Endoplasmic Reticulum Chaperone Glucose-Regulated Protein 94 Is Essential for Proinsulin Handling

Abstract

Although endoplasmic reticulum (ER) chaperone binding to mutant proinsulin has been reported, the role of protein chaperones in the handling of wild-type proinsulin is underinvestigated. Here, we have explored the importance of glucose-regulated protein 94 (GRP94), a prominent ER chaperone known to fold insulin-like growth factors, in proinsulin handling within b-cells. We found that GRP94 coimmunoprecipitated with proinsulin and that inhibition of GRP94 function and/or expression reduced glucosedependent insulin secretion, shortened proinsulin half-life, and lowered intracellular proinsulin and insulin levels. This phenotype was accompanied by postER proinsulin misprocessing and higher numbers of enlarged insulin granules that contained amorphic material with reduced immunogold staining for mature insulin. Insulin granule exocytosis was accelerated twofold, but the secreted insulin had diminished bioactivity. Moreover, GRP94 knockdown or knockout in b-cells selectively activated protein kinase R-like endoplasmic reticulum kinase (PERK), without increasing apoptosis levels. Finally, GRP94 mRNA was overexpressed in islets from patients with type 2 diabetes. We conclude that GRP94 is a chaperone crucial for proinsulin handling and insulin secretion.

A reference map of the human proinsulin biosynthetic interaction network

“…For the network design the criteria were quite stringent, requiring at least 2 fold increase in Intensity between 20G11 IP and IgG IPs, p< or = to 0.05 and MS/MS counts of at least 10 among the 6 human islet preparations as well as a minimum mRNA expression level in published mRNA seq studies from single beta cells (24). Our findings corroborate the previously identified proinsulin interacting proteins, BiP (HSPA5) and GRP94 (GSP90B1) that have both been shown to play essential roles in proinsulin folding (27) while extending those studies to provide an entire interaction network.…”

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

“…The rat insulinoma INS-1E cell line, a gift from Claes Wollheim and Pierre Maechler, University Medical Center, Geneva, Switzerland, was maintained as previously described [11]. The mouse insulinoma MIN6 cell line, was cultured in DMEM (Life Technologies, Naerum, Denmark) with 25 mM glucose, supplemented with 10% FBS, 0.1% Penicillin/Streptomycin (P/S), 50 uM β-mercaptoethanol and 2 mM L-glutamine.…”

“…immune cells [9], where they represent the dominant form. Formation of the proteolytic core of these specialized proteasomes involves substitution of the constitutively expressed catalytic β1, β2 and β5 subunits with the interferon (IFN)-γ-inducible β1i, β2i and β5i subunits (alternatively termed Psmb9/LMP2, Psmb10/MECL-1/LMP10 and Psmb8/LMP7, respectively) [6,10,11]. The immune-proteasome (i-proteasome) has an alternative 20S catalytic core where all βsubunits are replaced by IFN-γ inducible β-subunits and where the 20S-associated 19S can be replaced by the 11S (also termed PA28αβ) proteasome regulator [9,12,13].…”

The intermediate proteasome is constitutively expressed in pancreatic beta cells and upregulated by stimulatory, non-toxic concentrations of interleukin 1 β

“…The rat insulinoma INS-1E cell line, a gift from Claes Wollheim and Pierre Maechler, University Medical Center, Geneva, Switzerland, was maintained as previously described (11). The mouse insulinoma MIN6 cell line, was cultured in DMEM (Life Technologies, Nærum, Denmark) with 25 mM glucose, supplemented with 10 % FBS, 0.1 % Penicillin/Streptomycin (P/S), 50 uM β-mercaptoethanol and 2 mM L-glutamine.…”

“…immune cells (9), where they represent the dominant form. Formation of the proteolytic core of these specialized proteasomes involves substitution of the constitutively expressed catalytic β1, β2 and β5 subunits with the interferon (IFN)-γ-inducible β1i, β2i and β5i subunits (alternatively termed Psmb9/LMP2, Psmb10/MECL-1/LMP10 and Psmb8/LMP7, respectively) (6, 10, 11). The immune-proteasome (i-proteasome) has an alternative 20S catalytic core where all β-subunits are replaced by IFN-γ inducible β-subunits and where the 20S-associated 19S can be replaced by the 11S (also termed PA28αβ) proteasome regulator (9, 12, 13).…”