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Proinsulin folding and trafficking defects trigger a common pathological disturbance of endoplasmic reticulum homeostasis

par Arunagiri, Anoop;Alam, Maroof;Haataja, Leena;Draz, Hassan;Alasad, Bashiyer;Samy, Praveen;Sadique, Nadeed;Tong, Yue ;Cai, Ying ;Shakeri, Hadis ;Fantuzzi, Federica ;Ibrahim, Hazem;Jang, Insook;Sidarala, Vaibhav;Soleimanpour, Scott S.A.;Satin, Leslie LS;Otonkoski, Timo;Cnop, Miriam ;Itkin-Ansari, Pamela;Kaufman, Randal J;Liu, Ming;Arvan, Peter
Référence Protein science, 33, 4, e4949
Publication Publié, 2024-04-01

Article révisé par les pairs

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Résumé :

Primary defects in folding of mutant proinsulin can cause dominant-negative proinsulin accumulation in the endoplasmic reticulum (ER), impaired anterograde proinsulin trafficking, perturbed ER homeostasis, diminished insulin production, and β-cell dysfunction. Conversely, if primary impairment of ER-to-Golgi trafficking (which also perturbs ER homeostasis) drives misfolding of nonmutant proinsulin—this might suggest bi-directional entry into a common pathological phenotype (proinsulin misfolding, perturbed ER homeostasis, and deficient ER export of proinsulin) that can culminate in diminished insulin storage and diabetes. Here, we've challenged β-cells with conditions that impair ER-to-Golgi trafficking, and devised an accurate means to assess the relative abundance of distinct folded/misfolded forms of proinsulin using a novel nonreducing SDS-PAGE/immunoblotting protocol. We confirm abundant proinsulin misfolding upon introduction of a diabetogenic INS mutation, or in the islets of db/db mice. Whereas blockade of proinsulin trafficking in Golgi/post-Golgi compartments results in intracellular accumulation of properly-folded proinsulin (bearing native disulfide bonds), impairment of ER-to-Golgi trafficking (regardless whether such impairment is achieved by genetic or pharmacologic means) results in decreased native proinsulin with more misfolded proinsulin. Remarkably, reversible ER-to-Golgi transport defects (such as treatment with brefeldin A or cellular energy depletion) upon reversal quickly restore the ER folding environment, resulting in the disappearance of pre-existing misfolded proinsulin while preserving proinsulin bearing native disulfide bonds. Thus, proper homeostatic balance of ER-to-Golgi trafficking is linked to a more favorable proinsulin folding (as well as trafficking) outcome.