Regulation of the proteasome: Evaluating the lung proteasome as a new therapeutic target.

Significance: Lung diseases are on the second rank worldwide with respect to morbidity and mortality. For most respiratory diseases no effective therapies exist. While the proteasome has been successfully evaluated as a novel target for therapeutic interventions in cancer, neurodegenerative, and cardiac disorders, there is a profound lack of knowledge on the regulation of proteasome activity in chronic and acute lung diseases. Recent advances have identified various means of how the amount of active proteasome complexes in the cell can be regulated such as transcriptional regulation of proteasomal subunit expression, association with different regulators, assembly and half-life of proteasomes and regulatory complexes, as well as posttranscriptional modifications. It also becomes increasingly evident that proteasome activity is fine-tuned and depends on the state of the cell. We propose here that 20S proteasomes and their regulators can be regarded as dynamic building blocks, which assemble or disassemble in response to cellular needs. The composition of proteasome complexes in a cell may vary depending on tissue, cell type and compartment, stage of development, or pathological context. Critical Issues and Future Directions: Dissecting the expression and regulation of the various catalytic forms of 20S proteasomes, such as constitutive, immuno-, and mixed proteasomes, together with their associated regulatory complexes will not only greatly enhance our understanding of proteasome function in lung pathogenesis but will also pave the way to develop new classes of drugs that inhibit or activate proteasome function in a defined setting for treatment of lung diseases

In-gel proteasome assay to determine the activity, amount, and composition of proteasome complexes from mammalian cells or tissues.

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)

Regulation of 26S proteasome activity in pulmonary fibrosis.

RATIONALE: The ubiquitin-proteasome system is critical for maintenance of protein homeostasis by degrading polyubiquitinated proteins in a spatially and timely controlled manner. Cell and protein homeostasis are altered upon pathological tissue remodelling. Dysregulation of the proteasome has been reported for several chronic diseases of the heart, brain, and lung. We hypothesized that proteasome function is altered upon fibrotic lung remodelling, thereby contributing to the pathogenesis of idiopathic pulmonary fibrosis (IPF). METHODS: To investigate proteasome function during myofibroblast differentiation, we treated lung fibroblasts with TGF-β and examined proteasome composition and activity. For in vivo analysis, we used mouse models of lung fibrosis and fibrotic human lung tissue. MEASUREMENTS AND MAIN RESULTS: We demonstrate that induction of myofibroblast differentiation by TGF-β involves activation of the 26S proteasome, which is critically dependent on the regulatory subunit Rpn6. Silencing of Rpn6 in primary human lung fibroblasts counteracted TGF-β-induced myofibroblast differentiation. Activation of the 26S proteasome and increased expression of Rpn6 was detected during bleomycin-induced lung remodelling and fibrosis. Importantly, Rpn6 is overexpressed in myofibroblasts and basal cells of the brochiolar epithelium in lungs of patients with IPF, which is accompanied by enhanced protein polyubiquitination. CONCLUSION: Our study identifies Rpn6-dependent 26S proteasome activation as an essential feature of myofibroblast differentiation in vitro and in vivo and suggests an important role in IPF pathogenesis

Proteasome activator PA200 regulates myofibroblast differentiation.

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

Immunoproteasome dysfunction augments alternative polarization of alveolar macrophages.

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