1. Apoptosis Dr. Tania A. Shakoori

2. Apoptosis Apoptosis -programmed cell deathprogrammed cell death 3 stages – Initiation » (depending on where the the signal comes from) Extrinsic Intrinsic – Execution – Phagocytosis

3. Apoptosis everyday Between 50 and 70 billion cells die each day due to apoptosis in the average human adult. For an average child between the ages of 8 and 14, approximately 20 billion to 30 billion cells die a daybillion

4. Do you think apoptosis is good or bad??

5. Intrinsic initiation DNA lesions  activation of ATM  activates p53 ( tumor suppressor protein) P53 many functions. One is  activation of Bax protein  makes pores in outer membrane of mitochondria  leakage of protons, Ca++ and cytochrome C Cyt-C binds APAF  associate with procaspase 9-  activated to caspase. Caspase 9 cleaves more caspases (Caspase cascade) and breaks down proteins

6. Extrinsic initiation (cont.) Extracellular signals –commonly TNF-alpha secreted by many cells including killer T cells Binds receptors –TNFR which has a cytosolic death domain Cytosolic proteins with their own death domains bind and are activated – TNF receptor associated protein with death domain TRADD  fas …(FADD)  procaspase 8  caspase 8  CASPASE CASCADE

7. True/False Intrinsic pathway is mediated by caspase 8 Extrinsic pathway is mediated by caspase 9 DNA ligase is inhibited by_________

8. EXECUTION-DNA CLEAVAGE DNAse with inibitor Caspase cascade  caspase 3  Cleaves inhibitor Active DNAse cleaves DNA at regular intervals (about 80 bp) WHAT DO YOU THINK THE REASON IS? THINK?

9. EXECUTION: CLEAVAGE OF CYTOSKELETON Caspases breakdown cytoskeleton  what would happen. Cell would lose its structure Formation of apoptotic blebs. Most contain mitochondria and some DNA  what does this imply???

10. PHAGOCYTOSIS What is it? Blebs are phagocytized by macrophages Implication? Current theory on chronic inflammatory diseases-(efferocytosis)

11. Necrosis vs Apoptosis In contrast to necrosis, which is a form of traumatic cell death that results from acute cellular injury,. Unlike necrosis, apoptosis produces cell fragments called apoptotic bodies that phagocytic cells are able to engulf and quickly remove before the contents of the cell can spill out onto surrounding cells and cause damage.necrosisphagocytic cells

13. All nucleated animal cells contain the seeds of their own destruction, in the form of various inactive procaspases that lie waiting for a signal to destroy the cell. It is therefore not surprising that caspase activity is tightly regulated inside the cell to ensure that the death program is held in check until needed.caspase

15. Description of the previous figure (A) Extracellular activation. A killer lymphocyte carrying the Fas ligand binds and activates Fas proteins on the surface of the target cell. Adaptor proteins bind to the intracellular region of aggregated Fas proteins, causing the aggregation of procaspase-8 molecules. These then cleave one another to initiate the caspase cascade.lymphocyteligandcaspase (B) Intracellular activation. Mitochondria release cytochrome c, which binds to and causes the aggregation of the adaptor protein Apaf-1. Apaf-1 binds and aggregates procaspase-9 molecules, which leads to thecleavage of these molecules and the triggering of a caspase cascade. Other proteins that contribute to apoptosis are also released from the mitochondrial intermembrane space (not shown).cytochromeadaptor proteincleavageapoptosisintermembrane space

16. Bcl-2 Family Proteins and IAP Proteins Are the Main Intracellular Regulators of the Cell Death Program The Bcl-2 family of intracellular proteins helps regulate the activation of procaspases. – Some like Bcl-2 i, inhibit apoptosis, (partly by blocking the release of cytochrome c from mitochondria)apoptosiscytochrome – Others promote procaspase activation and cell death. some such as Bad, bind and inactivate the death-inhibiting members of the family, whereas others, like Bax and Bak, stimulate the release of cytochrome c from mitochondria. Another important family of intracellular apoptosis regulators  IAP (inhibitor of apoptosis) family. These proteins are thought to inhibit apoptosis in two ways:apoptosis – they bind to some procaspases to prevent their activation, – and they bind to caspases to inhibit their activity. – When mitochondria release cytochrome c to activate Apaf-1, they also release a protein that blocks IAPs, thereby greatly increasing the efficiency of the death activation process.cytochromeprotein

17. Summary Video link: https://drive.google.com/file/d/0B8lwCh5HN 0GTdExMT1pqVWh3UWs/view?usp=sharing https://drive.google.com/file/d/0B8lwCh5HN 0GTdExMT1pqVWh3UWs/view?usp=sharing CAN YOU ANSWER?? True about Apoptosis are all except : a) Inflammation is present b) Chromosomal breakage c) Clumping of chromatin d) Cell shrinkage

18. Candy question Ladder pattern of apoptosis on DNA electrophoresis is caused by a.DNAses b.Caspases c.Endonucleases d.Helicases