Apoptosis

Question 1

Define Necrosis.

Answer 1

Unregulated cell death associated with trauma, cellular disruption and an inflammatory response

Question 2

Define Apoptosis.

Answer 2

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 more permeable – the cell swells and the membrane ruptures

Proteases are released leading to dissolution and autodigestion of thecell There is localised inflammation

Question 4

What are the two phases of apoptosis? Describe them.

Answer 4

Latent phase

 Death pathways are activated, but cells appear morphologically the same

Execution phase:

 Loss of microvilli and intercellular junctions

 Cell shrinkage

 Loss of plasma membrane asymmetry

 Chromatin and nuclear condensation

 DNA fragmentation

 Formation of membrane blebs

 Fragmentation into membrane enclose apoptotic bodies (these are then taken up by macrophages)

Question 5

What is an important feature of apoptosis that distinguishes itfrom necrosis?

Answer 5

Plasma membrane remains intact – no inflammation

Question 6

What DNA modification is seen during apoptosis?

Answer 6

Fragmentation of DNA ladders (seen in agarose gel)

Formation of more ends, which are labelled by adding an extra fluorescently-labelled tag in a TUNEL assay

Question 7

What other types of cell death are there other than necrosis and apoptosis?

Answer 7

Apoptosis-like cell death

Necrosis-like cell death (sort of like an aborted apoptosis that ends up being necrosis)

NOTE: cell death is GRADED

Question 8

What are caspases?

Answer 8

Cysteine-dependent ASPartate-directed proteASES

They are the executioners of apoptosis

They are activated by cleavage (proteolysis cascade)

Question 9

Which caspases are effector caspases?

Answer 9

3, 6 and 7 - carry out apoptosis process

Question 10

Which caspases are initiator caspases?

Answer 10

2, 8, 9 and 10 - trigger apoptosis

Question 11

Describe the structure of effector caspases.

Answer 11

They are single chain polypeptides consisting of a small and large subunit

The subunits are released by proteolytic cleavage

Question 12

Describe the structure of initiator caspases.

Answer 12

They have the same two subunits found in effector caspases but they also have a targeting subunit (protein-protein interacting domain)

Question 13

What are the two types of targeting subunit that initiator caspases can have?

Answer 13

CARD – caspase recruitment domain

DED – death effector domain

Question 14

How are active caspases formed?

Answer 14

Cleavage of inactive procaspases is followed by the folding of 2 large and 2 small chains to form an active L2S2 heterotetramer

Question 15

What are the two mechanisms of apoptosis

Answer 15

Death by design (receptor-mediated)

Death by default (mitochondrial (intrinsic) death pathway)

Question 16

Describe the structure of death receptors.

Answer 16

Cysteine-rich extracellular domain

Transmembrane domain

Intracellular tail with a death domain (DD)

Question 17

What are the two important adaptor proteins in the death by design pathway and how are they different?

Answer 17

FADD – positive regulator that promotes cell death – DED + DD

FLIP – negative regulator – DED + DED - inhibits death pathway and allows regulation

Question 18

Describe signalling of apoptosis through Fas (extrinsic pathway).

Answer 18

1. Fas receptor is upregulated when cells need to apoptose
2. Fas ligand (on cytotoxic T-cells) binds to Fas receptor and the Fas receptors undergo trimerisation–> brings the three death domains (DDs) together.
3. The trimerised DDs recruit FADD (adaptor protein), which binds via its own DD
4. FADD then recruits and oligomerises procaspase 8 (through the DED (death effector domain) of procaspase 8)
5. Binding of procaspase 8 to FADD forms DISC (death-induced signalling complex)
6. DISC (death inducing signalling complex) formation results in cross-activation of procaspase 8—> caspase 8
7. Active caspase 8 is released, which then activates effector caspases

Question 19

Describe the important of oligomerisation in this pathway.

Answer 19

Some initiator caspases have intrinsic low catalytic activity

Oligomerisation brings them close enough together to allow transcleavage

Also, at least 2 procaspases are required to form an active caspase

Question 20

Describe how FLIP acts as an inhibitor of apoptosis

Answer 20

FLIP is evolutionarily related to caspases but has lost its catalytic activity

It has two DED domains and can compete with procaspase 8 to bind to the DED domains of FADD

It can incorporate into receptor-procaspase complexes and interfere with transcleavage

Question 21

As an overview, describe death by default.

Answer 21

Cellular stress (eg lack of GF)

–>causes a change in (loss of) mitochondrial membrane potential

–>This leads to release of cytochrome C from the mitochondrion

–>This stimulates formation of the apoptosome complex

Question 22

What does the apoptosome (aka wheel of death) consist of?

Answer 22

APAF-1 (apoptotic activating factor 1)

Cytochrome C

ATP

Procaspase 9

Question 23

Describe the domains found within APAF-1.

Answer 23

CARD domain

ATPase domain

WD-40 repeats (protein-protein interactions)

Question 24

Explain fully, how death by default leads to caspase activation.

Answer 24

The cytochrome C released from the mitochondria bind to the WD-40 repeats of APAF-1

—> make it form a heptamer structure (apoptosome)

This requires ATP

It has 7 CARD domains in the middle, which can interact with CARD domains of procaspase 9

Seven procaspase 9 bind via their CARD domains to the apoptosome

Their close contact allows them to cross-cleave each other to generate activate caspase 9’s

Question 25

What pro-apoptotic protein links the death by default and death by design pathways? Explain how it works.

Answer 25

Bid Caspase 8 (generated by the death by design pathway) cleaves Bid Bid travels to the mitochondrion and promotes the release of cytochrome C – thus triggering the mitochondrial death pathway

Question 26

How can energy levels of a cell show whether a cell is going through apoptosis or necrosis?

Answer 26

Apoptosis requires energy (ATP) whereas necrosis does not

Question 27

What is an important family of proteins that act as intrinsicmodulators of apoptosis?

Answer 27

Bcl-2 family

Question 28

There are three main groups of Bcl-2 proteins. What is common toall three groups?

Answer 28

All contain BH3 domain – this is a dimerisation motif, which allows members of the family to form dimers with each other Some contain Transmembrane (TM) domains

Question 29

What are two anti-apoptotic Bcl-2 proteins and where are they found?

Answer 29

Bcl-2 and Bcl-xL They are found localised on the mitochondrial membrane

Question 30

What are the pro-apoptotic Bcl-2 proteins and where are they found?

Answer 30

Bid, Bad, Bax, Bak These are found in the cytoplasm and in the mitochondrial membrane (move between the two)

Question 31

Other than Ras signalling, what other pathway does growth factor binding to growth factor receptors activate?

Answer 31

PI3-kinase

Question 32

What type of molecule is PI3-K?

Answer 32

Lipid kinase (not protein)

Question 33

What are the main subunits of PI3-K?

Answer 33

Adaptor subunit Targeting subunit Catalytic subunit

Question 34

What is the main action of PI3-K?

Answer 34

PI3-K converts PIP2 to PIP3--\> binds protein kinase B/Akt adaptor subunit

Question 35

What effect does this action have that leads to inhibition of apoptosis?

Answer 35

PIP3 is recognised by the adaptor subunit of Protein Kinase B (PKB/Akt) This allows PKB to move to the cell membrane where it becomes activated PKB phosphorylates and inactivates Bad (which is pro-apoptopic)

Question 36

Describe the arrangement of the anti-apoptotic and pro-apoptotic proteins when growth factor signalling and the PI3-K pathway is active.

Answer 36

This means PI3-K can produce PIP3 – so PKB/Akt is activated meaning that Bad is phosphorylated and inactivated Bad is held in an inactive heterodimer with 14-3-3 (PKB/Akt) On the mitochondrial membrane, Bak and Bax are held in inactive heterodimers with Bcl-2 and Bcl-xL

Question 37

Describe how loss of growth factor signalling can lead to apoptosis.

Answer 37

This means loss of activation of the PI3K pathway so less PIP3 produced so less activation of PKB/Akt Bad gets dephosphorylated and dissociated from its inactive heterodimer Bad then moves to the mitochondrial membrane and binds to the anti-apoptotic proteins (Bcl-2 and Bcl-xL) via its BH3 domain This displaces Bax and Bak from their inactive heterodimers So Bax and Bak then form a pore in the mitochondrial membrane allowing the release of cytochrome C from the mitochondrion – this leads to apoptosis

Question 38

Summarise the effects of PKB/Akt in promoting cell survival.

Answer 38

Phosphorylates and inactivates Bad Phosphorylates and inactivates caspase 9 Inactivates FOXO transcription factors (FOXOs promote the expression of apoptosis-promoting genes)

Question 39

Name two extrinsic regulators of apoptosis and describe their actions.

Answer 39

PTEN (Lipid phosphatase)- Counteracts the activation of PKB --\> Reduces cell survival and promotes apoptosis IAPs (Inhibitor of Apoptosis proteins) -Binds to procaspases and prevents their activation - Can bind to activated caspases and inhibit their activity

Question 40

Are the following tumour suppressor genes or oncogenes? a. Bcl-2 b. PTEN c. PKB/Akt

Answer 40

a. Bcl-2 Oncogene – increased activation would mean reduced likelihood of apoptosis (cancers are anti-apoptotic) b. PTEN Tumour suppressor gene –inactivation will mean reduced likelihood of apoptosis c. PKB/Akt Oncogene--overexpression--\>^cell survival--\>cancer