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(In)
Homeostasis
(Out)
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
EXECUTION (irreversible)
Dead cell
ENGULFMENT
DEGRADATION
Features of Apoptosis
• Apoptosis is a morphological distinct form of
programmed cell death (“cellular suicide”).
Sculpting
Deleting Structures
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
Withdrawal of cell adhesion
extracellular
survival factors DNA damage
extracellular
death factors/ligands
cellular stress
radicals
cell lineage
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)
Proenzyme DEXD↑
(inactive)
Caspase
(active)
Substrates
Caspases – key executioners of apoptosis
• 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.
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
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?
• 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
Proteolytic cleavage
Holoenzyme formation
Apoptosis initiation
Adaptor function
Type I cells
FADD
Binds to DD inTNFR’s
DEDDeath Effector Domain DDDeath Domain
N FADD
Type II cells
APAF-1-Apoptotic protease
activating factor 1
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
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
Life/Death Rheostat
A Cascade of Pro-Death Molecules Pro Death
Ligand
(FasL)
“Initiator” Caspase-8”
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
• 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