Glutathione Homeostasis & Glutathione S- Transferases

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Moris Banks
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1 Glutathione Homeostasis & Glutathione S- Transferases

2 So why do we even need to worry about ANTIoxidants? Oxidative damage has been linked with many human pathologies: aging; heart disease; diabetes; neurodegenerative syndromes; cancer Isn t oxygen critical to the Earth s existence?

3 Evolution of Oxygen Levels in Earth s Atmosphere Carboniferous Volume % Oxygen Precambrian Millions of Years before Present

4 Evolution of Oxygen Levels in Earth s Atmosphere Carboniferous Volume % Oxygen Precambrian Millions of Years before Present

5 Why is oxygen important? Glycolysis Glucose + 2 NAD P i + 2 ADP 2 pyruvate + 2 NADH + 2 ATP + 2 H H 2 O + heat Oxidative phosphorylation Reaction step ATP yield Source of ATP Glycolysis preparation -2 Phosphorylation of glucose and fructose 6 phosphate Glycolysis yield phase Oxidative decarboxylation of pyruvate Krebs cycle 4 Substrate phosphorylation 3 Oxidative phosphorylation: NADH yields 2 ATP following membrane transport 5 Oxidative phosphorylation 2 Substrate phosphorylation 15 Oxidative phosphorylation 3 Oxidative phosphorylation

6 Mitochondria and oxygen Electron transport chain Q cytochrome c reductase (complex III) produces a radical intermediate and can be leaky releasing electrons in the cell

7 What are free radicals? Although molecular oxygen (O 2 ) has two unpaired electrons in two different orbitals, it is not a free radical A free radical is an atom or molecule with a single unpaired electron.

8 The leaky mitochondrial system produces Reactive Oxygen Species that include: superoxide, (O 2.- ) hydrogen peroxide (H 2 O 2 ) hydroxyl radicals (. OH) Evolution has coopted some as important signaling molecules: Hydrogen peroxide (H 2 O 2 ) Nitric oxide (NO) Hydrogen sulfide (H 2 S) Carbon monoxide (CO)

9 Atomic Number: 16 Atomic Radius: 104 pm Atomic Weight: Electron Configuration: [Ne]3s23p4 Oxidation States: 6, 4, 2, -2 # cysteines in genome Biological complexity 200,000 cysteines in human proteome

10 Historical perspectives on Glutathione and GST GSH - First described as Philothion (love of sulfur) by Pailhade 1881 Crystallized by Hopkins 1921 and renamed glutathione Boyland first described liver GST s in the 1960 s Litwack and Ketterer coined the term Ligandin in the 1970 s

11 Glutathione Pathways S-glutathionylation

12 GSH Capacity and Turnover CAPACITY: 0.2 mm Glutathione transferase in liver + 5 mm GSH = 25 turnovers empties the liver of GSH (e.g. acetaminophen overdose). Theoretically this can happen in less than a second!!!! TURNOVER. Humans excrete 0.1 mmol glutathione conjugates per day = Equal to one turnover per enzyme every second day

13 GST - 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

14 Evolutionary relationships of GST s Methanobacteria Dechloromethane dehalogenase Theta ~5% identity Omega Zeta Mu Man Sigma ~25% identity Alpha Squid lens crystallin Pi

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

16 DIMER-STRUCTURE H-site G-site

17 MGST1 TRIMER 3-D model (3.2 Å)

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

19 Human soluble GSTs Gene family alpha mu theta pi zeta sigma kappa omega Genes A1- A5 M1- M5 T1,T2 P1, P2 Z1 S1 K1 O1 Chromosome 6p 1p 22q 11q 14q 4q 7q 10q Enzyme Nomenclature: GSTP1-1 or GSTA1-2.

20 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

21 GSTs Mechanism and Substrate specificity

22 GSH/GST Catalytic Detoxification H 2 O H 2 O 2 GPX MRP1 GSSG GSR GSH + Xenobiotic GSTP GS- Conjugate NADPH NADP +

23 Making GSH more reactive pk a GSH lowered from 9 to 6 in the enzyme GSH GS - +H + Tyrosine or Serine (Cys) OH Arg+ GS - thiolate is 10 9 times more reactive than the protonated thiol (Thiolate/CDNB 5 M -1 s -1 ; Selenolate/CDNB 23 M -1 s -1 )

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

25 GST pi in S-glutathionylation. Catalytic Cys The catalytic cysteine is near the surface of the substrate channel and appears more available as a GS - donor.

26 GST omega in deglutathionylation Catalytic Cys The catalytic cysteine is reasonably buried within the substrate Channel and may be better suited as a GS - acceptor

27 CDNB as substrate provides the generic enzyme assay Cl GS NO 2 NO 2 GSH + + HCl NO 2 NO 2

28 GSTP seems odd Signaling kinases S-glutathionylation

29 JNK1-GSTP Complex Atomic coordinates of JNK1 crystal structure and GSTp were used to dock. The best fit for monomers of JNK1 and GSTp. Secondary structure for JNK1: helices are in Magenta, strands Yellow, for GSTp: all elements are Green. Hydrophobic contact Polar contact The points of contact on pigst: Helix (polar) - Ala15a, Ala16a, Glu12a and Glu138A; Strands - Glu26a, Glu31a, Glu33a.

30 Decrease of JNK1-Alexa 546 fluorescence, % πgst A, KD=179 +/ nm πgst B, K D =219 +/ nm πgst C, K D =212 +/ nm πgst D, K D =209 +/ nm [GST-QSY 35], µm Purified JNK1 and πgst were labeled with Alexa 456 and QSY 35 chromophores correspondingly. JNK1-Alexa 456 complex was titrated with πgst-qsy 35 and decrease in Alexa 456 emission was recorded. 50% decrease in emission is corresponding to K D of complex.

31 Cys47 and Cys101 are required for GST:JNK protein interactions Hsp27 TRAF2 c-jun JNK GSTP c-jun JNK P c-jun P From: Townsend et al, JBC: 284, , 2009 APOPTOSIS PROLIFERATION

32 GSTP is a resident ER Protein ER Cytoplasm Nucleus SERCA2 Calnexin GAPDH Histone H3 GSTP Gstp1/p Ye et al, ARS 2016

33 GSTP co-localization with ER resident proteins. DAPI - nuclear Calnexin GSTP Merge - TRIO DAPI - nuclear BIP (GRP78) GSTP Merge - TRIO

34 Nrf2: a master regulator of antioxidant response

35 Is cancer chemoprevention a good idea? Isothiocyanates: sulforphane Nrf2 Tumeric/curcumin

36 KRas G12D /BRafV 619E /Myc ERT2 Electrophiles from curcumin or isothiocyanates MEK/ERK/Myc pathways Keap1-SH Nrf2 ARE driven antioxidant gene battery [and others] Cellular ROS TUMORIGENESIS Cell reduction potential GSH:GSSG Stimulation of protein S-glutathionylation?

37 Some scientists display an artificial certainty on some matters that might be interpreted as crossing into advocacy.

38 Yefim Manevich Danyelle Townsend Zhiwei Ye Christina Grek Jie Zhang Funding Sources: National Institutes of Health, NCI (CA08660 and CA117259) National Institute of General Medical Sciences (8 P20 GM ) South Carolina SmartState program.

GLUTATHIONE TRANSFERASES. Ralf Morgenstern Institute of Environmental Medicine Karolinska Institutet

GLUTATHIONE TRANSFERASES. Ralf Morgenstern Institute of Environmental Medicine Karolinska Institutet GLUTATHIONE TRANSFERASES Ralf Morgenstern Institute of Environmental Medicine Karolinska Institutet GSTs How many enzymes Structures THREE SUPERFAMILIES SOLUBLE GLUTATHIONE-TRANSFERASES (25 kda, dimers)