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

Hanneke Hoelen; Arnaud Zaldumbide; Wouter F van Leeuwen; Ellen C W Torfs; Marten A Engelse; Chopie Hassan; Robert Jan Lebbink; Eelco J de Koning; Maaike E Resssing; Arnoud H de Ru; Peter A van Veelen; Rob C Hoeben; Bart O Roep; Emmanuel J H J Wiertz

doi:10.1371/journal.pone.0128206

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

PLoS One. 2015 Jun 24;10(6):e0128206. doi: 10.1371/journal.pone.0128206. eCollection 2015.

Affiliations

¹ Department of Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands.
² Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands.
³ Department of Nephrology, Leiden University Medical Center, Leiden, The Netherlands.
⁴ Department of Immunohematology & Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands.
⁵ Department of Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands; Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands.

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Adenosine Triphosphatases / genetics*
Adenosine Triphosphatases / immunology
Amino Acid Sequence
Autoantigens / immunology
CD8-Positive T-Lymphocytes / immunology
CD8-Positive T-Lymphocytes / pathology
Diabetes Mellitus, Type 1 / immunology
Diabetes Mellitus, Type 1 / metabolism*
Diabetes Mellitus, Type 1 / pathology
Endoplasmic Reticulum-Associated Degradation / genetics
Endoplasmic Reticulum-Associated Degradation / immunology
Epitopes / genetics
Epitopes / immunology
Genes, MHC Class I / immunology
Humans
Insulin-Secreting Cells / immunology
Insulin-Secreting Cells / metabolism
Membrane Proteins / genetics*
Membrane Proteins / immunology
Nuclear Proteins / genetics*
Nuclear Proteins / immunology
Proinsulin / biosynthesis*
Proinsulin / genetics
Proinsulin / immunology
Proteasome Endopeptidase Complex / genetics
Proteasome Endopeptidase Complex / metabolism
Proteolysis
Ubiquitin-Protein Ligases / genetics*
Ubiquitin-Protein Ligases / immunology

Substances

Autoantigens
DERL2 protein, human
Epitopes
Membrane Proteins
Nuclear Proteins
Proinsulin
SYVN1 protein, human
Ubiquitin-Protein Ligases
Proteasome Endopeptidase Complex
Adenosine Triphosphatases
p97 ATPase

Grants and funding

This work was part of the "Diabetes Foundation Expert Center Beta Cell Protection" funded by the Dutch Diabetes Foundation (DFN 2008.04.001), to HH and EW (http://www.diabetesfonds.nl/). This work was also financially supported by the Dr. Valliant Foundation, to HH and EW (http://www.lvc-online.nl/dr-c-j-vaillantfonds). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.