Abstract
Preproinsulin (PPI) translocation across the membrane of the endoplasmic reticulum (ER) is the first and critical step of insulin biosynthesis. Inefficient PPI translocation caused by signal peptide (SP) mutations can lead to β-cell failure and diabetes. However, the effect of proinsulin domain on the efficiency of PPI translocation remains unknown. With whole exome sequencing, we identified a novel INS nonsense mutation resulting in an early termination at the 46th residue of PPI (PPI-R46X) in two unrelated patients with early-onset diabetes. We examined biological behaviors of the mutant and compared them to that of an established neonatal diabetes causing mutant PPI-C96Y. Although both mutants were retained in the cells, unlike C96Y, R46X did not induce ER stress or form abnormal disulfide-linked proinsulin complexes. More importantly, R46X did not interact with co-expressed wild-type (WT) proinsulin in the ER, and did not impair proinsulin-WT folding, trafficking, and insulin production. Metabolic labeling experiments established that, despite with an intact SP, R46X failed to be efficiently translocated into the ER, suggesting that proinsulin domain downstream of SP plays an important unrecognized role in PPI translocation across the ER membrane. The study not only expends the list of INS mutations associated with diabetes, but also provides genetic and biological evidence underlying the regulation mechanism of PPI translocation.
Highlights
Insulin is an essential hormone for maintaining glucose homeostasis of the body
Most dominant INS mutations are located in the proinsulin domain, impairing proinsulin oxidative folding and causing proinsulin misfolding in the endoplasmic reticulum (ER) [2, 16, 24, 34]
A study shows that misfolded proinsulin may impair intracellular trafficking and processing of the precursor of insulin receptor, causing an impairment of insulin signaling in b cells, which may contribute to b-cell failure caused by INS mutations [38]
Summary
Insulin is an essential hormone for maintaining glucose homeostasis of the body. In pancreatic bcells, the insulin biosynthesis starts from its precursor, preproinsulin (PPI), which is composed of the N-terminal signal peptide (SP) followed by the C-terminal proinsulin domain. Well-folded PI exits from the ER and traffics through the Golgi to the secretory granules where it is processed by prohormone convertase (PC1/3 and PC2) and carboxypeptidase E (CPE), forming mature insulin and Cpeptide [2,3,4]. It takes approximately 30–150 min to finish these intracellular processes. Recent evidence indicates that, as a small secretory protein with a suboptimal signal sequence, the fully synthesized PPI may not be efficiently recognized by SRP and cotranslationally translocated into the ER. The pathophysiological significance of inefficient PPI translocation is highlighted by recent evidence showing that deficiency of TRAPa [translocon associated protein alpha, a type 2 diabetes associated gene [8]], TRAPb, and/ or TRAPd impairs PPI translocation and insulin production [9, 10], and by the discoveries of PPI SP mutations that impair PPI translocation causing b-cell failure and diabetes in humans [2, 11,12,13,14]
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