Inflammasomes regulate innate immune responses by facilitating maturation of inflammatory cytokines, interleukin (IL)-1β and IL-18. NACHT, LRR and PYD domains-containing protein 7 (NALP7) is one inflammasome constituent, but little is known about its cellular handling. Here we show a mechanism for NALP7 protein stabilization and activation of the inflammasome by Toll-like receptor (TLR) agonism with bacterial lipopolysaccharide (LPS) and the synthetic acylated lipopeptide Pam3CSK4. NALP7 is constitutively ubiquitinated and recruited to the endolysosome for degradation. With TLR ligation, the deubiquitinase enzyme, STAM-binding protein (STAMBP) impedes NALP7 trafficking to lysosomes to increase NALP7 abundance. STAMBP deubiquitinates NALP7 and STAMBP knockdown abrogates LPS or Pam3CSK4-induced increases in NALP7 protein. A small-molecule inhibitor of STAMBP deubiquitinase activity, BC-1471, decreases NALP7 protein levels and suppresses IL-1β release after TLR agonism. These findings describe a unique pathway of inflammasome regulation with the identification of STAMBP as a potential therapeutic target to reduce pro-inflammatory stress.

We investigated the mechanisms by which chlorine (Cl 2 ) and its reactive byproducts inhibit Na ؉ -dependent alveolar fluid clearance (AFC) in vivo and the activity of amiloridesensitive epithelial Na ؉ channels (ENaC) by measuring AFC in mice exposed to Cl 2 (0 -500 ppm for 30 min) and Na ؉ and amiloride-sensitive currents (I Na and I amil , respectively) across Xenopus oocytes expressing human ␣-, ␤-, and ␥-ENaC incubated with HOCl (1-2000 M). Both Cl 2 and HOCl-derived products decreased AFC in mice and whole cell and single channel I Na in a dose-dependent manner; these effects were counteracted by serine proteases. Mass spectrometry analysis of the oocyte recording medium identified organic chloramines formed by the interaction of HOCl with HEPES (used as an extracellular buffer). In addition, chloramines formed by the interaction of HOCl with taurine or glycine decreased I Na in a similar fashion. Preincubation of oocytes with serine proteases prevented the decrease of I Na by HOCl, whereas perfusion of oocytes with a synthetic 51-mer peptide corresponding to the putative furin and plasmin cleaving segment in the ␥-ENaC subunit restored the ability of HOCl to inhibit I Na . Finally, I Na of oocytes expressing wild type ␣-and ␥-ENaC and a mutant form of ␤ENaC (S520K), known to result in ENaC channels locked in the open position, were not altered by HOCl. We concluded that HOCl and its reactive intermediates (such as organic chloramines) inhibit ENaC by affecting channel gating, which could be relieved by proteases cleavage.The balance of fluid covering the respiratory and alveolar epithelia is determined in part by the ability of these cells to transport sodium (Na ϩ ) and chloride (Cl Ϫ ) ions in a vectorial fashion. Active Na ϩ reabsorption across lung epithelia requires the coordinated entry of Na ϩ ions through cation-and Na ϩ -selective amiloride-sensitive channels (ENaC) 5 located at the apical membranes, their extrusion across the basolateral membranes by the electrogenic Na ϩ -K ϩ -ATPase, and the passive movement of K ϩ ions through basolateral K ϩ channels. The entry of Na ϩ ions through apical pathways is thought to be the rate-limiting step in this process (1-3). To preserve neutrality, Cl Ϫ ions follow Na ϩ ions both through transcellular and paracellular pathways (4, 5). The coordinated movement of Na ϩ and Cl Ϫ ions creates an oncotic gradient favoring the absorption of alveolar fluid.Injury to either apical or basolateral pathways by partially reduced intermediates may lead to impairment of fluid reabsorption, which in turn may result in pulmonary edema, hypoxemia, and eventually death from respiratory failure (6 -9). One such specie is hypochlorous acid (HOCl) 6 , which may be generated either endogenously or exogenously. Millimolar concentrations of HOCl may be generated by activated neutrophils and eosinophils by the catalytic actions of neutrophil-and eosinophil-derived myeloperoxidases on chloride (Cl Ϫ ) and hydrogen peroxide (H 2 O 2 ) in close proximity of the apical and basolatera...

We sought to determine the mechanisms by which influenza infection of human epithelial cells decreases cystic fibrosis transmembrane conductance regulator (CFTR) expression and function. We infected human bronchial epithelial (NHBE) cells and murine nasal epithelial (MNE) cells with various strains of influenza A virus. Influenza infection significantly reduced CFTR short circuit currents (Isc) and protein levels at 8 hours postinfection. We then infected CFTR expressing human embryonic kidney (HEK)‐293 cells (HEK‐293 CFTRwt) with influenza virus encoding a green fluorescent protein (GFP) tag and performed whole‐cell and cell‐attached patch clamp recordings. Forskolin‐stimulated, GlyH‐101‐sensitive CFTR conductances, and CFTR open probabilities were reduced by 80% in GFP‐positive cells; Western blots also showed significant reduction in total and plasma membrane CFTR levels. Knockdown of the influenza matrix protein 2 (M2) with siRNA, or inhibition of its activity by amantadine, prevented the decrease in CFTR expression and function. Lysosome inhibition (bafilomycin‐A1), but not proteasome inhibition (lactacystin), prevented the reduction in CFTR levels. Western blots of immunoprecipitated CFTR from influenza‐infected cells, treated with BafA1, and probed with antibodies against lysine 63‐linked (K‐63) or lysine 48‐linked (K‐48) polyubiquitin chains supported lysosomal targeting. These results highlight CFTR damage, leading to early degradation as an important contributing factor to influenza infection‐associated ion transport defects.—Londino, J. D., Lazrak, A., Noah, J. W., Aggarwal, S., Bali, V., Woodworth, B. A., Bebok, Z., Matalon, S. Influenza virus M2 targets cystic fibrosis transmembrane conductance regulator for lysosomal degradation during viral infection. FASEB J. 29, 2712–2725 (2015). http://www.fasebj.org

The human cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-activated chloride (Cl(-)) channel in the lung epithelium that helps regulate the thickness and composition of the lung epithelial lining fluid. We investigated whether influenza M2 protein, a pH-activated proton (H(+)) channel that traffics to the plasma membrane of infected cells, altered CFTR expression and function. M2 decreased CFTR activity in 1) Xenopus oocytes injected with human CFTR, 2) epithelial cells (HEK-293) stably transfected with CFTR, and 3) human bronchial epithelial cells (16HBE14o-) expressing native CFTR. This inhibition was partially reversed by an inhibitor of the ubiquitin-activating enzyme E1. Next we investigated whether the M2 inhibition of CFTR activity was due to an increase of secretory organelle pH by M2. Incubation of Xenopus oocytes expressing CFTR with ammonium chloride or concanamycin A, two agents that alkalinize the secretory pathway, inhibited CFTR activity in a dose-dependent manner. Treatment of M2- and CFTR-expressing oocytes with the M2 ion channel inhibitor amantadine prevented the loss in CFTR expression and activity; in addition, M2 mutants, lacking the ability to transport H(+), did not alter CFTR activity in Xenopus oocytes and HEK cells. Expression of an M2 mutant retained in the endoplasmic reticulum also failed to alter CFTR activity. In summary, our data show that M2 decreases CFTR activity by increasing secretory organelle pH, which targets CFTR for destruction by the ubiquitin system. Alteration of CFTR activity has important consequences for fluid regulation and may potentially modify the immune response to viral infection.

The cystic fibrosis transmembrane conductance regulator (CFTR) and the amiloride-sensitive epithelial sodium channels (ENaC) are located in the apical membranes of airway and alveolar epithelial cells. These transporters play an important role in the regulation of lung fluid balance across airway and alveolar epithelia by being the conduits for chloride (Cl) and bicarbonate ([Formula: see text]) secretion and sodium (Na) ion absorption, respectively. The functional role of these channels in the respiratory tract is to maintain the optimum volume and ionic composition of the bronchial periciliary fluid (PCL) and alveolar lining fluid (ALF) layers. The PCL is required for proper mucociliary clearance of pathogens and debris, and the ALF is necessary for surfactant homeostasis and optimum gas exchange. Dysregulation of ion transport may lead to mucus accumulation, bacterial infections, inflammation, pulmonary edema, and compromised respiratory function. Influenza (or flu) in mammals is caused by influenza A and B viruses. Symptoms include dry cough, sore throat, and is often followed by secondary bacterial infections, accumulation of fluid in the alveolar spaces and acute lung injury. The underlying mechanisms of flu symptoms are not fully understood. This review summarizes our present knowledge of how influenza virus infections alter airway and alveolar epithelial cell CFTR and ENaC function in vivo and in vitro and the role of these changes in influenza pathogenesis.

BackgroundThe ubiquitin-proteasome pathway, mediated in part, by ubiquitin E3 ligases, is critical in regulating cellular processes such as cell proliferation, apoptosis, and migration. FBXO17 was recently identified as an F-box protein that targets glycogen synthase kinase-3β to the E3 ubiquitin ligase protein complex for polyubiquitination and proteasomal degradation. Here, we identified that in several lung adenocarcinoma cell lines, FBXO17 cellular protein was detected at relatively high levels, as was expression in a subset of lung cancers. Hence, we investigated the effects of FBXO17 on cell proliferation.MethodsSingle cell RNA sequencing analysis was performed on a resection of a non-small cell lung carcinoma tumor to examine FBXO17 expression. Multiple lung cancer cell lines were immunoblotted, and The Cancer Genome Atlas was analyzed to determine if FBXO17 expression was amplified in a subset of lung cancers. A549 cells were transfected with empty vector or FBXO17-V5 plasmid and immunoblotted for Akt pathway mediators including PDK1, ERK1/2, ribosomal protein S6, and CREB. Cell proliferation and viability were analyzed by trypan blue exclusion, BrdU incorporation and an MTS-based fluorometric assay. Studies were also performed after transfecting with sifbxo17. Samples were used in an RNA microarray analysis to evaluate pathways affected by reduced FBXO17 gene expression.ResultsWe observed that overexpression of FBXO17 increased A549 cell proliferation coupled with Akt activation. Ectopically expressed FBXO17 also increased ERK1/2 kinase activation and increased phosphorylation of RPS6, a downstream target of mTOR. We also observed an increased number of cells in S-phase and increased metabolic activity of lung epithelial cells expressing FBXO17. FBXO17 knockdown reduced Akt Ser 473 phosphorylation approaching statistical significance with no effect on Thr 308. However, ERK1/2 phosphorylation, cellular metabolic activity, and overall cell numbers were reduced. When we analyzed RNA profiles of A549 cells with reduced FBXO17 expression, we observed downregulation of several genes associated with cell proliferation and metabolism.ConclusionsThese data support a role for FBXO17 abundance, when left unchecked, in regulating cell proliferation and survival through modulation of Akt and ERK kinase activation. The data raise a potential role for the F-box subunit in modulating tumorigenesis.Electronic supplementary materialThe online version of this article (10.1186/s12931-018-0910-0) contains supplementary material, which is available to authorized users.

Signal regulatory protein ␣ (SIRP␣) is a membrane glycoprotein immunoreceptor abundant in cells of monocyte lineage. SIRP␣ ligation by a broadly expressed transmembrane protein, CD47, results in phosphorylation of the cytoplasmic immunoreceptor tyrosine-based inhibitory motifs, resulting in the inhibition of NF-B signaling in macrophages. Here we observed that proteolysis of SIRP␣ during inflammation is regulated by a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), resulting in the generation of a membrane-associated cleavage fragment in both THP-1 monocytes and human lung epithelia. We mapped a charge-dependent putative cleavage site near the membrane-proximal domain necessary for ADAM10-mediated cleavage. In addition, a secondary proteolytic cleavage within the membrane-associated SIRP␣ fragment by ␥-secretase was identified. Ectopic expression of a SIRP␣ mutant plasmid encoding a proteolytically resistant form in HeLa cells inhibited activation of the NF-B pathway and suppressed STAT1 phosphorylation in response to TNF␣ to a greater extent than expression of wild-type SIRP␣. Conversely, overexpression of plasmids encoding the proteolytically cleaved SIRP␣ fragments in cells resulted in enhanced STAT-1 and NF-B pathway activation. Thus, the data suggest that combinatorial actions of ADAM10 and ␥-secretase on SIRP␣ cleavage promote inflammatory signaling.Inhibitory receptors including signal regulatory protein ␣ (SIRP␣), 2 CD33, SIGLECs (sialic acid-binding immunoglobulin-type lectins), CD66a, PD-1, CD31, PILRa (paired immunoglobin-like type 2 receptor ␣), CMRF35H, gp49B1, PECAM1, and others have been demonstrated to suppress the initiation of inflammatory signaling and contribute to the resolution of inflammation after infection (1, 2). SIRP␣ is membrane glycoprotein immunoreceptor that is expressed mainly in myeloid and neuronal cells (3). Ligation of SIRP␣ results in phosphorylation of the cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs), which recruit and activate SH2 domain-containing phosphotyrosine phosphatases SHP-1 and SHP-2 (4). SIRP␣ signaling results in reduced macrophage migration and phagocytosis and inhibition of NF-B signaling with reduction of release of NF-B-dependent cytokines in macrophages (5, 6). Phosphorylation of SIRP␣ ITIM domains also enhances JAK/STAT activation and NADPH oxidase expression and activity (7). Thus, SIRP␣ serves as an important modulator of the host adaptive immune response. Proteolysis and release of the SIRP␣ NH 2 -terminal domain has been demonstrated in primary cultured neurons, melanoma cells, and macrophage cell lines. Cleavage of murine SIRP␣ through an MMP inhibitor-sensitive pathway was first observed in cultured cells engineered to express both SIRP␣ and an active form of Ras (8). In these cells, blocking SIRP␣ proteolysis resulted in inhibited cell migration, cell spreading, and cytoskeletal reorganization. In addition, SIRP␣ was shown to regulate synaptic activity through activation of MMP inhibitor-sensitive prote...

No abstract