期刊
BIOMEDICINES
卷 10, 期 4, 页码 -出版社
MDPI
DOI: 10.3390/biomedicines10040814
关键词
ER stress; GRP94; proteasome; insulin; inflammation; MHC class-I
资金
- Punjab Educational Endowment Fund
- Department of Biomedical Sciences at the University of Copenhagen
- European Foundation for the Study of Diabetes/Lilly European Diabetes Research Programme
- National Health and Medical Research Council of Australia (NHMRC) Principal Research Fellowship [1137739]
- Vissing Fonden
The loss of GRP94 from the endoplasmic reticulum results in mishandling of proinsulin, ER stress, and activation of the immunoproteasome, leading to the sensitization of beta-cells to immune attack.
How immune tolerance is lost to pancreatic beta-cell peptides triggering autoimmune type 1 diabetes is enigmatic. We have shown that loss of the proinsulin chaperone glucose-regulated protein (GRP) 94 from the endoplasmic reticulum (ER) leads to mishandling of proinsulin, ER stress, and activation of the immunoproteasome. We hypothesize that inadequate ER proinsulin folding capacity relative to biosynthetic need may lead to an altered beta-cell major histocompatibility complex (MHC) class-I bound peptidome and inflammasome activation, sensitizing beta-cells to immune attack. We used INS-1E cells with or without GRP94 knockout (KO), or in the presence or absence of GRP94 inhibitor PU-WS13 (GRP94i, 20 mu M), or exposed to proinflammatory cytokines interleukin (IL)-1 beta or interferon gamma (IFN gamma) (15 pg/mL and 10 ng/mL, respectively) for 24 h. RT1.A (rat MHC I) expression was evaluated using flow cytometry. The total RT1.A-bound peptidome analysis was performed on cell lysates fractionated by reverse-phase high-performance liquid chromatography (RP-HPLC), followed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing protein (NLRP1), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (I kappa B alpha), and (pro) IL-1 beta expression and secretion were investigated by Western blotting. GRP94 KO increased RT1.A expression in beta-cells, as did cytokine exposure compared to relevant controls. Immunopeptidome analysis showed increased RT1.A-bound peptide repertoire in GRP94 KO/i cells as well as in the cells exposed to cytokines. The GRP94 KO/cytokine exposure groups showed partial overlap in their peptide repertoire. Notably, proinsulin-derived peptide diversity increased among the total RT1.A peptidome in GRP94 KO/i along with cytokines exposure. NLRP1 expression was upregulated in GRP94 deficient cells along with decreased I kappa B alpha content while proIL-1 beta cellular levels declined, coupled with increased secretion of mature IL-1 beta. Our results suggest that limiting beta-cell proinsulin chaperoning enhances RT1.A expression alters the MHC-I peptidome including proinsulin peptides and activates inflammatory pathways, suggesting that stress associated with impeding proinsulin handling may sensitize beta-cells to immune-attack.
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