Transcript Apoptosis pathways

Manifestation of Novel Social Challenges of the
European Union
in the Teaching Material of
Medical Biotechnology Master’s Programmes
at the University of Pécs and at the University
of Debrecen
Identification number: TÁMOP-4.1.2-08/1/A-2009-0011
Manifestation of Novel Social Challenges of the
European Union
in the Teaching Material of
Medical Biotechnology Master’s Programmes
at the University of Pécs and at the University
of Debrecen
Identification number: TÁMOP-4.1.2-08/1/A-2009-0011
Tímea Berki and Ferenc Boldizsár
Signal transduction
APOPTOSIS PATHWAYS
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„The process of natural
death”
• The word „apoptosis” (Greek spelling of
apoptosis) is used in Greek to describe the
„dropping off” or „falling off” of petals
from flowers, or leaves from trees
• Professor James Cormack of the Department of
Greek, University of Aberdeen, suggested
this term for the process of programmed cell
death in 1972
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Role of apoptosis
• Apoptosis, in general, confers advantages
during an organism's life cycle: one
appropriate response to a signal is for the
cell to commit suicide –presumably for the
good of the organism
• Between 50 and 70 billion cells die each day
due to apoptosis in the average human adult
• Programmed cell death is encoded in the
genome
• Apoptosis does not require new transcription
or translation, suggesting that the
molecular machinery required for cell death
lay dormant in the cell, and just requires
When would it be
advantageous to the
organism?
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• To „sculpt” an organism during development
such as during embryo development,
metamorphosis and tissue atrophy
• Regulate the total number of cells
• Defend and remove unwanted or dangerous
cells like tumor cells, virally infected
cells, or immune cells that recognize self
• Is required in the immune system for the
maturation, selection of lymphocytes
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The actual steps in cell
death require
• Condensing of the cell nucleus and
breaking it into pieces
• Condensing and fragmenting of
cytoplasm into membrane bound
apoptotic bodies
• Breaking chromosomes into fragments
containing multiple number of
nucleosomes (a nucleosome ladder)
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Apoptosis signals
Extracellular:
• A hormone - such as thyroxine which causes apoptosis
in tadpole tails
• Lack of a „survival” signal (which inhibits
apoptosis) such as a growth factor
• Cell-cell contact from an adjacent cell
• Toxins, nitric oxide, cytokines
• Increased intracellular calcium → calpain production
(calcium binding protease)
Intracellular:
• Ionizing radiation, heat, deprivation of nutrients
• Virus infection
• Oxidative damage from free radicals, hypoxia
• Glucocorticoids
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Mechanism of apoptosis:
caspases
• A whole family of proteases (about 10 in
humans) called caspases are required for
programmed cell
• Caspases: cys containing-asp specific
proteases
• They are endoproteases having an active site
Cys (C) and cleave at the C-terminal side of
Asp residues (asp)
• They are first synthesized as inactive procaspases
• These proteases are found in the cell in
inactive form which must undergo limited
proteolysis for activation
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Initiator caspases
• Initiator caspase can be activated if
they aggregate to a critical
concentration
• The prodomain of the initiator
caspases contain domains such as a
CARD domain (e.g. caspases-2 and -9)
or a death domain (DED) (caspases-8
and -10) that enables the caspases to
interact with other molecules that
regulate their activation
• The active initiator caspase activate
the effector caspases
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The caspase cascade can be
activated by
• Granzyme B: a serin protease (released
by cytotoxic T lymphocytes and NK
cells), which is known to activate
caspase-3 and -7
• Death receptors: Fas, TRAIL receptors
and TNF receptors, which can activate
caspase-8 and -10
• Apoptosome: is regulated by
cytochrome-c and the Bcl-2 family,
which activates caspase-9
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Apoptosis pathways
EXTRINSIC
INTRINSIC
Death ligands
(FasL, TRAIL, TNF)
Stimuli
(Cytokine deprivation, viral infection,
DNA damage, irradiation, cell stress)
Death receptors
(FasL, TRAIL, TNF)
FADD
DISC
BH3 only
molecules
Anti apoptopic
Bcl-2 family members
FLIP
ProCaspase-8
Kinase
Enzyme
Bax
Activated
Caspase-8
Bak
Mitochondria
Smac
Phosphatase
Caspase
Cyt
c
Pro-survival
Cyclin, pro-apoptotic
Transcription factor
Effector
Caspases
Apaf-1
Caspase-9
GAP/GEF
GTP-ase
Apoptosis
Apoptosome
XIAP
cIAP-1
IAPs
cIAP-2
Survivin
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Intrinsic apoptotic pathway
1 Involvement of mitochondria: opening of a
channel called a nonspecific inner membrane
permeability transition pore
2 Collapse of the electrochemical potential
across the inner membrane
3 Cytochrome C, Smac/DIABLO, Omi/HtrA2, AIF
and endonuclease G leaks out of the
intermembrane space and binds to a
cytoplasmic protein called Apaf-1 (apoptotic
protease activating factor-1)
4 This then activates an initiator caspase-9
in the cytoplasm
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Mitochondrial apoptosis
pathway
Apoptotic signals
Bcl-2
P
Bcl-2
Bad P
P
Bcl-2
Bad
P
Bad
Mitochondrion
P
P
Bax
Bax
Cyt
c
PT Pore
Cyt
c Apaf-1 Apoptosome
Caspase-9
Bcl-2
Bax
Caspase cascade
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Permeability transition
pore
• Outer membrane protein (porin, the voltagegated anion channel - VDAC)
• Inner membrane protein (adenine nucleotide
translocator – ant)
• This channel passes anything smaller than
molecular weight 1500.
Collapsing the
proton gradient uncouples oxidation and
phosphorylation in the mitochondria
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Apoptosome
Apoptosome formation
Cytc
Recruitment of Procaspase-9
Pro
caspase-9
Cytc
Cytc
Cytc
Apaf-11
Cytc
Cytc
Cytc
Caspase activation
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Bcl-family
BH4
BH3
BH1
BH2
TM
Anti-Apoptotic
Mcl1, CED9
BH4
BH3
BH3
BH4
Pro-Apoptotic
Bcl-2, Bcl-XL
BH3
BH4
BH1
A1, Bfl-1
BH2
BH1
BH2
BH1
BH2
BH3
BH3
BH3
TM
Bax, Bak
TM
Diva
TM
Bcl-Xs
TM
Bik, Bim
Bad, Bid, Egl-1
What causes all these
changes in the
mitochondria?
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• Disruption of ox-phos. and electron transport, caused
by irradiation and certain second messengers such as
ceramide
• Changes in cell redox potential and generation of
reactive oxygen species (ROS)
• Damage to DNA caused by radiation, ROS, etc. A
protein called p53 is often expressed in cells with
DNA damage. Expression of this protein results in
inhibition of cell division, or apoptosis, both of
which would keep the damaged cell from becoming a
tumor cell. Hence the p53 gene is a tumor suppressor
gene. It is inactivated by mutation in approximately
50% of all human tumor cells studied. p53 can induce
gene expression. Of the 14 different genes whose
expression are significantly altered by p53, many
seem to be used by cells to generate or respond to
Apoptosis pathways in
activated T cells
T-cell subgroup
Bulk activated T cells
Th1
Th2
Th17
Tc1
Tc2
Treg, gdT cells, NK, NKT
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Pathway
Fas/FasL
Fas/FasL
Granzyme B
Fas/FasL?
Fas/FasL/Granzyme B
?
?
Extrinsic apoptotic
pathway:
death receptors
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• Activated immune cells start expressing Fas
a few days after activation, targeting them
for elimination
• Some cells which have been stressed express
both Fas and Fas ligand and kill themselves
• Various cells express CD95 (Fas), but CD95L
(Fas-Ligand) is expressed predominately by
activated T cells
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Role of death receptors:
Fas
FAS receptor (also known as Apo-1 or CD95):
• FADD (Fas-associated death domain) binds to
the aggregated cytoplasmic domain (the death
domain) of CD95
• Recruits inactive caspase-8 and 10 to the
site → death-inducing signaling complex
(DISC)
TNF receptor mediated
apoptosis I
TNF
FasL
TNF
Fas/
CD95
TNFR-1
Daxx
ASK1
DAPK
FADD
Caspase-8,-10
RIP
TRADD
FADD
Caspase-8,-10
APO-3L/TWEAK
DR3
APO-3
TRADD
TRAF2
RIP
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FADD
Caspase-8,-10
RIP
RAIDD
TNFR2
c-IAP1/2 TRAF2
TRAF2
ASK1
APO-2L/TRAIL
DR4/5
FADD
Caspase-8,-10
TNF receptor mediated
apoptosis II
FasL
TNF-
TNF-
Fas/
CD95
Daxx
ASK1
APO-3L/TWEAK
TNFR-1
TNFR2
DAPK
c-IAP1/2 TRAF2
TRADD
FADD
RAIDD
Caspase-8,-10 FADD
UB
RIP
UB
RIP
TRAF2
ASK1
Caspaseindependent
cell death
Bid
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APO-2L/TRAIL
DR3
APO-3
TRADD
RIP
TRAF2
DR4/5
FADD
FADDCaspase-8,-10
NIK
FLIP
IKK
Smac
lB
tBi
d
MKK7
Bcl
-2
JNK
NFB
HtrA2
NFB
Cyt
c
Bcl-2
FLIPs
xIAPs
Apaf-1
Caspase-9
Caspase-6
Lamin A
Caspase-3
Actin
Fodrin
Gas2
Rock-1
Caspase-7
Acinus
ICAD
PARP
CAD
Cell shrinkage
Membrane blebbing
Chromatin
DNA
DNA repair
condensation fragmentation
Apoptosis
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TNFR signaling
TNF-R1 is expressed in most tissues → soluble
and membrane bound TNF
TNF-R2 is found only in cells of the immune
system → only membrane bound TNF
Effects:
• IKK → IB → NFkB → Transcription of
proteins involved in cell survival and
proliferation, inflammation, and antiapoptotic factors
• MKK7 → JNK → Ap-1 → Cell differentiation,
proliferation, pro-apoptotic
• Caspase-8 → Caspase 3 → Apoptosis induction
• Caspase-8 → Bid → Apoptosis induction
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Controlling apoptosis
• Apoptosis inhibitors: Bcl-2 and Bcl-X
• They have a hydrophobic tail and bind to the
outside surface of mitochondria and other
organelles like the nucleus and endoplasmic
reticulum
• Bcl-2 can also bind to Apaf-1 and inhibit its
activation of initiator caspase-9
• Overexpression of Bcl-2 can cause a cell to
become a tumor cell. Some virus make IAP’s
(Inhibitors of APoptosis)
• Bcl-xL inhibits the formation of the supermolecular holes by Bax, Bak, Bid and
cardiolipin.
• Another member of the family, BAX and BAD bind
BID a bridge between the
extarcellular and mitochondrial
apoptosis pathways
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• Activated caspase 8 causes the cleavage of
the amino terminal portion of the cytosolic
protein Bid to generate t-Bid that is
translocated into mitochondria during
apoptosis
• Bid = BH3 interacting domain death agonist,
is a pro-apoptotic member of the Bcl-2
protein family
• Bid interacts with Bax leading to the
insertion of Bax into the outer
mitochondrial membrane
• Bax is believed to interact with, and induce
the opening of the mitochondrial voltage-
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Effector molecules
1. Caspase activation → DNA endonuclease
activation → DNA damage
2. Caspase 3 cleaves gelsolin → cleaves actin
filaments → membrane changes
3. When cells undergo apoptosis,
Phosphatidyl-serine normally found only in
the inner leaftlet, is exposed to the
outside → It can then bind to receptors on
phagocytic cells
4. Caspase 3 activates p21-activated kinase 2
(PAK-2) → formation of apoptotic bodies
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Membrane lipid transport
with scramblases
• Scramblases are members of the general
family of transmembrane lipid transporters
known as flippases, they can transport
(scramble) the negatively-charged
phospholipids from the inner-leaflet to the
outer-leaflet, and vice versa
• Phosphatidyl-serine is translocated to the
outer membrane → providing a phagocytic
signal to the macrophages that engulf and
clear the apoptotic cells
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PS labelling with Annexin V
Annexin V
Apoptosis
Annexin V binding
Ca2+
Ca2+
Ca2+
Cytoplasmic
membrane
Phosphatidyl
serine
Normal cell
Apoptotic cell
Ca2+
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Efferocytosis
• The effect of efferocytosis is that dead
cells are removed before their membrane
integrity is breached and their contents
leak into the surrounding tissue.
• This prevents exposure of tissue to toxic
enzymes, oxidants and other intracellular
components such as proteases and caspase.
• Mediated by macrophages, DC, fibroblasts,
and epithelial cells
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Cell surface events also
can inhibit apoptosis
• Binding of "survival" factors (like growth
factors) to cell surface receptors can shut
of apoptotic pathways in the cells
• They are coupled to PI-3-kinase
(phosphoinositol-3-kinase) through the G
protein ras (p21) → produces PI-3,4-P2 and
PI-3,4,5-P3, which activates Akt, a Ser/Thr
protein kinase → phosphorylates the
proapoptotic-protein BAD, which then becomes
inactive
• Active Akt phosphorylates procapse → which
will not interact with cytochrome C, hence
inhibiting apoptosis