Proteasomal Degradation of Proinsulin Requires Derlin-2, HRD1 and p97

<div><p>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.</p></div

Generation of preproinsulin expressing K562 cells.

<p>(A) K562 cells were retrovirally transduced to express preproinsulin-IRES-GFP and sorted for the GFP positive population. Flow cytometry analysis of wild-type preproinsulin-expressing cells before transduction (left panel), after retroviral transduction but before sorting (middle panel) and after sorting (right panel). Sorting yielded a cell population that was approximately 95% GFP positive. (B) Human pancreatic islets cells and preproinsulin-expressing K562 cells were lysed and proteins were separated on 12% Nu-PAGE. Proinsulin levels were analyzed by Western blot. The number of cells used to prepare the lysates is indicated. (C) Schematic representation of the preproinsulin molecule including the three disulfide bonds. The epitopes eluted from MHC class I molecules are depicted in red.</p

Derlin2, p97 and HRD1 knockdown increases proinsulin steady state levels.

<p>(A) Schematic representation of the experimental setup for the knockdown of ERAD proteins with shRNAs. Preproinsulin-expressing K562 cells were transduced to express the respective shRNAs together with mOrange from a bicistronic lentiviral expression vector. mOrange expression levels were analyzed by flow cytometry either before transduction or on day 3 and day 7 after transduction; the latter includes 4 days of selection with puromycin. Flow cytrometry analysis of a representative transduction of K562 cells is shown. (B) Proinsulin-expressing K562 cells were transduced with the indicated shRNAs. Seven days after transduction, cell lysates were prepared and loaded onto 12% Nu-PAGE; Derlin-1, Derlin-2 and p97 protein levels were analyzed by Western blot. Actin was included as a loading control. Gels are representative for three different experiments. (C) K562 cells were transduced as described for B and proinsulin levels were analyzed by Western blot. Actin was included as a loading control. Gels are representative for three different experiments.</p

Epitopes eluted from MHC class I.

<p>Epitopes eluted from MHC class I.</p

Derlin-2 depletion delays insulin degradation.

<p>(A) K562 cells stably expressing preproinsulin were transduced to express either nonsense shRNA (left) or an shRNA targeting Derlin-1 (right). After pulse-labeling for 15 minutes with ³⁵S-methionine and cysteine, cells were chased for the indicated times. Proinsulin was immunoprecipitated from the lysates and analyzed using 15% SDS-PAGE. Quantification of the pulse chase experiment is shown on the right. Gels are representative for two different experiments. (B) Similar as described for A but with two shRNAs targeting Derlin-2. Quantification of the pulse chase experiment is shown on the right.</p

Derlin-2 overexpression decreases proinsulin steady state levels.

<p>K562 cells stably expressing preproinsulin were transduced with cDNA to overexpress Derlin-1 or Derlin-2 from a lentiviral expression vector. Seven days post transduction and selection, cell lysates were prepared and separated on 12% Nu-PAGE. Protein levels were analyzed by Western blot using antibodies against the indicated proteins and quantification of PI levels is shown. Gels are representative for three different experiments.</p