GLUTATHIONE TRANSFERASES

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GLUTATHIONE TRANSFERASES. Ralf Morgenstern Institute of Environmental Medicine Karolinska Institutet. Current themes. GSTs and intracellular signalling pathways MAPEG and eicosanoid signalling Redox regulation (Protein S-glutathionylation) Oxidative stress protection - PowerPoint PPT Presentation

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  • GLUTATHIONE TRANSFERASES

    Ralf MorgensternInstitute of Environmental MedicineKarolinska Institutet

  • Current themes GSTs and intracellular signalling pathwaysMAPEG and eicosanoid signallingRedox regulation (Protein S-glutathionylation) Oxidative stress protectionDrug resistance in tumorsChemo-preventionTools for bioengineering

  • THREE SUPERFAMILIES SOLUBLE GLUTATHIONE-TRANSFERASES (25 kDa, dimers) aerobic organisms MEMBRANE BOUND GLUTATHIONE- TRANSFERASEs (17 kDa, trimer) aerobic organisms FOSFOMYCIN RESISTANCE PROTEIN (Fos A) (16 kDa, dimer) bacterial

  • FOSFOMYCIN RESISTANCE (Fos A)Bacterial (plasmid or chromosomal)SpecificFosfomycin is a stable! epoxide that inhibits cell wall-synthesis in bacteriaFosfomycin (antibiotic)

  • CYTOSOLIC GLUTATHIONE TRANSFERASESSEVERAL FAMILIES: alfa, mu, pi, theta, sigma, zeta, omega, beta, phi (incl. 1)

    Form dimers:Within a familyhomo- and heterodimersMonomers:

  • Evolutionary aspectsThioredoxinfoldDomainadditionDomaininsertionGST ThetaMitochondrialGST KappaCytosolic GSTs Alpha, Mu, Pi,Sigma, Beta Zeta, Omega, Phi, Tau,Delta, etc

  • Human soluble GSTsEnzyme Nomenclature: GSTP1-1 or GSTA1-2.

    GenefamilyalphamuthetapizetasigmakappaomegaGenesA1-A5M1-M5T1,T2P1Z1S1K1O1Chromo-some6p1p22q11q14q4q7q10q

  • Tissue-distribution (human)1, Standard2, brain3, heart4, kidney5, liver6, lung7, pancreas8, prostate9, muscle10, intestine11, spleen12, testisSherratt et al., Biochem. J. (1997) 326, 837

  • DIMER-STRUCTUREG-siteH-site

  • GSH binding

  • Making GSH more reactiveGS- thiolate is 109 times more reactive than the protonated thiol(Thiolate/CDNB 5 M-1 s-1; Selenolate/CDNB 23 M-1 s-1)Arg+pKaGSH lowered from 9to 6 in the enzyme

  • GSH is bound in anExtended Conformationwhere all possibleinteractions are usedTyr-OHGS- thiolate

  • An model second substrate and convenient assay

  • New fluorogenic substratesGSTlander et al, Anal. Biochem. (2009) 390, 52.

  • The H-site

  • Multiple Functions

  • Reactive compounds are common in biologyCyanobacteria: microcystine

    GSH Mustard oil: allylisothiocyanate

  • Reactive compounds are formed continuously in the cellLipid peroxidation gives rise to:Hydroxyalkenals:Hydroperoxides:

  • Conjugate export and processingGSH conjugates are exported out of the cell by membrane transporters called MDR (multidrug resistance proteins)Conjugates are often processed to mercapturic acids before excretion in urine or bile

  • Knock-outsGSTP null mice are more susceptible to skin and lung cancerGSTA4 null mice are more susceptible to bacterial infection and oxidative stressGSTBeta null bacteria are more susceptible to oxidative stress

  • Genetic variation in human glutathione transferase MuEnglish45%Japanese48%Indian35%Micronesia100%Chinese58%French43%Scots62%% of population that are homozygous deleted for the gene.Persons that lack the gene are more susceptible to certain forms of cancer.

  • Drug resistanceBCNU (cytostatic drug)Up-regulation of GST seen in many tumours could contribute to resistance

  • GST protectionYang et al JBC276, 19220H2O2 is not a substrate for GTSs

  • GSTP knockout leads to increased c-Jun signalling= increased proliferationGSTPGSTPGSTPGSTPStress (H2O2)GSTPJNKJNKC-JunP

  • GSTP catalyses protein S-glutathionylation (H2O2 challenge)Townsend et al JBC 284, 436Tyr 7, and Cys 47/101

  • GSTP & Prdx6 = GSH Peroxidase

  • Regulation by InductionGLUTATHIONE-TRANSFERASE-ACTIVITYin butterfly larvae

    depends on dietand treatment withchemicals: e.g.Endosulfan (insecticide)DIET

  • Chemoprevention depends on Nrf2 regulationNrf2Reactive compoundsNrf2Antioxidant Response Element-SHCytosolGSTsQuinone reductaseGSH synthesisnucleiKeap

  • Multiple subcellular distributionMGST1: Endoplasmic reticulum, outer mitochondrial membrane and plasma membrane

    Soluble GSTs: Cytosol, mitochondria, nucleus and some forms show affinity for (plasma) membrane(s)

  • GSTP, Cytosolic and more

  • The MAPEG superfamilyMAPEG = Membrane Associated Proteins in Eicosanoid and Glutathione metabolism Membrane bound glutathione transferasesProstaglandin E2 synthaseLeukotriene C4 synthase5-Lipoxygenase activating protein

  • The MAPEG theme: reactive lipidOxygenated arachidonic acidProstaglandin ELeukotrienesPeroxidized lipidsDetoxification by Microsomal Glutathione Transferases (MGSTs) 1-3

  • MGST1 TRIMER 3-D model (3.2 )

  • Peroxidized lipid substratesAs GlutathionePeroxidases (GPX) Conjugation ofreactive lipidperoxidationproducts

  • cGST/GPX1PHGPX4MGST1Location, location, location....

  • Cellular protection by MGST1MGST1Trans-fected

  • MGST1 knockout flies display shorter life spanKnock-outsToba & Aigiki, Gene, 253, 179 (2000)

  • SPECIFIC FUNCTIONSStimuliAAFLAP*5-LOLTA4LTC4S*LTC4Airway-tonus(Asthma)AAPGH2COXPGES*PGE2FeverPainInflam-mation

    *MAPEG members

  • PGE synthaseRequires GSHOHOH

  • MGST2

    MGST3

    LeukotrieneC4 synthase5-Lipoxy-genaseactivatingproteinPGES

    Glutathione peroxidasesTissue distribution:WIDENARROWGST:sNARROWGSH-dep.oxido-reductase

  • MGST1 activation

  • MGST1 is activatedby sulfhydryl reagentsSHSHSH+ NEMSNEMSNEMSNEM2 mol/min mg30 mol/min mgAt the single cysteine-49 of the homo-trimer (subunits Mr 17 kDa)Activation does occur under toxic and oxidative stress in vivo!

  • Thiolate anion formation is activatedActivation increasesthe rate of thiolateanion formation(not the chemical step)

  • Activation of MGST1 by reactive intermediates in vivo (2-3 fold)Diethylmaleate (direct)CCl4 CCl3P450AcetaminophenReactive quinoneimineP450

  • Activation Mechanisms of MGST1

  • Activation of MGST1 by S-thiolationIn vitro by GSSGSies et al, ABB 322, 288

  • Capacity and throughput

  • Glutathione transferasesHighly abundant and diverse protection from reactive electrophilesNew functions in cell signalling and redox processesDynamic regulationDefined chemical transformations of important endogenous mediators and metabolitesRelevance to inflammation, drug development, drug resistance, anti-carcinogenesis, antibiotic resistance and agriculture.

  • Examples of drugs that are conjugated to GSHParacetamol (analgesic, antipyretic)Carbamazepine (analgesic)Indomethacin (anti-inflammary)

  • Paracetamol

  • Indomethacin

  • The nucleophile substrateMost Aerobic Organisms contain:

    mM concentrations of the low molecular weight nucleophile, Glutathione, g-L-Glu-L-Cys-Gly (GSH)

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