Dissecting the molecular effects of cigarette smoke on proteasome function.

Proteasome dysfunction is emerging as a novel pathomechanism for the development of chronic obstructive pulmonary disease (COPD), a major leading cause of death in the world. Cigarette smoke, one of the main risk factors for COPD, impairs proteasome function in vitro and in vivo. In the present study, we dissected the molecular changes induced by cigarette smoke on the proteasome in lung epithelial cells and mouse lungs. 26S proteasome pull-down, MS interactome, and stoichiometry analyses indicated that 26S proteasome complexes become instable in cigarette smoke-treated lung epithelial cells as well as in lungs of mice after three day smoke exposure. The interactome of the 26S was clearly altered in mouse lungs upon smoke exposure but not in cells after 24 h of smoke exposure. Using native MS analysis of purified 20S proteasomes, we observed some destabilization of 20S complexes purified from cigarette smoke-exposed cells in the absence of any dominant and inhibitory modification of proteasomal proteins. Taken together, our results suggest that cigarette smoke induces minor but detectable changes in the stability of 20S and 26S proteasome complexes which might contribute to imbalanced proteostasis in a chronic setting as observed in chronic lung diseases associated with cigarette smoking.

Cigarette smoke alters the secretome of lung epithelial cells.

Cigarette smoke is the most relevant risk factor for the development of lung cancer and chronic obstructive pulmonary disease. Many of its more than 4500 chemicals are highly reactive, thereby altering protein structure and function. Here, we used subcellular fractionation coupled to label-free quantitative MS to globally assess alterations in the proteome of different compartments of lung epithelial cells upon exposure to cigarette smoke extract. Proteomic profiling of the human alveolar derived cell line A549 revealed the most pronounced changes within the cellular secretome with preferential downregulation of proteins involved in wound healing and extracellular matrix organization. In particular, secretion of secreted protein acidic and rich in cysteine, a matricellular protein that functions in tissue response to injury, was consistently diminished by cigarette smoke extract in various pulmonary epithelial cell lines and primary cells of human and mouse origin as well as in mouse ex vivo lung tissue cultures. Our study reveals a previously unrecognized acute response of lung epithelial cells to cigarette smoke that includes altered secretion of proteins involved in extracellular matrix organization and wound healing. This may contribute to sustained alterations in tissue remodeling as observed in lung cancer and chronic obstructive pulmonary disease.

Impairment of Immunoproteasome Function by Cigarette Smoke and in Chronic Obstructive Pulmonary Disease.

RATIONALE: Patients with chronic obstructive pulmonary disease (COPD) and in particular smokers are more susceptible to respiratory infections contributing to acute exacerbations of disease. The immunoproteasome is a specialized type of proteasome destined to improve major histocompatibility complex (MHC) class I-mediated antigen presentation for the resolution of intracellular infections. OBJECTIVES: To characterize immunoproteasome function in COPD and its regulation by cigarette smoke. METHODS: Immunoproteasome expression and activity were determined in bronchoalveolar lavage (BAL) and lungs of human donors and patients with COPD or idiopathic pulmonary fibrosis (IPF), as well as in cigarette smoke-exposed mice. Smoke-mediated alterations of immunoproteasome activity and MHC I surface expression were analyzed in human blood-derived macrophages. Immunoproteasome-specific MHC I antigen presentation was evaluated in spleen and lung immune cells that had been smoke-exposed in vitro or in vivo. MEASUREMENTS AND MAIN RESULTS: Immunoproteasome and MHC I mRNA expression was reduced in BAL cells of patients with COPD and in isolated alveolar macrophages of patients with COPD or IPF. Exposure of immune cells to cigarette smoke extract in vitro reduced immunoproteasome activity and impaired immunoproteasome-specific MHC I antigen presentation. In vivo, acute cigarette smoke exposure dynamically regulated immunoproteasome function and MHC I antigen presentation in mouse BAL cells. End-stage COPD lungs showed markedly impaired immunoproteasome activities. CONCLUSIONS: 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.