| Autor: |
Khilji MS; Department of Biomedical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark.; Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, VIC 3168, Australia.; Department of Physiology, University of Veterinary and Animal Sciences, Lahore 54000, Pakistan., Faridi P; Department of Medicine, School of Clinical Sciences, Monash Univesity, Clayton, VIC 3168, Australia., Pinheiro-Machado E; Department of Pathology and Medical Biology, University Medical Center Groningen, University of Groningen, 9713 Groningen, The Netherlands., Hoefner C; Department of Biomedical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark., Dahlby T; Laboratory of Translational Nutrition Biology, Department of Health Sciences and Technology, Institute of Food, Nutrition and Health, ETH Zürich, 8603 Zürich, Switzerland., Aranha R; Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, VIC 3168, Australia., Buus S; Department of Immunology and Microbiology, University of Copenhagen, 2200 Copenhagen, Denmark., Nielsen M; Department of Health Technology, Section for Bioinformatics, Technical University of Denmark, 2800 Lyngby, Denmark.; Instituto de Investigaciones Biotecnológicas, Universidad Nacional de San Martín, San Martín CP1650, Argentina., Klusek J; Laboratory of Medical Genetics, Department of Surgical Medicine, Collegium Medicum, Jan Kochanowski University, 25-369 Kielce, Poland., Mandrup-Poulsen T; Department of Biomedical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark., Pandey K; Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, VIC 3168, Australia., Purcell AW; Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, VIC 3168, Australia., Marzec MT; Department of Biomedical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark.; Institute of Health Sciences, Collegium Medicum, Jan Kochanowski University, 25-002 Kielce, Poland. |
| Abstrakt: |
How immune tolerance is lost to pancreatic β-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 β-cell major histocompatibility complex (MHC) class-I bound peptidome and inflammasome activation, sensitizing β-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 µM), or exposed to proinflammatory cytokines interleukin (IL)-1β or interferon gamma (IFNγ) (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 (IκBα), and (pro) IL-1β expression and secretion were investigated by Western blotting. GRP94 KO increased RT1.A expression in β-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 IκBα content while proIL-1β cellular levels declined, coupled with increased secretion of mature IL-1β. Our results suggest that limiting β-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 β-cells to immune-attack. |
