Apoptosis Signaling Live??? Or Die???

Vijay Avin BR, Molecular Biomedicine Laboratory, Sahyadri Sceince College, Shimoga, Karnataka, India

Life of cell

• Mitosis checkpoints• Apoptosis will be

triggered to prevent cells from becoming cancers and harming the body

Cell death by injury

-Mechanical damage

-Exposure to toxic chemicals

Cell death by suicide

-Internal signals

-External signals

• Definition

Apo: apart

Ptosis: fallen– Shedding of leaves from tress

• During embriogenesis ------ occurs as PCD

• Post-embrional life------- as apoptosis

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!

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

NECROSIS Vs APOPTOSIS

Wilde, 1999

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.

• 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

• 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.

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

STAGES OF APOPTOSIS

Sherman et al., 1997

Induction of apoptosis related genes, signal transduction

membrane blebbing & changes

mitochondrial leakage

organelle

reduction

cell

shrinkage

nuclear fragmentation

chromatin condensation

APOPTOSIS: Morphology

Hacker., 2000

membrane blebbing & changes

mitochondrial leakage

organelle reduction

cell shrinkage

nuclear fragmentationchromatin condensation

APOPTOSIS: Morphological events

Bleb

Blebbing & Apoptotic bodies

The control retained over the cell membrane & cytoskeleton allows intact pieces of the cell to separate for recognition & phagocytosis by Ms

Apoptotic body

M M

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”

http://www.immunityageing.com/content/3/1/5/figure/F1?highres=y

• 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

MAJOR PLAYERS IN APOPTOSIS

• Caspases

• Adaptor proteins

• Bcl-2 family

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

• 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

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)

• NO exposure or activation may inhibit apoptosis in

Lymphocytes

Endothelial cells

Neurons

Hepatocytes

Kidney cells

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.

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….?

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

The bcl-2 family

BH4 BH3 BH1 BH2 TMN C

Receptor domain

phosphorylation

Raf-1calcineurin Pore

formation

Membraneanchor

Liganddomain

Group I

Group II

Group III

Bcl-2

bax

Badbidbik

Back

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)

Thank u