Apoptosis pathways
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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 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 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 TÁMOP-4.1.2-08/1/A-2009-0011 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 When would it be advantageous to the organism? TÁMOP-4.1.2-08/1/A-2009-0011 • 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 TÁMOP-4.1.2-08/1/A-2009-0011 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) TÁMOP-4.1.2-08/1/A-2009-0011 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 TÁMOP-4.1.2-08/1/A-2009-0011 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 procaspases • These proteases are found in the cell in inactive form which must undergo limited proteolysis for activation TÁMOP-4.1.2-08/1/A-2009-0011 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 TÁMOP-4.1.2-08/1/A-2009-0011 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 TÁMOP-4.1.2-08/1/A-2009-0011 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 ProCaspase-8 Kinase Enzyme Bax Activated Caspase-8 Bak Mitochondria Smac Phosphatase Caspase Cyt c Pro-survival Cyclin, pro-apoptotic Transcription factor Effector Caspases Apaf-1 Caspase-9 GAP/GEF GTP-ase Apoptosis Apoptosome XIAP cIAP-1 IAPs cIAP-2 Survivin TÁMOP-4.1.2-08/1/A-2009-0011 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 TÁMOP-4.1.2-08/1/A-2009-0011 Mitochondrial apoptosis pathway Apoptotic signals Bcl-2 P Bcl-2 Bad P P Bcl-2 Bad P Bad Mitochondrion P P Bax Bax Cyt c PT Pore Cyt c Apaf-1 Apoptosome Caspase-9 Bcl-2 Bax Caspase cascade TÁMOP-4.1.2-08/1/A-2009-0011 Permeability transition pore • Outer membrane protein (porin, the voltagegated 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 TÁMOP-4.1.2-08/1/A-2009-0011 Apoptosome Apoptosome formation Cytc Recruitment of Procaspase-9 Pro caspase-9 Cytc Cytc Cytc Apaf-11 Cytc Cytc Cytc Caspase activation TÁMOP-4.1.2-08/1/A-2009-0011 Bcl-family BH4 BH3 BH1 BH2 TM Anti-Apoptotic Mcl1, CED9 BH4 BH3 BH3 BH4 Pro-Apoptotic Bcl-2, Bcl-XL BH3 BH4 BH1 A1, Bfl-1 BH2 BH1 BH2 BH1 BH2 BH3 BH3 BH3 TM Bax, Bak TM Diva TM Bcl-Xs TM Bik, Bim Bad, Bid, Egl-1 What causes all these changes in the mitochondria? TÁMOP-4.1.2-08/1/A-2009-0011 • 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 Apoptosis pathways in activated T cells T-cell subgroup Bulk activated T cells Th1 Th2 Th17 Tc1 Tc2 Treg, gdT cells, NK, NKT TÁMOP-4.1.2-08/1/A-2009-0011 Pathway Fas/FasL Fas/FasL Granzyme B Fas/FasL? Fas/FasL/Granzyme B ? ? Extrinsic apoptotic pathway: death receptors TÁMOP-4.1.2-08/1/A-2009-0011 • 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 TÁMOP-4.1.2-08/1/A-2009-0011 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 TNF FasL TNF Fas/ CD95 TNFR-1 Daxx ASK1 DAPK FADD Caspase-8,-10 RIP TRADD FADD Caspase-8,-10 APO-3L/TWEAK DR3 APO-3 TRADD TRAF2 RIP TÁMOP-4.1.2-08/1/A-2009-0011 FADD Caspase-8,-10 RIP RAIDD TNFR2 c-IAP1/2 TRAF2 TRAF2 ASK1 APO-2L/TRAIL DR4/5 FADD Caspase-8,-10 TNF receptor mediated apoptosis II FasL TNF- TNF- Fas/ CD95 Daxx ASK1 APO-3L/TWEAK TNFR-1 TNFR2 DAPK c-IAP1/2 TRAF2 TRADD FADD RAIDD Caspase-8,-10 FADD UB RIP UB RIP TRAF2 ASK1 Caspaseindependent cell death Bid TÁMOP-4.1.2-08/1/A-2009-0011 APO-2L/TRAIL DR3 APO-3 TRADD RIP TRAF2 DR4/5 FADD FADDCaspase-8,-10 NIK FLIP IKK Smac lB tBi d MKK7 Bcl -2 JNK NFB HtrA2 NFB Cyt c Bcl-2 FLIPs xIAPs Apaf-1 Caspase-9 Caspase-6 Lamin A Caspase-3 Actin Fodrin Gas2 Rock-1 Caspase-7 Acinus ICAD PARP CAD Cell shrinkage Membrane blebbing Chromatin DNA DNA repair condensation fragmentation Apoptosis TÁMOP-4.1.2-08/1/A-2009-0011 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 → IB → NFkB → Transcription of proteins involved in cell survival and proliferation, inflammation, and antiapoptotic factors • MKK7 → JNK → Ap-1 → Cell differentiation, proliferation, pro-apoptotic • Caspase-8 → Caspase 3 → Apoptosis induction • Caspase-8 → Bid → Apoptosis induction TÁMOP-4.1.2-08/1/A-2009-0011 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 supermolecular holes by Bax, Bak, Bid and cardiolipin. • Another member of the family, BAX and BAD bind BID a bridge between the extarcellular and mitochondrial apoptosis pathways TÁMOP-4.1.2-08/1/A-2009-0011 • 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- TÁMOP-4.1.2-08/1/A-2009-0011 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 TÁMOP-4.1.2-08/1/A-2009-0011 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 TÁMOP-4.1.2-08/1/A-2009-0011 PS labelling with Annexin V Annexin V Apoptosis Annexin V binding Ca2+ Ca2+ Ca2+ Cytoplasmic membrane Phosphatidyl serine Normal cell Apoptotic cell Ca2+ TÁMOP-4.1.2-08/1/A-2009-0011 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 TÁMOP-4.1.2-08/1/A-2009-0011 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
