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Overview of oxidative stress and

antioxidant defence s ystem

Lee, Tae-Hoon, Ph.D

Lab. of Oral Biochemistry,

College of Dentistry, Chonnam Natl'

U niv.

Today's presentation

Oxidative stress

Eye

Vessels

Multi-organ

Lung Heart Skin Brain

Kidney

D e rmatitis

Psorias

i s

AngiopathyKeshan

disease

RheumatoidAthritis

AsthmaARDSHyperoxia

TraumaStrokeParkinson's diseaseAlzheimer's disease

RadiationAgingCancerDiabetes

VasospasmAtherosclerosis

Ischemic B

o wel

Endotoxin

Liver injury

Degenerative retinal disease

Ant Ant Ant Ant Ant Ant Ant Ant Ant Ant

Renal graftGlomerulonephritis

GI Joint

Oxidative Stress•

The O 2 is major promoters of radical reaction. • Oxidative stress is imposed on cells as a result of

one of three factors: 1) an increase in oxidant generation,2) a decrease in antioxidant protection, 3) a failure to repair oxidative damage.

Cell & tissue damage by ROS

ROS

NUCLEIC ACID

LIPID

PROTEIN

Degeneration

Disactivation

Peroxidation

Mutation

Damage of cell membrane & cellular components

Myocardial i

n farcti o n,

Cerebral apoplexy,

Arteriosclerosis

Ca ncer

Cellular aging

Redox b alance

Antioxidant defence

Oxidative stress

O 2 .- OH .- H 2 O 2 ONOO - GSSG

MDA/4-HNE

Etc...

SOD

Catalase

GSH peroxidase

Peroxiredoxin

¥á

-tocopherol

Ascorbic acid

GSH

Health

Survival

Ca ncer &

Apoptosis

European Journal of Biochemistry

267 (16):

4904.

ROS generation and detoxification

O 2

NADPH oxidaseRedox

cycling compounds UVA 1 O 2

QR, MT

Vitamin C and E

O 2 -• SOD

Catalase

H 2 O 2 OH •

Mono-oxigenases

h Q

Ferritin

HOx GPx

Glutathine

reductase J -Glutamylcysteine synthase

NAC, cysteine

GSSG GS H SOD

Fenton

reaction Fe 2+

Biochem. J. (1999) 342, 481.

Various factors e

l ic it intracellular ROS production

Metabolism

Phy s iological signaling

Exogenous str

ess Antio x id an t d e ficien cy (GSH depletion, enz y m e deficiency , etc)

Mitochondrial dy

sfunction In flammation

Phagocytosis

(m yelo peroxidase activit y ) Exocy t osis oxidants (e.g. peroxides) Redox c y c ling agents

UV irradiatio

Endotoxins

Intracellular

ROS production

Physical or

ch emical sen s ors In flammatorycytokines Other cytokines

Shear flo

ER overload

High H

2 O 2 IL -1 IL -2 IL -6 TNF D

GM-CSF

EGF PDGF TGF E

Homeostasis

modulation Hor m ons In sulin

Angiotensin

Biochem. J. (1999) 342, 481.

Adaptive response to oxidative stress

Adaptive

responses TF- activation (e.g . AP-1, Nf- N

B, Nrf2)

Immediately

early gen e induction (e.g . c- fo s )

Induction

of antioxidant genes : Gl utat hi one pe r o xi da se

CatalaseSODJ

-Glutamy lcy stein e sy nthase

Glutathine

reductase

ThioredoxinThioredoxin

reductase

Quinone

r eductase M eta ll ot hi one i n Heme oxygenase

Ferritin

Activation of several othe

r genes (e.g. cy tokines)

EPO gen

e expression TF inhibition (e.g . NFI)

Specific decrease in

RNA stability

CYPA1 repression Mito cho ndrial activity shutdown + Tran sferrin recep t or decrease

NADPHoxidaseinhibition

Oxidative stress

Activat

ion of antioxidant defence s

Repression

of ROS- producing systems

Biochem. J. (1999) 342, 481.

Reactive oxygen species

ROS generation

Cytokines

Growth factors,

GPCR ligands

Unknown

Lipoxygenases

NADPH oxidases

Mitochondria

O 2 O 2 .-

Topology of Nox

e nzymes Nox a ctivation signal

Nox-dependent signalings

in vascular cells

ROS targets: Requirement

• I t must have a low pKa and so highly reactive cysteine residue . • A cysteine residue should be able to be oxidized easily by either air or H 2 O 2 , which in turn causes a conformational change or an activity loss. Thiol

Cys-SH

Sulfenic

Sulfinic

Sulfonic

Cys-SOH

C ys-SO 2 H C ys-SO 3 H RS-SR

Antioxidants

Cys-S-S-Cys

Disulfide

ROS

Ways that ROS work

ASK1 PTP1B

PTENCDC25LMW-PTP

ASK1 Trx SHSH PTP1 B PTEN

SHSHSH

H 2 O 2 ASK1 Trx SS PTP1 B PTEN SS S H 2 O 2 H 2 O 2 +

ActiveInacti

v e

Inacti

v e SOH N ROS 2 nd messenger

Oxidative stress

Well-controlled

Uncontrollable

Receptor-mediated

External sources

Intracellular targets

Non-specific (carpet bombing)

Rapidly elimi

n ated

Usually followed by oxidati

v e burst

Localized action

Diffusible

ROS elimination

complex+2cyt-c(Fe 2+ )

¡ae

enzyme+2cyt-c(Fe 2+ ) +2OH -

Cytochrome

c peroxidase

Trx-(SH)

2 +Protein-S 2

¡à

Trx-S 2 +Protein-(SH) 2

Thioredoxin

NADPH +GSSG

¡à

NADP + +2GSH

Glutathione reductase

RX +GSH

¡ae

RSG +HX

Glutathione S-transferase

H 2 O 2 +2GSH

¡ae

GSSG +2H

2 O

LOOH +2GSH

¡ae

GSSG +H

O +LOH

Glutathione peroxidase

2H 2 O 2

¡ae

2H 2 O + O 2

Catalase

O 2 •- +O 2 •- +2H +

¡ae

2H 2 O 2 + O 2

Superoxide

dismutase

Reaction catalysed

Enzyme

Table 1. ROS scavenging and detoxifyi

n g enzymes

Peroxiredoxin

N M VI IV

Catalase

II I Gpx III Gpx P V I I II II IV O 2 .- H 2 O 2 S[O]H [AP-1][ NFKB ] VI OH . H 2 O Cell

PDGF-induced H

2 O 2 production in MEFs (-) PDGF (+) PDGF Prx I I +/+ Prx I I -/- (Kang et al., in Ehwa

Univ.)

Specific amplification of PLC

1 activation

Individual receptor-interacting proteins

Total phosphotyrosine

(Kang et al., in Ehwa

Univ.)

Prx I

I determines amplitude of PDGFR

-PLC

1 activation

pathway

PI3 kinase

PTPs(?

) H 2 O 2 PrxII - p

579581857

The identification of ROS target molecules

in PrxII-deficient mice

Generation of PrxII

d eficient mice 700
bp (N1 + N2) 250
bp (W1 + W2) BE E H E K

Endogenous Prx

II A

Homologous recombination

W1 W2 C

Western analysis

PCR analysis

W He Ho W He Ho probe B EE H E K neo

Targeted Prx

II N1 N2

Chronic hypertrophy in spleen

200x
AB D C +/ + +/ - -/- +/ + -/-

Heinz body formation in red blood cells

A +/ + -/- +/ - 0

1020304050

1 2 3 4 567
8 1 2 0

1020304050

1 2 3 4 567
8 1 2

Age(weeks)

Rate of Heinz Body formation (%)

B +/ + +/ - -/-

Table 1. Hematology and cation

contents of PrxII knock-out mice +/+ -/- (n=14) (n=18)

HematologyWBC (10

3 / P l) 5.1 r

2.5 5.7

r 2.9

RBC (10

6 / P l) 10.4 r

0.6 9.3

r 0.9

Hemoglobin (g/dl)

+ 16.5 r

0.9 14.7

r 1.5

Hematocrit(%)

+ 54.4
r

3.1 48.8

r 5.8

Reticulocyte(%)

++ 1.8 r

0.7 3.5

r 1.0

MCV(fL)

52.0
r

2.6 52.2

r 2.4

Spleen weight(SW/BW) 0.002

0.0002 0.003

0.0003

+, P<0.0 5 ; ++, P<0.00 0 1

RBC abnormality in PrxII

-/- mice

H7H6H5H4H3H2H1

W7W6W5

-/- + /+ H7 H6 H5 H4 H3 H2 R R B Sc Sp Sp Sp W7 W6 W5

Myeloid Cell Comparison in Bone Marrow

A Wild (+/+) WH W H W H WH 35
8 1 0 (weeks)

EPO level in serum

B Homo. (-/-)

Protein target validation in PrxII

KO mice

Thiol

Sulfenic

Sulfinic

Sulfonic

Cys-SH

ROS

Cys-SOH

C ys-SO 2 H C ys-SO 3 H

Antioxidants

RS-SR

Cys-S-S-Cys

Disulfide

175

836247.532.525Actin

+/+ -/- A B +/+ -/- Detecting membrane proteins that contain sensitive cysteineresidues to oxidative stress . (A) T he BIAM indicates a biotin labeled iodoactic a mide. The

BIAM was decreased in PrxIIdeficient RBC.

(B) T he 14 C-IAM also showed that oxidized proteins were increased in RBC of PrxII-deficient mice.

Comparison of oxidized proteins

+/+ - /- KDa

8049362621

11 2

Selection of oxidized proteins

KDa112

12 3 4 5

8049362621

PI 10 to 3

Identification of oxidized proteins

Decreased IAB

labelling (

WT > HO)

25
1 5

Decreased IAB

labelling (

WT > HO)

50
2 4

Decreased IAB

labelling (

WT > HO)

45
5 3

Decreased IAB

labelling (

WT > HO)

45
5 2

Decreased IAB

labelling (

WT > HO)

45
5 1 Th e r eason th at the spot is selected

Approxi

m ate size (KDa)

Approxi

m ate quantity*

Number

actin

EFGST P

¡Ö

100 ng

Cytoskeletal

network 3 3 4 . 1 4 . 1 4 . 1 Actin

Tropomyosin

H 2 O 2 H 2 O 2 H 2 O 2 H 2 O 2

Spectrin

(Lee et al., Blood, 2003) PrxII +/+

RNA Isolation

Reverse

Transcription

Subtraction

Hybridiz

e under C overslip Scan Cy-3 Cy-5

DNA chip

PrxII -/-

Differentially expressed genes in PrxII

-/- BMCs Tr an s c r iptio n fa cto r K i nase Ph os p h a t ase Tr an sport

BindingRibosom

e ( P r ote i n) bio s y n the s i s

Cell adhesion

mol e cul e &ECM Su r f ace mo lecu le &recept o r E2 F

Foxo1My

c E2 F-4

FosPparbp TSC-22

Nfx

TK1Mnk1Cs

nk 1a Cs nk 2a2 JA k 3 Ak t

Ptpr Pt

p n16 PT P 3 5 pr otein ANT 2

ATPase-like vaculoa

r-proton cha nnel Aqp 1 eIF-p44 hn R N P

G3BP Zfp

m 1 DJ -1 Ps m c 4 Rbm 3 Z f p12 7 Rp s 5 Rp l 2 1 Rp l 3

Spp1Prg

T hym ic epitheli a l cell surface antigen

Mea1 CD

151

Ryr1Srb1Rarg

-1 5 - 1 0 -5 05 F o ld decre a s e

Fold incre

a se

List of

differentially regulated genes in PrxII -/- BM cells (1) -1 5 - 1 0 -5 0 5 F o ld decre ase F old incre a se

Structure m

o lecul e D e fe nse/Immu n i t y

Actin-cy

tos k e l e ton bindi ng protein Ce ll cy cl e or prolifer ation r e gul ator RO S r e l a t e d

Etc. en

zy me Etc. dematinKerati n comp lex 2, basic, g ene 1 Sy n1 Pp l M H C ca l s s

III regio

n R D g e n e B2m Kea p 1

Mad2 D52MPOTr

x I Xa nth i n e de hy drogena se As ns D p m 2

Gpi1 GlnsP5

40
A S L MM P1 4 M o cs2

Shd CI

R P Pl u n c dbp A H r b

Psme1SH

3 P

9

p16 k gen e for 16K

Da prote

i n Spo p Cc t 5

List of

differentially regulated genes in PrxII -/- BM cells (2)

Decreased : 20 genesIncreased : 47 genes

A. Increased candidate in Prx

-/- bone marrow cells Ppl Defen s e / Immunity Keap 1 RO

S-related

HrbPs me1

Aqp1Slc25a5

Transport

B2mMMP14Ps

mc4

Protease

Ptpr

Phosphatase

MAPK8Csnk1a1TK1

Kinase

My c

Foxo1Idh

3 b Tran scription factor -Actin -/- + /+

Etc..Enz

y me R

NA helicase

p16kDa proteinTh ymic epithelial cell surface antigen Mea1 Su rface molecule

DbpAAsnsMad

2 L Cirbp

Cell cy

cle

PluncPrg

Ce l l a dhes i on

Rpl3Rps5

Ribosome biosy

n thesis

Rbm3DJ1Zfpm1G3bphnRNPH1eIF3s4

Binding

-Actin -/- + /+

B. Decreased candidate in Prx

-/- bone marrow cells

Bin3Shd

Etc.. Mocs2 Enz y me

Gpi1Xanthine

dehy drogenase RO

S-related

Structure molecu

SpopSrb1

Su rface molecule Spp1 Ce l l a dhes i on Sy n1

MPOPtpn16

Phosphatase

Serine/Threonine

protein kinase

PrxIICsnk2a2Mnk1

Kinase

RARgGilzPparbpFos

Tran scription factor -Actin -/- + /+

C. Real-time PCR in bone marrow cells of Prx

-/- & Prx +/+ mice 0 0. 51
1. 52
2. 53
3. 5 Pr x - /- Pr x + / + m R N A le v e l o f F o x o 1 / b - a c t i n 0 0. 51
1. 52
2. 53
3. 5 P r x- / - P r x+ / + mR N A l e v e l o f M y c / b - a c t i n 0 0. 51
1. 52
2. 53
3. 54
4. 5 Pr x - /- Pr x + / + mR N A l e v e l o f Z f p m 1 / b - a c t i n 0 0. 51
1. 52
2. 53
3. 54
Pr x - / - Pr x + / + m R N A le v e l o f G3 b p / b - a ct in PI3K -/- + /+