RESUMEN
AIMS: Transfusion with stored red blood cells (RBCs) is associated with increased morbidity and mortality. Peroxiredoxin-2 (Prx-2) is a primary RBC antioxidant that limits hydrogen peroxide (H2O2)-mediated toxicity. Whether Prx-2 activity is altered during RBC storage is not known. RESULTS: Basal and H2O2-induced Prx-2 activity was measured in RBCs (stored for 7-35 days). Basal Prx-2 thiol oxidation increased with RBC age, whereas H2O2-dependent formation of dimeric Prx-2 was similar. However, reduction of Prx-2 dimers to monomers became progressively slower with RBC storage, which was associated with increased H2O2-induced hemolysis. Surprisingly, no change in the NADPH-dependent thioredoxin (Trx)/Trx-reductase system, which recycles dimeric Prx-2, was observed in stored RBCs. Using mouse RBCs expressing human wild type (ß93Cys) or hemoglobin (Hb) in which the conserved ß93Cys residue is replaced by Ala (ß93Ala), a role for this thiol in modulating Prx-2 reduction was demonstrated. Specifically, Prx-2 recycling was blunted in ß93Ala RBC, which was reversed by carbon monoxide-treatment, suggesting that heme autoxidation-derived H2O2 maintains Prx-2 in the oxidized form in these cells. Moreover, assessment of the oxidative state of the ß93Cys in RBCs during storage showed that while it remained reduced on intraerythrocytic Hb in stored RBC, it was oxidized to dehydroalanine on hemolyzed or extracellular Hb. INNOVATION: A novel mechanism for regulated Prx-2 activity in RBC via the ß93Cys residue is suggested. CONCLUSION: These data highlight the potential for slower Prx-2 recycling and ß93Cys oxidation in modulating storage-dependent damage of RBCs and in mediating post-transfusion toxicity.
Asunto(s)
Conservación de la Sangre , Eritrocitos/enzimología , Peroxirredoxinas/metabolismo , Animales , Monóxido de Carbono/farmacología , Glucosa/metabolismo , Hemoglobinas/metabolismo , Humanos , Peróxido de Hidrógeno/farmacología , Cinética , Masculino , Ratones , Oxidación-ReducciónRESUMEN
The ß93 cysteine (ß93Cys) residue of hemoglobin is conserved in vertebrates but its function in the red blood cell (RBC) remains unclear. Because this residue is present at concentrations more than 2 orders of magnitude higher than enzymatic components of the RBC antioxidant network, a role in the scavenging of reactive species was hypothesized. Initial studies utilizing mice that express human hemoglobin with either Cys (B93C) or Ala (B93A) at the ß93 position demonstrated that loss of the ß93Cys did not affect activities nor expression of established components of the RBC antioxidant network (catalase, superoxide dismutase, peroxiredoxin-2, glutathione peroxidase, GSH:GSSG ratios). Interestingly, exogenous addition to RBCs of reactive species that are involved in vascular inflammation demonstrated a role for the ß93Cys in hydrogen peroxide and chloramine consumption. To simulate oxidative stress and inflammation in vivo, mice were challenged with lipopolysaccharide (LPS). Notably, LPS induced a greater degree of hypotension and lung injury in B93A versus B93C mice, which was associated with greater formation of RBC reactive species and accumulation of DMPO-reactive epitopes in the lung. These data suggest that the ß93Cys is an important effector within the RBC antioxidant network, contributing to the modulation of tissue injury during vascular inflammation.
Asunto(s)
Antioxidantes/metabolismo , Cisteína/metabolismo , Eritrocitos/metabolismo , Hemoglobinas/química , Hemoglobinas/metabolismo , Pulmón/metabolismo , Pulmón/patología , Animales , Cisteína/química , Eritrocitos/química , Eritrocitos/efectos de los fármacos , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/farmacología , Inflamación/metabolismo , Inflamación/patología , Lipopolisacáridos/farmacología , Pulmón/efectos de los fármacos , Masculino , Ratones , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacosRESUMEN
The cellular response to viral infection often includes activation of pathways that shut off protein synthesis and thereby inhibit viral replication. In order to enable efficient replication, many viruses carry genes such as the E3L gene of vaccinia virus that counteract these host antiviral pathways. Vaccinia virus from which the E3L gene has been deleted (VVDeltaE3L) is highly sensitive to interferon and exhibits a restricted host range, replicating very inefficiently in many cell types, including human fibroblast and U373MG cells. To determine whether human cytomegalovirus (CMV) has a mechanism for preventing translational shutoff, we evaluated the ability of CMV to complement the deficiencies in replication and protein synthesis associated with VVDeltaE3L. CMV, but not UV-inactivated CMV, rescued VVDeltaE3L late gene expression and replication. Thus, complementation of the VVDeltaE3L defect appears to depend on de novo CMV gene expression and is not likely a result of CMV binding to the cell receptor or of a virion structural protein. CMV rescued VVDeltaE3L late gene expression even in the presence of ganciclovir, indicating that CMV late gene expression is not required for complementation of VVDeltaE3L. The striking decrease in overall translation after infection with VVDeltaE3L was prevented by prior infection with CMV. Finally, CMV blocked both the induction of eukaryotic initiation factor 2alpha (eIF2alpha) phosphorylation and activation of RNase L by VVDeltaE3L. These results suggest that CMV has one or more immediate-early or early genes that ensure maintenance of a high protein synthetic capacity during infection by preventing activation of the PKR/eIF2alpha phosphorylation and 2-5A oligoadenylate synthetase/RNase L pathways.