Lecture 7 - activating apoptosis

Question 1

Intrinsic vs extrinsic apoptosis

Answer 1

Intrinsic - internal signals (ROS, etc)

Extrinsic - Signals from the extracellular environment (ligands, etc)

Question 2

Initiator caspase activations

Answer 2

• Inactive caspases are pro-enzymes in healthy cells
• Appropriate apoptotic signal leads to the recruitment of the initiator caspase to an activating multi-protein platform

Question 3

Caspase-activating platforms: what are they and what causes their formation?

Answer 3

Multiprotein complexes that assemble in response to the caspase-activating signal

Question 4

Initiator caspases: what is their structure when inactive and active, is cleavage required for activation, and what are the initiator caspases?

Answer 4

Inactive - monomeric
Active - dimeric

Unlike executioner caspases, cleavage is not required

Caspase 8 - , extrinsic pathway
Caspase 9 - activates caspase 3/7, intrinsic pathway
Caspase 2

Question 5

Initiator vs executioner caspases

Answer 5

Initiator:
* Large, small, and pro-domains
* Monomeric when inactivated
* Cleavage nis ot necessary for activation
* Dimer upon activation

Executioner
* Large and small domain
* Dimeric (when inactivated?)
* Cleavage is necessary for activation
* Dimer upon activation

Question 6

Caspase 9

Answer 6

Initiator caspase

• BID cleaved by granzymes
• Truncated BID disrupts MOM
• Cytochrome C released
• Apaf-1 activated
• 7 APAF-1 form the apoptosome and their CARD domain binds the CARD domain of caspase 9
• Caspase 9 activated through dimerisation that occurs at the apoptosome
• Activated caspase 3/7
• DNA cleavage - cell death induced

Question 7

Caspase 8

Answer 7

Initiator caspase - extrinsic apoptosis pathway

Primarily initiated by:
* The formation of the Death-Inducing Signalling Complex (DISC), which is formed when death receptors (Fas/TRAIL) interact with their ligands
* DISC recruits FAS-associated protein with death domain (FADD) and then procaspase 8
* Procaspase 8 undergoes auto-cleavage to form its active form as a dimer
* Caspase 8 triggers apoptosis by activating downstream executioner caspases

Question 8

Cytotoxic T-cells: how may they signal cells to die?

Answer 8

Cytotoxic T-cells display Fas ligand - signals the cell to die

Question 9

Apoptosome: how is it formed?

Answer 9

CARD domain of APAF-1 molecules recruits caspase-9 molecules

Question 10

Caspase pro-domain: why is it necessary?

Answer 10

Essential for activating caspase - the caspase must be recruited to its activation complex

Question 11

XIAP: what is it, what does it do, and why is it necessary?

Answer 11

X-linked inhibitor of apoptosis protein

Inhibits dimerisation of caspases (different between caspases - inhibits caspase 9 dimerisation via a distinct domain to that which inhibits caspase 3)

Uncontrolled apoptosis is bad

Question 12

MOMP: what is it, what does it do, and what caspases does it interact with?

Answer 12

Mitochondrial outer membrane permeabilisation

Activating switch for most apoptotic cell death:
* Releases cytochrome c and Smac at the same time
* Cytochrome c promotes caspase activation
* Smac inhibits the inhibitors of apoptosis
* Large, transient, and non-selective pores are formed

Caspase 3, 7, and 9

Question 13

Cytochrome c: what is it, what does it do, and how?

Answer 13

Essential protein for the electron transport chain

Causes oligomerization of APAF-1:
* Cytochrome c binds the regulatory regions of APAF-1 which prevent oligomerization
* Cytochrome c causes movement of the regulatory regions, allowing dATP hydrolysis
* dADP exchanges for dATP, allowing oligomerisation

Question 14

APAF-1: what is it, what does it do, and what is it activated by?

Answer 14

Apoptotic protease activating factor-1

Promotes apoptosis:
* Recruits pro-caspase 9 through its exposed CARD domain
* Caspase 9 becomes activated and promotes apoptosis

Cytochrome c binding to its regulatory region and allowing oligomerisation

Question 15

Smac: what is it and what does it do?

Answer 15

Second mitochondria-derived activator of caspase

Inhibit IAPs and XIAPs - relieves any inhibition of caspase 9 activation that XIAP can do (competes with them for binding - blocking them)

Question 16

Bcl-2: what is it, and what does it do?

Answer 16

B-cell lymphoma 2

Regulate apoptosis:
* Pro-apoptotic family - promote OMM permeabilisation
* Anti-apoptotic family - block OMM permeabilisation (suppress Bax and Bak)
* BH3 proteins - regulate other Bcl proteins (both pro- and anti-apoptotic families)

Question 17

Pro-apoptotic Bcl-2 proteins: what do they do, and how are they generated?

Answer 17

Promote OMM permeabilisation

Generated by defective VDJ rearrangements during B-cell development (pro-apoptotic family)

Question 18

Anti-apoptotic Bcl-2 proteins: what do they do and where do they act?

Answer 18

Block OMM permeabilisation (suppress Bax and Bak)

Act at the surface of mitochondria

Question 19

BH3-only proteins: what do they do and what are the types?

Answer 19

Regulate other Bcl proteins (both pro- and anti-apoptotic families)

• Activators - interact directly with Bax/Bak to promote apoptosis
• Sensitiser - interact directly with Bcl-2/Bcl-X to promote apoptosis

Question 20

PUMA: what is it, how is it activated, and what does it do?

Answer 20

P53 upregulated modulator of apoptosis

DNA damage - p53 is activated in response to DNA damage and p53 activates PUMA

PUMA causes ???

Question 21

p53: what is it, how is it regulated, and what does it do?

Answer 21

Tumour suppressor protein P53

Transcriptionally - only transcribed in response to survival signals

Activates PUMA - promotes apoptosis

Question 22

Bad: what is it, how is it regulated, and what does it do?

Answer 22

Sensitiser - a type of BH3 only protein

Post-translationally modified - growth factor signalling binds to Bad and prevents it from promoting apoptosis

Interact directly with Bcl-2/Bcl-X to promote apoptosis

Question 23

Bim: what is it, what is it produced by, and what does it do?

Answer 23

Activator - a type of BH3 only protein

Growth factor signalling inhibits Bim production - further away from apoptosis

Interact directly with Bax/Bak to promote apoptosis

Question 24

Apoptotic priming: what is it, what is it regulated by, and why is being too far/close to it bad?

Answer 24

How close a particular cell is to MOMP

Dependent on survival signals (through sensitisers) and death signals (through activations)

If either side is overexpressed, it may result in issues like unkillable cells or cells that die rapidly

Question 25

Apoptotic priming: what may it be like in cancer cells and how may treatment account for this?

Answer 25

Overexpression of Bcl-2 prevents MOMP resulting in a cell that doesn't die by apoptosis Extracting cancer cells and treating them with Bcl-2 proteins to understand the level of priming can allow treatment plans using proteins that can counteract the degree of priming and promote apoptosis

Question 26

BH3-domain mimetics ''

Answer 26

High-affinity binding molecules that can bind Bcl-2 proteins Used to swing the apoptotic priming away from the survival signals in cases where Bcl-2 is overexpressed (this is done by the molecules binding the Bcl-2 proteins and preventing them from causing their anti-apoptotic effects) Platelet apoptosis - platelet lifestyle (~4 weeks) is dependent on BclXL amount

Question 27

LO's:

Answer 27

Initiator caspases are activated on multi-protein platforms: - That they are inactive as monomers - How they are recruited via their pro-domains - That activation occurs by induced dimerisation MOMP is the activating switch for most apoptotic cell death: - Explain how cytochrome c functions - How MOMP represents the point of cell death Bcl-2 proteins control MOMP in response to stress: - Know how the different Bcl-2 proteins work - Understand the concept of priming and its relation to cancer - How Bcl-2 proteins can be targeted therapeutically