Redox proteomics of the inflammatory secretome identifies a common set of redoxins and other glutathionylated proteins released in inflammation, influenza virus infection and oxidative stress

Protein cysteines can form transient disulfides with glutathione (GSH), resulting in the production of glutathionylated proteins, and this process is regarded as a mechanism by which the redox state of the cell can regulate protein function. Most studies on redox regulation of immunity have focused on intracellular proteins. In this study we have used redox proteomics to identify those proteins released in glutathionylated form by macrophages stimulated with lipopolysaccharide (LPS) after pre-loading the cells with biotinylated GSH. Of the several proteins identified in the redox secretome, we have selected a number for validation. Proteomic analysis indicated that LPS stimulated the release of peroxiredoxin (PRDX) 1, PRDX2, vimentin (VIM), profilin1 (PFN1) and thioredoxin 1 (TXN1). For PRDX1 and TXN1, we were able to confirm that the released protein is glutathionylated. PRDX1, PRDX2 and TXN1 were also released by the human pulmonary epithelial cell line, A549, infected with influenza virus. The release of the proteins identified was inhibited by the anti-inflammatory glucocorticoid, dexamethasone (DEX), which also inhibited tumor necrosis factor (TNF)-α release, and by thiol antioxidants (N-butanoyl GSH derivative, GSH-C4, and N-acetylcysteine (NAC), which did not affect TNF-α production. The proteins identified could be useful as biomarkers of oxidative stress associated with inflammation, and further studies will be required to investigate if the extracellular forms of these proteins has immunoregulatory functions

Redox regulation of the influenza hemagglutinin maturation process: a new cell mediated strategy for antinfluenza therapy

L’emoagglutinina (HA), glicoproteina del virus influenzale presente sotto forma di omotrimero nell’envelope virale, è la principale responsabile dell’adesione e dell’entrata del virus nella cellula ospite. Il folding dell’HA avviene nel reticolo endoplasmatico (RE) dove la formazione dei legami disulfidici è favorita da condizioni ossidanti. La proteina così foldata passa nel Golgi dove subisce ulteriori processi di glicosilazione. Una variazione dello stato redox intracellulare in senso pro-ossidante è stata riscontrata in varie infezioni virali, tra cui quella provocata dal virus influenzale. Il nostro gruppo di ricerca ha dimostrato che la somministrazione di gluttaione ridotto (GSH) corregge lo stress ossidativo ed inibisce la replicazione di virus a RNA e DNA sia in vitro che in vivo. Più recentemente è stata descritta l’attività antivirale del GSH-C4, derivato butanoile del GSH con aumentate proprietà idrofobiche, contro il virus Sendai e Herpes Simplex-1. Obiettivo dello studio è stato quello di valutare l’efficacia del GSH-C4 sulla replicazione del virus influenzale A/PR8/H1N1 studiando in particolare il suo effetto nel processo di maturazione dell’HA. I risultati ottenuti hanno dimostrato che il GSH-C4 ha inibito la replicazione virale di diversi ceppi di virus influenzale (ceppo umano A/PR8 H1N1, ceppo umano A/NWS H1N1 e ceppo aviario A/Parrot/ULSTER/73 H7N1) in maniera dose-dipendente. In particolare, il titolo virale, nelle cellule trattate con GSH-C4 10 mM, era ridotta del 90% rispetto al controllo, senza alcun effetto tossico sulle cellule non infettate. L’analisi in western blot delle proteine virali ha dimostrato che nelle cellule trattate con GSH-C4 l’inibizione del titolo virale era associata ad una diminuzione del peso molecolare dell’emoagglutinina rispetto alle cellule di controllo. Tale effetto non era dovuto ad un effetto diretto sulla glicosilazione o sul trasporto dell’HA come dimostrato da esperimenti effettuati con inibitori degli enzimi coinvolti nella glicosilazione e inibitori del trasporto. Western blot effettuati in condizioni non riducenti, in presenza o in assenza di NEM, hanno dimostrato che in cellule NCI infettate e trattate con GSH-C4 l’espressione dell’HA in forma trimerica era diminuita rispetto alle cellule di controllo e la proteina era prevalentemente in una forma monomerica ridotta. Inoltre, PDI, un’ossidoreduttasi responsabile della formazione del legame disulfidico nel RE, era mantenuta in forma ridotta. Il trattamento degli estratti cellulari con Endo H ha evidenziato che in presenza di GSH-C4 l’HA era prevalentemente nel RE. Allo stesso tempo l’osservazione mediante immunofluorescenza ha evidenziato che la localizzazione della HA sulla membrana cellulare, fenomeno normalmente osservato 8 ore dopo l’infezione nelle cellule di controllo, è fortemente diminuita nelle cellule trattate con GSH-C4. I risultati ottenuti suggeriscono che la minima diminuzione di peso molecolare dell’HA indotta dal GSH-C4 si riflette in un accumulo della proteina immatura e fornisce l’evidenza che il GSH-C4 inibisce la replicazione del virus influenzale interferendo con il processo di maturazione dell’HA.Hemagglutinin (HA) is a homotrimeric integral membrane glycoprotein of influenza virus that is responsible for the binding of viral particles to sialic acid-containing receptor on cell surface. Folding of HA starts in the endoplasmic reticulum (ER), where the oxidizing conditions and the presence of lectins and oxidoreductases supports N-linked glycosylation and disulfide bond formation. Then, the folded HA moves through the Golgi apparatus to the cell membrane. We previously reported that glutathione (GSH) inhibited replication of several viruses by restoring intracellular reducing conditions altered by infection. The present study was aimed at determining whether: i) the GSH derivative GSH-C4 inhibits influenza virus replication; ii) this effect is mediated by an alteration in HA maturation. In MDCK cells, influenza virus replication was dose-dipendently inhibited by 1-15 mM GSH-C4. At 10 mM, GSH-C4 reduced by 90-95% the replication of different strains of influenza virus (human A/PR8/H1N1, human A/NWS/H1N1 and avian A/Parrot/ULSTER/73/H7N1), without producing toxic effects on uninfected cells. Western blot of viral proteins extracted from GSH-C4-treated human pulmonary NCI-H292 cells demonstrated that this inhibition was associated with a slight decrease in the apparent molecular weight of HA. By comparing the effects of GSH-C4 with that of HA glycosylation- and transport- inhibitors, we demonstrated that this event was not due to a direct alteration of HA glycosylation and transport. Western blot performed in non reducing conditions, with or without NEM, showed that GSH-C4 caused a reduction of HA dimeric and trimeric forms along with an increase of its reduced form. Consistently, PDI, an oxidoreductase responsible for disulfide bond formation in the ER, was predominantly found in the reduced form, that physiologically prevents its function. Moreover, HA extracted from GSH-C4 treated cells was sensitive to Endo H digestion thus confirming that the protein is kept in the ER. Finally, HA immunolabelling showed that GSH-C4 markedly reduced insertion of the protein in the cell membrane. Altogether, these results suggest that the slight decrease in the HA molecular weight induced by GSH-C4 reflects an accumulation of the immature HA and provide the evidence that GSH-C4 inhibits influenza virus replication by interfering with the maturation process of HA

Influenza virus and redox mediated cell signaling: a complex network of virus/host interaction

Several viruses, including influenza, induce an imbalance of intracellular redox state toward pro-oxidant conditions. Through different mechanisms these alterations contribute both to influenza virus replication and to the pathogene- sis of virus-induced disease. At the same time, influenza virus activates several intracellular signaling pathways involved in important physiological functions of the cell. Interestingly, many of these pathways are finely regulated by small changes in intracellular redox state, and the virus-induced redox imbalance might also control viral replication through this mechanism. Here we review the main intracellular redox-sensitive pathways activated upon influenza infection and involved in reg- ulating viral replication