Inhibition of Proteasome Activity Induces Formation of Alternative Proteasome Complexes.

The proteasome is an intracellular protease complex consisting of the 20S catalytic core and its associated regulators, including the 19S complex, PA28αß, PA28γ, PA200, and PI31. Inhibition of the proteasome induces autoregulatory de novo formation of 20S and 26S proteasome complexes. Formation of alternative proteasome complexes, however, has not been investigated so far. We here show that catalytic proteasome inhibition results in fast recruitment of PA28γ and PA200 to 20S and 26S proteasomes within 2-6 h. Rapid formation of alternative proteasome complexes did not involve transcriptional activation of PA28γ and PA200 but rather recruitment of preexisting activators to 20S and 26S proteasome complexes. Recruitment of proteasomal activators depended on the extent of active site inhibition of the proteasome with inhibition of ß5 active sites being sufficient for inducing recruitment. Moreover, specific inhibition of 26S proteasome activity via siRNA-mediated knockdown of the 19S subunit RPN6 induced recruitment of only PA200 to 20S proteasomes, whereas PA28γ was not mobilized. Here, formation of alternative PA200 complexes involved transcriptional activation of the activator. Alternative proteasome complexes persisted when cells had regained proteasome activity after pulse exposure to proteasome inhibitors. Knockdown of PA28γ sensitized cells to proteasome inhibitor-mediated growth arrest. Thus, formation of alternative proteasome complexes appears to be a formerly unrecognized but integral part of the cellular response to impaired proteasome function and altered proteostasis.

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 temporally controlled manner. Cell and protein homeostasis are altered upon pathological tissue remodeling. 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 remodeling, thereby contributing to the pathogenesis of idiopathic pulmonary fibrosis (IPF). OBJECTIVES: To investigate proteasome function during myofibroblast differentiation. METHODS: We treated lung fibroblasts with transforming growth factor (TGF)-ß 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-ß 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-ß-induced myofibroblast differentiation. Activation of the 26S proteasome and increased expression of Rpn6 were detected during bleomycin-induced lung remodeling and fibrosis. Importantly, Rpn6 is overexpressed in myofibroblasts and basal cells of the bronchiolar epithelium in lungs of patients with IPF, which is accompanied by enhanced protein polyubiquitination. CONCLUSIONS: We identified Rpn6-dependent 26S proteasome activation as an essential feature of myofibroblast differentiation in vitro and in vivo, and our results suggest it has an important role in IPF pathogenesis.

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).

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.