Apoptosis

Question 1

Why does programmed cell death occur? •

Answer 1

Harmful cells
• e.g. DNA damaged.

Developmentally defective cells
• e.g. self-antigen B-cells.

Excess cells
• e.g. sculpting of hands during embryonic development (get rid of webbing).

Obsolete cells
• e.g. mammary epithelium at the end of lactation.

Exploitation
• e.g. chemotherapeutic killing of cells.

Question 2

Necrosis?

Answer 2

Unregulated cell death associated with
 • trauma
 • cellular disruption
&
 • INFLAMMATORY response

Question 3

Apoptosis?

Answer 3

Programmed (regulated) cell death
• controlled disassembly of cellular contents without disruption
&
• NO inflammatory response

Question 4

Steps associated with necrosis?

Answer 4

1. Plasma membrane becomes permeable.
2. Cell swelling and rupture.
3. Release of proteases leading to auto-digestion.
4. Localised inflammation.

Question 5

Steps associated with apoptosis?

Answer 5

LATENT PHASE
• death pathways are activated but cell stays morphologically the same

EXECUTION PHASE
1. Loss of microvilli and inter-cellular junctions.
2. Cell shrinkage.
3. Loss of plasma membrane asymmetry.
4. Chromatin and nuclear condensation.
5. DNA fragmentation.
6. Membrane bleb formations.
7. Fragmentation into membrane-enclosed apoptotic bodies.
• So, no inflammation as the plasma membrane remains intact

Question 6

DNA modification can also occur during apoptosis - what can this lead to?

Answer 6

Fragmentation of DNA ladders
• seen in agarose gel

AND

Formation of more “ends”
• labelled with an extra fluorescently-tagged base

Question 7

Other than necrosis and apoptosis, what are other types of cell death and mechanism?

Answer 7

Apoptosis-like PCD (Programmed Cell Death)
• some, but not all, features of apoptosis
• display of phagocytic recognition molecules BEFORE plasma membrane lyses

Necrosis-like PCD
• variable features of apoptosis BEFORE cell lyses; “Aborted apoptosis”

Question 8

The fact that other forms of cell death also exist apart from necrosis and apoptosis, what does this suggest?

Answer 8

The fact that these other forms exist suggests a GRADED response of cell death.

Question 9

4 broad mechanisms of apoptotic cell death?

Answer 9

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

Question 10

What are caspases and their MoA?

Answer 10

Caspases = Cysteine-dependent ASPartate-directed proteASES

• a cysteine residue in the active site is required for their activity
• they cut proteins after their aspartate residue
• activated by a proteolysis cascade

Question 11

There are 2 major classes of caspases - what are they and explain it?

Answer 11

Initiator caspases 
 • 2, 9, 8, 10 (TRIGGER APOPTOSIS)
 • 2 subunits – p20 and P10.
 • They have an extra targeting subunit:
  - CARD – Caspase Recruitment Domain
  - DED – Death Effector Domain
  (these direct them to a location)

Effector caspases
• 3, 6, 7 (CARRY OUT APOPTOSIS PROCESS)

Question 12

How do caspases mature?

Answer 12

Procaspases are single-chain polypeptides
• to activate, procaspases undergo proteolytic cleavage to form LARGE and SMALL subunits.
• Initiator caspases also cleave the targeting subunits (DED and CARD)

After cleavage, there is a folding of the 2 large and 2 small chains
• to form an active L2S2 HETEROTETRAMER

Question 13

Explain the caspases cascade (aka. the main purpose)

Answer 13

Main purpose of the caspase cascade is for:
• amplification
• divergent responses
• regulation

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

Question 14

What do the effector capcases do?

Answer 14

Execute the APOPTOTIC PROGRAMME

In 2 ways:

1. Cleave & INACTIVE proteins/complexes
   • e.g. nuclear lamins lead to nuclear breakdown
2. ACTIVATING enzymes by direct cleavage or cleavage of inhibitors
   • e.g. nucleases (CAD)

Question 15

What are the 2 broad mechanisms for caspase activation?

Answer 15

Death by design
• Receptor-mediated (extrinsic) pathways

Death by default
• Mitochondrial (intrinsic) death pathway

Question 16

What are death receptors?

Answer 16

(extrinsic pathway)

ALL CELLS have death receptors which consist of:
• EC cysteine domains
• Transmembrane domain
• Cytoplasmic tail (death domain)

Question 17

What can interact with death receptors?

Answer 17

ADAPTOR PROTEINS can interact with these receptors

• FADD (ACTIVATION)

• +VE regulator
• promotes cell death
• DED + DD

• FLIP (INHIBITION)

• -VE regulator
• inhibits death pathway (allowing for regulation)
• DED + DED

Question 18

Explain and describe the signalling through the death receptors

Answer 18

1. Fas receptor is upregulated when the cell needs to apoptose
2. Fas ligand on cytotoxic T-cells binds to the Fas receptor and Fas receptor trimerises
3. Trimerised DD domains recruit adaptor proteins such as FADD
4. FADD binding causes recruitment and oligomerisation (links monomers to form dimers/trimers/etc.) of procaspase 8 through DED –> FADD DED
5. Procaspase 8 + FADD –> DISC (Death-inducing-signalling-complex)
6. DISC cross-activates other procaspase 8 molecules
7. Active caspase 8 is released to cleave effector caspases

Question 19

Explain the oligomerisation of procaspase 8

Answer 19

Occurs to allow procaspase 8 for cleavage and activation (as some initiator procaspases have low catalytic activity)

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

Question 20

Explain how FLIP functions in relation to the death receptors.

Answer 20

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 (competes with procaspase 8)

• It can still bind to the DED regions on FADD however*

Question 21

Why is Caspase 8 important?

Answer 21

It is the INITIATOR CASPASE

Can go on to activate the other EFFECTOR caspases that then carry out the apopotic process

(onenote!!)

Question 22

Explain and describe the intrinsic (mitochondrial) pathway

Answer 22

1. Cellular stresses
   • e.g. lack of GFs, DNA damage (p53)
2. Loss of mitochondrial membrane potential
3. Release of cytochrome C
   • and OTHER apoptosis-inducing factors
4. Stimulation of formation of “apoptosome complex”

Question 23

What does the Apoptosome (“wheel of death”) consist of?

Answer 23

APAF-1
• Apoptotic Factor 1

Cytochrome C

ATP

Procaspase 9

Question 24

What do the different parts of the Apoptosome do and how do they relate together?

Answer 24

APAF1 is composed of:
 • CARD
 • ATPase 
and 
 • WD-40 repeats

When cytochrome C binds to WD-40 repeats on APAF-1, it forms a heptamer (the apoptosome) which requires ATP

The CARD domain (also on APAF-1) then binds to CARD on procaspase 9
• so multiple procaspase 9s (x7) can bind to one apoptosome
• proximity of procaspases then allows cross-cleaving

Activated caspase 9 is then released to trigger apoptosis.

Question 25

What does apoptosis require, determining if this pathway is chosen?

Answer 25

ENERGY - APT Energy levels in the cell may therefore determine whether death is by • necrosis (no ATP) • apoptosis (ATP)

Question 26

Following on from both the extrinsic & intrinsic pathway, explain the principal mechanisms of apoptosis

Answer 26

Bid links the extrinsic (receptor) and intrinsic (mitochondrial) pathways together • Caspase 8 from the extrinsic pathway can cleave Bid --> enhances activation of the intrinsic pathway • Bid releases Cytochrome C from the mitochondrion which triggers the death pathway Major difference – intrinsic mitochondrial pathway requires energy (ATP) • Apoptosis always uses intrinsic to some degree so will always use ATP • Remember that necrosis uses MUCH LESS ATP.

Question 27

What are the intrinsic modulators of apoptosis?

Answer 27

Bcl-2 family proteins 3 main groups • ALL contain BH3 domains (required for dimerisation betw. one another) • SOME contain TM domains

Question 28

What are the 2 main categories of Bcl-2 proteins?

Answer 28

Anti-apoptotic • Bcl-2 • Bcl-xL • Localised in mitochondrial membrane ``` Pro-apoptotic • Bid • Bad • Bax • Bak • Move between cytosol & mitochondrial membrane ```

Question 29

What are the 2 GF pathways associated with anti-apoptotic effects

Answer 29

1. Ligand binds --> dimerisation --> cross-phosphorylation --> signal transduction and docking of adapter proteins (e.g. Grb2) to adapt pathway direction (e.g. activating Ras --> MAPK/ERK cascade) 2. Another phosphorylation site on tyrosine kinase receptors triggers the PI3-kinase pathway --> cell survival and anti-apoptotic effects.

Question 30

What does the PI3-kinase pathway lead to?

Answer 30

Cell survival HENCE Anti-apoptotic effects

Question 31

Explain the PI3-kinase pathway

Answer 31

PI3-K = Phosphatidylinositol 3-kinase = a lipid (not protein) kinase Three main subunits: • Targeting subunit • Adapter subunit • Catalytic subunit PI3-K phosphorylates PIP2 --> PIP3 • which then binds PKB/Akt (protein kinase B) adapter subunit • PKB is then recruited to the cell membrane and it is activated - it has anti-­apoptotic effects

Question 32

Explain how PKB has anti-apoptotic effects

Answer 32

PKB has anti-apoptotic effects by: * Phosphorylating and inactivating Bad (a Bcl-2) * Phosphorylating and inactivating caspase 9 * Inactivating FOXO (promote expression of apoptosis-promoting genes) transcription factors. * Other – stimulates ribosome production.

Question 33

What are 2 models of regulation of apoptosis by Bcl-2 family proteins via. BH3 heterodimerisation

Answer 33

A - PKB/Akt (PI3-K, GF pathways) B - GF absent

Question 34

Explain the A model of apoptosis of Bcl-2 family proteins via. BH3 heterodimerisation

Answer 34

Pro-apoptotic proteins (e.g. Bax, Bak) are held INACTIVE in their heterodimers to Bcl-2 and Bcl-xL on the mitochondrial membrane • by the BH3 domain As the pro-apoptotic are held in the inactive heterodimers, cell proliferation and survival is promoted

Question 35

Explain the B model of apoptosis of Bcl-2 family proteins via. BH3 heterodimerisation

Answer 35

GF is ABSENT!! No PI3-K pathway --> PIP3 not generated --> PKB not recruited --> Bad NOT phosphorylated and so is released from heterodimer with 14-3-3 Bad therefore goes to mitochondrial membrane and binds to Bcl-2 and Bcl-xL • displacing the pro-apoptotic Bcl-2 family proteins Bcl-2 family proteins (Bax, Bak) then form a pore in the mitochondrial membrane to allow cytochrome C to escape to induce apoptosis

Question 36

What can counteract PI3-K?

Answer 36

PTEN (lipid phosphatase) Counteracts PI3-K signalling and production of PKB SO PROMOTES APOPTOSIS

Question 37

What are IAPs and what do they do?

Answer 37

Inhibitor of Apoptosis Proteins Regulate programmed cell death by binding to: • Procaspases - preventing activation • Caspases - inhibiting activity ALL extrinsically (as dealing w. caspases)

Question 38

Summarise the anti-apoptotic pathways

Answer 38

Bcl-2 & Bcl-xL • via the intrinsic pathway FLIP & IAPs • via the extrinsic pathway Growth factor pathways • via PI3-K and PKB/Akt

Question 39

How can cancer cells avoid apoptosis?

Answer 39

Apoptosis regulators as oncogenes or TSGs Bcl-2 • Oncogene – as overexpression of Bcl-2 results in cancer PKB/Akt • Oncogene – as overexpression of PKB leads to increased cell survival and thus cancer PTEN • TSG – as inactivation raises the PI3-K pathway and thus allows increased survival of cancer.