Apoptosis

Question 1

Why do we need programmed cell death?

Answer 1

To remove:

1. Harmful cells (e.g. cells with viral infection, DNA damage)
2. Developmentally defective cells (e.g. B lymphocytes expressing antibodies against self-antigens)
3. Excess/unnecessary cells:
   
   • Embryonic development e.g. brain to eliminate excess neurons; liver regeneration; sculpting of digits and organs
4. Obsolete organs (e.g. mammary epithelium at the end of lactation)
5. Exploitation - chemotherapeutic killing of cells

Question 2

Compare Necrosis vs Apoptosis, what is their main difference?

Answer 2

• Necrosis - unregulated cell death associated with trauma, cellular disruption and an INFLAMMATORY RESPONSE
• Apoptosis (Programmed Cell Death) - regulated cell death; controlled disassembly of cellular contents without disruption - NO INFLAMMATORY RESPONSE

Question 3

Describe the process of Necrosis

Answer 3

• The plasma membrane becomes permeable
• There is cell swelling and rupture of cellular membranes
• Proteases are released leading to autodigestion and dissolution of the cell
• Localised inflammation

Question 4

Describe the process of Apoptosis, what are the two stages?

Answer 4

• Latent Phase - death pathways are activated, but cells appear morphologically the same
• Execution Phase
  
  1. Loss of microvilli and intercellular junctions
  2. Cell shrinkage
  3. Loss of plasma membrane asymmetry (phosphatidylserine lipid appears in outer leaflet)
  4. Chromatin and nuclear condensation
  5. DNA fragmentation
  6. Formation of membrane blebs
  7. Fragmentation into membrane-enclosed apoptotic bodies
     
     • IMPORTANT FEATURE OF APOPTOSIS: plasma membrane remains INTACT - so there is NO inflammation
  8. Once the cells have broken down in apoptosis, the apoptotic bodies are taken up by macrophages

Question 5

What happens during apoptosis and how can it be identified?

Answer 5

DNA modification occurs during apoptosis, this leads to:

1. Fragmentation of DNA ladders (seen in agarose gel)
2. Formation of more ‘ends’, which are labelled by adding an extra fluorescently-tagged base in a TUNEL assay

What are some other types of cell death?

1. Apoptosis-like programmed cell death(PMC) - has some, but not all, features of apoptosis. Display of phagocytic recognition molecules before plasma membrane lysis
2. Necrosis-like programmed cell death - displays variable features of apoptosis before cell lysis - this is like an ‘aborted’ apoptosis that ends up being necrosis
   
   • So cells quite often die of something that is in between necrosis and apoptosis - it is a graded response

Question 6

What are the main mechanisms of cell death?

Answer 6

1. Caspase cascade – the executioners.
2. Death response – death receptors and mitochondria.
3. Bcl-2 family.
4. Stopping the death programme.

Question 7

Describe the function of Caspases, how they are activated and their different classes

Answer 7

• Caspase - Cysteine-dependent aspartate-directed proteases
• They have a cysteine residue in their active site that is required for their activity
• They cut proteins just after their aspartate residue
• They are activated by proteolysis
• They take part in a cascade of activation

Question 8

Classes of Caspases

Answer 8

• Effector Caspases (3, 6 and 7)
  
  • They start of as a single chain polypeptide with TWO subunits (large and small)
  • The subunits are released by proteolytic cleavage during maturation
• Initiator Caspases (2, 8, 9 and 10)
  
  • These also have the same two subunits that are found in effector caspases (p20 and p10)
  • They also have an extra targeting subunit (protein-protein interacting domain)
  • The targeting subunit directs them to a particular location
  • Targeting subunits:
    
    • CARD - Caspase Recruitment Domain
    • DED - Death Effector Domain

Question 9

Caspase Maturation

Answer 9

• Procaspases (zymogens) are single chain polypeptides
• To become activated, the procaspases must undergo proteolytic cleavage to form large and small subunits (proteolytic is cut)
• NOTE: initiator caspases must also be cleaved to release the targeting subunit (DED,CARD)
• These cleavages are done by the caspases themselves
• After the cleavage, you get folding of 2 large and 2 small chains to form an active L2S2 hetero-tetramer

Question 10

Caspase Cascades

Answer 10

• Main purposes of the caspase cascades:

1. Amplification
2. Divergent responses
3. Regulation

• Once apoptosis is triggered, the initiator caspases cleave and activate the effector caspases

Question 11

What are the ways of action for Effector caspases?

Answer 11

• Effector caspases carry out the apoptotic programme in TWO ways:
  
  • Cleaving and inactivating various proteins and complexes (e.g. nuclear lamins leading nuclear breakdown)
  • Activating enzymes by direct cleavage, or cleavage of inhibitor molecules (e.g. protein kinases, nucleases such as Caspase-activated Dnase (CAD))

Question 12

Which are the Mechanisms of Caspase Activation?

Answer 12

• Death by design - receptor-mediated (extrinsic) pathways
• Death by default - mitochondrial (intrinsic) death pathway

Question 13

Explain the extrinsic design of Caspase activation

Answer 13

• All cells have death receptors on their surface
• Death receptors consist of:
  
  • Extracellular cysteine-rich domain
  • Single transcellular domain
  • Cytoplasmic tail (with a death domain)
• These receptors are only activated when they encounter secreted or transmembrane trimeric ligands (e.g. TNF-alpha or Fas) - these are called death ligands

Two ADAPTER PROTEINS are very important in this pathway:

• FADD - POSITIVE regulator (required for the death pathway to become activated) and promotes cell death
• FLIP - negative regulator (inhibits the death pathway and allows it to be regulated)

Question 14

Explain the different structures of the ADAPTER PROTEINS

Answer 14

• FADD and FLIP are different in structure:
  
  • FADD = DED + DD
  • FLIP = DED + DED
• NOTE:
  
  • DED = Death Effector Domain
  • DD = Death Domain

Question 15

Signalling through death receptors e.g. Fas/Fas-ligand

Answer 15

Fas is a death receptor that is upregulated if apoptosis is required e.g. if a cell is infected by a virus

1. The Fas ligand binds to the Fas receptor on the surface of cytotoxic T lymphocytes
2. The Fas receptors then undergo trimerisation, which brings the three cytoplasmic DD domains together
3. The trimerised death domains recruit the positive adapter protein FADD by its own DD
4. The binding of FADD causes recruitment and oligomerisation of procaspase 8 through its DED to the FADD DED
5. The binding of procaspase 8 to FADD forms a Death-Inducing Signalling Complex (DISC)
6. DISC formation results in cross-activation of procaspase 8, whereby they cleave each other within the complex (due to close proximity)
7. The active caspase 8 is then released, and it cleaves effector caspases to execute the death programme

Oligomerisation = a chemical process that links monomeric compounds (e.g. amino acids, nucleotides or monosaccharides) to form dimers, trimers, tetramers, or longer chain molecules (oligomers)

Question 16

Describe Procaspase 8 Oligomerisation and its deactivation

Answer 16

1. Initiator procaspases bind to FADD (DEDàDED).
   
   1. DED regions bind to DED regions.
2. This brings procaspases into close contact to allow cleavage.
3. Active initiator caspase 8 tetramers release.

• Death receptor activation of procaspase 8 is inhibited by FLIP (negative regulator).
• FLIP incorporates into the trimer but it has NO PROTEOLYTIC ACTIVITY and so cannot cleave the other procaspases.
  
  • It can still bind to the DED regions on FADD though*.
  • So it can compete with procaspase 8 to bind to the DED domains of FADD

Question 17

What does Caspase activate?

Answer 17

Active caspase 8 can then go on to activate the other effector caspases that then carry out the apoptotic process.

• The effector caspases go on to carry out the apoptotic programme

Question 18

Describe the intrinsic pathway of Caspase activation

Answer 18

1. Cellular stresses – e.g. lack of/overstimulation growth factor, DNA damage.
2. Loss of mitochondrial membrane potential.
3. Release of cytochrome C (and other apoptosis-inducing factors).
4. Stimulation of formation of “apoptosome complex”.

Question 19

Describe the structure of Apoptosome, what does it connect with ?

Answer 19

• The apoptosome consists of:
  
  • APAF-1 (apoptotic activating factor 1)
  • Cytochrome C
  • ATP
  • Procaspase 9
• APAF1 is composed of CARD, ATPase and WD-40 repeats.
  
  • At one end, APAF-1 contains a number of repeats that are involved in protein-protein interactions
    
    • There is also an ATPase domain within APAF-1
  • At the other end of APAF-1 there is a caspase recruitment domain (CARD), which is also found in some initiator caspases (e.g. caspase 9)
• When cytochrome C binds to the WD-40 repeats on APAF-1, it forms a heptamer (the apoptosome)
  
  • This process also requires ATP
• The CARD domains at the centre of the apoptosome can interact with the CARD domains on procaspase-9 (so seven procaspase 9s can bind to the apoptosome)
  
  • The close proximity of the procaspase 9s that bind to the CARD domains of the apoptosome can cross-cleave and activate each other to produce caspase 9
• The activated caspase 9 is then released, which is able to trigger the caspase cascade, which leads to apoptosis

Question 20

Describe how the intrinsic and extrinsic pathways are connected

Answer 20

• Bid links the receptor-mediated and mitochondrial death pathways
• When one pathway is triggered, it can trigger the other pathway
• Caspase 8 from the receptor-mediated pathway can cleave Bid, which enhances release of mitochondrial proteins, thus engaging the intrinsic pathway
• The difference between the two mechanisms is that the mitochondrial pathway requires ATP
• Bid promotes the release of cytochrome C from the mitochondrion, which triggers the mitochondrial death pathway

NOTE: apoptosis is an ACTIVE process, which requires energy so the energy levels of a cell may determine whether death is by necrosis (less ATP) or apoptosis (more ATP)

Question 21

Which are the Modulators of Apoptosis?

Answer 21

• : Bcl-2 Family Proteins
• These are intrinsic modulators of apoptosis
• There are THREE main groups of Bcl-2 proteins, all of which contain BH3 domains
• Some of the proteins contain other domains including a transmembrane domain
• BH3 is a dimerisation motif (for protein-protein interaction) that allows proteins in the Bcl-2 family to associate and dimerise with each other

Question 22

In which categories are the Bcl-2 proteins divided to?

Answer 22

• Anti-apoptotic proteins - localised to the mitochondrial membrane and INHIBIT apoptosis (– Bcl-2, Bcl-xL.)
• Pro-apoptotic proteins - move between the cytosol and the mitochondrial membrane and they PROMOTE apoptosis (- Bid, Bad, Bax, Bak)

Question 23

Describe the PI3’-Kinase Signalling Pathway in the Cell Cycle and Apoptosis Regulation

Answer 23

• Growth factors may activate TWO growth factor pathways associated with anti-apoptotic effects
• Ligand binding causes dimerisation and cross-phosphorylation of the tyrosine kinase receptors
• Ligand binds à dimerisation à cross-phosphorylation à signal transduction and docking of adapter proteins (e.g. Grb2) to adapt pathway direction (e.g. activating Ras à MAPK/ERK cascade).
• Another phosphorylation site on the tyrosine kinase receptors triggers the PI3-Kinase pathway, which is involved in cell survival and has anti-apoptotic effects

Question 24

Describe the PI3-Kinase pathway

Answer 24

• Phosphatidylinositol 3-kinase (PI3-K) is a lipid kinase (not a protein kinase) involved in growth control and cell survival
• It has three main subunits:

1. Targeting subunit
2. Adapter subunit
3. Catalytic subunit

• It phosphorylates PIP2 to PIP3, which is then recognised by the adapter subunit of PKB/Akt (protein kinase B)
• PKB is then recruited to the cell membrane and it is activated - it has anti-apoptotic effects
  
  1. PKB phosphorylates and INACTIVATES Bad (part of the Bcl-2 family)
  2. Phosphorylating and inactivating caspase 9.
  3. Inactivating FOXO (promote expression of apoptosis-promoting genes) transcription factors.
  4. Other – stimulates ribosome production

Question 26

**Describe the process of apoptosis by Bcl-2 via BH3 heterodimerisation**

Answer 26

1. Other pro-apoptotic proteins (such as **Bax** and **Bak**) are held in their _inactive_ heterodimers (by their **BH3** domains) to the anti-apoptotic Bcl-2/xL proteins 2. As the pro-apoptotic proteins are held in the inactive heterodimers, **cell survival** and **proliferation** are promoted 3. When growth factors are **ABSENT**, the PI3-kinase pathway is not activated, so PIP3 is _NOT_ generate and, hence, PKB is _NOT_ recruited to the cell membrane and activated 4. This means that Bad can _NOT_ be phosphorylated and held in an inactive heterodimer (with PKB) 5. So the Bad is **dephosphorylated** and **released** from the heterodimer 6. Bad can then go to the mitochondrial membrane, where it can bind through its **BH3 domain** to the BH3 domains of the anti-apoptotic Bcl-2 family members thus **DISPLACING the pro-apoptotic Bcl-2 family members** 7. Once the pro-apoptotic Bcl-2 family members (e.g. Bax and Bak) are released from inhibition by the anti-apoptotic Bcl-2 family members, they _form a pore_ in the mitochondrial membrane, which **allows cytochrome C to _escape into the cytosol_** and induce apoptosis

Question 27

**Summarize the action of PKB/Akt on Cell Survival**

Answer 27

* Phosphorylates and _inactivates_ Bad * Phosphorylates and _inactivates_ caspase 9 * _Inactivates_ FOXO transcription factors (FOXOs promote the expression of apoptosis-promoting genes) * Other e.g. stimulates

Question 28

**Summarize the extrinsic control of apoptosis**

Answer 28

* **PTEN** is a _lipid phosphatase_ that counteracts the production of PKB, therefore reducing the regulation of cell survival and promoting apoptosis * **IAPs (Inhibitor of Apoptosis Proteins)** * bind to procaspases and prevent activation * IAPs also bind to active caspases and inhibit their activity

Question 29

**What are the Anti-Apoptotic Pathways?**

Answer 29

* Bcl-2, Bcl-xL = intrinsic pathway * FLIP, IAPs = extrinsic pathway * Growth factor pathways via PI3-kinase and PKB/Akt

Question 30

**Which are the Proto-oncogenes/Tumour suppressors associated with apoptosis**

Answer 30

* Bcl-2 (oncogene - because over-expression of Bcl-2 will promote cancer) * PKB/Akt (oncogene - because over-expression of PKB/Akt will promote cancer) * PTEN (tumour suppressor - because inactivation of this gene promotes cancer) NOTE: apoptosis is essential for removing harmful (oncogenic) cells - this forms the basis of chemotherapeutic killing of tumour cells, involving, for example, DNA cleavage (dexamethasone stimulates DNA cleavage)