Reactive Oxygen Species II Free radicals and cell signaling Pathological conditions that involve ROS Measurements of free radicals Free Radicals and cell function Environmental factors Endogenous mediators Normal metabolism Signal transduction Regulated sequence of biochemical steps through which a stimulant conveys a message resulting in a physiologic response Agonist Receptor Primary effectors enzymes or channels Second messenger s Secondary effectors enzymes other molecules Oxidants can act to modify signal transduction 1 Can 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 How free radicals can be involved in signaling Heme oxidation Oxidation of iron sulfur centers in proteins Changes in thiol disulfide redox state of the cell Change in conformation 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 adducts NO signaling in physiology Nitric Oxide Synthase O2 NO ONOO Binds to heme moiety of guanylate cyclase Conformational change of the enzyme Increased activity production of cGMP Modulation of activity of other proteins protein kinases phosphodiesterases ion channels Physiological response relaxation of smooth muscles inhibition of platelet aggregation etc 2 Iron sulfur proteins and free radical signaling Active protein Fe Inactive protein ROS Fe Sulfide cysteine thiolate groups in non heme iron proteins Mammalian 4Fe 4S aconitase citric acid cycle citrate isocitrate 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 cysteine residues 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 ROS Endogenous sources of ROS and RNS Environmental factors Oxidant antioxidant balance From Dr ge W 2002 Physiol Rev 82 47 95 Thiol disulfide redox state and signaling by ROS Bacterial OxyR is a model case of a redox sensitive signaling protein that may be activated by either H2O2 or changes in intracellular GSH content From Dr ge 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 OxyR 3 Thiol disulfide redox state and signaling by ROS Mixed disulfide formation ROS SH Disulfide formation SH S S ROS Alteration of proteinprotein interactions Change in protein conformation by ROS ROS delete add H2O2 Sensor yAP 1 Change in conformation Cys rich domain at C terminus overexpress No sensor Translocation to the nucleus No binding DNA binding No change in gene expression Changes in gene expression SOD Grx Catalase etc No protection Protection Redox signaling by protein degradation Hypoxia Inducible Factor 1 HIF 1 Nuclear Factor B Cytokines other signals ROS I B p65 p65 I B Polyubiquitylation p50 p65 p50 Degradation Transcription From Dr ge W 2002 Physiol Rev 82 47 95 4 Redox signaling by DNA damage Redox signaling by lipid peroxidation DNA adducts Protein adducts MDA Membrane lipids and their role in signaling Nature Reviews Molecular Cell Biology 1 31 39 2000 Models of how signalling could be initiated through raft s A In these models signalling occurs 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 5 Lipid peroxidation products and receptor signaling ROS sensitive targets in signaling cascades Lipid particle mediated activation of macrophages ROS NF B Cytokines From Dr ge W 2002 Physiol Rev 82 47 95 Alterations of Ca2 signaling by ROS Ca Channel Na Ca antiport Ca2 Binding Protein Ca ATPase Ca2 i Ca ATPase Endoplasmic Reticulum Ca2 IP3 R Oxidants can directly increase Ca2 i Oxidants can alter Ca signaling PKC cysteines in regulatory domain 6 Bi functional and mono functional inducers Bi functional inducers Compounds that can induce both Phase I monooxygenases and Phase II GST NQO1 enzymes Mono functional inducers Compounds that can only regulate Phase II enzymes GST NQO1 Modified from Whitlock et al 1996 FASEB J 10 809 818 Antioxidant Response Element ARE Transcriptional regulation of the rat GSTA2 and NQO1 genes by bifunctional and monofunctional inducers The bifunctional inducers and the dioxin TCDD bind to and activate the AhR which then translocates into the nucleus and associates with ARNT to activate transcription through the XRE The bifunctional inducers can also activate transcription through the ARE via a separate pathway following their biotransformation