L12 - Apoptosis

Question 1

What is apoptosis?

Answer 1

Programmed cell death – cellular suicide

Derived from a Greek word ‘to fall away’

Occurs by a precise mechanism

Accompanied by defined morphological changes

Different to cell death by necrosis

Question 2

What happens during necrosis?

Answer 2

• The cell swells
• Mitochondria dilate & other organelles dissolve
• Plasma membrane ruptures releasing cytoplasm
• Often elicits an inflammatory response

Question 3

What happens during apoptosis?

Answer 3

• The cytoplasm shrinks
• Chromatin condenses & nucleus fragments
• The plasma membrane blebs
• The cell fragments
• Membrane doesn’t rupture – contents of cytoplasm aren’t released so no inflammatory response
• Apoptotic bodies are cleared by phagocytotic cells

Question 4

Why is apoptosis important?

Answer 4

Some cells need to die for multiple reasons:

1. Organism maintenance – old & dysfunctional cells
2. Response to damage
   For example:
   • Excessive UV-mediated DNA damage – sunburn & peeling
   • Invasion of plant by pathogen – hyposensitive response
3. Development

Question 5

Studies with C. elegans

Answer 5

Of 947 non gonadal cells (somatic cells), 131 die & undergo apoptosis during development

Proper development requires the 131 cells to die during development

Doomed cells = cells that usually undergo apoptosis

CONCLUSIONS
• Ced-3&4 promote apoptosis
• Ced-9 inhibits apoptosis
• Ced-9 is upstream of Red-3

Question 6

Apoptotic pathway conservation

Answer 6

C. elegans has direct counterparts in vertebrates
Similar pathways & similar conserved structure

Ced-9 homologue in vertebrates is Bcl-2

Question 7

What is Bcl-2 involved in?

Answer 7

Isolated during studies on from human follicular lymphomas (blood cancer)

In these cancer cells a chromosomal translocation results in the overexpression of Bcl2

This overexpression of Bcl2 prevents apoptosis (keeps cancer cells alive when they should have been programmed to die)

Question 8

What are caspases?

Answer 8

They bring about apoptosis (& can be involved in inflammation)

Cysteinyl-aspartate-specific proteases

Have a cysteine residue at active site

Different caspases have different target preferences

Question 9

Human caspases

Answer 9

There are 11 human caspases but not all of them are involved in cell death

Can be divided into 2 groups

Question 10

2 groups of human caspases

Answer 10

Caspase 1 family
• Not involved in apoptosis
• Involved in cytokine processing

CED-3 family
• Involved in apoptosis
• Can be divided further into 2 more groups

Question 11

What are the 2 categories of CED-3 family caspases?

Answer 11

Initiator caspases
• Activate executioner caspases

Executioner caspases
• Cleave substrates to bring about apoptosis

Question 12

Activation of initiator caspases

Answer 12

When activated they bring about cell death so have to be carefully controlled

Present as inactive monomers (procaspases) – have a big pro domain at their N terminal

Apoptosis inducing signals result in interaction with an adaptor protein and dimerization

Cleavage of adjacent procaspase results in the formation of an active dimer

Pro domain is released

Active caspase made of the large subunit and the small subunit

Initiator caspase then cleave and activate executioner caspases

Question 13

Activation of executioner caspases

Answer 13

Present as inactive dimers

These are cleaved by initiator caspases to bring about the formation of an active dimer

Cleaved between the large & small subunits

Active dimers cleave their substrate proteins

Question 14

Caspase cascade

Answer 14

One initiator caspase can act on many downstream executioner caspases

These can then active many more caspases

Amplification signal

Important that the cell controls the activation of the initiator caspase very closely

Question 15

What is the difference between the intrinsic & the extrinsic pathway in apoptosis?

Answer 15

In the intrinsic pathway the cell kills itself because it senses cell stress

In the extrinsic pathway the cell kills itself because of signals from other cells

Weak external signals may also activate the intrinsic pathway of apoptosis

Question 16

The intrinsic pathway in apoptosis

Answer 16

The central role of the mitochondria
• Responsible for ATP production

Also plays a key role in the decision of cell to live or die by apoptosis

In response to adverse stimuli mitochondria become permeablized

Leads to the release of proteins that promote apoptosis

A key protein is cytochrome c

Question 17

Activation of the intrinsic pathway of apoptosis

Answer 17

1. Cytochrome C is present in the inter-membrane space of the mitochondria
2. Cytochrome C is released when an apoptotic stimulus occurs
3. When released in binds to an adaptor protein Apaf1 & once bound, there is the assembly of the apoptosome which is made up of 8 sections
4. Apoptosome recruits caspase 9
5. Caspase 9 becomes activated & then cleaves & activates executioner caspases

Question 18

What is CARD?

Answer 18

Caspase recruitment domain

Question 19

What do the Bcl-2 family of proteins regulate?

Answer 19

Regulate mitochondria permeabilisation

Some INHIBIT apoptosis – pro-survival
Some PROMOTE apoptosis – pro-apoptosis

Question 20

Pro-survival members of the Bcl-2 family of proteins

Answer 20

Pro-survival members all have a transmembrane domain & different BH domains

Have BH4, BH3, BH1 & BH2

Question 21

Pro-apoptotic members of the Bcl-2 family of proteins

Answer 21

Pro-apoptotic members can be split into 2 groups:
– Effectors – have a BH3, BH1 & BH2 domain
– BH3 only – only have a BH3 domain

Question 22

Regulation of mitochondrial permeability by Bcl proteins

NORMAL CELL

Answer 22

No apoptotic signals

Pro survival Bcl-2 proteins prevent the pro-apoptotic Bcl proteins from forming a pore in the membrane

Question 23

Regulation of mitochondrial permeability by Bcl proteins

STRESSED CELL

Answer 23

Apoptotic signals

BH3-only proteins inhibit the pro-survival Bcl-2 proteins

This allows pro-apoptotic Bcl proteins (Bak, Bax) to form a pore in the membrane

Once pores have formed, cytochrome C can be released from mitochondria

p53 will upregulate specific BH3 only genes

Question 24

What are IAPs?

Answer 24

Inhibitors of apoptosis

A mechanism to ensure caspases are activated only when appropriate

Question 25

Action of IAPs

Answer 25

IAPs have BIR (baculovirus IAP repeat) domains BIR domains allow binding to and inhibition of activated caspases Anti-IAPs such as Hid (Drosophila) Smac/Diablo & Omi (mammals) overcome this inhibition

Question 26

The extrinsic pathway of apoptosis

Answer 26

Relies of specific receptors at plasma membranes called death receptors

Question 27

What are death receptors?

Answer 27

Involved in the extrinsic pathway of apoptosis They are a subgroup of the TNF receptor (TNF-R) family

Question 28

Structure of death receptors

Answer 28

All have a transmembrane domain Ligand binding section (CRD) is extracellular The death domain is intracellular & controls intracellular signalling

Question 29

Signalling from death receptors

Answer 29

Ligand binding results in trimerisation and activation of receptor Facilitates the binding of adaptor proteins to death domain Adaptor proteins bind through their own death domains Adaptor proteins facilitate the recruitment of initiator caspases Active initiator caspases activate executioner caspases and thus apoptosis

Question 30

What are Fas ligands?

Answer 30

Transmembrane protein that belongs to the tumor necrosis factor (TNF) family Its binding with its receptor induces apoptosis Fas ligand/receptor interactions play an important role in the regulation of the immune system and the progression of cancer

Question 31

Fas signalling

Answer 31

When stimulated Fas induces apoptosis in the Fas-bearing cell. The ligand for Fas, (called FasL), is chiefly expressed on cytotoxic T-cells and TH1 (T-helper) cells. Apoptosis triggered by Fas is involved in an array of immune-system functions For example: – Elimination of used effector cells – Elimination of peripheral autoreactive lymphocytes

Question 32

What happens if you have mutations in Fas or its ligand?

Answer 32

Autoimmune disease

Question 33

Fas signalling pathway

Answer 33

1. Killer lymphocyte expresses the Fas ligand 2. Target cell is expressing the death receptor at its surface 3. Killer lymphocyte interacts with target cell through ligand interacting with receptor 4. Trimerisation of the receptor 5. Allows binding of adaptor proteins called FADD 6. FADD has its own death domain & death effecter domain 7. Death domain on FADD interacts with death domain in death receptor 8. Allows assembly of DISC 9. Initiator caspases bind to the adaptor domain through the death domain 10. Cleavage & activation of caspase 8 11. Caspase 8 is an initiator caspase so goes on & activates downstream caspases 12. It also brings about apoptosis

Question 34

How does caspase activation promote DNA fragmentation?

Answer 34

Executioner caspases cause the CAD to become active Once CAD is active it breaks up DNA between nucleosomes Leads to fragmentation

Question 35

What does CAD do?

Answer 35

Breaks up DNA between nucleosomes Leads to fragmentation

Question 36

What are extracellular survival factors that inhibit apoptosis?

Answer 36

Survival factors Require signalling from other cells to avoid apoptosis – pro-survival signalling Can inhibit apoptosis by a variety of mechanisms

Question 37

What is the apoptosome?

Answer 37

Large quaternary protein structure formed in the process of apoptosis Its formation is triggered by the release of cytochrome c from the mitochondria in response to an internal (intrinsic) or external (extrinsic) cell death stimulus.