apoptosis pathways
DESCRIPTION
Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s P rogrammes at the University of Pécs and at the University of Debrecen Identification number : TÁMOP-4.1.2-08/1/A-2009-0011. - PowerPoint PPT PresentationTRANSCRIPT
Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat the University of Pécs and at the University of DebrecenIdentification number: TÁMOP-4.1.2-08/1/A-2009-0011
APOPTOSIS PATHWAYS
Tímea Berki and Ferenc BoldizsárSignal transduction
Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat the University of Pécs and at the University of DebrecenIdentification number: TÁMOP-4.1.2-08/1/A-2009-0011
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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 appropriate activation.
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When would it be advantageous to the organism? • 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 pro-caspases• These proteases are found in the cell in inactive form
which must undergo limited proteolysis for activation• These caspases form a cascade
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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 pathwaysDeath ligands
(FasL, TRAIL, TNF)Stimuli
(Cytokine deprivation, viral infection,DNA damage, irradiation, cell stress)
Pro-Caspase-8
FADD
Caspase-9
Cytc
Apaf-1
Smac
FLIP
ActivatedCaspase-8
EffectorCaspases
Bax Bak
BH3 onlymolecules
Anti apoptopicBcl-2 family members
Death receptors(FasL, TRAIL, TNF)
Mitochondria
Apoptosome
DISC
IAPs
Apoptosis
INTRINSICEXTRINSIC
XIAPcIAP-1cIAP-2
Survivin
Kinase
Phosphatase
Enzyme
Cyclin, pro-apoptotic
Pro-survival
GTP-ase
GAP/GEF
Caspase
Transcription factor
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Intrinsic apoptotic pathway1 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
Cytc
Bcl-2Bcl-2
Bcl-2Bad
BaxBax
Bcl-2Bax
Caspase-9
Cytc Apaf-1 Apoptosome
Mitochondrion
BadP
PP
Bad
Caspase cascade
Apoptotic signals
PT Pore
PP
P
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Permeability transition pore • Outer membrane protein (porin, the voltage-gated
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
Apaf-1Apaf-1
Apaf
-11 Apaf-1
Apaf-1
Apaf-1
Apaf-1
CytcCytc
Cytc
Cytc
CytcCytc
Cytc
Apoptosome formation Recruitment of Procaspase-9
Caspase activation
Procaspase-9
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Bcl-family
Anti-ApoptoticBH4 BH3 BH1 BH2
BH4 BH3 BH1 BH2 TM Bcl-2, Bcl-XL
BH3 BH1 BH2 TM Bax, Bak
BH4 BH3 BH1 BH2 TM Diva
BH4 BH3 TM Bcl-Xs
BH3 TM Bik, Bim
BH3 Bad, Bid, Egl-1
Pro-Apoptotic
Mcl1, CED9A1, Bfl-1
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What causes all these changes in the mitochondria? • 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 oxidative stress. Cells undergo p53 apoptosis through oxidative damage.
• Increases in intracellular calcium ions through signal transduction
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Apoptosis pathways in activated T cells
T-cell subgroup Pathway
Bulk activated T cells Fas/FasL
Th1 Fas/FasL
Th2 Granzyme B
Th17 Fas/FasL?
Tc1 Fas/FasL/Granzyme B
Tc2 ?
Treg, gdT cells, NK, NKT ?
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Extrinsic apoptotic pathway:death receptors• 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: FasFAS 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)
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TNF receptor mediated apoptosis I
FADD TRADD
FasL TNF
Fas/CD95
TNFR-1 TNFR2
TNF
ASK1RIP Caspase-8,-10
Daxx
RAIDDTRAF2
RIPFADD
ASK1
DAPK c-IAP1/2 TRAF2
Caspase-8,-10
FADDTRADD
APO-3L/TWEAK APO-2L/TRAIL
DR4/5
Caspase-8,-10
DR3APO-3
FADDTRAF2
RIPCaspase-8,-10
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TNF receptor mediated apoptosis II
TRADD FADDFADD TRADD
FasL TNF- APO-3L/TWEAK APO-2L/TRAIL
Fas/CD95
TNFR-1 TNFR2 DR3APO-3
DR4/5
TNF-
FADDASK1
DAPKRAIDDTRAF2
RIP TRAF2RIP
NIK
Caspase-8,-10
Daxx
FADD
Caspase-6Caspase-9
Caspase-7
MKK7
IKK
Lamin A Actin Gas2Fodrin Rock-1 ICAD
CAD
Acinus PARP
CytcNFB
Cell shrinkageMembrane blebbing
DNAfragmentation
Chromatincondensation
DNA repair
FLIPs
Caspase-independent
cell death
Bcl-2
tBid
Bid
Apaf-1
Caspase-3
FLIP
lBNFB
ASK1
TRAF2
Apoptosis
Bcl-2
HtrA2
Smac
xIAPs
UBUBRIP
JNK
c-IAP1/2
Caspase-8,-10
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TNFR signalingTNF-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 TNFEffects: • IKK → IB → NFkB → Transcription of proteins
involved in cell survival and proliferation, inflammation, and anti-apoptotic 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 super-molecular holes by Bax, Bak, Bid and cardiolipin.
• Another member of the family, BAX and BAD bind to mitochondria and facilitate apoptosis by stimulating cytochrome C release
TÁMOP-4.1.2-08/1/A-2009-0011BID a bridge between the extarcellular and mitochondrial apoptosis pathways• 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-dependent anion channel, VDAC
• The anti-apoptotic Bcl-2 proteins may inhibit apoptosis by sequestering BID, leading to reduced Bax activation
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Effector molecules1. Caspase activation → DNA endonuclease activation
→ DNA damage2. Caspase 3 cleaves gelsolin → cleaves actin
filaments → membrane changes3. 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
Normal cell
Cytoplasmicmembrane
Phosphatidylserine
Apoptosis
Apoptotic cell
Annexin V bindingCa2+ Ca2+ Ca2+
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