4. Discussion
4.3 Immunoproteasome deficiency has no influence on NF‐kB activation
Two decades of immunoproteasome research solidified the hypothesis of immunoproteasome particles shaping the immunopeptideome presented on MHC class I molecules (Basler et al., 2011; Basler et al., 2006; Basler et al., 2004; Kincaid et al., 2012; Osterloh et al., 2006).
Moreover, it became evident that the immunoproteasome has, apart from antigen processing, additional immunological functions. Immunoproteasome deficiency or inhibition affects T cells survival, expansion, and differentiation (Basler et al., 2004; Chen et al., 2001; Kalim et al., 2012;
Moebius et al., 2010; Muchamuel et al., 2009; Zaiss et al., 2008), cytokine production (Basler et al., 2011; Basler et al., 2010; Basler et al., 2014; Muchamuel et al., 2009), and progression of autoimmune conditions (Basler et al., 2015). The impact of the immunoproteasome on various immunological aspects made this complex to an emerging pharmacolocigal target for various diseases and raised an even higher interest in understanding its exact cellular role.
The inherent difficulty of studying the function of immunoproteasome subunits is the fact that deficiency in one or several subunits can affect assembly of core particles (Bai et al., 2014; De et al., 2003; Joeris et al., 2012). In order to effectively mature into precursor particles, the chaperone‐like function of the propeptides of β‐subunits is required (Ramos et al., 1998;
Schmidt et al., 1999). During full maturation of particles the propeptides are then autocatalytically cleaved, which finally activates the catalytic sites of these subunits (Groettrup et al., 2010). Griffin et al. could show that preparticles containing MECL‐1 and LMP2 need LMP7 for efficient maturation (Griffin et al., 1998). On the other hand, MECL‐1 depends on LMP2 for incorporation (De et al., 2003). Moreover, LMP7 exerts higher chaperone activity compared to β5 and hence, has a higher capacity to promote particle maturation (Heink et al., 2005; Joeris et al., 2012). The combination of subunits present in the particles of knockout cells could affect various aspects of proteasomal activity. Discrimination between artifacts potentially caused by
“mixed” proteasomes and effects of immunoproteasome subunit deficiency is crucial yet difficult to put into practice. Under inflammatory conditions, cells devoid of LMP7 could furthermore have an overall decreased proteasomal capacity due to less efficient particle maturation. Decreased capacity and/or compensatory mechanisms to balance decreased proteasomal capacity could both influence the results obtained with knockout cells.
The last part of this study was focused on the activation of the transcription factor NF‐κB in MEFs and pMΦs derived from L7M‐/‐ and LMP2‐/‐. In order to define the proteasomal composition of these cells, a combined approach of proteasome immunoprecipitation and 2D‐
gel electrophoresis was tested for its applicability. The possibility to better assess the proportion of immunoproteasome expression relative to total proteasome content constitutes the clear advantage of this technique compared to western blot analysis. Immunorecipitation of proteasomes proved to be an easy way to rapidly enrich particles, especially if cells to be analyzed are limited in number. However, the visualization of proteins on 2D‐gels still has to
be optimized in order to detect all relevant subunits. A fluorescent dye applied to the enriched proteasomes before the electrophoretic separation could be an approach to be tested in the future. It would then be interesting to apply this technique for the analysis of proteasome composition of different mouse and human tissues.
Expression of different proteasome subunits was also determined by SDS‐PAGE and western blot. After stimulation with IFN‐γ, both pMΦs and MEFs expressed immunoproteasomes. Still, expression of constitutive subunits was not completely downregulated indicating a mix of constitutive and immunoproteasomes present in the cells. Due to the assembly defect in L7M‐
/‐ cells the particles present are almost exclusively composed of constitutive subunits. In contrast, LMP2‐/‐ cells seem to express LMP7 but due to the defect in MECL‐1 incorporation most likely contain constitutive proteasomes as well as mixed particles containing β1, β2, and LMP7. It would be interesting to further determine the proteasomal activity of the cells to potentially rule out differences in the overall degradative capacity between the knockout cells.
The literature about the role of the immunoproteasome in NF‐κB activation is controversial. An early report by Hayashi and Faustman suggested that LMP2 is required for the generation of p50 from the p105 precursor (Hayashi and Faustman, 1999). This finding could, however, not be reproduced by two other groups (Kessler et al., 2000; Runnels et al., 2000). Along the same line, Visekruna et al. proposed enhanced processing of p105 and degradation of IkBα in cytosolic extracts or purified proteasomes derived from mucosa of patients with Crohn’s disease or ulcerative colitis, which both differ in the proportion of immunoproteasome expressed (Visekruna et al., 2006). Unfortunatley, poor experimental setup and missing controls leave these data impossible to interpret. A follow‐up study by Hayashi and Faustman found reduced IkBα degradation in LMP2‐/‐ lymphocytes upon stimulation with TNF‐α (Hayashi and Faustman, 2000). This finding could be confirmed with LPS‐stimulated B cells derived from
LMP2‐/‐ mice, although the effect observed was rather minor (Hensley et al., 2010). In contrast,
a recent report by Maldonado and colleagues claimed that activation of the canonical pathway was not affected in LMP2‐/‐ and L7M‐/‐ cells while the alternative pathway seemed to be
“aberrant” in LMP2‐/‐ cells (Maldonado et al., 2013). However, data presented in this study is inconsistent and rather an overinterpretation of minor effects.
The results presented here clearly argue against an influence of immunoproteasomes on the canonical pathway of NF‐κB activation. Two different types of primary cells prepared from LMP2‐/‐, L7M‐/‐, and wild type mice displayed no differences in the extent and kinetic of IκBα degradation when stimulated with LPS or TNF‐α. Consistent with this finding, the amount of
active NF‐κB dimers in the nucleus of knockout cells as well as the transactivation activity was normal. Although generation of p50 from the p105 precursor was not analyzed here, these results do clearly not indicate a deficit in NF‐κB subunits in the knockout cells. A study conducted with small molecule inhibitors targeting LMP2 or LMP7 further supports the model of NF‐κB activation being independent of proteasome composition. Inhibition of LMP2, LMP7, or even both had no influence on IκBα degradation in cells stimulated with TNF‐α (Jang et al., 2012).
Until now, the immunological impact of immunoproteasomes is still not fully understood. It has been suggested that the immunoproteasome has a higher capacity to clear ubiquitylated proteins accumulating after stimulation with IFN‐γ (Seifert et al., 2010). However, this finding could not be reproduced by others (Nathan et al., 2013). Taken together, more research is needed to improve the mechanistic understanding of immunoproteasomes in immune regulation.
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