1. 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 /1/A

2. Apoptosis pathways Tímea Berki and Ferenc Boldizsár
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 /1/A
Tímea Berki and Ferenc Boldizsár
Signal transduction
Apoptosis pathways

3. „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

4. 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.

5. 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

6. 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)

7. 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

8. 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

9. 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

10. 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

11. 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
Pro-
Caspase-8
Bax
Bak
Kinase
Phosphatase
Enzyme
Cyclin, pro-apoptotic
Pro-survival
GTP-ase
GAP/GEF
Caspase
Transcription factor
Activated
Caspase-8
Smac
Mitochondria
Cytc
XIAP
cIAP-1
cIAP-2
Survivin
Apaf-1
Apoptosome
IAPs
Effector
Caspases
Caspase-9
Apoptosis

12. 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

13. Mitochondrial apoptosis pathway
Apoptotic signals
Bcl-2
Bad P
Bad
Bcl-2 P
Bad P P
Mitochondrion
Bax
Cytc
Caspase-9
Cytc
Apaf-1
Apoptosome
PT Pore
Bcl-2
Bax
Caspase cascade

14. 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   Collapsing the proton gradient uncouples oxidation and phosphorylation in the mitochondria

15. Recruitment of Procaspase-9
Apoptosome
Apoptosome formation
Recruitment of Procaspase-9
Apaf-1
Apaf-11
Cytc
Pro
caspase-9
Caspase activation

16. Bcl-family Anti-Apoptotic Bcl-2, Bcl-XL Bax, Bak Diva Bcl-Xs Bik, Bim
BH4
BH3
BH1
BH2 TM
Bcl-2, Bcl-XL
Bax, Bak
Diva
Bcl-Xs
Bik, Bim
Bad, Bid, Egl-1
Pro-Apoptotic
Mcl1, CED9
A1, Bfl-1

17. 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

18. Apoptosis pathways in activated T cells
T-cell subgroup
Pathway
Bulk activated T cells
Fas/FasL
Th1
Th2
Granzyme B
Th17
Fas/FasL?
Tc1
Fas/FasL/Granzyme B
Tc2 ?
Treg, gdT cells, NK, NKT

19. 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 

20. 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)

21. TNF receptor mediated apoptosis I
FADD
TRADD
FasL
TNF
Fas/
CD95
TNFR-1
TNFR2
ASK1
RIP
Caspase-8,-10
Daxx
RAIDD
TRAF2
DAPK
c-IAP1/2
APO-3L/TWEAK
APO-2L/TRAIL
DR4/5
DR3
APO-3

22. TNF receptor mediated apoptosis II
FasL
TNF-
TNF-
APO-3L/TWEAK
APO-2L/TRAIL
Fas/
CD95
TNFR-1
TNFR2
DR3
APO-3
DR4/5
Daxx
DAPK
c-IAP1/2
TRADD
TRAF2
FADD
TRADD
FADD
ASK1
Caspase-8,-10
RAIDD
FADD
RIP UB
RIP
FADD
Caspase-8,-10
RIP
TRAF2 UB
TRAF2
ASK1
NIK
Caspase-
independent
cell death
FLIP
Bid
IKK
Smac
lB
NFB
tBid
MKK7
HtrA2
Bcl-2
NFB
FLIPs
JNK
Cytc
Bcl-2
xIAPs
Apaf-1
Caspase-9
Caspase-6
Caspase-3
Caspase-7
Lamin A
Actin
Fodrin
Gas2
Rock-1
Acinus
ICAD
PARP
CAD
Cell shrinkage
Membrane blebbing
Chromatin
condensation
DNA
fragmentation
DNA repair
Apoptosis

23. 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 → IkB → 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

24. 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

25. BID 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

26. Effector molecules
Caspase activation → DNA endonuclease activation → DNA damage
Caspase 3 cleaves gelsolin → cleaves actin filaments → membrane changes
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
Caspase 3 activates p21-activated kinase 2 (PAK-2) → formation of apoptotic bodies

27. 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

28. PS labelling with Annexin V
Apoptosis
Annexin V
Annexin V binding
Ca2+
Cytoplasmic
membrane
Phosphatidyl
serine
Normal cell
Apoptotic cell

29. 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

30. 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