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Cancer > Apoptosis > Flashcards

Apoptosis Flashcards

1
Q

Define Necrosis.

A

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

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2
Q

Define Apoptosis.

A

Regulated cell death; controlled disassembly of cellular contents without disruption – no inflammatory response

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3
Q

Describe the process of necrosis.

A
  • Plasma membrane becomes permeable
  • Cell swelling and rupture of cellular membranes
  • Release of proteases leading to autodigestion and dissolution of the cell
  • Localised inflammation
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4
Q

What are the two phases of apoptosis?

A

Latent phase

Execution phase

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5
Q

Describe the latent phase of apoptosis

A

Death pathways are activated, but cells appear morphologically the same

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6
Q

Describe the Execution phase of apoptosis

A
  • Loss of microvilli and intercellular junctions
  • Cell shrinkage
  • Loss of plasma membrane asymmetry (phosphatidylserine lipid appears in outer leaflet)
  • Chromatin and nuclear condensation
  • DNA fragmentation
  • Formation of membrane blebs
  • Fragmentation into membrane enclose apoptotic bodies (these are then taken up by macrophages)
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7
Q

What is an important feature of apoptosis that distinguishes it from necrosis?

A

Plasma membrane remains intact – no inflammation

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8
Q

How is DNA fragmentation observed in apoptosis?

A
  • Fragmentation of DNA ladders (seen in agarose gel)

- TUNEL assay = DNA fragmentation leads to more “ends” which are labelled by adding an extra fluorescently-tagged base

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9
Q

What other types of cell death are there other than necrosis and apoptosis? Describe the nature of cell death in reality

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

Cell death is a GRADED response i.e. shows features of both apoptosis and necrosis

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10
Q

What are caspases?

A

Cysteine-dependent aspartate-directed proteases that bring about apoptosis

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11
Q

Which caspases are the ‘initiator caspases’? What are they responsible for?

A

2, 8, 9 and 10

They initiate the apoptosis signal

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12
Q

Which caspases are the ‘effector caspases’? What are they responsible for?

A

3, 6 and 7

They carry out the mass proteolysis that leads to apoptosis

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13
Q

Describe the structure of effector caspases.

A

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

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14
Q

Describe the structure of initiator caspases.

A

They also have a large and small subunit found in effector caspases but they also have a targeting subunit (protein-protein interacting domain also called the pro domain)

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15
Q

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

A

CARD – caspase recruitment domain; e.g. Caspase-2, -9

DED – death effector domain; e.g. Caspase-8, -10

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16
Q

Describe the activation of initiator Caspases

A
  • Initiator Caspases normally exist as inactive procaspase monomers.
  • Their activation is initiated by dimerisation, which is facilitated by binding to adaptor proteins via the protein–protein interacting domains.
  • Both pro-caspases undergo cleavage by autocatalysis. This leads to removal of the prodomain and cleavage of the linker region between the large and small subunit.
  • An active heterotetramer is formed
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17
Q

Describe the activation of effector Caspases. What do they do once they are activated?

A
  • Inappropriate activation of the effector caspases is prevented by their production as inactive procaspase dimers that must be cleaved by initiator caspases.
  • This cleavage is between the large and small subunits
  • The resulting small and large subunit of each Caspase will associate, resulting in an active heterotetramer.
  • Once activated, a single effector caspase can cleave and activate other effector caspases, leading to an accelerated feedback loop of caspase activation.
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18
Q

More specifically, what can effector Capsases go on to do to execute the apoptotic programme?

A
  • Cleave for example nuclear lamins leading to nuclear breakdown
  • Activate kinase and nuclease enzymes by direct cleavage, or
    cleavage of inhibitory
    molecules; e.g. Caspase-Activated DNase (CAD)
19
Q

What are the two mechanisms of Caspase activation, and hence apoptosis

A
  1. Death by design or extrinsic pathway (receptor-mediated)

2. Death by default or intrinsic mitochondrial death pathway

20
Q

Describe the three domains of the death receptor mediating the extrinsic pathway

A
  • Extracellular cysteine rich domain (N-terminus)
  • Transmembrane domain
  • Intracellular tail with a Death Domain, DD (C-terminus)
21
Q

What are the two important adaptor proteins in the death by design pathway and how are they different, in terms of their function and important domains?

A
  1. FADD = positive regulator that promotes cell death – has Death Effector Domain (DED) and DD
  2. FLIP = negative regulator – has DED + DED
22
Q

Describe signalling of apoptosis through Fas.

A
  • Fas ligand on CTLsbinds to Fas receptor and the Fas receptors undergo trimerisation, which brings the three DDs together
  • The trimerised DDs recruit FADD, which binds via its own DD
  • FADD then recruits and oligomerises procaspase 8 through the DED of procaspase 8
  • Binding of procaspase 8 to FADD forms DISC (death-induced signalling complex)
  • DISC formation results in cross-activation of procaspase 8
  • Active caspase 8 is released, which then activates effector caspases
23
Q

Describe the importance of oligomerisation in this pathway.

A

Some initiator caspases have intrinsic low catalytic activity
Oligomerisation brings them close enough together to allow transcleavage (auto-catalytic cleavage)
Also, at least 2 procaspases are required to form an active caspase

24
Q

Describe how FLIP acts as an inhibitor of apoptosis

A

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 receptor tails or FADD via its DED domains
It can incorporate into receptor-procaspase complexes and interfere with transcleavage

25
Q

As an overview, describe death by default.

A

Cellular stresses (e.g. lack of or overstimulation by growth factors, DNA damage, ROS) causes a change in mitochondrial membrane potential
=> Release of cytochrome C and other apoptosis-
inducing factors from the mitochondrion
=> Formation of the apoptosome complex

26
Q

What does the apoptosome consist of?

A

APAF-1 (apoptotic activating factor 1)
Cytochrome C
ATP
Procaspase 9

27
Q

Describe the domains found within APAF-1.

A

CARD domain
ATPase domain
WD-40 repeats (protein-protein interactions)

28
Q

How does the APAF-1, Cyt c and ATP form the apoptosome

A

The cytochrome C released from the mitochondria bind to the WD-40 repeats of APAF-1 and causing formation of a heptamer (x7 APAF-1); this requires ATP

  • The 7 CARD domains are in the middle, each of which can interact with a CARD domain of procaspase 9
  • Seven procaspase 9 bind via their CARD domains to the APAF-1 heptamer and their close contact allows them to cross-cleave each other to generate activate caspase 9
29
Q

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

A 
Bid 
Caspase 8 (generated by the death by design pathway) cleaves Bid, which travels to the mitochondrion and promotes the release of cytochrome C – thus triggering the mitochondrial death pathway
30
Q

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

A

Apoptosis requires ATP whereas necrosis does not

31
Q

State the important family of proteins that act as intrinsic modulators of apoptosis?

A

Bcl-2 family

32
Q

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

A

BH3 domain – this is a dimerisation motif, which allows members of the family to form dimers with each other

33
Q

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

A

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

34
Q

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

A 
Bid 
Bad 
Bax 
Bak 
These are found in the cytoplasm and in the mitochondrial membrane
35
Q

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

A

PI3-kinase pathway

36
Q

What type of molecule is PI3-K?

A

Lipid kinase (so NOT a protein kinase); = Phosphatidylinositol 3’-kinase

37
Q

What are the main subunits of PI3-K?

A

Adaptor subunit
Targeting subunit
Catalytic subunit

38
Q

What is the main action of PI3-K?

A

PI3-K catalyses the convertion PIP2 to PIP3

39
Q

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

A

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

40
Q

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

A

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
On the mitochondrial membrane, Bak and Bax are held in inactive heterodimers with Bcl-2 and Bcl-xL

41
Q

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

A

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

42
Q

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

A

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

43
Q

Name two extrinsic regulators of apoptosis and describe their actions.

A

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 activate caspases and inhibit their activity

44
Q

Are the following tumour suppressor genes or oncogenes?

a. Bcl-2
b. PTEN
c. PKB/Akt

A

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

Cancer (15 decks)

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