1Reactive Oxygen Species II:Reactive Oxygen Species II:• Free radicals and cell signaling• Pathological conditions that involve ROS• Measurements of free radicalsEndogenous mediatorsEnvironmental factorsFree Radicals and cell functionNormal metabolismSignal transduction:Regulated sequence of biochemical steps through which a stimulant conveys a message, resulting in a physiologic responseAgonistReceptorPrimary effectors (enzymes or channels)Second messenger(s)Secondary effectors (enzymes, other molecules)Oxidants can act to modify signal transduction2Can free radicals be second messengers?Second messengers should be:• Short lived (concentrations can change rapidly)• Enzymatically generated in response to stimulant• Enzymatically degraded• Specific in action (?)Some free radicals fit these criteria!O2.-, H2O2, NO., ONOO-• Heme oxidation• Oxidation of iron-sulfur centers in proteins• Changes in thiol/disulfide redox state of the cell• Change in conformation Æ change in activity• Oxidative modification of proteins: degradation, loss of function, or gain of function• Oxidative modification of DNA: activation of repair, and/or apoptosis• Oxidative modification of lipids: disruption of membrane-associated signaling, DNA damage, and formation of protein adductsHow free radicals can be involved in signaling?Nitric Oxide SynthaseNO•Binds to heme moiety of guanylate cyclaseConformational change of the enzymeIncreased activity (production of cGMP)Modulation of activity of other proteins (protein kinases, phospho-diesterases, ion channels)Physiological response (relaxation of smooth muscles, inhibition of platelet aggregation, etc.)NO• signaling in physiologyO2-•ONOO-3Iron-sulfur proteins and free radical signalingFeFeActive protein Inactive proteinROSSulfide, cysteine thiolate groups in non-heme iron proteinsMammalian (4Fe-4S) aconitase (citric acid cycle, citrate Æisocitrate):• Contains non-heme iron complex Fe-S-Cys• ROS and RNS disrupt Fe-S clusters and inhibit aconitase activity• Binding site is exposed: binding to ferritin mRNA and transferrin receptor • Participates in regulation of iron homeostasis (iron-regulatory protein-1)Bacterial SoxR protein:• Contains two stable (2Fe-2S) centers that are anchored to four cysteineresidues near carboxy-terminus of the protein• Under normal conditions these iron-sulfur centers are reduced • When oxidized, SoxR changes configuration and is able to activate transcription of oxidative stress-related proteins (MnSOD, G6PDH, etc.)Thiol/disulfide redox state and signaling by ROSOxidant - antioxidant balanceEndogenous sources of ROS and RNSEnvironmental factors[+][+][-][-]From Dröge W. (2002) Physiol Rev 82:47-95Bacterial OxyR is a model case of a redox-sensitive signaling protein that may be activated by either H2O2or changes in intracellular GSH contentFrom Dröge W. (2002) Physiol Rev 82:47-95• No new protein synthesis occurs to activate OxyR, but it acts by ↑transcription (MnSOD, catalase, GSH reductase, glutaredoxin 1, thioredoxin, etc.)• Both reduced and oxidized OxyR can bind promoter regions of these genes but they exhibit different binding characteristics: oxidized much more active• OxyR response is autoregulated since glutaredoxin and thioredoxin can reverse formation of disulfide bonds thus inactivating OxyRThiol/disulfide redox state and signaling by ROS4Mixed disulfide formationDisulfide formationSH SHROSS-SAlteration of protein-protein interactionsROSThiol/disulfide redox state and signaling by ROSChange in protein conformation by ROSROSSensor (yAP-1)Change in conformation(Cys-rich domain at C-terminus)↑DNA bindingChanges in gene expression(SOD, Grx, Catalase, etc.)↑ProtectionTranslocation to the nucleusadd H2O2overexpressdeleteNo sensorNo bindingNo change in gene expressionNo protectionFrom Dröge W. (2002) Physiol Rev 82:47-95Redox signaling by protein degradationHypoxia-Inducible Factor 1 (HIF-1)ROSCytokines, other signalsp65IκBIκBp50p65p65p50TranscriptionPolyubiquitylationDegradationNuclear Factor κB5Redox signaling by DNA damageRedox signaling by lipid peroxidationMDADNA adducts Protein adductsModels of how signalling could be initiated through raft(s). A | In these models, signallingoccurs in either single rafts (Model 1) or clustered rafts (Model 2). Following dimerization the protein becomes phosphorylated (blue circle) in rafts. In single rafts this can occur by activation a | within the raft, or b | by altering the partitioning dynamics of the protein. B | In the second model we assume that there are several rafts in the membrane, which differ in protein composition (shown in orange, purple or blue). Clustering would coalesce rafts (red), so that they would now contain a new mixture of molecules, such as crosslinkers and enzymes. Clustering could occur either extracellularly, within the membrane, or in the cytosol (a–c, respectively). Raft clustering could also occur through GPI-anchored proteins (yellow), either as a primary or co-stimulatory response. Notably, models 1 and 2 are not mutually exclusive. For instance, extracellular signals could increase a protein's raft affinity (for example, similar to the effect of single versus dual acylation) therefore drawing more of the protein into the raft where it can be activated and recruit other proteins, such as LAT, which would crosslink several rafts. Nature Reviews Molecular Cell Biology 1; 31-39 (2000)Membrane lipids and their role in signaling6Lipid peroxidation products and receptor signalingFrom Dröge W. (2002) Physiol Rev 82:47-95ROS-sensitive targets in signaling cascadesLipid particle-mediated activation of macrophagesROSCytokinesNF-κBAlterations of Ca2+signaling by ROS•Oxidants can directly increase [Ca2+]i•Oxidants can alter Ca++signalingPKC cysteines in regulatory domainCa2+BindingProtein[Ca2+]iCa2+IP3RCa-ATPaseCa-ChannelNa/Ca antiportCa-ATPaseEndoplasmicReticulum7Bi-functional and mono-functional inducersBi-functional inducers:Compounds that can induce both Phase I (mono-oxygenases) and Phase II (GST, NQO1) enzymesMono-functional inducers:Compounds that can only regulate Phase II enzymesGSTNQO1Modified from Whitlock et al. (1996) FASEB J. 10:809-818Antioxidant Response Element (ARE)Transcriptional regulation of the rat GSTA2 and NQO1 genes by bifunctional and monofunctionalinducers. The bifunctional inducers and the dioxin TCDD bind to and activate the AhR, which then translocates into the