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YR2: MCD Cancer > Apoptosis > Flashcards

Apoptosis Flashcards

1
Q

Define Necrosis.

A

Unregulated cell death associated with trauma, cellular disruption + 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 more permeable: the cell swells + membrane ruptures
Proteases are released leading to dissolution + autodigestion of the cell
There is localised inflammation

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4
Q

What are the two phases of apoptosis? Describe them.

A

Latent phase: Death pathways are activated, but cells appear morphologically the same
Execution phase:
Loss of microvilli + intercellular junctions
Cell shrinkage
Loss of plasma membrane asymmetry
Chromatin + nuclear condensation
DNA fragmentation
Formation of membrane blebs
Fragmentation into membrane enclosed apoptotic bodies (digested by macrophages)

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5
Q

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

A

Plasma membrane remains intact so there is no inflammation

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6
Q

What DNA modification is seen during apoptosis?

A

Fragmentation of DNA ladders (seen in agar gel)

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

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7
Q

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

A

Cell death is GRADED:
Apoptosis-like cell death (display phagocytic recognition molecules before membrane lysis)
Necrosis-like cell death (features of apoptosis before lysis, like an aborted apoptosis)

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8
Q

What are caspases?

A

Cysteine-dependent aspartate-directed proteases
Executioners of apoptosis
Activated by cleavage (Proteolysis)

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9
Q

Which caspases are effector caspases?

A

3, 6 + 7

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10
Q

Which caspases are initiator caspases?

A

2, 9, 8 + 10

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11
Q

Describe the structure of effector caspases.

A

Single chain polypeptides consisting of a small (p10)+ large (p20) subunit
Subunits are released by proteolytic cleavage

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12
Q

Describe the structure of initiator caspases.

A

They have the same 2 subunits found in effector caspases but they also have a targeting subunit (for homotypic protein-protein interactions)

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13
Q

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

A

CARD: caspase recruitment domain (Caspase 2 + 9)
DED: death effector domain (Caspase 8 + 10)

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14
Q

How are active caspases formed?

A

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

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15
Q

What are the two mechanisms of apoptosis

A

Death by design (receptor-mediated, extrinsic)

Death by default (mitochondrial (intrinsic))

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16
Q

Describe the structure of death receptors.

A

Cysteine-rich extracellular domain
Transmembrane domain
Intracellular tail with a death domain (DD)

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17
Q

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

A

FADD: positive regulator that promotes cell death (DED + DD)
FLIP: negative regulator (DED + DED)

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18
Q

Describe signalling of apoptosis through Fas.

A

Fas ligand (on CTLs) binds to Fas receptor
Fas receptors undergo trimerisation, which brings the 3 DDs together
Trimerised DDs recruit FADD, which binds via its own DD
FADD recruits + 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

19
Q

Describe the importance of oligomerisation in the death by design pathway

A

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

20
Q

Describe how FLIP acts as an inhibitor of apoptosis

A

FLIP has 2 DED domains but no proteolytic activity so can compete with procaspase 8 to bind to the DED domains of FADD
It can incorporate into receptor-procaspase complexes + interfere with transcleavage

21
Q

As an overview, describe death by default.

A

Cellular stress causes change in mitochondrial membrane potential
Mitochondrion releases cytochrome C + other apoptotic inducing factors
This stimulates formation of the heptameric apoptosome complex

22
Q

What does the apoptosome consist of?

A

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

23
Q

Describe the domains found within APAF-1.

A

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

24
Q

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

A

Cytochrome C released from mitochondria binds to WD-40 repeats of APAF-1 + leading to formation of a heptamer structure (apoptosome)
This requires ATP
It has 7 central CARD domains, which can interact with CARD domains of procaspase 9
7 procaspase 9’s bind via their CARD domains to the apoptosome
Their close contact allows them to cross-cleave each other to generate active caspase 9, which initiates a caspase cascade, resulting in apoptosis

25
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 death by design pathway) cleaves Bid, which travels to the mitochondrion + promotes release of cytochrome C, thus triggering the mitochondrial death pathway
26
Q

What may determine whether a cell undergoes apoptosis or necrosis?

A

Apoptosis requires energy (ATP) whereas necrosis does not

27
Q

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

A

Bcl-2 family

28
Q

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

A

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

29
Q

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

A

Bcl-2
Bcl-xL
Found localised on the mitochondrial membrane

30
Q

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

A 
Bid 
Bad 
Bax 
Bak 
Found in the cytoplasm + in the mitochondrial membrane
31
Q

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

A

PI3-kinase

32
Q

What type of molecule is PI3-K?

A

Lipid kinase

33
Q

What are the main subunits of PI3-K?

A 
Adaptor domain (binds to tyrosine phosphorylated receptor)
Kinase domain
34
Q

What is the main action of PI3-K?

A

PI3-K converts PIP2 to PIP3

35
Q

What effect does conversion of PIP2 to PIP3 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
Has mitogenic + anti-apoptotic signal

36
Q

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

A

PKB/Akt is activated meaning Bad is phosphorylated + inactivated (held in an inactive heterodimer with 14-3-3 protein)
On the mitochondrial membrane, Bak + Bax are held in inactive heterodimers with Bcl-xL + Bcl-2

37
Q

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

A

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

38
Q

Name two extrinsic regulators of apoptosis and describe their actions.

A

PTEN (Lipid phosphatase):
Counteracts the activation of PKB
Reduces cell survival + promotes apoptosis
IAPs (Inhibitor of Apoptosis proteins):
Binds to procaspases + prevents their activation
Can bind to activate caspases + inhibit their activity

39
Q

Are the following tumour suppressor genes or oncogenes?

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

A

Bcl-2
Oncogene: increased activation would mean reduced likelihood of apoptosis (cancers are anti-apoptotic)
PTEN
Tumour suppressor gene: inactivation will mean reduced likelihood of apoptosis
PKB/Akt
Oncogene

40
Q

What cells would require programmed cell death?

A 
Harmful cells (damaged DNA/ Infected)
Developmentally defective cells (B cells expressing antibodies against self)
Excess cells (sculpting of digits + organs)
Obsolete cells (mammary epithelial at end of lactation)
41
Q

What are caspase cascades? What do they result in?

A

Initiator caspases trigger apoptosis by cleaving + activating effector caspases, which carry out the apoptotic programme
Cascades generate amplification of signal, divergent responses + points for regulation

42
Q

What actions can caspases have?

A

Cleave + inactivate proteins/ complexes e.g. nuclear lamins for nuclear breakdown
Activate enzymes e.g. protein kinases by direct cleavage or cleavage of inhibitory molecules

43
Q

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

A

Phosphorylates + inactivates Bad + caspase 9
Inactivates FOXO transcription factors (which promote expression of apoptosis promoting genes)
Stimulates ribosome production + protein synthesis

44
Q

List the cytoprotective anti-apoptotic pathways

A

Bcl-2, Bcl-xL (intrinsic pathway)
FLIP, IAPs (extrinsic pathway)
Growth factor pathways via PI3’-K + PKB/Akt

YR2: MCD Cancer (18 decks)

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