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Apoptosis signalling


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Apoptosis signalling by Vijay Avin BR

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Apoptosis signalling

  1. 1. Apoptosis Signaling Live??? Or Die??? Vijay Avin BR, Molecular Biomedicine Laboratory, Sahyadri Sceince College, Shimoga, Karnataka, India
  2. 2. Life of cell • Mitosis checkpoints • Apoptosis will be triggered to prevent cells from becoming cancers and harming the body
  3. 3. Cell death by injury -Mechanical damage -Exposure to toxic chemicals Cell death by suicide -Internal signals -External signals
  4. 4. • Definition Apo: apart Ptosis: fallen – Shedding of leaves from tress • During embriogenesis ------ occurs as PCD • Post-embrional life------- as apoptosis
  5. 5. apoptosis • Apoptosis is used as a synonymous for PCD but PCD is physiological death, occurs only during embriogenesis. • It is a functional death and it is a good mechanism to eliminate wasted, useless, unwanted, or crippled cells!
  6. 6. Necrosis vs. Apoptosis • Cellular condensation • Membranes remain intact • Requires ATP • Cell is phagocytosed, no tissue reaction • Ladder-like DNA fragmentation • In vivo, individual cells appear affected • Cellular swelling • Membranes are broken • ATP is depleted • Cell lyses, eliciting an inflammatory reaction • DNA fragmentation is random, or smeared • In vivo, whole areas of the tissue are affected Necrosis Apoptosis
  7. 7. NECROSIS Vs APOPTOSIS Wilde, 1999
  8. 8. Why have we developed such a self-destructive system? • A. PCD allows a constant selection for the fittest cell in a colony • Every cell carries the molecular machinery to do PCD! • Cells that are sensitive to extracellular signals will survive, cell that cannot compete with their more vital sisters will undergo apoptosis.
  9. 9. • PCD machinery is silent until signals arrive to start PCD: • Signals: • damage to DNA • Activation of membrane receptors. Ligands are: peptides, cytokines, ATP, ROS etc
  10. 10. • Fas receptor? Receptors for growth factors, cytokines and hormones • Membrane alterations cause apoptosis. What kind of membrane alterations ?? Phospholipid redistributions, changes in membrane charge, carbohydrate and surface markers.
  11. 11. Proteins involved in apoptosis • Fas ligand (FasL or CD95L) is a type-II transmembrane protein that belongs to the tumor necrosis factor (TNF) family • Fas-Associated protein with Death Domain (FADD) is an adaptor molecule that bridges the Fas-receptor, and other death receptors, • Apoptotic protease activating factor 1, also known as APAF1 • Bcl-2 (B-cell lymphoma 2) is the founding member of the Bcl-2 family of apoptosis regulator proteins encoded by the BCL2gene Caspases • Inflammatory Caspases: -1, -4, and -5 • Initiator Caspases: -2, -8, -9, and -10 – Long N-terminal domain – Interact with effector caspases • Effector Caspases: -3, -6, and -7 – Little to no N-terminal domain – Initiate cell death • The Mitochondrial Apoptosis-Induced Channel (or MAC), • BAK: Bcl-2 homologous antagonist killer • BAX: Bcl-2 associated x protein • BID: BH3 interacting domain death agonist, a pro-apoptotic protein • BAD: The Bcl-2-associated death promoter (BAD) protein is a pro-apoptotic member of the Bcl-2 gene family which is involved in initiating apoptosis. BAD is a member of the BH3-only family
  12. 12. STAGES OF APOPTOSIS Sherman et al., 1997 Induction of apoptosis related genes, signal transduction
  13. 13. membrane blebbing & changes mitochondrial leakage organelle reduction cell shrinkage nuclear fragmentation chromatin condensation APOPTOSIS: Morphology Hacker., 2000
  14. 14. membrane blebbing & changes mitochondrial leakage organelle reduction cell shrinkage nuclear fragmentation chromatin condensation APOPTOSIS: Morphological events
  15. 15. Bleb Blebbing & Apoptotic bodies The control retained over the cell membrane & cytoskeleton allows intact pieces of the cell to separate for recognition & phagocytosis by MΦs Apoptotic body MΦ MΦ
  16. 16. Apoptosis: Pathways Death Ligands Effector Caspase 3 Death Receptors Initiator Caspase 8 Cell death DNA damage & p53 Mitochondria/ Cytochrome C Initiator Caspase 9 “Extrinsic Pathway” “Intrinsic Pathway”
  17. 17. • Binding of Fas by FasL induces recruitment of FADD to the cytoplasmic tail of Fas • The opposite end of FADD contains a death effector domain (hatched boxes); recruitment of either procaspase-8 or c-FLIP • Caspase-8 can cleave Bid • truncated Bid (tBid) can inactivate Bcl-2 in the mitochondrial membrane. • This allows the escape of cytochrome c, which clusters with Apaf-1 and caspase-9 in the presence of dATP to activate caspase-9. • Smac/DIABLO is also released from the mitochondria and inactivates inhibitors of apoptosis (IAPs). • breakdown of several cytoskeletal proteins and degradation of the inhibitor of caspase-activated DNase (ICAD). Extrinsic or Death Receptor Pathway
  18. 18. MAJOR PLAYERS IN APOPTOSIS • Caspases • Adaptor proteins • Bcl-2 family
  19. 19. Modulation of apoptosis • Apoptotic cell death can be switched to necrosis during oxidative stress by 2 mechanisms: Inactivation of caspases due to oxidation of their active site thiol group by oxidants Decrease in ATP due to failure of mitochondrial energy production by oxidants
  20. 20. • NO can also have dual effects on apoptosis NO is reactive, unstable free radical gas that can easily cross cell membranes. L-Arg------ NO Low NO: Neurotransmitter, regulator in vasodilation and platelet aggregation. High NO: Cytotoxicity
  21. 21. NO may also mediate apoptosis: How??? • Formation of iron-nitrosyl complexes with FeS-containing enzymes: This leads to impairment of mitochondrial function ATP depletion. • NO may directly damage DNA- mutagenesis • Generation of OONO- Apoptosis • NO may inactivate several antioxidant enzymes (CAT, GPx, SOD etc)
  22. 22. • NO exposure or activation may inhibit apoptosis in Lymphocytes Endothelial cells Neurons Hepatocytes Kidney cells
  23. 23. How?? • Direct inhibition of caspase (S-nitrosylation of the active site Cys) • R-S-NO is important component of signal transduction cascades. • S-nitrosylation can regulate many proteins: • Enzymes • Ion channels • G-proteins • Transcription factors • NO may act as a modular switch to control protein function via –SH groups.
  24. 24. For example, S-nitrosylation was shown to occur in: • Calpain • NF-KB • AP-1 These are all implicated in the regulation of apoptosis. • Nitrosylation/denitrosylation- may serve as a regulatory mechanism just like….?
  25. 25. Importance of Apoptosis • Important in normal physiology / development – Development: Immune systems maturation, Morphogenesis, Neural development – Adult: Immune privilege, DNA Damage and wound repair. • Excess apoptosis – Neurodegenerative diseases • Deficient apoptosis – Cancer – Autoimmunity
  26. 26. The bcl-2 family BH4 BH3 BH1 BH2 TMN C Receptor domain phosphorylation Raf-1 calcineurin Pore formation Membrane anchor Ligand domain Group I Group II Group III Bcl-2 bax Bad bid bik Back
  27. 27. P53 & Apoptosis p53 first arrests cell growth between G1 → S This allows for DNA repair during delay If the damage is too extensive then p53 induces gene activation leading to apoptosis (programmed cell death)
  28. 28. Thank u