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The environmental, genetic, and/or age-related changes in proteostasis induce inflammation, oxidative stress, and apoptosis. We quantified the correlation of protein expression of critical proteostasis mediators to severity of chronic lung disease using lung tissue samples from control and chronic obstructive pulmonary disease (COPD) subjects (GOLD stage 0–IV) and cigarette smoke (CS)-induced murine model. The human bronchial epithelial cells, HEK-293, and Beas2B cells were used for in vitro experiments to verify the mechanisms. Our data verifies the correlation of higher expression of valosin-containing protein (VCP) retrograde translocation complex (VCP-Rma1-gp78) with severity of emphysema in COPD lung tissues and over-expression of inflammatory, ER stress and apoptotic mediators like NFκB, GADD-153/CHOP, and p-eIF2α. Moreover, subjects with severe emphysema had a higher accumulation of ubiquitinated proteins and deubiquitinating enzyme, UCHL-1, indicating towards the aggregation of misfolded or damaged proteins. The modulation of both protein degradation and synthesis rates by CS-extract substantiates the pathogenetic role of proteostasis-imbalance in emphysema and COPD. We identified that VCP also mediates proteasomal degradation of HDAC2 and Nrf2, as a potential mechanism for increased oxidative stress and corticosteroid resistance in COPD subjects with emphysema. Next, we confirmed that higher VCP expression associates with increased inflammation and apoptosis using in vitro and murine models. Our data clearly shows aberrant proteostasis in COPD subjects with severe emphysema. In addition, we evaluate therapeutic efficacy of salubrinal (ER stress inhibitor) to correct the proteostasis-imbalance based on its ability to control VCP expression and ubiquitin accumulation. Overall, our data demonstrate for the first time the critical role of proteostasis-imbalance in pathogenesis of severe emphysema.

BackgroundDysfunctional CFTR in the airways is associated with elevated levels of NFκB mediated IL-8 signaling leading to neutrophil chemotaxis and chronic lung inflammation in cystic fibrosis. The mechanism(s) by which CFTR mediates inflammatory signaling is under debate.Methodology/Principal FindingsWe tested the hypothesis that wt-CFTR down-regulates NFκB mediated IL-8 secretion. We transiently co-expressed wt-CFTR and IL-8 or NFκB promoters driving luciferase expression in HEK293 cells. Wt-CFTR expression in HEK293 cells suppresses both basal and IL1β induced IL-8, and NFκB promoter activities as compared to the control cells transfected with empty vector (p<0.05). We also confirmed these results using CFBE41o- cells and observed that cells stably transduced with wt-CFTR secrete significantly lower amounts of IL-8 chemokine as compared to non-transfected control cells. To test the hypothesis that CFTR must be localized to cell surface lipid rafts in polarized airway epithelial cells in order to mediate the inflammatory response, we treated CFBE41o- cells that had been stably transduced with wt-CFTR with methyl-β-cyclodextrin (CD). At baseline, CD significantly (p<0.05) induced IL-8 and NFκB reporter activities as compared to control cells suggesting a negative regulation of NFκB mediated IL-8 signaling by CFTR in cholesterol-rich lipid rafts. Untreated cells exposed to the CFTR channel blocker CFTR-172 inhibitor developed a similar increase in IL-8 and NFκB reporter activities suggesting that not only must CFTR be present on the cell surface but it must be functional. We verified these results in vivo by comparing survival, body weight and pro-inflammatory cytokine response to P. aeruginosa LPS in CFTR knock out (CFKO) mice as compared to wild type controls. There was a significant (p<0.05) decrease in survival and body weight, an elevation in IL-1β in whole lung extract (p<0.01), as well as a significant increase in phosphorylated IκB, an inducer of NFκB mediated signaling in the CFKO mice.Conclusions/SignificanceOur data suggest that CFTR is a negative regulator of NFκB mediated innate immune response and its localization to lipid rafts is involved in control of inflammation.

Ceramide accumulation mediates the pathogenesis of chronic obstructive lung diseases. Although an association between lack of cystic fibrosis transmembrane conductance regulator (CFTR) and ceramide accumulation has been described, it is unclear how membrane-CFTR may modulate ceramide signaling in lung injury and emphysema. Cftr+/+ and Cftr−/− mice and cells were used to evaluate the CFTR-dependent ceramide signaling in lung injury. Lung tissue from control and chronic obstructive pulmonary disease patients was used to verify the role of CFTR-dependent ceramide signaling in pathogenesis of chronic emphysema. Our data reveal that CFTR expression inversely correlates with severity of emphysema and ceramide accumulation in chronic obstructive pulmonary disease subjects compared with control subjects. We found that chemical inhibition of de novo ceramide synthesis controls Pseudomonas aeruginosa-LPS–induced lung injury in Cftr+/+ mice, whereas its efficacy was significantly lower in Cftr−/− mice, indicating that membrane-CFTR is required for controlling lipid-raft ceramide levels. Inhibition of membrane-ceramide release showed enhanced protective effect in controlling P. aeruginosa-LPS–induced lung injury in Cftr−/− mice compared with that in Cftr+/+ mice, confirming our observation that CFTR regulates lipid-raft ceramide levels and signaling. Our results indicate that inhibition of de novo ceramide synthesis may be effective in disease states with low CFTR expression like emphysema and chronic lung injury but not in complete absence of lipid-raft CFTR as in ΔF508-cystic fibrosis. In contrast, inhibiting membrane-ceramide release has the potential of a more effective drug candidate for ΔF508-cystic fibrosis but may not be effectual in treating lung injury and emphysema. Our data demonstrate the critical role of membrane-localized CFTR in regulating ceramide accumulation and inflammatory signaling in lung injury and emphysema.

BackgroundValosin-containing protein (VCP)/p97 is an AAA ATPase molecular chaperone that regulates vital cellular functions and protein-processing. A recent study indicated that VCP expression levels are correlated with prognosis and progression of non-small cell lung carcinoma (NSCLC). We not only verified these findings but also identified the specific role of VCP in NSCLC pathogenesis and progression.Methodology/Principal FindingsOur results show that VCP is significantly overexpressed in non-small cell lung carcinoma (NSCLC) as compared to normal tissues and cell lines (p<0.001). Moreover, we observed the corresponding accumulation of ubiquitinated-proteins in NSCLC cell lines and tissues as compared to the normal controls. VCP inhibition by si/shRNA or small-molecule (Eeyarestatin I, EerI) significantly (p<0.05, p<0.00007) suppressed H1299 proliferation and migration but induced (p<0.00001) apoptosis. Cell cycle analysis by flow cytometry verified this data and shows that VCP inhibition significantly (p<0.001, p<0.003) induced cell cycle arrest in the G0/G1 phases. We also found that VCP directly regulates p53 and NFκB protein levels as a potential mechanism to control tumor cell proliferation and progression. Finally, we evaluated the therapeutic potential of VCP inhibition and observed significantly reduced NSCLC tumor growth in both in vitro and xenograft murine (athymic-nude) models after EerI treatment (p<0.05).Conclusions/SignificanceThus, targeting VCP in NSCLC may provide a novel strategy to restore p53 and NFκB levels and ameliorate the growth and tumorigenicity, leading to improved clinical outcomes.

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is a recently emerged virus that has caused a number of human infections and deaths, primarily in the Middle East. The transmission of MERS-CoV to humans has been proposed to be as a result of contact with camels, but evidence of human-to-human transmission also exists. In order to work with MERS-CoV in a laboratory setting, the US Centers for Disease Control and Prevention (CDC) has determined that MERS-CoV should be handled at a biosafety level (BSL) 3 (BSL-3) biocontainment level. Many processes and procedures used to characterize MERS-CoV and to evaluate samples from MERS-CoV infected animals are more easily and efficiently completed at BSL-2 or lower containment. In order to complete experimental work at BSL-2, demonstration or proof of inactivation is required before removal of specimens from biocontainment laboratories. In the studies presented here, we evaluated typical means of inactivating viruses prior to handling specimens at a lower biocontainment level. We found that Trizol, AVL buffer and gamma irradiation were effective at inactivating MERS-CoV, that formaldehyde-based solutions required at least 30 minutes of contact time in a cell culture system while a mixture of methanol and acetone required 60 minutes to inactivate MERS-CoV. Together, these data provide a foundation for safely inactivating MERS-CoV, and potentially other coronaviruses, prior to removal from biocontainment facilities.

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BackgroundThe mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene results in CF. The most common mutation, ΔF508-CFTR, is a temperature-sensitive, trafficking mutant with reduced chloride transport and exaggerated immune response. The ΔF508-CFTR is misfolded, ubiquitinated, and prematurely degraded by proteasome mediated- degradation. We recently demonstrated that selective inhibition of proteasomal pathway by the FDA approved drug PS-341 (pyrazylcarbonyl-Phe-Leuboronate, a.k.a. Velcade or bortezomib) ameliorates the inflammatory pathophysiology of CF cells. This proteasomal drug is an extremely potent, stable, reversible and selective inhibitor of chymotryptic threonine protease-activity. The apprehension in considering the proteasome as a therapeutic target is that proteasome inhibitors may affect proteostasis and consecutive processes. The affect on multiple processes can be mitigated by nanoparticle mediated PS-341 lung-delivery resulting in favorable outcome observed in this study.ResultsTo overcome this challenge, we developed a nano-based approach that uses drug loaded biodegradable nanoparticle (PLGA-PEGPS-341) to provide controlled and sustained drug delivery. The in vitro release kinetics of drug from nanoparticle was quantified by proteasomal activity assay from days 1-7 that showed slow drug release from day 2-7 with maximum inhibition at day 7. For in vivo release kinetics and biodistribution, these drug-loaded nanoparticles were fluorescently labeled, and administered to C57BL6 mice by intranasal route. Whole-body optical imaging of the treated live animals demonstrates efficient delivery of particles to murine lungs, 24 hrs post treatment, followed by biodegradation and release over time, day 1-11. The efficacy of drug release in CF mice (Cftr-/-) lungs was determined by quantifying the changes in proteasomal activity (~2 fold decrease) and ability to rescue the Pseudomonas aeruginosa LPS (Pa-LPS) induced inflammation, which demonstrates the rescue of CF lung disease in murine model.ConclusionWe have developed a novel drug delivery system to provide sustained delivery of CF "correctors" and "anti-inflammatories" to the lungs. Moreover, we demonstrate here the therapeutic efficacy of nano-based proteostasis-modulator to rescue Pa-LPS induced CF lung disease.