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

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-4.1.2-08/1/A-2009-0011 Tímea Berki and Ferenc Boldizsár Signal transduction Apoptosis pathways

„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.

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

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 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

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 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

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

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

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

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

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

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 

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 ASK1 RIP Caspase-8,-10 Daxx RAIDD TRAF2 DAPK c-IAP1/2 APO-3L/TWEAK APO-2L/TRAIL DR4/5 DR3 APO-3

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

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

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

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

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

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

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

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