THE MOLECULAR BASIS

OF APOPTOSIS
Oleh

Prof. dr. Purnomo Suryohudoyo

APOPTOSIS & NECROSIS

APOPTOSIS

NECROSIS

1. Occurrence

Physiological of
Pathological

Always Pathological

2. Cells
Involvement

Active

Passive

3. Inflammation

Intact
Shrinking, become
more rounded,
loose contact with
neigbouring cells
Apoptotic bodies

Damaged
Swollen

4. Horphological
features
Cell membrane
Cell shape

Final outcome
5. Cell remnant
removal

Neigbouring cells
resident MO

Cell lysas

Inflammatory
phagocyt es NO,
PMN stimult
Apoptosis can be induced by extracellular as well as intracellular

APOPTOTIC PATHWAYS
EXTRINSIC PATHWAY

INITIATED FROM OUTSIDE THE CELL

REQUIRE:
SIGNALING MOLECULE :
DEATH LIGAND
AND A SPECIAL RECEPTOR :
DEATH RECEPTOR

INTRINSIC PATHWAY

INITIATED FROM INSIDE THE CELL

REQUIRE THE PRESENCE OF NITOCHONDRIA
THESE 2 PATHWAYS MAYBE INTERCONNECTED

CASPASE ACTIVATION(1)
DEATH LIGAND
DEATH RECEPTOR

TRANSDUCTOR NOLECULES
INITIATOR CASPASE
(Caspase 8)
(FLICE)

EXCECUTOR CASPASES
Cleaved protein substrate

APOPTOSIS
FLICE : Fas like interleukin converting enzyme

CASPASE ACTIVATION(1)
DAMAGING STIMULI
Ondants
Bax
Ceramide
Ca ++

HITOCHONDRIA

OPENING OF PT-PORE
Cytochrome C
Apof-1
d-ATP

INITIATOR CASPASE (Caspase 9)

EXCECUTOR CASPASES
APOPTOSIS

PT : Permeability transition

FADD
DD
DED
TNF
TNF-R1

: DEATH DOMAIN
: DEATH EFFECTOR DOMAIN
: TUMOR NECROSIS FACTOR
: TNF-RECEPTOR 1

: Fas ASSOCIATED DEATH

DOMAIN (PROTEIN)
TRADD : TNF-R ASSOCIATED DEATH
DOMAIN (PROTEIN)
FLICE
: FADD-LIKE ICE
ICE
: INTERLEUKIN 1B
CONVERTING ENZYME

DEATH LIGANDS, DEATH RECEPTOR &

TRANSDUCTORS
LIGAND (L)
1. Fas L
(CD95L)

2. TNF
3. Apo 3 L

4. TRAIL

RECEPTOR (R)
Fas
(CD 95)
(Apo 1)
(DR 2)
TNFR
(DR 1)
Apo 3
(TRAMP)
(DR 3)
TRAIL-R1
(DR 4)
TRAIL-R2
(DR 5)
TRAIL-R3
(TRID)
(Dc R1)
TRAIL-R4
(TRUNDD)
(Dc R2)

TRANSDUCTOR
FADD

TRADD, FADD
TRADD, FADD

?
?
- (decoy
receptor)
- (decoy
receptor)

Abbreviations :
Fas : Fibroblast associated, CD : Cluster of
differentiation, APO : Apoptosis, DR : Death
receptor, DcR : decoy receptor, FADD : Fas
assoctiated death domein, TNF : tumor
necrosis factor, TRADD : TNF associated
death domain, TRAMP : TNF related
apoptosis mediating protein, TRAIL : TNF
related apoptosis inducing ligand, TRID :
TRAIL receptor without intracellular domain,
TRUNDD : truncated death domain

APOPTOSIS : ROLE OF HITOCHONDRIA

CASPASES
CASPASES
PRO-APTOPTOTIC
PROINFLAMMATORY
ANITIATOR
CASPASE 2 (?)
CASPASE 2 (?)
9
9
8
8
10 (?)
10 (?)
EFFECTOR
CASPASE 2 (?)
3
6
7
PROCASPASE
LOW
ACTIVITY
CASPASE
HIGH ACTIVITY

CASPASE SUBSTRATES
SUBSTRATES
1.
2.
3.
4.
5.
6.

ICAD
Nuclear Lamins
Gelsolin
FAK
PAK-2
DNA-PK

EFFECTS
Release of CAD
Depolymerisation

Active gelsolin
Cleavage
Cleavage
Cleavage

RESULTS
DNA Cleavage
Dissappearance of nuclear
membrane, chromatin
condensation
Actin filament degradation

Inhibition of DNA repair

Approximately 40 proteins are known substrates of caspases, however the

relationship of their cleavage to the specific morphologic changes and cell death
remain for most of them unknown.
Abbreviations :
CAD
: Caspase activated deoxy-rivonucleare
ICAD
: Inhibitor of CAD
FAK
: Focal adhesion kinase
PAK-2
: P21 activated kinase-2
DNA-PK : DNA activated protein kinase

MODULATION OF APOPTOSIS (1)

MOLECULES
1.
2.
3.
4.
5.
6.
7.
8.
9.

EFFECT

} INHIBIT

Decoy receptors (DcR)

Osteoprotegerin (?)
FLIP
IAP
Survivin (?)
AIF
Bcl-2
Bax
P53

MECHANISM
Compete with death receptor (DR)

INHIBIT

Compete with Caspase 8

INHIBIT

Inhibit Excecutor Caspases

ACCELERATE
INHIBIT
ACCELERATE
ACCELERATE

Activate Excecutor caspases

Inhibit Apaf-1
Inhibit Bcl-2
Activate Bax

Bcl FAMILY :
PRO-APOPTOSIS
Bax
Bak
Bok
Bad

Bid
Bik
Blk
MimL

ANTI-APOPTOSIS
Bcl-2
Bcl-XL
Bcl-W
CED-9

MODULATION OF APOPTOSIS

Abbreviations :
DL
FLIP
FLICE
IAP
Apaf-1
AIF

:
:
:
:
:
:

Death ligands, DR : Death receptors, DcR : Decoy receptors

FADD-like ICE inhibitory protein
FADD-like interleukin converting enzyme (FADD-like ICE)
Inhibitor of apoptosis protein
Apoptosis activating factor 1
Apoptosis inducing factor

BIOLOGICAL ROLE OF APOPTOSIS

1. REMOVAL OF UNNEEDED CELLS
(e.g. during embryonal development)
2. REMOVAL OF SENESCENT CELLS
(e.g. during tissue renewal)
3. REMOVAL OF DNA-DAMAGED CELLS
(e.g. due to genotoxic agents)
4. IMMUNE RESPONSE MODULATION
5. GRAFT REJECTION
6. MAINTENANCE OF IMMUNOPRIVILEGED SITES
Too much apoptosis leads to excessive cell death leading to degenerative disorders
Too little apoptosis leads to excessive cell proliferation leading to cancer

CASPASE
INDEPENDENT
APOPTOSIS

NMDA
: N methyl D aspartate
NOS
: Nitrogen oxide synthetase (n = neuronal)
PARP
: Poly (ADP-ribose) polymerase
PARG
: Poly (ADP-ribose) glycohydrolase
AIF : Apoptosis inducing factor
PAR
: Poly (ADP-ribose)

APOPTOSIS
TOO MUCH :
DEGENERATIVE DISEASES
E.G. : ALZHEIMER DISEASES
: C.V.A. (STROKE)
TOO LITTLE :
CANCER
MUTATION IF EXTENSIVE P53 APOPTOSIS
IF P53 IS INACTIVATED NO APOPTOSIS
CANCER

R.O.S.

(REACTIVE OXYGEN SPECIES)

Oleh

Prof. dr. Purnomo Suryohudoyo

R.O.S. (1)

ARE OXIDANTS DERIVED FROM O2

CAN DAMAGE TISSUES AND HAS
BEEN IMPLICATED IN SEVERAL
PATHOLOGICAL STATES SUCH AS :
1. CARDIOVASCULAR DISEASES
(e.g. : HEART INFARCTION)
2. RESPIRATORY DISEASES
(e.g. : EMPHYSEMA)
3. CARCINOGENESIS
4. INFLAMMATION
5. AGING (?)

R.O.S. (2)
SOURCE :
NORMAL INTRACELLULAR
- OXIDATIVE PHOSPHORYLATION
- OXYGENATION OF Hb

RELEASED BY INFLAMMATORY CELLS

(GRANUCOCYTES & MACROPHAGES) AS A

DEFENCE AGAINST MICRO-ORGANISM
EXPOSURE TO HIGH ENERGY RADIATION
- X RAYS
- RAYS

* INHALED OR INGESTED OXIDANTS (DRUGS, POLLUTANTS) OTHER

THAN ROS CAN GIVE THE SAME EFFECT AS ROS

R.O.S. (3)
NON-RADICAL
HYDROGEN PEROXIDE

H2O2

HYPOCHLORITE ION
SINGLET OXYGEN

ClO
O12

O H
HO

Cl OI
OI

OH
OOH
O2

HO

H O O

O
IO

FREE RADICAL
HYDROXYL RADICAL
PEROXYL RADICAL
SUPEROXIDE ION

O2 (GROUND STATE)
WATER (H2O)
HYDROXYL ION (OH)

**

FREE RADICALS :

O
O

H O
H
H OI

ATOMS / MOLECULES POSESSING ONE

OR MORE UNPAIRED (LONE) ELECTRON

R.O.S. (4)
1. THE REDUCTION OF O2 TO H2O INVOLVES THE
TRANSFER OF 4 (FOUR) ELECTRONS
O2 + 4 e + 4 H+ 2 H2O
2. THESE TRANSFERS OCCUR ONE ELECTRON
EACH TIME
3. ALL R.O.S. EXCEPT ClO & 1O2 CAN BE
CONSIDERED AS BEING THE RESULT OF
INCOMPLETE OXYGEN REDUCTION
1. ONE ELECTRON :

O2 + e O2 (SUPEROXIDE)
O2 + e + H+ OOH (PEROXYL RADICAL)

2. TWO ELECTRONS :

O2 + 2 e + 2 H+ H2O2 (HYDROGEN PEROXIDE)

3. THREE ELECTRONS :

O2 + 3 e + 3 H+ H2O + OH (HYDROXYL RADICAL)

OXIDANTS & FREE RADICALS

OXIDANTS : ARE ELECTRON ACCEPTORS
i.e. IT ATTRACTS ELECTRONS
Fe +++ + e Fe ++
FREE RADICALS :
BECAUSE OF THE PRESENCE OF AN UNPAIRED ELECTRON
ALSO SHOW A TENDENCY TO ATTRACT ELECTRONS AND CAN
THUS BE CONSIDERED OXIDANTS
a. ELECTRON DONOR ANOTHER RADICAL
R1 + R2 R1:R2 (R1 R2)
b. ELECTRON DONOR A NON RADICAL
R1 + R2:H (R2-H) R1:H (R1-H) + R2
(RADICAL) (NON-RADICAL)
(NON-RADICAL)
(RADICAL)
THE NEWLY FORMED RADICAL CAN THEN ATTACK OTHER (NON-RADICAL) MOLECULES

FREE RADICALS ARE OXIDANTS

BUT
NOT ALL OXIDANTS ARE FREE RADICALS

OXIDANTS & FREE RADICALS

1. FREE RADICALS ARE MORE DANGEROUS THAN
NON-RADICALS OXIDANTS BECAUSE THE CAN
TRIGGER CHAIN REACTION
2. WHEN A FREE RADICAL ATTACKS A NON-RADICAL, IT WILL RESULT
IN THE FORMATION OF A NEW RADICAL :
R1 + R2 H
R1 H + R2
3. THE NEWLY FORMED RADICAL CAN AGAIN ATTACK
A NON-RADICAL :
R2 + R3 H
R2 H + R3
4. THIS PROCESS CAN BE REPEATED AGAIN AND AGAIN RESULTING IN
A CHAIN REACTION :
R3 + R4 H
R3 H + R4
R4 + R5 H
R4 H + R5 ETC
5. SUCH A CHAIN REACTION WILL ONLY STOP WHEN 2 RADICALS
MEET :
R1 + R1
R1 R1
R1 + R2
R1 R2
R2 + R2
R2 R2
ETC

FORMATION OF R.O.S.
RELATIONSHIP BETWEEN R.O.S.
(SUMMARY)

O2 & H2O2 ARE PRIMARY R.O.S. FROM

WHICH ALL THE OTHERS ARE DERIVED

FORMATION OF OTHER R.O.S.

OOH
O 2 + H +

OOH

OH
O2 + H2O2

Fe+ + +
(Cu++ )

H2O2 + Fe++ /Cu+

ClO
1

H2O2 + Cl

O2 + OH + OH
HABER-WEISS REACTION

Fe+++ /Cu++ + OH + OH
(FENTON REACTION)

(MYELOPEROXIDASE)

O2

H2O + ClO
1

H2O2 + ClO

(MYELOPEROXIDASE)

H2O + Cl + O2

FORMATION OF PRIMARY R.O.S.

O2
TRANSITION METALS :
O2 + Fe++ (Cu+)
O2 + Fe+++ (Cu++ )
NADPH OXIDASE (INFLAMMATORY CELLS)
2 O2 + NADPH
2 O2 + NADP+ + H+
XANTHINE OXIDASE (REPERFUSION INJURY)
2 O2 + XH + H2O

2 O2 + 2 H+ + X OH

(XANTHINE)

(URIC ACID)

BY PRODUCTS OF NORMAL PROCESSES OXIDATIVE

PHOSPHORYLATION HEMOGLOBIN OXYGENATION
(FORMATION OF Hb O2)
H 2O 2
OXIDASES (ENDOPLAS MIC RETICULUM)
( PEROXISOME)
RH2 + O2
R + H2O2

EFFECT OF R.O.S.
1. ROS CAN REACT WITH MANY
SUBSTANCES :
- SIMPLE COMPOUNDS :
AMINO ACIDS, FATTY ACIDS,
- COMPLEX SUBSTANCES :
PROTEINS, DNA

CHOLESTEROL SUGAR

2. THE MOST DAMAGING EFFECT OF ROS

HOWEVER, IS ON 3 IMPORTANT
COMPONENTS OF CELLS :
-

MEMBRANE LIPIDS
PROTEINS
DNA

EFFECT OF ROS ON MEMBRANE LIPIDS

EFFECT OF ROS ON PROTEINS

EFFECT OF ROS ON DNA(1)

EFFECT OF ROS ON DNA(2)

EFFECT OF ROS ON DNA(3)

ANTI-OXIDANTS(1)
1. ORIGINAL DEFINITION IN CHEMISTRY :
ANTI-OXIDANTS = ELECTRON DONORS
EXAMPLE : Cu+
Cu2 ++ e
2. IN BIOLOGY (8 MEDICINE :
ANTI-OXIDANT = ANY SUBSTANCE THAT
PREVENTS THE FORMATION OR ACTIVITY OF
OXIDANTS
3. THIS BROADER DEFINITION IN BIOLOGY INCLUDES
NOT ONLY ELECTRON DONORS BUT OTHER
SUBSTANCES SUCH AS :
- METAL BINDING PROTEINS
- ENZYMES

ANTI-OXIDANTS(2)
ANTI-OXIDANTS CAN BE CLASSIFIED ACCORDING
A. ITS MODE OF ACTION
1. PREVENTIVE ANTI-OXIDANTS
PREVENT UNDUE ACCUMULATION OF OXIDANTS
2. CHAIN BREAKING ANTI-OXIDANTS
PREVENT PROPAGATION OF CHAIN REACTIONS
INITIATED BY FREE RADICALS
B. ITS SOLLUBILITY
1. LIPOPHILIC ANTI-OXIDANTS
FAT SOLLUBLE MOLECULES THAT ACT
IN CELL MEMBRANES :
- TOCOPHEROLS (VITAMIN E)
- CAROTENE (PROVITAMIN A)
2. HYDROPHILIC ANTI-OXIDANTS
WATER SOLLUBLE MOLECULES, ACT
IN THE CYTOSOL AND E.C.T :
- ASCORBIC ACID (VITAMIN C)
- GLUTATHIONE
- CYSTEINE
- OTHERS (e.g. URIC ACID)

PREVENTIVE ANTI-OXIDANTS
1.

2.

METAL BINDING PROTEINS

Fe : TRANSFERIN AND FERRITIN
Cu : CERULOPLASMIN AND ALBUMIN
PREVENTS : HABER-WEISS REACTION :
O2 + H2O2
O2 + OH
OH

+ OH

Fc+++ (Cu++)
FENTON REACTION :
H2O2 + Fe++ / Cu+
Fe+++ / Cu++ + OH
OH + O
ENZYMES
a. SUPEROXIDE DISMUTASE :
2 O2 + 2H+
H2O2 + O2
b. CATALASE :
2 H2O2
2 H2O + O2
c. GLUTATHIONE PEROXIDASE (GPx) :
H2O2 + 2 GSH
G-S-S-G + 2 H2O
(RED)
(OX)
ANTI-OXIDANT COMPOUNDS :
GLUTATHIONE : 2 GSH + 2 OH
G-S-S-G + 2 H2O
(RED)
(OX)
CYSTEINE :
2 CYS SH + 2 OH
CYS S S CYS + 2 H2O
(CYSTEINE)
(CYSTEINE)
ASCORBIC ACID : Asc H2 + OH
Asc + H+ + H2O
(ASCORBIC ACID)
2 Asc + 2 H+

(ASCORBYC RADICAL)
Asc H2
+ Asc
(ASCORBIC ACID)
(DEGYDRO ARCOBIC ACID, DHAA)

CHAIN BREAKING ANTI-OXIDANT

VITAMIN E (TOCOPHEROLS) : TocH :
1. TocH
+
L
LH
+ Toc
(TOCOPHEROL)

(LIPID RADICAL)

(LIPID)

(TOCOPHERYL RADICAL)

2. TOCOPHERYL RADICAL (Toc) IS RELATIVELY STABLE

BUT MUST EVENTUALLY BE ELIMINATED :
Toc + H2O
TQ
(TOCOQUINONE)
(NON-RADICAL)

OR
Toc + Asc H2

TocH + Asc + H+

(ASCORBIC ACID)

2 Asc + 2 H+
ASCORBIC

(ASCORBYL RADICAL)

Asc H2 + Asc
(ASCORBIC
ACID)

(DEHYDROACID, DHAA)

TOCOPHEROL (VITAMIN E)

ASCORBIC ACID & ITS

DERIVATIVES

G6PD

ANTI-OXIDANT
DEFENSE SYSTEM

GLUTATHIONE PLAYS AN IMPORTANT ROLE IN ANTI-OXIDANT DEFENSE,

IT CAN REACT DIRECTLY WITH OH (HYDROXYL RADICAL) :
2 GSH + 2 OH

GSSG + 2 H20

OR WITH H2O2 (HYDROGEN PEROXIDE) CATALYZED BY GLUTATHION

PEROXIDASE (GPX) :
2 GSH + H2O2

GSSG + 2 H2O
(GPX)

THE RATIO : (GSH : GSSG) IN NORMAL CELLS ARE ALWAYS HIGH, SO

THAT THERE MUST BE A MECHANISM TO REGERATE GSH FROM GSSG.
THIS IS DONE BY THE ACTION OF 2 ENZYMES :
GLUTATHIONE REDUCTASE AND GLUCOSE -G- PHOSPHATE
DEHYDROGENASE (G6PD)
GSSG + NADPH + H+

2 GSH + NADP+
(GLUTATHIONE
REDUCTASE)

GLUCOSE 6 PHOSPHATE + NADP +

6 PHOSPHOGLUCONATE
(G6PD)
+ NADPH + H+