1. Apoptosis
Many functions for programmed cell death, many consequences for failure of its regulation
Apoptosis vs. necrosis
Intrinsic and extrinsic pathways for survival vs. proliferation vs. apoptosis
Caspases: activation by proteolysis
Mitochondria and cytochrome c release
BCL-2 proteins
Jason Kahn: Apoptosis

2. What is Apoptosis? Greek: Falling away
A.k.a. Programmed Cell Death or PCD
Distinct from Necrosis, which is unplanned cell death
Apoptosis is actually the default pathway for metazoan cells: they must constantly be told to keep living
Sources:
Voet and Voet Chapter 34
Helmreich Chapter 13
Gomperts Chapter 14
Danial and Korsmeyer (2004), “Cell Death: Critical Control Points.” Cell 116, 205–219.
Jiang and Wang (2004), “Cytochrome C-Mediated Apoptosis.” Annu. Rev. Biochem. 73,
From
Jason Kahn: Apoptosis

3. Apoptosis vs. Necrosis
Necrotic cells lose their ability to handle osmotic pressure, swell, and burst, much like cell lysis done in the lab. Their contents spill into the surroundings, tending to produce inflammation. DNA is largely intact, possibly capable of being taken up by neighboring cells (…bad).
Apoptosis is characterized by contraction of the cell and “blebbing” (blistering) of membrane patches, DNA degradation, mitochondrial disruption and fission, and eventual disintegration into encapsulated blobs of stuff. These capsules (apoptotic bodies) are readily absorbed by macrophages or other neighboring cells without leading to inflammation.
Apoptosis is an orderly process, often part of normal development: many pathways were first worked out in C. elegans, in which 131 out of 1090 cells die during the development of the mature worm.
Jason Kahn: Apoptosis

4. Progression of Apoptosis
Cell is committed to apoptosis upon cytochrome c release, even though it takes a couple of hours to complete the process
This is not a process that has been selected to be one that can be aborted half way through!
Hence we will see rather aggressive signalling, such as proteolysis, membrane pores
Compare to blood clotting, cell cycle progression: also unidirectional.
Jason Kahn: Apoptosis

5. Functions for Apoptosis
Development: Normal development requires transient tissues. For example, mammalian embryos have webbed feet. Self-reactive immature T cells are eliminated. Neurons that have not made enough synapses are eliminated.
Apoptosis is part of the body’s defenses against viral invasion: infected cells can be induced to apoptose by T cells that recognize displayed antigens, excessive DNA damage leads to apoptosis.
The mouse on the right lacks Caspase 9
V+V 3/e, Chapter 34
Jason Kahn: Apoptosis

6. Removal of Apoptotic Bodies
Exposure of phosphatidyl serine is the “eat me” signal
Jason Kahn: Apoptosis

7. Proliferation vs. apoptosis
Typically the number of cells that divide each day is equal to the number that die (steady state).
Thus there is a delicate balance between proliferation and apoptosis, and many signalling pathways (e.g. c-myc) activate both, so that rapidly dividing cells are often short-lived.
Jason Kahn: Apoptosis

8. What Can Go Wrong?
Excessive apoptosis is seen in autoimmune diseases like Type I diabetes, in neurodegenerative disorders like Parkinson’s and Alzheimer’s, and in infertility and immunodeficiency
Insufficient apoptosis can lead to autoimmunity and to cancer: apoptosis is supposed to be the last resort for cells that have lost the ability to respond to their surroundings, but if the apoptotic pathways themselves break down… For example, BCL-2 = B-cell lymphoma 2 is an antiapoptotic protein. It is overexpressed after a common chromosomal translocation event, and the resulting cells are resistant to apoptosis and hence prone to uncontrolled proliferation if other mutations occur that dysregulate cell division.
Viruses have strategies to block apoptotic signalling
Cancer cells are often found to overexpress “IAP” proteins = inhibitors of apoptosis.
Jason Kahn: Apoptosis

9. What Can Go Wrong II
Tumor Necrosis Factor family members control apoptosis, proliferation, and other cascades
Aggarwal (2003). “Signalling pathways of the TNF superfamily: a double-edged sword.” Nature Reviews in Immunology, 3 , 745
Jason Kahn: Apoptosis

10. Apoptosis Is Complex
(Roche web site)
Jason Kahn: Apoptosis

11. Intrinsic vs. Extrinsic Pathways
Extrinsic: signals from outside the cell can lead to survival, proliferation, or apoptosis
Many tissue culture cells die unless they are attached to a surface (the extracellular matrix or ECM). The antiapoptotic signals are mediated through integrins associated with focal adhesion sites.
Cytotoxic T cells express ligands (e.g. the Fas ligand) which activate “death receptors” on the surface of target cells to induce apoptosis.
Intrinsic: signals from inside the cell, or lack of survival signals from outside
When DNA is damaged, activation of p53 leads to increased DNA repair and cell cycle arrest, but higher levels of damage activate apoptosis.
This is also the default pathway for apoptosis in the absence of the growth factors, cytokines, or contacts to the ECM that are needed to keep the cell alive.
Intrinsic and extrinsic pathways are mediated through the mitochondria: cytochrome c is released.
Both types of pathway end up activating Caspases (Cysteine-ASPartate-Proteases), which are cysteine proteases that cut after DEVD sequences.
Jason Kahn: Apoptosis

12. Caspases DED = death effector domain
Very specific proteases. They are synthesized as zymogens (procaspases), activated by proteolytic cleavage. They can then activate other caspases or cleave other targets.
Initiator caspases (-8, -9, -10) can self-activate slowly. They have effector caspases as their targets. Confers amplification and irreversibility.
Effector caspases (-3, -6, -7) cut up many other targets (~60), including:
Inhibitors of apoptosis
The ICAD inhibitor of the CAD DNase, which destroys chromosomal DNA (easy assay ->)
BCL-2, an antiapoptotic protein
Cytoskeletal proteins and the nuclear lamin
DNA replication and repair proteins like RFC (the eukaryotic clamp loader)
Repair enzymes and splicing factors
Transcription factors and kinases
DED = death effector domain
Jason Kahn: Apoptosis

13. Extrinsic Pathways
Trimerization of receptors upon ligand binding leads to trimerization of cytoplasmic death domains, which bind adaptors, which recruit caspase 8 through its DED domains
TNF is the founding extracellular ligand, acting through the TNFR1 receptor
Jason Kahn: Apoptosis

14. Intrinsic Pathway
Release of cytochrome c from the mitochondria is the signal: it’s never found outside otherwise. It assembles with the adapter APAF-1 to make the “apoptosome,” which activates Caspase-9, which then cleaves Caspase 3.
The antiapoptotic BCL-2 blocks Cytochrome c release.
Jason Kahn: Apoptosis

15. APAF-1/Cytochrome C + Caspase-9
Figure b Cryoelectron microscopy–based images of the apoptosome at 27-Å resolution. (b) The apoptosome in complex with a noncleavable mutant of procaspase-9 in oblique top view.
Jason Kahn: Apoptosis

16. Mechanism of Cytochrome C Release
Mechanism of release is controversial. There is a family of BCL-2 related proteins. Group I: antiapoptotic, Groups 2 + 3: proapoptotic.
Some BCL-2 related proteins form pores in membranes. But are they big enough? And why would antiapoptotic factors act as pores? Bax/Bak may aggregate to form a large pore.
Interaction between Bax and/or Bak and VDAC = voltage-dependent anion channel might form pores?
Simple dissipation of protein gradient or introduction of other molecules?
Pro-apoptotic proteins may also act by sequestering BCL-2 or BCL-XL: there are several proteins that can interact with each other through their BH3 domains:
Jason Kahn: Apoptosis

17. More on BCL-2
BCL-2 sequesters APAF-1 either directly or by preventing cytochrome c release, thereby keeping the procaspases bound to APAF-1 inactive.
Inhibitors of BCL-2 (such as Bad), either compete with APAF-1 for binding to BCL-2 or promote release of cytochrome c, leading to APAF-1 activation via cytochrome c binding, multimerization of APAF-1, and caspase activation
Jason Kahn: Apoptosis