Abstract
Patients with type 1 diabetes (T1D) suffer from beta-cell destruction by CD8+ T-cells that have preproinsulin as an important target autoantigen. It is of great importance to understand the molecular mechanism underlying the processing of preproinsulin into these CD8+ T-cell epitopes. We therefore studied a pathway that may contribute to the production of these antigenic peptides: degradation of proinsulin via ER associated protein degradation (ERAD). Analysis of the MHC class I peptide ligandome confirmed the presentation of the most relevant MHC class I-restricted diabetogenic epitopes in our cells: the signal peptide-derived sequence A15-A25 and the insulin B-chain epitopes H29-A38 and H34-V42. We demonstrate that specific silencing of Derlin-2, p97 and HRD1 by shRNAs increases steady state levels of proinsulin. This indicates that these ERAD constituents are critically involved in proinsulin degradation and may therefore also play a role in subsequent antigen generation. These ERAD proteins therefore represent interesting targets for novel therapies aiming at the reduction and possibly also prevention of beta-cell directed auto-immune reactions in T1D.
Highlights
In type 1 diabetes patients (T1D), pancreatic beta cells are destroyed by autoreactive CD8+ T-cells that have preproinsulin as their most important target antigen [1]
Elution of peptides from MHC class I molecules isolated from these cells confirms the presentation of the most relevant MHC class I diabetogenic epitopes: the signal peptide-derived sequence A15-A25 and the insulin B-chain epitopes H29-A38 and H34-V42. We demonstrate in these cells that specific silencing of Derlin-2, p97 and HRD1 by shRNA increases steady state levels of proinsulin, indicating that these ER associated protein degradation (ERAD) proteins are involved in proinsulin degradation and subsequent antigen generation
While proinsulin was abundantly present in the human islet cells, only a small quantity was detectable in the K562 cells, indicating that proinsulin is expressed in the surrogate βcells, but at levels that are much lower compared to those in human islet cells
Summary
In type 1 diabetes patients (T1D), pancreatic beta cells are destroyed by autoreactive CD8+ T-cells that have preproinsulin as their most important target antigen [1]. The importance of these T-cells is emphasized by their presence in insulitic lesions and in peripheral blood of T1D patients [2, 3]. CD8+ T-cells were found to recognize several different sequences within the preproinsulin protein. Some CD8+ T-cell antigens originate from the signal sequence of preproinsulin [5], but the majority of the epitopes identified originate from the proinsulin protein itself [1]. In view of the dominant role of proinsulin as an autoantigen, it is of great importance to understand
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