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GLUTATHIONE TRANSFERASES

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

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GLUTATHIONE TRANSFERASES

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  1. GLUTATHIONE TRANSFERASES Ralf Morgenstern Institute of Environmental Medicine Karolinska Institutet

  2. Current themes • GSTs and intracellular signalling pathways • MAPEG and eicosanoid signalling • Redox regulation (Protein S-glutathionylation) • Oxidative stress protection • Drug resistance in tumors • Chemo-prevention • Tools for bioengineering

  3. 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

  4. FOSFOMYCIN RESISTANCE (Fos A) • Bacterial (plasmid or chromosomal) • Specific • Fosfomycin is a stable! epoxide that inhibits cell wall-synthesis in bacteria Fosfomycin (antibiotic)

  5. CYTOSOLIC GLUTATHIONE TRANSFERASES • SEVERAL FAMILIES: alfa, mu, pi, theta, sigma, zeta, omega, beta, phi (incl. ≥1) Monomers: Form dimers: Within a family homo- and heterodimers

  6. Evolutionary aspects Domain addition GST Theta Cytosolic GSTs Alpha, Mu, Pi, Sigma, Beta Zeta, Omega, Phi, Tau, Delta, etc Thioredoxin fold Mitochondrial GST Kappa Domain insertion

  7. Human soluble GSTs Enzyme Nomenclature: GSTP1-1 or GSTA1-2.

  8. Tissue-distribution (human) 1, Standard 2, brain 3, heart 4, kidney 5, liver 6, lung 7, pancreas 8, prostate 9, muscle 10, intestine 11, spleen 12, testis Sherratt et al., Biochem. J. (1997) 326, 837

  9. DIMER-STRUCTURE H-site G-site

  10. GSH binding

  11. GSH GS- +H+ OH Tyrosine or Serine (backbone amide?) Making GSH more reactive Arg+ pKaGSH lowered from 9 to ≈ 6 in the enzyme GS- thiolate is 109 times more reactive than the protonated thiol (Thiolate/CDNB ≈ 5 M-1 s-1; Selenolate/CDNB ≈ 23 M-1 s-1)

  12. GSH is bound in an Extended Conformation where all possible interactions are used GS- thiolate Tyr-OH

  13. Cl GS NO2 NO2 GSH + + HCl NO2 NO2 An model second substrate and convenient assay

  14. New fluorogenic substrates GST Ålander et al, Anal. Biochem. (2009) 390, 52.

  15. The H-site

  16. Aflatoxin (carcinogen) BCNU (cytostatic) Atrazine (herbicide) Multiple Functions

  17. Reactive compounds are common in biology • Cyanobacteria: microcystine • Mustard oil: allylisothiocyanate GSH

  18. Reactive compounds are formed continuously in the cell Lipid peroxidation gives rise to: Hydroxyalkenals: Hydroperoxides:

  19. N-Acetylation g-L-Glu-L-Cys-Gly L-Cys-Gly L-Cys N-Ac-L-Cys SX SX SX SX g-L-Glu Gly Conjugate export and processing • GSH 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

  20. Knock-outs • GSTP null mice are more susceptible to skin and lung cancer • GSTA4 null mice are more susceptible to bacterial infection and oxidative stress • GSTBeta null bacteria are more susceptible to oxidative stress

  21. Genetic variation in human glutathione transferase Mu English 45% Japanese 48% Indian 35% Micronesia 100% Chinese 58% French 43% Scots 62% % of population that are homozygous deleted for the gene. Persons that lack the gene are more susceptible to certain forms of cancer.

  22. Drug resistance BCNU (cytostatic drug) Up-regulation of GST seen in many tumours could contribute to resistance

  23. GST protection H2O2 is not a substrate for GTSs Yang et al JBC276, 19220

  24. GSTP GSTP knockout leads to increased c-Jun signalling= increased proliferation GSTP Stress (H2O2) GSTP GSTP P C-Jun GSTP JNK JNK

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

  26. GSTP & Prdx6 = GSH Peroxidase

  27. + endosulfan + endosulfan Willov leaves Apple leaves Regulation by Induction GLUTATHIONE- TRANSFERASE- ACTIVITY in butterfly larvae depends on diet and treatment with chemicals: e.g. Endosulfan (insecticide) DIET

  28. O S N C S Sulphoraphane Glukosinolat Chemoprevention depends on Nrf2 regulation Reactive compounds Keap -SH Nrf2 GSTs Quinone reductase GSH synthesis Cytosol Nrf2 Antioxidant Response Element nuclei

  29. Multiple subcellular distribution • MGST1: Endoplasmic reticulum, outer mitochondrial membrane and plasma membrane • Soluble GSTs: Cytosol, mitochondria, nucleus and some forms show affinity for (plasma) membrane(s)

  30. GSTP, Cytosolic and more

  31. The MAPEG superfamily • MAPEG = Membrane Associated Proteins in Eicosanoid and Glutathione metabolism • Membrane bound glutathione transferases • Prostaglandin E2 synthase • Leukotriene C4 synthase • 5-Lipoxygenase activating protein

  32. The MAPEG theme: reactive lipid Oxygenated arachidonic acid Peroxidized lipids Detoxification by Microsomal Glutathione Transferases (MGSTs) 1-3 Prostaglandin E Leukotrienes

  33. MGST1TRIMER3-D model(3.2 Å)

  34. Peroxidized lipid substrates Conjugation of reactive lipid peroxidation products As Glutathione Peroxidases (GPX)

  35. Location, location, location.... cGST/GPX1 MGST1 PHGPX4

  36. Cellular protection by MGST1 MGST1 Trans- fected

  37. MGST1 knockout flies display shorter life span Toba & Aigiki, Gene, 253, 179 (2000) Knock-outs

  38. SPECIFIC FUNCTIONS Airway- tonus (Asthma) LTC4 LTA4 LTC4S* AA 5-LO PGE2 FLAP* Stimuli Fever Pain Inflam- mation PGH2 PGES* AA COX *MAPEG members

  39. PGH2 PGE2 PGE synthase OH Requires GSH OH

  40. MGST1 Tissue distribution: NARROW WIDE NARROW MGST2 MGST3 5-Lipoxy- genase activating protein PGES Leukotriene C4 synthase GSH-dep. oxido- reductase GST:s Glutathione peroxidases

  41. MGST1 activation

  42. N O O MGST1 is activated by sulfhydryl reagents SH SH SH SNEM SNEM SNEM + NEM 2 µmol/min mg 30 µmol/min mg At the single cysteine-49 of the homo-trimer (subunits Mr ≈ 17 kDa) Activation does occur under toxic and oxidative stress in vivo!

  43. Thiolate anion formation is activated Activation increases the rate of thiolate anion formation (not the chemical step)

  44. Activation of MGST1 by reactive intermediates in vivo (2-3 fold) Diethylmaleate (direct) Acetaminophen P450 P450 CCl4 • CCl3 Reactive quinoneimine

  45. Activation Mechanisms of MGST1

  46. In vivo by hydroperoxide GSSG/GSH ratio = 50 at half maximal activation Activation of MGST1 by S-thiolation In vitro by GSSG Sies et al, ABB 322, 288

  47. CAPACITY: 0.2 mM Glutathione transferase in liver + 5 mM GSH = 25 turnovers empties the liver of GSH (e.g. paracetamol overdose) Theoretically this can happen in less than a second!!!! THROUGHPUT: Humans excrete 0.1 mmol glutathione conjugates per day = Equal to one turnover per enzyme every second day CONCLUSION Glutathione dependent protection has to be highly abundant and efficient to serve as an interception system Capacity and throughput

  48. Glutathione transferases • Highly abundant and diverse protection from reactive electrophiles • New functions in cell signalling and redox processes • Dynamic regulation • Defined chemical transformations of important endogenous mediators and metabolites • Relevance to inflammation, drug development, drug resistance, anti-carcinogenesis, antibiotic resistance and agriculture.

  49. Examples of drugs that are conjugated to GSH • Paracetamol (analgesic, antipyretic) • Carbamazepine (analgesic) • Indomethacin (anti-inflammary)

  50. Paracetamol

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