Apoptosis signalling by Vijay Avin BR

1. Apoptosis Signaling
Live??? Or Die???
Vijay Avin BR, Molecular Biomedicine Laboratory, Sahyadri
Sceince College, Shimoga, Karnataka, India

2. Life of cell
• Mitosis checkpoints
• Apoptosis will be
triggered to prevent
cells from
becoming cancers
and harming the
body

3. Cell death by injury
-Mechanical damage
-Exposure to toxic chemicals
Cell death by suicide
-Internal signals
-External signals

4. • Definition
Apo: apart
Ptosis: fallen
– Shedding of leaves from tress
• During embriogenesis ------ occurs as
PCD
• Post-embrional life------- as apoptosis

5. apoptosis
• Apoptosis is used as a synonymous for
PCD but PCD is physiological death,
occurs only during embriogenesis.
• It is a functional death and it is a good
mechanism to eliminate wasted, useless,
unwanted, or crippled cells!

6. Necrosis vs. Apoptosis
• Cellular condensation
• Membranes remain intact
• Requires ATP
• Cell is phagocytosed, no
tissue reaction
• Ladder-like DNA
fragmentation
• In vivo, individual cells
appear affected
• Cellular swelling
• Membranes are broken
• ATP is depleted
• Cell lyses, eliciting an
inflammatory reaction
• DNA fragmentation is
random, or smeared
• In vivo, whole areas of
the tissue are affected
Necrosis Apoptosis

7. NECROSIS Vs APOPTOSIS
Wilde, 1999

8. Why have we developed such a
self-destructive system?
• A. PCD allows a constant selection for the
fittest cell in a colony
• Every cell carries the molecular machinery
to do PCD!
• Cells that are sensitive to extracellular
signals will survive, cell that cannot
compete with their more vital sisters will
undergo apoptosis.

9. • PCD machinery is silent until signals arrive
to start PCD:
• Signals:
• damage to DNA
• Activation of membrane receptors.
Ligands are: peptides, cytokines, ATP,
ROS etc

10. • Fas receptor?
Receptors for growth factors, cytokines
and hormones
• Membrane alterations cause apoptosis.
What kind of membrane alterations ??
Phospholipid redistributions, changes in
membrane charge, carbohydrate and
surface markers.

12. Proteins involved in apoptosis
• Fas ligand (FasL or CD95L) is a type-II transmembrane protein that belongs to the
tumor necrosis factor (TNF) family
• Fas-Associated protein with Death Domain (FADD) is an adaptor molecule that
bridges the Fas-receptor, and other death receptors,
• Apoptotic protease activating factor 1, also known as APAF1
• Bcl-2 (B-cell lymphoma 2) is the founding member of the Bcl-2 family of apoptosis
regulator proteins encoded by the BCL2gene
Caspases
• Inflammatory Caspases: -1, -4, and -5
• Initiator Caspases: -2, -8, -9, and -10
– Long N-terminal domain
– Interact with effector caspases
• Effector Caspases: -3, -6, and -7
– Little to no N-terminal domain
– Initiate cell death
• The Mitochondrial Apoptosis-Induced Channel (or MAC),
• BAK: Bcl-2 homologous antagonist killer
• BAX: Bcl-2 associated x protein
• BID: BH3 interacting domain death agonist, a pro-apoptotic protein
• BAD: The Bcl-2-associated death promoter (BAD) protein is a pro-apoptotic
member of the Bcl-2 gene family which is involved in initiating apoptosis. BAD is a
member of the BH3-only family

13. STAGES OF APOPTOSIS
Sherman et al., 1997
Induction of apoptosis related genes, signal transduction

14. membrane
blebbing &
changes
mitochondrial
leakage
organelle
reduction
cell
shrinkage
nuclear
fragmentation
chromatin
condensation
APOPTOSIS: Morphology
Hacker., 2000

15. membrane blebbing & changes
mitochondrial leakage
organelle reduction
cell shrinkage
nuclear fragmentation
chromatin condensation
APOPTOSIS: Morphological events

16. Bleb
Blebbing & Apoptotic bodies
The control retained over the cell
membrane & cytoskeleton allows intact
pieces of the cell to separate for
recognition & phagocytosis by MΦs
Apoptotic body
MΦ MΦ

17. Apoptosis: Pathways
Death
Ligands
Effector
Caspase 3
Death
Receptors
Initiator
Caspase 8
Cell
death
DNA damage & p53
Mitochondria/
Cytochrome C
Initiator
Caspase 9
“Extrinsic Pathway”
“Intrinsic Pathway”

20. • Binding of Fas by FasL
induces recruitment of FADD
to the cytoplasmic tail of Fas
• The opposite end of FADD
contains a death effector
domain (hatched boxes);
recruitment of either
procaspase-8 or c-FLIP
• Caspase-8 can cleave Bid
• truncated Bid (tBid) can
inactivate Bcl-2 in the
mitochondrial membrane.
• This allows the escape of
cytochrome c, which clusters
with Apaf-1 and caspase-9 in
the presence of dATP to
activate caspase-9.
• Smac/DIABLO is also released
from the mitochondria and
inactivates inhibitors of
apoptosis (IAPs).
• breakdown of several
cytoskeletal proteins and
degradation of the inhibitor of
caspase-activated DNase
(ICAD).
Extrinsic or
Death Receptor
Pathway

22. MAJOR PLAYERS IN
APOPTOSIS
• Caspases
• Adaptor proteins
• Bcl-2 family

23. Modulation of apoptosis
• Apoptotic cell death can be switched to
necrosis during oxidative stress by 2
mechanisms:
Inactivation of caspases due to oxidation
of their active site thiol group by oxidants
Decrease in ATP due to failure of
mitochondrial energy production by
oxidants

24. • NO can also have dual effects on
apoptosis
NO is reactive, unstable free radical gas that
can easily cross cell membranes.
L-Arg------ NO
Low NO: Neurotransmitter, regulator in
vasodilation and platelet aggregation.
High NO: Cytotoxicity

25. NO may also mediate
apoptosis:
How???
• Formation of iron-nitrosyl complexes with
FeS-containing enzymes: This leads to
impairment of mitochondrial function
ATP depletion.
• NO may directly damage DNA-
mutagenesis
• Generation of OONO- Apoptosis
• NO may inactivate several antioxidant
enzymes (CAT, GPx, SOD etc)

26. • NO exposure or activation may inhibit
apoptosis in
Lymphocytes
Endothelial cells
Neurons
Hepatocytes
Kidney cells

27. How??
• Direct inhibition of caspase (S-nitrosylation of the active
site Cys)
• R-S-NO is important component of signal transduction
cascades.
• S-nitrosylation can regulate many proteins:
• Enzymes
• Ion channels
• G-proteins
• Transcription factors
• NO may act as a modular switch to control protein
function via –SH groups.

28. For example, S-nitrosylation was shown to occur in:
• Calpain
• NF-KB
• AP-1
These are all implicated in the regulation of apoptosis.
• Nitrosylation/denitrosylation- may serve as a
regulatory mechanism just like….?

29. Importance of Apoptosis
• Important in normal physiology / development
– Development: Immune systems maturation,
Morphogenesis, Neural development
– Adult: Immune privilege, DNA Damage and wound
repair.
• Excess apoptosis
– Neurodegenerative diseases
• Deficient apoptosis
– Cancer
– Autoimmunity

30. The bcl-2 family
BH4 BH3 BH1 BH2 TMN C
Receptor domain
phosphorylation
Raf-1
calcineurin Pore
formation
Membrane
anchor
Ligand
domain
Group I
Group II
Group III
Bcl-2
bax
Bad
bid
bik
Back

31. P53 & Apoptosis
p53 first arrests cell growth between G1 → S
This allows for DNA repair during delay
If the damage is too extensive then p53
induces gene activation leading to
apoptosis (programmed cell death)

32. Thank u