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

at the University of Pécs and at the University of Debrecen

Identification number: TÁMOP-4.1.2-08/1/A-2009-0011

APOPTOSIS PATHWAYS

Tímea Berki and Ferenc Boldizsár Signal transduction

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

„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

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.

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

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)

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

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

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

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

Apoptosis pathways

Death 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

Activated Caspase-8

Effector Caspases

Bax Bak

BH3 only molecules

Anti apoptopic Bcl-2 family members Death receptors

(FasL, TRAIL, TNF)

Mitochondria

Apoptosome DISC

IAPs

Apoptosis

INTRINSIC EXTRINSIC

XIAP cIAP-1 cIAP-2 Survivin

Kinase

Phosphatase Enzyme

Cyclin, pro-apoptotic Pro-survival

GTP-ase GAP/GEF Caspase

Transcription factor

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

Mitochondrial apoptosis pathway

Cytc Bcl-2

Bcl-2

Bcl-2 Bad

Bax Bax

Bcl-2 Bax

Caspase-9

CytcApaf-1 Apoptosome Mitochondrion

Bad

P P P

Bad

Caspase cascade Apoptotic signals

PT Pore

P P P

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

Apoptosome

Apaf-11

Cytc Cytc

Cytc

Cytc

Cytc

Cytc Cytc

Apoptosome formation Recruitment of Procaspase-9

Caspase activation

caspase-9 Pro

Bcl-family

Anti-Apoptotic

BH4 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, CED9 A1, Bfl-1

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

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 ?

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

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

FADD TRADD

FasL TNF

Fas/

CD95

TNFR-1 TNFR2

TNF

ASK1

RIP Caspase-8,-10 Daxx

RAIDD TRAF2 FADD RIP

ASK1 DAPK

c-IAP1/2 TRAF2

Caspase-8,-10

FADD TRADD

APO-3L/TWEAK APO-2L/TRAIL

DR4/5

Caspase-8,-10

DR3 APO-3

FADD TRAF2

RIP

Caspase-8,-10

TNF receptor mediated apoptosis II

TRADD FADD

FADD TRADD

FasL TNF- APO-3L/TWEAK APO-2L/TRAIL

Fas/

CD95

TNFR-1 TNFR2 DR3

APO-3

DR4/5 TNF-

FADD ASK1

DAPK

RAIDD TRAF2

RIP TRAF2

RIP NIK

Caspase-8,-10 Daxx

FADD

Caspase-6

Caspase-9

Caspase-7 MKK7

IKK

Lamin A Actin Fodrin Gas2 Rock-1 ICAD

CAD

Acinus PARP

Cytc

NFB

Cell shrinkage Membrane blebbing

DNA fragmentation Chromatin

condensation

DNA repair FLIPs

Caspase- independent

cell death

Bcl-2 tBid Bid

Apaf-1

Caspase-3 FLIP

lB NFB ASK1

TRAF2

Apoptosis

Bcl-2

HtrA2 Smac

xIAPs

UB UB

RIP

JNK

c-IAP1/2

Caspase-8,-10

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 anti-

apoptotic factors

• MKK7 → JNK → Ap-1 → Cell differentiation, proliferation, pro-apoptotic

• Caspase-8 → Caspase 3 → Apoptosis induction

• Caspase-8 → Bid → Apoptosis induction

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

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

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

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

PS labelling with Annexin V

Annexin V

Normal cell

Cytoplasmic membrane

Phosphatidyl serine

Apoptosis

Apoptotic cell Annexin V binding

Ca²⁺ Ca²⁺ Ca²⁺

Ca²⁺

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

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