APOPTOSIS cell and molecular biology.pptx

apoptosis

1. APOPTOSIS Presented by – Vivek Giri parinita giri Msc . Microbiology (previous)

2. Contents Introduction What is apoptosis ? Need for apoptosis Reasons for apoptosis Apoptosis vs necrosis Caspases Mechanisms of apoptosis

3. What is Apoptosis? comes from a Greek word meaning “falling off,” of leaves from a tree. Apoptosis or Programmed cell death is an energy dependent process In 1972, John F. Kerr, Andrew H. Wyllie and A. R. Currie, coined the term “apoptosis”

4. Need for Apoptosis

5. Elimination of cells whose function is over and no longer required Development of mammalian nervous system Maintain size and shape of tissue and organ

6. Apoptosis is needed to kill : Cells infected with virus Cells with DNA damage Cancer cell

7. Morphological changes during Apoptosis Cell shrinkage Nuclear fragmentation Chromatin condensation Formation of cytoplasmic blebs and apoptotic bodies Phagocytisis

8. Biochemical changes during Apoptosis Activation of caspases Proteolysis of cytoskeletal protein Fragmentation of nuclear chromatin by activation of nucleases Membrane alteration Recognition by phagocytes

9. Apoptosis of T Lymphocytes in Systemic Sclerosis - Scientific Figure on ResearchGate . Available from: https://www.researchgate.net/figure/Morphological-cell-changes-during-apoptosis-42_fig1_221923701 [accessed 16 Aug, 2022]

10. History of Apoptosis Research - Scientific Figure on ResearchGate . Available from: https://www.researchgate.net/figure/A-diagram-describing-the-morphology-of-apoptosis-Adapted-from-Kerr-et-al-1972-with_fig1_262013868 [accessed 21 Aug, 2022]

11. Biochemical features of apoptosis Endogenous Dnases , which cut the internucleosomal regions into double-stranded DNA fragments of 180–200 base pairs ( bp ) DNA fragmentation factor (DFF40), caspase activated Dnase (CAD) Present in normal cells as inactive heterodimers with the inhibitor proteins DFF45 [40] and ICAD (inhibitor of CAD) Activated upon cleavage by caspase 3 Exposure of nuclei to activated CAD or DFF40 is sufficient to induce the nuclear morphologic changes typical of apoptosis

12. More than a dozen protein kinases , including focal adhesion kinase  ( FAK ), PKB, PKC, and Raf1. Inactivation of FAK, for example, disrupt cell adhesion, leading to detachment of the apoptotic cell from its neighbors. Inactivation of certain other kinases , such as PKB, serves to disrupt prosurvival signaling pathways. Caspases also disrupt the generation of survival signals by inactivating the NF‐ kB pathway  Lamins , which make up the inner lining of the nuclear envelope. Cleavage of lamins leads to the disassembly of the nuclear lamina and shrinkage of the nucleus. Proteins of the cytoskeleton, intermediate filaments, actin , tubulin , and gelsolin . Cleavage and consequent inactivation of these proteins lead to changes in cell shape

13. Removal of apoptotic cells by phagocytes Chemical changes on the surface of the apoptotic cell, which displays signals that recruit phagocytic cells. negatively charged phospholipid phosphatidylserine on the cell surface, normally located exclusively in the inner leaflet of the lipid bilayer of the plasma membrane It flips to the outer leaflet in apoptotic cells. The external exposure of phosphatidylserine depends on the caspase cleavage of some protein involved in phospholipid distribution in the membrane A variety of soluble “bridging” proteins interact with the exposed phosphatidylserine on the apoptotic cell. These bridging proteins also interact with specific receptors on the surface of a neighboring cell or macrophage, triggering cytoskeletal and other changes that initiate the engulfment process.

14. Macrophages do not phagocytose healthy cells in the animal Healthy cells express signal proteins on their surface that interact with inhibitory receptors on macrophages that block phagocytosis Apoptotic cells lose or inactivate these “don’t eat me” signals that block phagocytosis .

15. Apoptosis Necrosis Morphological changes Membrane blebbing with loss of membrane integrity Loss of membrane integrity Aggregation and margination of chromatin Random fragmentation of chromatin Cellular shrinkage Cellular swelling Formation of apoptotic bodies Cell lysis Persistence of apoptotic bodies Swelling and disintegration of organelles Biochemical changes Genetically controlled activation of enzymes Loss of ion homeostasis ATP-dependent process Passive process (no energy requirement) Generation of non-random oligonucleosomes of DNA (ladder pattern on agarose gel ) Random digestion of DNA (DNA smear on agarose gel) Early pre- lytic DNA fragmentation Late post-lytic DNA fragmentation Physiological significance Death of individual or small groups of cells Death of large contiguous groups of cells or organ segments Evoked by physiological stimuli Evoked by pathological stimuli Phagocytosis by macrophages or neighboring cells Phagocytosis by macrophages No inflammation Acute inflammation

16. The Role of GnRH Analogues in Endometriosis-Associated Apoptosis and Angiogenesis - Scientific Figure on ResearchGate . Available from: https://www.researchgate.net/figure/Comparison-of-the-cellular-changes-that-occur-during-apoptosis-and-necrosis_fig1_51434475 [accessed Aug 20, 2022]

17. Caspases : The key player Caspases stand for cysteine -dependent aspartate -directed protease Have cysteine at their active site Cleave their target proteins at specific aspartic acid Activated in a cascade manner There are two subclasses of apoptotic caspases : Initiator caspases Executioner caspases

18. Initiator caspases Exists as inactive, soluble monomer in cytosol Carries the apoptotic signal Dimerisation Activate executioner caspase Examples – Caspases 8, Caspases 9

19. Executioner caspases Normally present as inactive dimers Cleaved by an initiator caspase at a site in the protease domain Catalyze widespread protein clevage Kill the cell Examples – Caspases 3, Caspases 6, Caspases 7

20. Activation of caspases

21. Pathways for Apoptosis

22. Extrinsic pathway Cell surface death receptors transmembrane protein Composed of three components: Extracellular ligand -binding domain, A single transmembrane domain An intracellular death domain Homotrimers TNF Receptor family and Fas death receptor The ligands that activate the death receptors are also homotrimers ; they are structurally related to one another and belong to the TNF family of signal protein

23. Binding of Death signal receptor and homologous trimeric ligand Cytoplasmic adaptor protein exhibiting corresponding death domain binds to the receptor. In case of FasL / FasR and TNF-α/TNFR1 the adaptor protein are FADD and TRADD respectively Association of FADD with procaspase-8 via dimerization of the death effector domain. death-inducing signaling complex (DISC) is formed, resulting in the auto-catalytic activation of procaspase-8

24.   death-inducing signaling complex (DISC) is formed, resulting in the auto-catalytic activation of procaspase-8 Activation of executioner caspase by initiator caspases

25. Regulation of extrinsic pathway Inhibition of death-receptor mediated apoptosis Protein c-FLIP bind to FADD and Caspase-8, rendering them ineffective. Protein Toso block Fas -induced apoptosis in T cells via inhibition of caspase-8 processing.

26. Apoptosis in Cancer: Key Molecular Signaling Pathways and Therapy Targets - Scientific Figure on ResearchGate . Available from: https://www.researchgate.net/figure/The-extrinsic-pathway-of-apoptosis-and-its-regulation-Death-receptor-signaling-can-be_fig3_26280189 [accessed 18 Aug, 2022]

27. Intrinsic Pathway Irreparable genetic damage, lack of oxygen (hypoxia), extremely high concentrations of cytosolic Ca 2+ , viral infection, ER stress, or severe oxidative stress trigger apoptosis by the intrinsic pathway Pathway begins with the release of cytochrome c into the cytosol Bind to adaptor protein Apaf1 (apoptotic protease activating factor-1 ) Apaf1 oligomerize into a wheel-like heptamer called an apoptosome Recruitment of initiator caspase-9 proteins The activated caspase-9 molecules then activate downstream executioner caspases to induce apoptosis

28. Assembly of the mammalian apoptosome

29. The intrinsic pathway of apoptosis. Intracellular apoptotic stimuli cause mitochondria to release cytochrome c, which interacts with Apaf1. The binding of cytochrome c causes Apaf1 to unfold partly, exposing a domain that interacts with the same domain in other activated Apaf1 molecules. Seven activated Apaf1 proteins form a large ring complex called the apoptosome . Each Apaf1 protein contains a caspase recruitment domain (CARD), and these are clustered above the central hub of the apoptosome . The CARDs bind similar domains in multiple caspase-9 molecules, which are thereby recruited into the apoptosome and activated. The mechanism of caspase-9 activation is not clear: it probably results from dimerization and cleavage of adjacent caspase-9 proteins, but it might also depend on interactions between caspase-9 and Apaf1. Once activated, caspase-9 cleaves and thereby activates downstream executioner caspases . Note that the CARD is related in structure and function to the death effector domain of caspase-8

30. Bcl-2 protein: The major regulator Control the release of cytochrome c and other intermembrane mitochondrial proteins into the cytosol Bcl-2 homology domain 3 types: Anti-apoptotic Bcl2 family protein Pro-apoptotic effector Bcl2 family protein BH3-only protein

31. Anti-apoptotic Bcl2 family protein Located on the cytosolic surface of the outer mitochondrial membrane Prevent inappropriate release of intermembrane proteins Inhibit apoptosis Bind to pro-apoptotic Bcl2 family protein Examples: Bcl-2 and Bcl -X L

32. Pro-apoptotic effector Bcl2 family protein Induces the release of cytochrome c from mitochondrial inner membrane Examples- Bax and Bak protein Bak is bound to the mitochondrial outer membrane and Bax is mainly located in the cytosol Death inducing stimuli – Bax move from the cytosol to the outer mitochondrial membrane- oligomerize - Bax homodimers - influx of ions through the mitochondrial membrane

33. BH3-only protein Largest subclass Inactivate Bcl-2 protein Promote Apoptosis BH3-only proteins Puma and Noxa - cells with DNA damage BH3- only protein Bid connecting link between extrinsic and intrinsic pathway

34. The role of pro-apoptotic effector Bcl2 family proteins (mainly Bax and Bak ) in the release of mitochondrial intermembrane proteins in the intrinsic pathway of apoptosis. When activated by an apoptotic stimulus, the effector Bcl2 family proteins aggregate on the outer mitochondrial membrane and release cytochrome c and other proteins from the intermembrane space into the cytosol by an unknown mechanism

35. How pro-apoptotic BH3-only and anti-apoptotic Bcl2 family proteins regulate the intrinsic pathway of apoptosis. (A) In the absence of an apoptotic stimulus, anti-apoptotic Bcl2 family proteins bind to and inhibit the effector Bcl2 family proteins on the mitochondrial outer membrane (and in the cytosol —not shown). (B) In the presence of an apoptotic stimulus, BH3-only proteins are activated and bind to the anti-apoptotic Bcl2 family proteins so that they can no longer inhibit the effector Bcl2 family proteins; the latter then become activated, aggregate in the outer mitochondrial membrane, and promote the release of intermembrane mitochondrial proteins into the cytosol . Some activated BH3-only proteins may stimulate mitochondrial protein release more directly by binding to and activating the effector Bcl2 family proteins. Although not shown, the anti-apoptotic Bcl2 family proteins are bound to the mitochondrial surface.

36. Inhibitors of apoptosis (IAPs) First identified in Baculoviruses They have BIR ( baculovirus IAP repeat) domains, which enable them to bind to and inhibit activated caspases Some IAPs also polyubiquitylate caspases , marking the caspases for destruction by proteasomes Anti-IAP neutralize the effect of IAP Examples: In Droshophila Reaper, Grim, and Hid, have N-terminal, IAP-binding motif, which binds to the BIR domain of IAPs, preventing the domain from binding to a caspase

37. In mammals, anti-IAP Smac /Diablo and Omi are released from the mitochondrial intermembrane space when the intrinsic pathway of apoptosis is activated, blocking IAPs in the cytosol and thereby promoting Apoptosis

38. Dai, Yao et al. “Overcoming cancer therapy resistance by targeting inhibitors of apoptosis proteins and nuclear factor-kappa B.” American journal of translational research vol. 1,1 (2009): 1-15.

39. Regulation of Apoptosis Proposed intracellular pathways leading tocell death by apoptosis or to trophic factor-mediated cell survival in mammalian cells. (a) In the absence of a trophic factor, the soluble pro-apoptotic protein Bad binds to the anti-apoptotic proteins Bcl-2 and Bcl -xl, which are inserted into the mitochondrial membrane ( tr ). Bad binding preventshe anti-apoptotic proteins from interacting with Bax , a membrane-bound pro-apoptotic protein. As a consequence, Bax forms homo- oligomeric channels in the membrane that mediate ion flux [Z] Through an as-yet-unknown mechanism, this flux leads to the release of cytochrome c into the cytosol , where it binds to the adapter protein Apaf-1 (p), promoting a caspase cascade that leads to cell death (Z|) (b) In some cells, binding of a trophic factor, such as NGF (Il) stimulates Pl-3 kinase activity, leading to the downstream activation of protein kinase B (PKB), which phosphorylates Bad Phosphorylated Bad then forms a complex with the 14-3-3 protein (U ). With Bad sequestered in the cytosol , the anti-apoptotic Bcl-2lBcl-xl proteins can inhibit the activity of Bax (B), thereby preventing the release of cytochrome c and activation of the caspase cascade. [Adapted from B Pettman and C E Henderson,1998, Neuron 20:633 l

40. Extracellular survival factors inhibit Apoptosis Three ways that extracellular survival factors can inhibit apoptosis. (A) Some survival factors suppress apoptosis by stimulating the transcription of genes that encode anti-apoptotic Bcl2 family proteins such as Bcl2 itself or BclXL . (B) Many others activate the serine/ threonine protein kinase Akt , which, among many other targets, phosphorylates and inactivates the pro-apoptotic BH3-only protein Bad When not phosphorylated , Bad promotes apoptosis by binding to and inhibiting Bcl2; once phosphorylated , Bad dissociates, freeing Bcl2 to suppress apoptosis. Akt also suppresses apoptosis by phosphorylating and inactivating transcription regulatory proteins that stimulate the transcription of genes encoding proteins that promote apoptosis (C) In Drosophila, some survival factors inhibit apoptosis by stimulating the phosphorylation of the anti-IAP protein Hid. When not phosphorylated , Hid promotes cell death by inhibiting IAPs. Once phosphorylated , Hid no longer inhibits IAPs, which become active and block apoptosis. MAP kinase , mitogen -activated protein kinase .

41. Apoptosis gone wrong

42. p 53-A tumor suppressor p53 is a protein which is responsible for activating cell death When a cell is injured, p53 is activated, and in turn causes the activation of the cell’s apoptotic proteins P53 therefore is a “controller” of abnormal cells, halting their growth and proliferation