Cigarette smoke (CS) exposure is known to induce proteostasis imbalance that can initiate accumulation of ubiquitinated proteins. Therefore, the primary goal of this study was to determine if first-and secondhand CS induces localization of ubiquitinated proteins in perinuclear spaces as aggresome bodies. Furthermore, we sought to determine the mechanism by which smoke-induced aggresome formation contributes to chronic obstructive pulmonary disease (COPD)-emphysema pathogenesis. Hence, Beas2b cells were treated with CS extract (CSE) for in vitro experimental analysis of CSinduced aggresome formation by immunoblotting, microscopy, and reporter assays, whereas chronic CS-exposed murine model and human COPD-emphysema lung tissues were used for validation. In preliminary analysis, we observed a significant (P , 0.01) increase in ubiquitinated protein aggregation in the insoluble protein fraction of CSE-treated Beas2b cells. We verified that CS-induced ubiquitin aggregrates are localized in the perinuclear spaces as aggresome bodies. These CS-induced aggresomes (P , 0.001) colocalize with autophagy protein microtubule-associated protein 1 light chain-3B 1 autophagy bodies, whereas U.S. Food and Drug Administration-approved autophagy-inducing drug (carbamazepine) significantly (P , 0.01) decreases their colocalization and expression, suggesting CS-impaired autophagy. Moreover, CSE treatment significantly increases valosin-containing protein-p62 protein-protein interaction (P , 0.0005) and p62 expression (aberrant autophagy marker; P , 0.0001), verifying CSimpaired autophagy as an aggresome formation mechanism. We also found that inhibiting protein synthesis by cycloheximide does not deplete CS-induced ubiquitinated protein aggregates, suggesting the role of CS-induced protein synthesis in aggresome formation. Next, we used an emphysema murine model to verify that chronic CS significantly (P , 0.0005) induces aggresome formation. Moreover, we observed that autophagy induction by carbamazepine inhibits CS-induced aggresome formation and alveolar space enlargement (P , 0.001), confirming involvement of aggresome bodies in COPD-emphysema pathogenesis. Finally, significantly higher p62 accumulation in smokers and severe COPD-emphysema lungs (Global Initiative for Chronic Obstructive Lung Disease Stage III/IV) as compared with normal nonsmokers (Global Initiative for Chronic Obstructive Lung Disease Stage 0) substantiates the pathogenic role of autophagy impairment in aggresome formation and COPD-emphysema progression. In conclusion, CS-induced aggresome formation is a novel mechanism involved in COPD-emphysema pathogenesis.Keywords: cigarette smoke; chronic obstructive pulmonary disease; ubiquitin; aggresomes; autophagy Clinical RelevanceWe anticipate that these research findings will have a great impact on development of novel therapeutics for treatment of chronic obstructive pulmonary disease-emphysema and other diseases involving impaired proteostasis or autophagy. In addition to the therapeutic application, thes...

Proteostasis is a critical cellular homeostasis mechanism that regulates the concentration of all cellular proteins by controlling protein- synthesis, processing and degradation. This includes protein-conformation, binding interactions and sub-cellular localization. Environmental, genetic or age-related pathogenetic factors can modulate the proteostasis (proteostasis-imbalance) through transcriptional, translational and post-translational changes that trigger the development of several complex diseases. Although these factors are known to be involved in pathogenesis of chronic obstructive pulmonary disease (COPD), the role of proteostasis mechanisms in COPD is scarcely investigated. As a proof of concept, our recent data reveals a novel role of proteostasis-imbalance in COPD pathogenesis. Briefly, cigarette- and biomass- smoke induced proteostasis-imbalance may aggravate chronic inflammatory-oxidative stress and/or protease-anti-protease imbalance resulting in pathogenesis of severe emphysema. In contrast, pathogenesis of other chronic lung diseases like ΔF508-cystic fibrosis (CF), α1-anti-trypsin-deficiency (α-1 ATD) and pulmonary fibrosis (PF) is regulated by other proteostatic mechanisms, involving the degradation of misfolded proteins (ΔF508-CFTR/α1-AT- Z variant) or regulating the concentration of signaling proteins (such as TGF-β1) by the ubiquitin-proteasome system (UPS). The therapeutic strategies to correct proteostasis-imbalance in misfolded protein disorders such as ΔF508-CF have been relatively well studied and involve strategies that rescue functional CFTR protein to treat the underlying cause of the disease. While in the case of COPD-emphysema and/or PF, identification of novel proteostasis-regulators that can control inflammatory-oxidative stress and/or protease-anti-protease balance is warranted.