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Normal Tissue

(In)

Homeostasis

(Out)

Diseases of Disordered Cell Death


Proliferation

Neurodegeneration
Immunodeficiency Cancer
Infertility Autoimmunity

Death
Diseases associated with the induction or inhibition of apoptosis
TYPES OF CELL DEATH

NECROSIS APOPTOSIS
TYPES OF CELL DEATH
Morphological
NECROSIS APOPTOSIS
• loss of membrane • Membrane blebbing, no loss
integrity of integrity
• Aggregation of chromatin at
• swelling of cytoplasm nuclear membrane
& mitochondria • Shrinking of cytoplasm &
nuclear condensation
• total cell lysis • Fragmentation into smaller
bodies
• disintegration of • Formation of apoptotic
organelles bodies
• Mitochondria become leaky
physiological
NECROSIS APOPTOSIS
• Affects groups of • Affects individual cells
cells
• Evoked by non • Induced by physiological
physiological stimuli
disturbances
• Phagocytosis by
macrophages
• Significant
inflammatory
• No inflammatory response
response
“Eat me” signals of the apoptotic cell.
Nuclear morphology in HL-60 cells
(Mlejnek 2001)

I) II)

III) I) Control

II) Apoptosis

III) Necrosis
STAGES OF APOPTOSIS
Healthy cell

DEATH SIGNAL

Commitment to die (reversible)

EXECUTION (irreversible)

Dead cell
ENGULFMENT

DEGRADATION
Features of Apoptosis
• Apoptosis is a morphological distinct form of
programmed cell death (“cellular suicide”).

• Vast numbers of cells die by apoptosis


during normal development and tissue
homeostasis.
Programmed Cell Death in Development

Sculpting

Deleting Structures

Adjusting Cell Eliminating


Numbers Dangerous Cells

Jacobson et al., 1997 Cell 88, 347


Morphogenesis (eliminates excess cells):
APOPTOSIS: important in adults
Tissue remodeling (eliminates cells no longer needed):

Apoptosis
Virgin mammary gland Late pregnancy, lactation Involution
(non-pregnant, non-lactating)

- Testosterone
Apoptosis

Prostate gland
APOPTOSIS: important in adults
Tissue remodeling (eliminates cells no longer needed):

Apoptosis

Resting lymphocytes + antigen (e.g. infection) - antigen (e.g. recovery)

Steroid immunosuppressants: kill


lymphocytes by apoptosis

Lymphocytes poised to die by apoptosis


•The regulation of apoptosis involves
many different signals from both
within and outside the doomed cell.
Many signals trigger apoptosis

Withdrawal of cell adhesion
extracellular
survival factors DNA damage

extracellular
death factors/ligands
cellular stress

steroid hormones Apoptosis heat

radicals
cell lineage

cell­cell conflicting errors in development


interactions signals and differentiation
Many distinct death-inducing
signals lead to stereotypic
morphological and
biochemical changes that are
common to many different
cell types, suggesting the
activation of a common
apoptotic death program.
•Caspases (“cysteine aspartases), an unusual
class of proteases, play a central role in the
execution of apoptosis.

•All the components that are necessary to


carry out apoptosis are expressed in all cells
at all times.
Caspases induced apoptosis, main features:

•Caspases share similarities in amino acid


sequence, structure and Substrate specificity.
Caspases (cysteine aspartases) have an active­
site cysteine

and cleave substrates after aspartic acid residues:   
Asp­X­x­x­
A caspase’s distinct substrate specificity is determined by the 
three amino acids terminal to the Asp cleavage site (e.g. 
DEVD for caspase­3). –
WT D|EVD

+ caspase

cleaved target protein

Mutant: AEVD

+ caspase
Caspases are executors of apoptosis

Proenzyme DEXD↑
(inactive)

Caspase
(active)

DEXD↑ DEXD↑ DEXD↑


Substrates
A B C
Caspases are executors of apoptosis

Proenzyme DEXD↑
(inactive)

Caspase
(active)

Substrates
Caspases – key executioners of apoptosis

(cysteinyl aspartate specific


proteases)

Highly conserved proteases


14 homolgues
inactive zymogens

Caspases divided into

• Initiator caspases:
Caspases 2, 8,9,10 or

2) Effector caspases:
caspase 3,6,
Major Proteolytic Events in the Apoptotic Cascade

Signal

Signal

Initiator Caspase

Amplifier Caspase
Execution Caspase

Substrate cleavage
•Caspases are synthesized as precursors that have
little if any catalytic activity.

-They are converted to active enzymes, either by


another caspase or autocatalytic activity.
caspase activation

active
protease
(enzyme)

inactive
protease
(proenzyme)

inactive
protein
fragment
Caspase structure
3 domains
1) highly variable
NH2 domain
2) large subunit
(~20kD)
3) small subunit
( ~10kD)

Highly specific
absolute requirement for cleavage after aspartic acid

recognition of at least 4 amino acids NH2 terminals to


the cleavage site
Caspases are activated in cascades that
lead to extensive cellular degradation
How do caspases kill a cell?
•Inactivation of molecules which protect from death

Example: ICAD/CAD
The molecular biology of DNA fragmentation in apoptosis

• Stimulation of apoptosis ⇒ caspase­3 activation ⇒ cleave and 
inactivate ICAD ⇒ free CAD to cleave the DNA.

Caspase­3 DNA

ICAD
CAD
The molecular biology of DNA fragmentation in apoptosis

• Stimulation of apoptosis ⇒ caspase­3 activation ⇒ cleave and 
inactivate ICAD ⇒ free CAD to cleave the DNA.

Caspase­3 DNA

ICAD
CAD
The molecular biology of DNA fragmentation in apoptosis

• Stimulation of apoptosis ⇒ caspase­3 activation ⇒ cleave and 
inactivate ICAD ⇒ free CAD to cleave the DNA.

Caspase­3 DNA

CAD
The molecular biology of DNA fragmentation in apoptosis

• Stimulation of apoptosis ⇒ caspase­3 activation ⇒ cleave and 
inactivate ICAD ⇒ free CAD to cleave the DNA.

Caspase­3 DNA

CAD
How do caspases kill a cell?

Example: destruction of Nuclear lamina-by cleavage of lamins


which leads to lamina collapse, and nuclear shrinkage.
Caspases also reorganize cell structures indirectly by cleaving
Several proteins involved in cytoskeletal regulation.
Such as : Gelsolin
FAK (foacl adhesion kinase)
Actin
How do caspases kill a cell?

•Inactivation or deregulation of proteins involved in DNA repair


and DNA replication.
Caspases participate in apoptosis in a manner reminiscent of
a well planned military operation;

•Re-organize the cytoskeleton.


•Shut down DNA replication and repair.
•Interrupt RNA splicing.
•Destroy DNA (fragmentation).
•Disrupt the nuclear structure.
•Induce the cells to display signals that mark it
for phagocytosis.
•Disintegrate the cell into apoptotic bodies.
Antibodies against activated caspases
The molecular biology of DNA fragmentation in apoptosis

• A hallmark of apoptosis is DNA fragmentation: “DNA ladders” 
because DNA are cut to lengths corresponding to multiple 
integers of approximately 180 base pairs.

DNA

Apoptosis Gel
DNA Ladder
Marker    Normal cells    Apoptotic cells
Degradation of nuclear DNA in HL-60
cells
(Mlejnek 2001)
M 1 2 3 4 5 6 M

M – DNA size standards

1 - DNA from untreated cells,


(control)

2-6 - DNA from treated


(apoptotic) cells
Type I
Cells
Type II Cells
How are caspases activated?
More than one way to skin a cat

Proteolytic cleavage

activation of downstream, effector caspases


How are caspases activated?
Induced proximity theory

aggregation of multiple procaspase-8 molecules into


close proximity somehow results in cross-activation
How are caspases activated?
More than one way to skin a cat

Holoenzyme formation

Activation of caspase-9 is mediated by means of


conformational change, not proteolysis
-Activity of caspases is triggered either by
adaptor/co-factor or by removal of
inhibitors.
Adaptor molecules enable activation of initiator caspases
External signals
driven by death receptors (DR)
e.g. CD95 (or Fas/Apo)

Each CD95L trimer binds to 3 CD95


leading to DD clustering.

FADD ( Fas associated death domain/


Mort 1) binds via its own DD

Caspase –8 oligomerisation drives


activation through self cleavage

Caspase –8 then activates downstream


effector caspases

Apoptosis initiation
Adaptor function

Type I cells

FADD

Binds to DD inTNFR’s

DED­Death Effector Domain DD­Death Domain

N FADD

Binds to DED in Casp.8, Casp.10


Adaptor function

Type II cells

APAF-1-Apoptotic protease
activating factor 1

CARD-Caspase Recruitment domain


WD repeat domain (12­13 repeats)

binds CytoC
Ced4 homology domain
Mediates oligomerization of APAF-1 in the
presence of ATP
Regulation of caspapses;
Regulation through adaptors/co-factors.

Adaptors

Inhibitors

•Compartmentaliozation
•Relocation with apoptotic trigger
How is it that caspases are 
only activated in cells that 
are doomed to die?
Apoptosis trigger

Cytochrome C
IAP -Antagonists
IAP’s inhibit caspase activation Cell survival

Ubiquitination of Apoptosis
IAP’s
Inhibitors of apoptosis: IAPs
BIR BIR BIR CARD RING

Originally identified as proteins


from viruses infecting insect cells

Cells tried to induce apoptosis -


virus prevents activation of
apoptosis
Inhibitors of apoptosis: IAPs
BIR BIR BIR CARD RING

BIR motif:
-Multiple N-terminal domains
-binds to caspase surface
-seq. Between BIR motifs blocks
active site
Inhibitors of apoptosis: IAPs
BIR BIR BIR CARD RING

RING motif:
-C-terminal
-can act as ubiqutin ligase
-caspases are targets of Ub ligase
activity
XIAP Caspase 3 Caspase 9
Apoptosis trigger

Cytochrome C
IAP -Antagonists
What is Bcl-2?
 Family of proteins that includes promoters
and inhibitors of apoptosis
 Localized to outer mitochondrial
membrane
 Can form homo- and heterodimers
Humans contain a large family of Bcl­2 related 
proteins (the “Bcl­2 family”).  Members of this 
family share at least one of several conserved 
BH (Bcl­2­homology) domains.

Some of these proteins (including Bcl­2, Bcl­
XL) have largely anti­apoptotic function, but 
others (including Bax, Bak, Bok, Bid) promote 
apoptosis.
Bcl-2 family

Prevents cyto c release


Disrupts membrane potential
An Evil Twin: BAX

Life/Death Rheostat
A Cascade of Pro-Death Molecules Pro Death
Ligand
(FasL)

“Initiator” Caspase-8”

BH3 Death Receptors


(Fas)

Procaspase-8

Apoptosome
“Inactive” BH3
BID
Cytochrome c
“Inactive”
BAK HIT
&
BH3 RUN

“Active”
tBID “Active”
BAK

DEATH

MITOCHONDRION
Bcl­2 family members interact with each other (form 
homodimers and heterodimers) to regulate apoptosis.  The 
composition of the dimers determines whether the cell survives 
or undergoes apoptosis (ratio of death promoter to the death 
suppressor)
­ mutagenesis experiments that prevented Bcl­2 from associating 
with Bax also stopped Bcl­2 from blocking cell death.
Life Death

Bax Bcl­2 Bax Bax

• How exactly this is done is not very clear (see functions 
of Bcl­2 below).
BCL-2 in HUMANS

-B-cell lymphoma
-Characteristic chromosomal
rearrangement
-over-proliferation of B cells
Bcl-2 translocation
• Portion of chrom. 18
with Bcl-2 with portion
of chr.14 with Ab
heavy chain
• Bcl-2 close to
enhancer region
• Active enhancer in B-
cells = increased Bcl-2
production
• Burkitt’s Lymphoma:
translocation with c-
myc producing
cancerous B’s
Model of Human Lymphoma
18 14
Transgene
BCL-2 Antibody Gene

i de
iv

Di
D

e
BCL-2
B Cell
Normal Transgenic
Mouse Mouse
Fas L
Fas
FADD
Pro­caspase­8
Fas L
Fas
FADD
Caspase­8

Pro­caspase­3
Fas L
Fas
FADD
Caspase­8

Caspase­3

Apoptosis
Fas L
Fas
FADD
Caspase­8

Bid

Pro­caspase­3
Fas L
Fas
FADD
Caspase­8

tBid

Pro­caspase­3

Cyto c
IAP­antagonists
Smac/DIABLO
Omi/HTRA
ARTS

Bcl­2
Bcl­XL
Fas L
Fas
FADD
Caspase­8

tBid

Pro­caspase­3
“Apoptosome”
Apaf1
Pro­caspase­9

Bcl­2
Bcl­XL
Fas L
Fas
FADD
Caspase­8

tBid

Pro­caspase­3
“Apoptosome”
Apaf1
Caspase­9

Bcl­2
Bcl­XL
Fas L
Fas
FADD
Caspase­8

tBid

Caspase­3
“Apoptosome”
Apaf1
Caspase­9

Bcl­2
Apoptosis Bcl­XL
Fas L
Fas
FADD
Caspase­8

tBid

Caspase­3
“Apoptosome”
Apaf1
Caspase­9

IAP
Bcl­2
Apoptosis Bcl­XL
Fas L
Fas
FADD DNA damage
Caspase­8
p53
tBid
Bax

Caspase­3
“Apoptosome”
Apaf1
Pro­caspase­9

IAP
Bcl­2
Apoptosis Bcl­XL
Deregulated
cell
proliferation
NORMAL NEOPLASTIC
CELL PROGRESSION
Suppressed
cell death
APOPTOSIS: Role in Disease
Cancer

Apoptosis eliminates damaged cells


(damage => mutations => cancer

Most cancer cells defective in apoptotic response

High levels of anti-apoptotic proteins


or
Low levels of pro-apoptotic proteins
===> CANCER