Cells Deploy a Two-Pronged Quality Control Strategy to Degrade Misfolded Proinsulin Mutants

Abstract

There are over 30 missense mutations found in the human insulin gene responsible for a newly-characterized diabetic syndrome called Mutant INS-gene-induced Diabetes of Youth (MIDY). In MIDY, mutations in the insulin gene lead to toxic misfolding of mutant proinsulin in the endoplasmic reticulum (ER). Importantly, MIDY proinsulin mutants bind to and sequester wild-type (WT) proinsulin via mixed disulfide bonds in the ER, resulting in decreased insulin maturation and secretion. These mixed disulfide-bonded WT-mutant complexes oligomerize into high molecular weight (HMW) species, eventually forming toxic insoluble aggregates that lead to Beta-cell death. To alleviate the disease, we posit that the efficient degradation of the MIDY mutant proinsulins should allow WT proinsulin to properly fold in the ER and mature into insulin poised for secretion.

In this thesis, we uncover two ER-dependent protein quality control pathways that are used to eliminate the classic MIDY mutant proinsulin Akita. We first reveal that Akita is degraded via the ER-associated degradation (ERAD) pathway. In this pathway, the ER-resident chaperone Grp170 acts to prevent aggregation of the Akita HMW complex, while the PDI redox enzyme reduces the HMW species to generate ERAD-competent smaller oligomers of Akita. These smaller Akita oligomers in turn translocate across the ER membrane via the Hrd1-Sel1L membrane proteins and are extracted into the cytosol by the p97 ATPase. Upon reaching the cytosol, Akita is delivered to the proteasome for destruction. Strikingly, we found that enhancing Grp170-dependent ERAD degradation of Akita led to restoration of WT insulin secretion. In a second and distinct protein quality control pathway, we discovered that the Akita aggregates (that are formed despite the action of Grp170) are in fact removed by a RTN3-dependent ER-coupled autophagy (ER-phagy) pathway. Importantly, RTN3-dependent clearance of aggregated Akita also stimulates WT insulin secretion.

In sum, my thesis unveils a two-pronged quality control strategy – ERAD and ER-phagy – that are strategically deployed against a mutant proinsulin molecule. Our results raise the possibility that enhancing the activities of key components within the ERAD and ER-phagy pathways that stimulate degradation of mutant proinsulins and concomitantly promote WT insulin secretion may provide a rational therapeutic approach to combat MIDY.