Immunoproteasome Function in Lymphocyte Activation

The immunoproteasome constitutes a specialized form of the proteasome, a multimeric protein complex with important functions in eukaryotic cells. Within a 20S core particle three different protease types cleave the polypeptide chains destined for degradation. Each of the three proteases constitutes one position per half-core particle. The standard proteasome active subunits β1c, β2c and β5c are replaced by alternative subunits in immunoproteasomes: LMP2 is incorporated at the position of β1c, MECL-1 substitutes for β2c and LMP7 substitutes for β5c. The immunoproteasome is expressed in cells of hematopoietic origin as well as in peripheral tissues under the influence of pro-inflammatory cytokines, mainly interferon-γ. Besides a well characterized role in MHC-I antigen processing, a potential role of the immunoproteasome as a drug target for treatment of autoimmune diseases was shown, as immunoproteasome inhibition proved to have disease ameliorating effects in several pre-clinical models for autoimmunity. Since the description of the immunoproteasome-selective inhibitor ONX 0914 in 2009, the underlying mechanism in the course of these autoimmune-diseases has remained largely elusive. Previous studies have shown that intrinsic effects in activated T cells, which are involved in many autoimmune disaeses, are likely to be important for the underlying mechanism. Based on previous work, which was performed during the course of my master’s thesis 2013, the aim of this study was to characterize the effects of ONX 0914 treatment at the molecular level in more detail in order to improve our understanding of the underlying mechanism of immunoproteasome inhibition. In this work, primary T and B cells from mice and humans were functionally investigated. Previous results from my master’s thesis like ameliorated CD69 up-regulation upon activation after ONX 0914 treatment were corroborated and additionally the effect was shown in B cells, in human cells and in antigenspecifically activated T cells in vivo. Furthermore, the effects were not only phenotypically characterized but substantiated by analysis at the mRNA level. An extended investigation into canonical signaling pathways of T cell activation indicated a reduction in phosphorylation of the kinase ERK after treatment with ONX 0914. This reduction was corroborated using quantitative near-infrared-dye based immunoblotting, flow cytometry and finally by confocal microscopy. However, the direct up-stream kinase was not found to be affected. Therefore, dual specificity phosphatases were identified as potential candidates for a functional involvement and several dual specificity phosphatases were analyzed at the protein level. Two of the analyzed phosphatases were affected by ONX 0914 treatment. The dual specificity phosphatase DUSP5 was less expressed, while DUSP6 accumulated at protein level. In combined cycloheximid and radioactive labelling approaches it was shown that DUSP6 degradation was impaired, but not fully blocked by ONX 0914 in T cell activation. Therfore, the possible involvement of DUSP6 for impaired T cell activation was investigated using DUSP6-deficient mice. However, it was found that DUSP6 was not responsible for the observed effects in a non-redundant manner and 11 compensation by other phosphatases cannot be ruled out. Therefore, the mechanism leading to reduced ERK-phosphorylation could not be fully unraveled so far. Apart from altered T cell signaling, the second focus of this work was set on proteostasis regulation during T cell activation after ONX 0914 treatment. Unlike previously investigated T cell lines, primary T cells and B cells showed ubiquitin-conjugate accumulation after activation when cells had been pre-treated with ONX 0914. Two factors were identified likely underlying this effect: First, it was found that almost all proteasomes in T cells and B cells constituted of LMP7-containing immunoproteasomes or mixed proteasomes. Second, it was found that the reportedly LMP7-selective inhibitor ONX 0914 co-inhibited LMP2 as well. As activated lymphocytes show marked metabolic and proteomic re-organization, these features render the cells susceptible to proteostasis stress after immunoproteasome inhibition. Consequently, the effect of enhanced proteostasis stress on activation and cell viability was characterized. It was found that T cells could alleviate the enhanced ubiquitin-conjugates within 20 hours of activation without significant induction of the integrated stress response or apoptosis. Accompanying, an enhanced neosynthesis of the standard proteasome subunit β5c and accumulation of soluble Nrf1 were detected in cell lysates. These results indicate that T cells survive mild proteostasis stress after ONX 0914 treatment likely via Nrf1-mediated proteasome up-regulation, while functional T cell activation is impaired at the same time. Similar effects were found in B cells, but induction of apoptosis-markers after ONX 0914 treatment was detected in B cells. Taken together, this work provides evidence that ONX 0914 treatment, but not immunoproteasomedeficiency, results in mild proteostasis stress in activated lymphocytes, impeding their functional capacity. This effect is likely attributed to the particularly high dependency of primary lymphocytes on LMP7- containing proteasomes and co-inhibition of LMP2 by ONX 0914. The provided mechanism hence likely explains at least parts of the anti-inflammatory effects of immunoproteasome inhibitors and why immunoproteasome inhibition shows high clinical potential with less overall toxicity as compared to broad-spectrum proteasome inhibitors.publishe