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

TÁMOP-4.1.2-08/1/A-2009-0011

„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