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apoptosis

1. APOPTOSIS Vivek Giri Msc . Microbiology Semester- 1 P.G. Department Of Biosciences Sardar Patel University

2. Contents What is apoptosis ? Need for apoptosis Reasons for apoptosis Morphological changes during Apoptosis Apoptosis vs necrosis Caspases Pathways for apoptosis Bcl 2 protein Inhibitors of apoptosis (IAPs) Regulation of apoptosis Biochemical features of apoptosis Apoptosis gone wrong

3. What is Apoptosis? Apoptosis or Programmed cell death is an energy dependent naturally occuring process which helps the cell to destroy itself in response to appropriate signals Apoptosis is a Greek name describing falling of leaves The average human loses about 5 x 10 11 blood cells every day due to apoptosis

4. In 1972, John F. Kerr, Andrew H. Wyllie and A. R. Currie, coined the term “apoptosis” Study of the development of the nematode Caenorhabditis elegans by H. Robert Horvitz in 1986 Death of 131 out of 1090 cells – identification of genes ced- 3 , ced- 4 and ced- 9

5. Reasons for apoptosis

6. Morphological changes during Apoptosis 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]

7. Cell death

8. 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

9. 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]

10. Targets for caspases More than a dozen protein kinases , including focal adhesion kinase ( FAK ), PKB, PKC, and Raf1. Inactivation of FAK, for example, is presumed to 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 , such as those of intermediate filaments, actin , tubulin , and gelsolin . Cleavage and consequent inactivation of these proteins lead to changes in cell shape. An endonuclease called caspase activated DNase (CAD ), which is activated following caspase cleavage of an inhibitory protein. Once activated, CAD translocates from the cytoplasm to the nucleus where it attacks DNA, severing it into fragments.

11. Activation of caspases Caspase activation during Apoptosis An initiator caspase contains a protease domain and a small protein interaction Domain inactive, monomeric form, sometimes called procaspase . Apoptotic signals trigger the assembly of adaptor proteins carrying multiple binding sites for the caspase The initiator caspases dimerize and are thereby activated, leading to cleavage of protease domains. Each protease domain is then rearranged into a large and small subunit. Executioner caspases are initially formed as inactive dimers . Upon cleavage at a site in the protease domain by an initiator caspase , the executioner caspase dimer undergoes an activating conformational change. The executioner caspases then cleave a variety of key proteins, leading to the controlled death of the cell.

12. Pathways for Apoptosis

13. Extrinsic pathway The extrinsic pathway of apoptosis activated through Fas death receptors . Trimeric Fas ligands interact with trimeric Fas receptors on the surface of the target cell, leading to clustering of several ligand -bound receptor trimers . Receptor clustering activates death domains on the receptor tails, which interact with similar domains on the adaptor protein FADD (FADD stands for Fas -associated death domain). Each FADD protein then recruits an initiator caspase (caspase-8) via a death effector domain on both FADD and the caspase , forming a death-inducing signaling complex (DISC). Within the DISC, two adjacent initiator caspases interact and cleave one another to form an activated protease dimer , which then cleaves itself in the region linking the protease to the death effector domain. This stabilizes and releases the active caspase dimer into the cytosol , where it activates executioner caspases by cleaving them.

14. Intrinsic Pathway

15. Assembly of the mammalian apoptosome

16. Intrinsic pathway of apoptosis cytochrome c 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 . Apaf1 protein contains a caspase recruitment domain (CARD) 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

17. Biologydictionary.net Editors. "Apoptosis."  Biology Dictionary , Biologydictionary.net, 06 Jun. 2017, https://biologydictionary.net/apoptosis/

18. 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

19. 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 ( B) In the presence of an apoptotic stimulus, BH3-only proteins are activated and bind to the anti-apoptoticBcl2 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 antiapoptotic Bcl2 family proteins are bound to the mitochondrial surface.

20. 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.

21. Regulation of Apoptosis Proposed intracellular pathways leading tocell death by apoptosis or to trophic factor-mediated cell survival in mammalian cells. [ Adapted from B Pettman and C E Henderson,1998, Neuron 20:633 l

22. 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 antiapoptotic Bcl2 family proteins such as Bcl2 itself or BclXL . ( B) others activate the serine/ threonine protein kinase Akt , 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 .

23. Biochemical features of apoptosis

24. Removal of apoptotic cells by phagocytes Phagocytosis of apoptotic cells Apoptotic cells and cell fragments are recognized and engulfed by phagocytic cells. One of the signals recognized by phagocytes is phosphatidylserine on the cell surface. In normal cells, phosphatidylserine is restricted to the inner leaflet of the plasma membrane, but it becomes expressed on the cell surface during apoptosis.

25. Apoptosis gone wrong

26. Refrences Cooper, G., M., Hausman , R. E., (2015). The Cell: A Molecular Approach. 7 th Edn . Sinauer Associates Inc, United States Carp , G., (2013). Cell Biology. 7 th Edn . Wiley, United States Albert , B., Johnson, A., Lewis, J., Raff, M., Robert, K., Walter, P., (2014). Molecular Biology of the Cell. 6 th Edn . Garland Science, United States Lodish , H., Berk , A., Kaiser, C., A., (2007). Molecular Cell Biology. 6 th Edn . W. H. Freeman & Co Ltd, South Asia Elmore, Susan. “Apoptosis: a review of programmed cell death.” Toxicologic pathology vol. 35,4 (2007): 495-516. Doi:10.1080/01926230701320337

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