1/7/2024 0 Comments Er lumen oxidizing![]() ![]() What is known is that in vitro refolding of proteins of the insulin/IGF superfamily proceeds stepwise via one- and two-disulfide intermediates, in which a molten globule containing the B19-A20 disulfide bond is critical ( 17– 19). Nevertheless, few studies have actually examined the pathway(s) of proinsulin folding and misfolding within the β-cell ER. It is recognized that all three disulfide bonds of insulin, the two “interchain” disulfide bonds called Cys(B7)-Cys(A7) and Cys(B19)-Cys(A20) as well as the intra-A chain disulfide Cys(A6)-Cys(A11)-all of which form within proinsulin-must be intact for optimal insulin bioactivity on the insulin receptor ( 16). The process of proinsulin disulfide bond formation in vivo is complicated by the rapid time scale (<1 min) for proinsulin translation/translocation into the ER lumen ( 11), the timing of preproinsulin signal peptide cleavage ( 12), the regulation of the ER lumenal redox environment ( 13, 14), and activity of ER oxidoreductases ( 15). ![]() To the best of our knowledge, all MIDY mutations perturb the native disulfide pairing of proinsulin ( 9, 10). The clearest model of β-cell failure triggered by proinsulin misfolding currently comes from a cluster of autosomal dominant INS gene mutations that produce the syndrome we call MIDY (mutant INS gene–induced diabetes of youth) ( 8). These data suggest possible therapeutic avenues to ameliorate ER stress and diabetes. MIDY mutations inhibit Cys(B19)-Cys(A20) formation, but treatment to force oxidation of this disulfide bond improves folding and results in a small but detectable increase of proinsulin export. The three most common proinsulin disulfide mispairings in the ER appear to involve Cys(A11)-Cys(A20), Cys(A7)-Cys(A20), and Cys(B19)-Cys(A11), each disrupting the critical Cys(B19)-Cys(A20) pairing. Interestingly, formation of these two “interchain” disulfide bonds demonstrates cooperativity, and together, they are sufficient to confer intracellular transport competence to proinsulin. In the absence of preexisting proinsulin disulfide pairing, Cys(B19)-Cys(A20) (a major determinant of ER stress response activation and proinsulin stability) preferentially initiates B-A chain disulfide bond formation, whereas Cys(B7)-Cys(A7) can initiate only under oxidizing conditions beyond that existing within the ER of β-cells. Herein, we have undertaken a molecular dissection of proinsulin disulfide bond formation, using bioengineered proinsulins that can form only two (or even only one) of the native proinsulin disulfide bonds. Proinsulin folding within the endoplasmic reticulum (ER) remains incompletely understood, but it is clear that in mutant INS gene–induced diabetes of youth (MIDY), progression of the (three) native disulfide bonds of proinsulin becomes derailed, causing insulin deficiency, β-cell ER stress, and onset of diabetes. ![]()
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