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Apoptosis signalling
1) Apoptosis, or programmed cell death, is a natural and important process by which cells self-destruct in response to internal or external signals. It plays a key role in development, tissue homeostasis, and defense against infection and cancer. 2) There are two main pathways of apoptosis - the extrinsic or death receptor pathway, which involves cell surface death receptors, and the intrinsic or mitochondrial pathway, which is activated by intracellular stresses. Both pathways activate caspases, cysteine proteases that dismantle the cell in an orderly manner. 3) Apoptosis is tightly regulated by a network of pro- and anti-apoptotic Bcl-2 family proteins that control mitochondrial membrane permeability and
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Apoptosis signalling
- 1. Apoptosis Signaling Live??? OrDie??? Vijay Avin BR, Molecular Biomedicine Laboratory, Sahyadri Sceince College, Shimoga, Karnataka, India
- 2. Life of cell •Mitosis checkpoints • Apoptosis will be triggered to prevent cells from becoming cancers and harming the body
- 3. Cell death byinjury -Mechanical damage -Exposure to toxic chemicals Cell death by suicide -Internal signals -External signals
- 4. • Definition Apo: apart Ptosis:fallen – Shedding of leaves from tress • During embriogenesis ------ occurs as PCD • Post-embrional life------- as apoptosis
- 5. apoptosis • Apoptosis isused 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. 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.
- 8. Why have wedeveloped 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. • PCD machineryis silent until signals arrive to start PCD: • Signals: • damage to DNA • Activation of membrane receptors. Ligands are: peptides, cytokines, ATP, ROS etc
- 10. • Fas receptor? Receptorsfor growth factors, cytokines and hormones • Membrane alterations cause apoptosis. What kind of membrane alterations ?? Phospholipid redistributions, changes in membrane charge, carbohydrate and surface markers.
- 12. Proteins involved inapoptosis • 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
- 13. STAGES OF APOPTOSIS Shermanet al., 1997 Induction of apoptosis related genes, signal transduction
- 14.
- 15. membrane blebbing &changes mitochondrial leakage organelle reduction cell shrinkage nuclear fragmentation chromatin condensation APOPTOSIS: Morphological events
- 16. Bleb Blebbing & Apoptoticbodies 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
- 17. Apoptosis: Pathways Death Ligands Effector Caspase 3 Death Receptors Initiator Caspase8 Cell death DNA damage & p53 Mitochondria/ Cytochrome C Initiator Caspase 9 “Extrinsic Pathway” “Intrinsic Pathway”
- 20. • Binding ofFas 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
- 22. MAJOR PLAYERS IN APOPTOSIS •Caspases • Adaptor proteins • Bcl-2 family
- 23. 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
- 24. • NO canalso 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
- 25. NO may alsomediate 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)
- 26. • NO exposureor activation may inhibit apoptosis in Lymphocytes Endothelial cells Neurons Hepatocytes Kidney cells
- 27. How?? • Direct inhibitionof 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.
- 28. For example, S-nitrosylationwas 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….?
- 29. 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
- 30. The bcl-2 family BH4BH3 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
- 31. P53 & Apoptosis p53first 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)
- 32.
Editor's Notes
- #3 For some this may be a review but this is a diagram showing the importance of cell apoptosis in checkpoints during mitosis. Before entering a phase of mitosis, checkpoints are set up that inspect that everything is going according to plan. If it is, the cell proceeds on its path to reproduction, however if everything is not as it should be the caspase cascade is triggered and the cell is destroyed.
- #12 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 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
- #18 http://www.youtube.com/watch?v=9KTDz-ZisZ0
- #19 In a healthy cell, the outer membranes of its mitochondria display the protein Bcl-2 on their surface. Internal damage to the cell (e.g., from reactive oxygen species) causes Bcl-2 to activate a related protein, Bax, which punches holes in the outer mitochondrial membrane, causing cytochrome c to leak out. The released cytochrome c binds to the protein Apaf-1 (&quot;apoptotic protease activating factor-1&quot;). Using the energy provided by ATP, these complexes aggregate to form apoptosomes. The apoptosomes bind to and activate caspase-9. Caspase-9 is one of a family of over a dozen caspases. They are all proteases. They get their name because they cleave proteins — mostly each other — at aspartic acid (Asp) residues). Caspase-9 cleaves and, in so doing, activates other caspases (caspase-3 and -7). The activation of these &quot;executioner&quot; caspases creates an expanding cascade of proteolytic activity (rather like that in blood clotting and complement activation) which leads to digestion of structural proteins in the cytoplasm, degradation of chromosomal DNA, and phagocytosis of the cell.
- #26 Superoxide dismutase SOD Glutathione peroxidase GPx or plasma glutathione peroxidase Catalase
- #29 A calpain (pronounced /ˈkælpeɪn/;[1] EC 3.4.22.52, EC 3.4.22.53) is a protein belonging to the family of calcium-dependent, non-lysosomal cysteine proteases (proteolytic enzymes) In the field of molecular biology, the activator protein 1 (AP-1) is a transcription factor which is a heterodimeric protein composed of proteins belonging to the c-Fos, c-Jun, ATF and JDP families NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) is a protein complex that controls the transcription of DNA and Receptor activator of nuclear factor kappa B (RANK), which is a type of TNFR, is a central activator of NF-κB. Osteoprotegerin (OPG), which is a decoy receptor homolog for RANK ligand, inhibits RANK by binding to RANKL, and, thus, osteoprotegerin is tightly involved in regulating NF-κB activation