The proteasome system degrades more than 80% of intracellular proteins into small peptides. Accordingly, the proteasome is involved in many essential cellular functions, such as protein quality control, transcription, immune responses, cell signaling, and apoptosis. Moreover, degradation products are loaded onto major histocompatibility class I molecules to communicate the intracellular protein composition to the immune system. The standard 20S proteasome core complex contains three distinct catalytic active sites that are exchanged upon stimulation with inflammatory cytokines to form the so-called immunoproteasome. Immunoproteasomes are constitutively expressed in immune cells and have different proteolytic activities compared with standard proteasomes. They are rapidly induced in parenchymal cells upon intracellular pathogen infection and are crucial for priming effective CD8(+) T-cell-mediated immune responses against infected cells. Beyond shaping these adaptive immune reactions, immunoproteasomes also regulate the function of immune cells by degradation of inflammatory and immune mediators. Accordingly, they emerge as novel regulators of innate immune responses. The recently unraveled impairment of immunoproteasome function by environmental challenges and by genetic variations of immunoproteasome genes might represent a currently underestimated risk factor for the development and progression of lung diseases. In particular, immunoproteasome dysfunction will dampen resolution of infections, thereby promoting exacerbations, may foster autoimmunity in chronic lung diseases, and possibly contributes to immune evasion of tumor cells. Novel pharmacological tools, such as site-specific inhibitors of the immunoproteasome, as well as activity-based probes, however, hold promises as innovative therapeutic drugs for respiratory diseases and biomarker profiling, respectively.

We here show that the activity of the immunoproteasome is impaired by cigarette smoke resulting in reduced MHC I antigen presentation. Regulation of immunoproteasome function by cigarette smoke may thus alter adaptive immune responses and add to prolonged infections and exacerbations in COPD and IPF.

The proteasome is a central regulatory hub for intracellular signaling by degrading numerous signaling mediators. Immunoproteasomes are specialized types of proteasomes involved in shaping adaptive immune responses, but their role in innate immune signaling is still elusive. Here, we analyzed immunoproteasome function for polarization of alveolar macrophages, highly specialized tissue macrophages of the alveolar lung surface. Classical activation (M1 polarization) of primary alveolar macrophages by LPS/IFNγ transcriptionally induced all three immunoproteasome subunits, low molecular mass protein 2 (LMP2), LMP7 and multicatalytic endopeptidase complex-like 1, which was accompanied by increased immunoproteasome activity in M1 cells. Deficiency of LMP7 had no effect on the LPS/IFNγ-triggered M1 profile indicating that immunoproteasome function is dispensable for classical alveolar macrophage activation. In contrast, IL-4 triggered alternative (M2) activation of primary alveolar macrophages was accompanied by a transcriptionally independent amplified expression of LMP2 and LMP7 and an increase in immunoproteasome activity. Alveolar macrophages from LMP7 knockout mice disclosed a distorted M2 profile upon IL-4 stimulation as characterized by increased M2 marker gene expression and CCL17 cytokine release. Comparative transcriptome analysis revealed enrichment of IL-4-responsive genes and of genes involved in cellular response to defense, wounding and inflammation in LMP7-deficient alveolar macrophages indicating a distinct M2 inflammation resolving phenotype. Moreover, augmented M2 polarization was accompanied by amplified AKT/STAT6 activation and increased RNA and protein expression of the M2 master transcription factor interferon regulatory factor 4 in LMP7 − / − alveolar macrophages. IL-13 stimulation of LMP7-deficient macrophages induced a similar M2-skewed profile indicative for augmented signaling via the IL-4 receptor α (IL4Rα). IL4Rα expression was generally elevated only on protein but not RNA level in LMP7 − / − alveolar macrophages. Importantly, specific catalytic inhibition with an LMP7-specific proteasome inhibitor confirmed augmented IL-4-mediated M2 polarization of alveolar macrophages. Our results thus suggest a novel role of immunoproteasome function for regulating alternative activation of macrophages by limiting IL4Rα expression and signaling. The immunoproteasome is a specialized type of proteasome and constitutively active in immune cells, whereas standard proteasomes are expressed in all non-immune cells. Similar to the standard 20S proteasome, immunoproteasomes are composed of two outer α-rings and two inner β-rings of seven subunits each 1 but contain a different set of IFNγ-inducible

Summary This protocol describes an easy and reliable in-gel proteasome assay to quantify the activity and composition of different proteasome complexes in cells and tissues. The assay works well with limited amounts of total cell protein lysates. Although this assay is optimized specifically for the proteasome chymotrypsin-like activity, it can be expanded to other proteasome activities as well. Using antibodies that detect distinct proteasome subunits or regulators, we can determine the composition and relative quantity of active proteasome complexes. For complete details on the use and execution of this protocol, please refer to Meul et al. (2020) .

The proteasome is essential for the selective degradation of most cellular proteins and is fine-tuned according to cellular needs. Proteasome activators serve as building blocks to adjust protein turnover in cell growth and differentiation. Understanding the cellular function of proteasome activation in more detail offers a new strategy for therapeutic targeting of proteasomal protein breakdown in disease. The role of the proteasome activator PA200 in cell function and its regulation in disease is unknown. In this study, we investigated the function of PA200 in myofibroblast differentiation and fibrotic tissue remodeling. PA200 was upregulated in hyperplastic basal cells and myofibroblasts of fibrotic lungs from patients with idiopathic pulmonary fibrosis. Increased expression of PA200 and enhanced formation of PA200-proteasome complexes was also evident in experimental fibrosis of the lung and kidney in vivo and in activated primary human myofibroblasts of the lung in vitro. Transient silencing and overexpression revealed that PA200 functions as a negative regulator of myofibroblast differentiation of human but not mouse cells. Our data thus suggest an unexpected and important role for PA200 in adjusting myofibroblast activation in response to pro-fibrotic stimuli, which fails in idiopathic pulmonary fibrosis.

Immune cells contain a specialised type of proteasome, i.e. the immunoproteasome, which is required for intracellular protein degradation. Immunoproteasomes are key regulators of immune cell differentiation, inflammatory activation and autoimmunity. Immunoproteasome function in peripheral immune cells might be altered by smoking and in COPD thereby affecting immune cell responses.We here analysed the expression and activity of proteasome complexes in peripheral blood mononuclear cells (PBMC) isolated from healthy male young smokers as well as from patients with severe COPD and compared them to matching controls. Proteasome expression was upregulated in COPD patients as assessed by RT-qPCR and mass spectrometry-based proteomics analysis. Proteasome activity was quantified using activity-based probes and native gel analysis. We observed distinct activation of immunoproteasomes in the peripheral blood cells of young male smokers and severely ill COPD patients. Native gel analysis and linear regression modeling confirmed robust activation and elevated assembly of 20S proteasomes, which correlated significantly with reduced lung function parameters in COPD patients. The immunoproteasome was distinctly activated in COPD patients upon inflammatory cytokine stimulation of PBMCs in vitro. Inhibition of the immunoproteasome reduced proinflammatory cytokine expression in COPD-derived blood immune cells.Given the crucial role of chronic inflammatory signalling and the emerging involvement of autoimmune responses in COPD, therapeutic targeting of the immunoproteasome might represent a novel therapeutic concept for COPD.

No abstract

Dysfunction of the immunoproteasome has been implicated in cardiovascular and pulmonary diseases. Its potential as a biomarker for predicting disease stages, however, has not been investigated so far and population-based analyses on the impact of sex and age are missing. We here analyzed the activity of all six catalytic sites of the proteasome in isolated peripheral blood mononuclear cells obtained from 873 study participants of the KORA FF4 study using activity-based probes. The activity of the immuno- and standard proteasome correlated clearly with elevated leukocyte counts of study participants. Unexpectedly, we observed a strong sex dimorphism for proteasome activity with significantly lower immunoproteasome activity in women. In aging, almost all catalytic activities of the proteasome were activated in aged women while maintained upon aging in men. We also noted distinct sex-related activation patterns of standard and immunoproteasome active sites in chronic inflammatory diseases such as diabetes, cardiovascular diseases, asthma, or chronic obstructive pulmonary disease as determined by multiple linear regression modeling. Our data thus provides a conceptual framework for future analysis of immunoproteasome function as a bio-marker for chronic inflammatory disease development and progression.