11 background. C Crystallographic T-state protomer (Protein Databank entry 4INS, 2-Zn molecule-1) with B-chain in blue, A-chain in red, sulfur atoms engaged in three cystines (B7-A7, B19-A20, and A6-A11) shown in gold, and sites of mutation within the insulin moiety of proinsulin considered in this study denoted (the C carbon of GlyB8 is shown as a red ball) comparable to that of WT proinsulin [19]. We recognize that MIDY mutant mice bearing the heterozygous Ins2-Munich allele—encoding proinsulin–C(A6)S—develop severe insulin-deficient diabetes [20], indicating that an unpaired proinsulin–Cys(A11) creates an impediment to proinsulin trafficking through disulfide mispairings [21] that can result in catastrophic proinsulin misfolding [19]
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