Glutathione Metabolism

&

Glutathione S-transferase

Danyelle M. Townsend Director,

Analytical Redox Biochemistry

Nebraska, June 11, 2019

https://www.medicalnewstoday.com/articles/318652.php

• 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

Historical perspective on GSH and GST

• Thiol within cysteine and gamma-glutamyl bond are key !!!!

• High concentration (5 mM) in cells

• Reduced (GSH) and oxidized forms (GSSG)

– 10-100:1 ratio in most cells

Glutathione (GSH)

Reduced form shown

https://www.google.com/search?q=glutathione+reductase&client=firefox-b-

1&source=lnms&tbm=isch&sa=X&ved=0ahUKEwjo09CZgvPXAhWCYd8KHfq6CvAQ_AUICigB&biw=1803&bih=1135#imgrc=dm_o7qk6RUJz-M:

Maintaining GSH pools

http://physiologyonline.physiology.org/content/nips/18/5/201/F2.large.jpg

GSH/GSSG levels determine cell fate

glutamate + cysteine + ATP g-glutamylcysteine

+ ADP + Pi

g-glutamylcysteine

synthetase

g-glutamylcysteine + glycine + ATP

glutathione

synthetaseglutathione

+ ADP + Pi

• First step is the rate-limiting step *

• ATP is required to form the peptide bonds

Glutathione synthesis

(De-novo / Intracellular process)

Fig. 1 GSH is synthesized in the

cytoplasm through two sequential

enzymatic ATP-dependent

reactions: glutamate cysteine ligase

(GCL) links the cysteine to glutamate

and glutathione synthetase (GS)

catalyzes the bond between glycine

and γ-glutamyl-cysteine to produce

GSH. After synthesis, cytosolic GSH

is transported into subcellular

compartments.

Synthesis and localization of glutathione

Scire et al, Biofactors, 45 (2); 152-168, 2019

• Also known as g-glutamyl peptidase

• Transmembrane protein in high concentrations in liver, kidney, and pancreas

• Salvage pathway for GSH

• Elevated in liver disease

• **Diagnostic marker **

g-Glutamyl transferase (GGT)

https://www.oumedicine.com/images/ad-cell-biology/haniganfigure.jpg?sfvrsn=2

GGT is released in circulation

following liver damage (Biomarker)

Biol Chem. 2009 March ; 390(3): 191–214. doi:10.1515/BC.2009.033

GSH Dependent Reactions

• Example: assists in disulfide bond formation of

newly synthesized proteins

– Mediated by chaperone proteins

Disulfide Bond Formation:

Protein Structure & Conformation

GSHGSSG

Properly folded,

functional protein

Newly synthesized,

partially folded protein

13

Most abundant cellular

thiol

Involved in:

Detoxification

Transport

Metabolism

Co-factor / substrate

Protein Structure

S-glutathionylation

Summary: GSH

GSH/GST Catalytic Detoxification

GSH

GSSG

GPX

NADP+NADPH

GSTP+ Xenobiotic

GS-

ConjugateGSR

H2O2H2O

MRP1

But for anticancer drugs, Kcat values are bad

Zeta

Dechloromethane

dehalogenase

Mu (M)

Theta

Alpha (A)

Pi (P)

Sigma~25%

identity

~5%

identity

Methanobacteria

Squid lens crystallin

Man

Omega

mGST

Thioredoxin

fold

Evolutionary relationships of GSTs

Glutathione S-Transferase family

• Isozyme families 22 to 29kD (catalysis as dimers)

• Catalyze GSH conjugation, thioether bond formation

• Promiscuous substrates; weak binding affinities

• High levels in many solid tumors (drug resistant)

• Have significant ligand binding properties (first described by Boyland as “ligandin” for heme/bilirubin)

• Human polymorphisms prevalent

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

• Homo-trimmer;

• Highly expressed membrane bound protein

• (3- 5% of proteins in the membrane of ER or Mitochondria);

• Selenium-independent phospholipid peroxidase that has overlapping substrates with GPx4.

Microsomal GST (mGST)

Microsomal glutathione transferase 1: mechanism and functional roles: Ralf

Morgenstern, Jie Zhang & Katarina Johansson: Drug Met Reviews:

• Knocking down Mgst1 is embryonically lethal (day E10) in mice and causes failure of cardiac development, and lipid peroxidation and protein carbonyl contents are increased in heterozygotes;

• Knock-down experiments in zebrafish embryos revealed a critical role for MGST1 in hematopoiesis;

Bräutigam et al

Redox Biology 2018

MGST1 & Hematopoiesis

zfMGST1 influences both the myeloid and

lymphoid lineages in hematopoiesis

96 hpf

Knock-down of zfMGST1 leads to

reduction of melanocytes

48 hpf

0

0.2

0.4

0.6

Control UV250 uv500

Tyors

inas

e ac

tivit

y

B16-f1 Non-…

0

0.05

0.1

Control UV250 UV500

Tyro

sinas

e ac

tivit

y

UVR effect on 1205Lu cellsEV

0

0.01

0.02

0.03

0.04

0.05

Non-Target Mgst1 KD

SK-Mel-28

Control UV

MGST1 knockdown diminishes Tyrosinase activity &

enhances sensitivity to UV radiation in mouse and human

melanoma cells

NonTarget Mgst1 KD

0 h 16 h 0 h 16 h

MNT-1

B16-f1

SK-Mel-28

MGST1 knockdown suppresses migration in melanoma cells

Summary: MGST• MGST knockdown suppresses hematopoiesis

• Reduces melanocytes, heme and melanin

• Melanin is a critical midline antioxidant in zebrafish

• In human melanomas, MGST knockdown also

reduces melanin and sensitizes B16 to drugs

• Cysteinyldopa is likely a product of MGST

catalysis, altering pheomelanin.

CYTOSOLIC GSTs

• SEVERAL FAMILIES:

alpha, mu, pi, theta, sigma, zeta, omega,

beta, phi (incl. ≥1)

Form dimers:

Within a family

homo- and

heterodimers

DIMER-STRUCTURE

G-site

H-site

GSH/GST Catalytic Detoxification

GSH

GSSG

GPX

NADP+NADPH

GSTP+ Xenobiotic

GS-

ConjugateGSR

H2O2H2O

MRP1

But for anticancer drugs, Kcat values are bad

28

Drug-Metabolizing Enzymes

Pharmacogenomics: Translating Functional Genomics into Rational Therapeutics. Evans and

Relling Science 1999

Most DMEs have clinically relevant polymorphisms

Those with changes in drug effects are separated from pie.

Cytosolic GST 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

GSTs are induced via Nrf2

regulation (and several other pathways)

Nrf2

Reactive compounds

Nrf2

Antioxidant

Response Element

-SH

Cytosol

GSTs

Quinone reductase

GSH synthesis

S

O

N C S

Sulphoraphane

Glukosinolat

nuclei

Keap

Drug Metabolism Reviews 43

Cytosolic GST Knockout Mice

Gstp1/p2-/- mice

HPC : hematopoietic progenitor cells

`✓

GSTP knockout mice

Functional Roles of GSTP

20th century• Ligand binding/ transport (e.g. steroids; bilirubin; heme; NO)

• Enzymatic catalysis/ detoxification

21st century• Protein:protein interactions/ chaperones?

• Endogenous regulation of kinase mediated signaling

• Drug target linked to proliferative pathways

• Forward reaction in post-translational S-glutathionylation

• GGT and peptidase cleave g-

Glu and Gly

• Acetylation

Electrophile Detoxification

• Overdose of acetaminophen depletes GSH, liver

damage occurs

• N-Acetylcysteine as antidote

– Protects liver by restoring glutathione levels

– Most effective within 8-10 h of overdose

Figure From: Basic Concepts in Medicinal Chemistry, Harrold and Zavod, Fig 8-24, p. 306

APOPTOSIS PROLIFERATION

The role of GSTπ and Kinase Signaling

P

JNK

c-jun

GSTπ

P

S-glutathionylated

oligomer

c-jun

JNK

c-jun

GSTπ

S-glutathionylation

of multiple targets

GSTπ

Townsend, DM, 2005, Mol Interventions

HSC HSC

ROS

Thiol

Low High

Quiescent

Self-renew

Bone Marrow

Peripheral

blood

Osteoblastic nicheVascular niche

HSC : hematopoietic stem cells;

ROS: reactive oxygen species

Differentiation

proliferation

Increased cell proliferation in Gstp1/p2−/− bone marrow cell populations.

Brd

Up

osi

tive

cell

s

(%o

fW

Tce

lls)

0

200

250

WT

Gstp1/p2-/-

*

1

5

0

*

1

0

0

5

0

P<0.05Lin(-) cells BMDDCs

PloS One. 2014; 9(9).Glutathione S-transferase P influences redox and migration pathways in bone marrow.

Townsend et al JBC 284, 436

GSTP catalyses S-glutathionylation

Tyr 7, and Cys 47/101

PEROXIREDOXINSperoxidases that catalyze the reduction of ROS

Peurto-Galan et al., Front. Plant Sci., 19 August2013

Heterodimerization and S-glutathionylation of 1-cysPrx.

Y. Manevich et al. PNAS 2004;101:3780-3785

©2004 by National Academy ofSciences

•GSTP1*A: Ile105∞Ala114

•GSTP1*B: Val105∞Ala114

•GSTP1*C: Val105∞Val114

•GSTP1*D: Ile105∞Val114

Human Polymorphisms of GSTP

Post-translational regulation of Prdx

Y. Manevich, S. Hutchens, K.D. Tew, D.M. Townsend

Free Radical Biology and Medicine, Volume 54, 2013, 62–70

Allelic variants of GSTP differentially mediate the peroxidase function of

peroxiredoxin VI and alter membrane lipid peroxidation

GSTP & Prdx6 = GSH Peroxidase

Manevich, Townsend, Tew et al, FRBM 51, 299.

Activation of TLK199 to TLK117, a GST inhibitor.

Source: Drug Metabolism, Goodman & Gilman's: The Pharmacological Basis of Therapeutics, 13e

Citation: Brunton LL, Hilal-Dandan R, Knollmann BC. Goodman & Gilman's: The Pharmacological Basis of Therapeutics, 13e; 2017 Available at:

http://accessmedicine.mhmedical.com/content.aspx?sectionid=167889421&bookid=2189&jumpsectionID=172473810&Resultclick=2

Copyright © 2017 McGraw-Hill Education. All rights reserved

GSTP activated prodrugs

• Highly expressed in tumors & drug resistant cells – although the drugs are not substrates for GSTP

• C-jun kinase regulation through protein-protein interaction

• Reactivates peroxiredoxins

• Modulates hematopoiesis

• Mediates S-glutathionylation of redox sensitive cysteine containing proteins

Summary: GST pi

Questions???

Glutathione Pathways

Post-translational

glutathionylation

Capacity and throughput

CAPACITY:

0.2 mM GST 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!!!!

Humans excrete 0.1 mmol GSH conjugates/ day = Equal to one

turnover per enzyme every second day