MilliporeSigma
  • Reduction in ATP levels triggers immunoproteasome activation by the 11S (PA28) regulator during early antiviral response mediated by IFNβ in mouse pancreatic β-cells.

Reduction in ATP levels triggers immunoproteasome activation by the 11S (PA28) regulator during early antiviral response mediated by IFNβ in mouse pancreatic β-cells.

PloS one (2013-02-06)
Wieke Freudenburg, Madhav Gautam, Pradipta Chakraborty, Jared James, Jennifer Richards, Alison S Salvatori, Aaron Baldwin, Jill Schriewer, R Mark L Buller, John A Corbett, Dorota Skowyra
ABSTRACT

Autoimmune destruction of insulin producing pancreatic β-cells is the hallmark of type I diabetes. One of the key molecules implicated in the disease onset is the immunoproteasome, a protease with multiple proteolytic sites that collaborates with the constitutive 19S and the inducible 11S (PA28) activators to produce immunogenic peptides for presentation by MHC class I molecules. Despite its importance, little is known about the function and regulation of the immunoproteasome in pancreatic β-cells. Of special interest to immunoproteasome activation in β-cells are the effects of IFNβ, a type I IFN secreted by virus-infected cells and implicated in type I diabetes onset, compared to IFNγ, the classic immunoproteasome inducer secreted by cells of the immune system. By qPCR analysis, we show that mouse insulinoma MIN6 cells and mouse islets accumulate the immune proteolytic β1(i), β2(i) and β5(i), and 11S mRNAs upon exposure to IFNβ or IFNγ. Higher concentrations of IFNβ than IFNγ are needed for similar expression, but in each case the expression is transient, with maximal mRNA accumulation in 12 hours, and depends primarily on Interferon Regulatory Factor 1. IFNs do not alter expression of regular proteasome genes, and in the time frame of IFNβ-mediated response, the immune and regular proteolytic subunits co-exist in the 20S particles. In cell extracts with ATP, these particles have normal peptidase activities and degrade polyubiquitinated proteins with rates typical of the regular proteasome, implicating normal regulation by the 19S activator. However, ATP depletion rapidly stimulates the catalytic rates in a manner consistent with levels of the 11S activator. These findings suggest that stochastic combination of regular and immune proteolytic subunits may increase the probability with which unique immunogenic peptides are produced in pancreatic β-cells exposed to IFNβ, but primarily in cells with reduced ATP levels that stimulate the 11S participation in immunoproteasome function.

MATERIALS
Product Number
Brand
Product Description

Octoxinol 10, European Pharmacopoeia (EP) Reference Standard
Sigma-Aldrich
Triton X-100 solution, BioUltra, for molecular biology, ~10% in H2O
Sigma-Aldrich
Triton X-102
Sigma-Aldrich
Triton X-305 solution, 70% in H2O
Sigma-Aldrich
Anti-Ubiquitin antibody produced in rabbit, whole antiserum, lyophilized powder
Sigma-Aldrich
Monoclonal Anti-polyHistidine antibody produced in mouse, clone HIS-1, ascites fluid
Sigma-Aldrich
Triton X-45
Sigma-Aldrich
Triton X-100, for molecular biology
Sigma-Aldrich
Triton X-100, peroxide- and carbonyl-free
Sigma-Aldrich
Triton X-100, laboratory grade
Sigma-Aldrich
Triton X-100, BioXtra