APOPTOSIS

Can the cell be programmed

to suicide?
PRESENTED BY :

 ARSHIYA JAHAN
DEEPAK TANWAR
DIVYARAJ ZALA
KRATI TIWARI
VOMIKA SONI
(B.TECH – BIOTECH [III])
 Classification of cell Death

Cell death

Physiological
Necrotic
apoptosis autophagic other

Caspase-dependent Caspase-independent

receptor-caspase 8 mitochondria-caspase 9
What makes a cell decide to commit
suicide?
Withdrawal of positive (Growth) signals
◦ growth factors for neurons
◦ Interleukin-2 (IL-2)

Receipt of negative (Death) signals

◦ increased levels of oxidants within the cell

◦ damage to DNA by oxidants
◦ death activators :
 Tumor necrosis factor alpha (TNF-)
 Lymphotoxin (TNF-β)
 Fas ligand (FasL)
Morphological changes
associaated with
programmed cell death
The term was used by
Wyllie and his
colleagues.
It is from the Greek
meaning “dropping
away”
as the leaves from the
trees.
 APOPTOSIS

 Active cell death

Requires energy and RNA and protein synthesis
Characteristic morphological features
DNA cleaved, chromatin condenses
Cells shrink
Formation of apoptotic body
Cleared by phagocytosis
No inflammation=no tissue damage
Introduction
Elimination of unwanted cells in a
morphologically distinct manner is described
as Programmed cell Death or Apoptosis.
 Excessive cell death may cause
NEURODEGENERATIVE disease and
IMMUNODEFICIENCY.
In mutants, it leads to CANCER or
AUTOIMMUNE diseases.If the cells
destined for cell death survive.
 C. elegans has played a key role in
our understanding of Apoptosis
1090 total cells
131 die
Ced-3=no death ced-1 mutant
Ced-4=no death (No engulfment)
Ced-9=all die

ced-1/ced-3
(No cells die)
Examples of Apoptosis
Apoptosis is needed for proper development

◦ The resorption of the tadpole tail

◦ The formation of the fingers and toes of the mouse fetus
◦ The sloughing off of the inner lining of the uterus
◦ The formation of the proper connections between neurons in the
brain

Apoptosis is needed to destroy cells

◦ Cells infected with viruses

◦ Cells of the immune system
◦ Cells with DNA damage
◦ Cancer cells
Death in the Mouse’s Paw

Fluorescence
microscopy :
the dark green region
represents apoptotic cells
The resorption of Tadpole Tail
Formation of proper connections between
neurons in the brain
MAJOR PLAYERS IN APOPTOSIS

 Caspases
 Death signals e.g. TNF & TNFR
 p53
 BAX
 Bcl-2 family
Caspases
Apoptosis depends on an Intracellular proteolytic
cascade that is mediated by caspases.
Caspases are Cysteine Aspartic Acid Proteases.
They are of two types:-
INITIATOR CASPASES:
– Caspases –8 and –9 are “initiator” caspases
EFFECTOR CASPASES:
– Caspases –3 is the “effector” caspase
 Caspase activation requires a stimulus
They proteolyse cellular proteins to carry out cell death
program
Procaspase activation by cleavage
Procaspase activation by cleavage
Each caspase is initially made as an inactive
proenzyme known as procaspase.
Initiator procaspases are activated by adaptor
proteins.
Procaspases cleave each other due to the
presence of cleavage sites.
Cleavage serves the purpose of stabilizing the
active caspase
Procaspases are activated by proteolytic
cleavage by an active caspase.
Two cleaved fragments from each of the
two procaspases associate to form an
active caspase.
This active caspase is a tetramer of two
large and two small subunits & the
prodomains are discarded.
Caspase cascade
Caspase Cascade
The first activated procaspase are called
initiator active caspase.
They cleave and activate many
executioner caspase molecules producing
an amplifying chain reaction.
The executioner caspases then cleave a
variety of key proteins in the cell like:
- specific cytosolic proteins
- nuclear lamins
Apoptosis: PATHWAYS
 “Extrinsic Pathway”
 Deat  Deat  Initiator
h h Caspase
Liga Rece 8
nds ptor
 Effect
or
PC
 “Intrinsic Pathway”
Caspa D
 DNA
se 3
dam  Mitochon  Initiat
age dria/Cyto or
chrome C Caspa
&
p53 se 9
Extrinsic Pathway
Extrinsic Pathway
FAS ligand on the surface of a killer
lymphocyte activates FAS death receptors
on the surface of the target cell.
FAS mediates recruitment of adaptor
proteins FADD (FAS associated death
domain) & procaspase8 or 10.
Each FADD protein then recruits an
initiator procaspase forming a death
inducing signalling complex(DISC).
Extrinsic Pathway
Within the DISC the initiator procaspase
molecules activates initiator caspase
which further activates executioner
caspases, thus triggering caspase cascade.
This leads to Apoptosis.
The receptor involved in this pathway
belong to TNF(Tumor Necrosis Factor)
receptor family.
Intrinsic Pathway
 Cell Cycle
 DNA
DNA damage p53 p21  Repair
 Arrest
 Nuclear
 Mitochondrial
 BAX
(proapoptotic)  Cyt C
 Apaf-1
bcl- 2  Initiator
 Anti-apoptotic  Caspase-8E / 9I
 Aspartate- AA  Effector
 proteolysis  Caspase- 3
CAD
DNA (DNA-ase)
nucleoso
Intrinsic Pathway
Intrinsic Pathway
Apoptosis can also occur in response to injury
or other stresses such as DNA damage or lack of
O2, nutreints or extracellular survival signals.
Such intracellular activation occurs via the
intrinsic pathway of apoptosis.
This pathway depends upon mitochondria.
Cytochrome c protein is released from
mitochondria during intrinsic pathway.
It is a water soluble component of mitochondrial
electron transport chain
Release of Cytochrome c from
mitochondria during apoptosis
 The release of cytochrome c from the
mitochondria activates Apaf1(Apoptotic protease
activating factor1)
 The binding of cytochrome c causes Apaf1 to
hydrolyse its bound dATP to dADP.
 Apaf1 & cytochrome c aggregate to form
apoptosome.
 The apoptosome causes recruitment and
activation of procaspase9 through a
CARD(caspase recriutment domain) in each
protein.
It cleaves and thereby activates
executioner procaspases which form
caspase cascade leading to apoptosis.
Bcl2 proteins regulate the intrinsic
pathway of apoptosis by controlling
release of cytochrome c and other
mitochondrial proteins into the cytosol.
Classes of Bcl2 proteins
 The bcl-2 family
N BH4 BH3 BH1 BH2 TM C
Receptor domain Membrane
Raf-1 Ligand
calcineurin domain Pore anchor
formation
phosphorylation

 Group I  Bcl-2
 Group II  bax
 Bad
 Group III
bik
 bid
Classes of Bcl2
Anti apoptotic Bcl2 protein Inhibits apoptosis by blocking the
Eg. Bcl2, Bcl-Xl release of cytochrome c

Pro Apoptotic BH 123 protein Promote apoptosis by enhancing the

Eg. Bax, bak. release.

Pro Apoptotic BH3- only protein It mediates the direct interactions

Eg. Bad, bim between proapoptotic &anti
apoptotic family members.
Classes of Bcl2
The pro and anti apoptotic Bcl2 proteins
can bind to each other in various
combinations to form heterodimers, in
which the two proteins inhibit each
other’s function.
The balance between pro apoptotic and
anti apoptotic Bcl2 proteins determines
whether a mammalian cell lives or dies by
the intrinsic pathway of apoptosis.
Absence of Apoptotic Stimulus
Function of IAPs
Abscence of Apoptotic Stimulus
In the absence of an apoptotic stimulus
anti apoptotic Bcl2 proteins bind to and
inhibit the BH123 proteins on the
mitochondrial outer memebrane.
IAP’s prevent accidental apoptosis.
These are located in cytosol and bind to
caspase and inhibit any spontaneusly
activated caspase.
Activation of Intrinsic pathway in the
prescence of Apoptotic Stimulus
Function of Anti-IAPs
Activation of Intrinsic pathway in the
prescence of Apoptotic Stimulus
In the prescence of an apoptotic stimulus BH3- only
proteins are activated and bind to the anti-apoptotic Bcl2
proteins.
Now BH123 proteins become activated and aggregate in
the outer mitochondrial membrane & promote release of
inter membrane mitochondrial proteins inro cytosol.
Some activated BH3-only proteins may stimulate
mitochondrial proteins release by binding with BH123
proteins.
Anti-IAP proteins are released which bind to & block
the inhibitory activity of the IAPs which helps in
apoptosis.
Conclusion of Intrinsic Pathway
Occurs due to the DNA damage & other factors
Release of cytochrome c leads to the formation
of Caspase cascade leading to apoptosis.
The release of cytochrome c is governed by Bcl2
protein family.
In the absence of IAPs binds to the caspase
leading to its deactivation.
In the prescence of Anti-IAPs binds to IAPs &
inhibit their action leading to Apoptosis.
Inhibition of Apoptosis
The three ways that extracellular survival
factors can inhibit apoptosis:-

Increased production of anti apoptotic

Bcl2 protein.
Inactivation of pro apoptotic BH3 only
Bcl2 protein.
Inactivation of anti IAP’s
Inhibition of Apoptosis
CANCER causing p53 gene &bcl2
protein
Bcl2 gets its name from B cell lymphoma
High level of Bcl2 proteins in the
lymphocytes leads to cancer by inhibiting
apoptosis.
Similarly if tumour suppresor protein p53
is mutated, apoptosis is not triggered &
the cell with damaged DNA reenters the
cell cycle causing cancer
Importance of apoptosis
 Important in normal physiology / development
 Development: Immune systems maturation, Neural
development
 Adult: Immune privilege, DNA Damage and wound
repair.

 Excess apoptosis
 Neurodegenerative diseases

 Deficient apoptosis
 Cancer
 Autoimmunity
WHERE can APOPTOSIS be
ENCOUNTERED ?
... Growth of Embrio
... Tissue Homeostasis
... Immunology
... Chronic viral diseases
... Neurodegenerative diseases
... Reperfusion injury
... Insuline-dependent Diabetes
... Atheroschlerosis
... AIDS
... Development and Treatment of Malignancies
FUTURE PERSPECTIVES
The biological roles of newly identified death
receptors and ligands need to be studied

Need to know whether defects in these ligands and

receptors contribute to disease
CONCLUSION
an important process of cell death

Extrinsic Pathway
It can be initiated extrinsically through death ligands(e.g.
TRAIL, FasL) activating initiator caspase 8 through
induced proximity.

Intrinsic Pathway
can be initiated intrinsically through DNA damage,
cytochrome c releases, activating initiator caspase9.

Initiator caspases 8 and 9 cleave and activate

effector caspase 3, which leads to cell death.
REFERENCES:
Alberts Book
THANKS