FK506-Binding Protein 2 Participates in Proinsulin Folding.

Apart from chaperoning, disulfide bond formation, and downstream processing, the molecular sequence of proinsulin folding is not completely understood. Proinsulin requires proline isomerization for correct folding. Since FK506-binding protein 2 (FKBP2) is an ER-resident proline isomerase, we hypothesized that FKBP2 contributes to proinsulin folding. We found that FKBP2 co-immunoprecipitated with proinsulin and its chaperone GRP94 and that inhibition of FKBP2 expression increased proinsulin turnover with reduced intracellular proinsulin and insulin levels. This phenotype was accompanied by an increased proinsulin secretion and the formation of proinsulin high-molecular-weight complexes, a sign of proinsulin misfolding. FKBP2 knockout in pancreatic ß-cells increased apoptosis without detectable up-regulation of ER stress response genes. Interestingly, FKBP2 mRNA was overexpressed in ß-cells from pancreatic islets of T2D patients. Based on molecular modeling and an in vitro enzymatic assay, we suggest that proline at position 28 of the proinsulin B-chain (P28) is the substrate of FKBP2's isomerization activity. We propose that this isomerization step catalyzed by FKBP2 is an essential sequence required for correct proinsulin folding.

MCPIP1 is a novel link between diabetogenic conditions and impaired insulin secretory capacity.

During diabetes development insulin production and glucose-stimulated insulin secretion (GSIS) are defective due to inflammation-related, yet not fully understood mechanisms. MCPIP1 (monocyte chemotactic protein-induced protein-1) is a strong regulator of inflammation, and acts predominantly as a specific RNase. The impact of MCPIP1 on insulin secretory capacity is unknown. We show that the expression of the ZC3H12A gene, which encodes MCPIP1, was induced by T1DM- and by T2DM-simulating conditions, with a stronger effect of cytokines. The number of MCPIP1-positive pancreatic islet-cells, including beta-cells, was significantly higher in diabetic compared to nondiabetic individuals. In the 3'UTR regions of mRNAs coding for Pdx1 (pancreatic and duodenal homeobox 1), FoxO1 (forkhead box protein O1), and of a novel regulator of insulin handling, Grp94 (glucose-regulated protein 94), MCPIP1-target structures were detected. Overexpression of the wild type MCPIP1wt, but not of the mutant MCPIP1D141N (lacking the RNase activity), decreased the expression of genes involved in insulin production and GSIS. Additionally INS1-E-MCPIP1wt cells exhibited a higher Ire1 (inositol-requiring enzyme 1) expression. MCPIP1wt overexpression blunted GSIS and glucose-mediated calcium influx with no deleterious effects on glucose uptake or glucokinase activity. We identify MCPIP1 as a new common link between diabetogenic conditions and beta-cell failure. MCPIP1 may serve as an interesting target for novel beta-cell protective approaches.

The inducible ß5i proteasome subunit contributes to proinsulin degradation in GRP94-deficient ß-cells and is overexpressed in type 2 diabetes pancreatic islets.

Proinsulin is a misfolding-prone protein, and its efficient breakdown is critical when ß-cells are confronted with high-insulin biosynthetic demands, to prevent endoplasmic reticulum stress, a key trigger of secretory dysfunction and, if uncompensated, apoptosis. Proinsulin degradation is thought to be performed by the constitutively expressed standard proteasome, while the roles of other proteasomes are unknown. We recently demonstrated that deficiency of the proinsulin chaperone glucose-regulated protein 94 (GRP94) causes impaired proinsulin handling and defective insulin secretion associated with a compensated endoplasmic reticulum stress response. Taking advantage of this model of restricted folding capacity, we investigated the role of different proteasomes in proinsulin degradation, reasoning that insulin secretory dynamics require an inducible protein degradation system. We show that the expression of only one enzymatically active proteasome subunit, namely, the inducible ß5i-subunit, was increased in GRP94 CRISPR/Cas9 knockout (KO) cells. Additionally, the level of ß5i-containing intermediate proteasomes was significantly increased in these cells, as was ß5i-related chymotrypsin-like activity. Moreover, proinsulin levels were restored in GRP94 KO upon ß5i small interfering RNA-mediated knockdown. Finally, the fraction of ß-cells expressing the ß5i-subunit is increased in human islets from type 2 diabetes patients. We conclude that ß5i is an inducible proteasome subunit dedicated to the degradation of mishandled proinsulin.

The intermediate proteasome is constitutively expressed in pancreatic beta cells and upregulated by stimulatory, low concentrations of interleukin 1 ß.

A central and still open question regarding the pathogenesis of autoimmune diseases, such as type 1 diabetes, concerns the processes that underlie the generation of MHC-presented autoantigenic epitopes that become targets of autoimmune attack. Proteasomal degradation is a key step in processing of proteins for MHC class I presentation. Different types of proteasomes can be expressed in cells dictating the repertoire of peptides presented by the MHC class I complex. Of particular interest for type 1 diabetes is the proteasomal configuration of pancreatic ß cells, as this might facilitate autoantigen presentation by ß cells and thereby their T-cell mediated destruction. Here we investigated whether so-called inducible subunits of the proteasome are constitutively expressed in ß cells, regulated by inflammatory signals and participate in the formation of active intermediate or immuno-proteasomes. We show that inducible proteasomal subunits are constitutively expressed in human and rodent islets and an insulin-secreting cell-line. Moreover, the ß5i subunit is incorporated into active intermediate proteasomes that are bound to 19S or 11S regulatory particles. Finally, inducible subunit expression along with increase in total proteasome activities are further upregulated by low concentrations of IL-1ß stimulating proinsulin biosynthesis. These findings suggest that the ß cell proteasomal repertoire is more diverse than assumed previously and may be highly responsive to a local inflammatory islet environment.