1
Q
Define Necrosis
A
- Unregulated cell death associated with trauma, cellular disruption and an inflammatory response
2
Q
Define Apoptosis
A
- Regulated cell death; controlled disassembly of cellular contents without disruption – no inflammatory response
3
Q
Describe the process of necrosis
A
- The plasma membrane becomes more permeable – the cell swells and the membrane ruptures
- Proteases are released leading to dissolution and autodigestion of the cell
- There is localised inflammation as immune cells are attracted
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 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)
5
Q
What is an important feature of apoptosis that distinguishes it from necrosis?
A
- Plasma membrane remains intact – no inflammation
6
Q
What DNA modification is seen during apoptosis and by what techniques can you observe these changes?
A
- 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
7
Q
What other types of cell death are there other than necrosis and apoptosis?
A
- Apoptosis-like cell death - some but not all features of apoptosis. There may be a display of phagocytic recognition molecules, even before cell surface membrane lysis
- Necrosis-like cell death (sort of like an aborted apoptosis that ends up being necrosis)
- NOTE: cell death is GRADED
8
Q
What are caspases?
A
- Cysteine-dependent aspartate-directed proteases
- They are the executioners of apoptosis
- They are activated by cleavage
9
Q
Which caspases are effector caspases?
A
- 3, 6 and 7
10
Q
Which caspases are initiator caspases?
A
2, 8, 9 and 10
11
Q
Describe the structure of effector caspases
A
- They are single chain polypeptides consisting of a small and large subunit
- The subunits are released by proteolytic cleavage
12
Q
Describe the structure of initiator caspases
A
- They have the same two (one large and one small) subunits found in effector caspases
- … but they also have a targeting subunit (protein-protein interacting domain)
- They have CARD and DED motifs that effector caspases do not have
13
Q
How do effector caspases actually carry out the apoptotic programme - give 2 example mechanisms?
A
- Cleave or inactivate various proteins and complexes - e.g. nuclear lamins leading to nuclear breakdown
- Activating enzymes by cleavage or indirect cleavage of inhibitor molecules - e.g. Caspase Activated DNAases which break down the DNA
14
Q
What are the two types of targeting subunit that initiator caspases can have?
A
- CARD – caspase recruitment domain
- DED – death effector domain
15
Q
How are active caspases formed?
A
- Cleavage of inactive procaspases is followed by the folding of 2 large and 2 small chains to form an active L2S2 heterotetramer
16
Q
What are the two mechanisms of apoptosis?
A
- Extrinsic - Death by design (receptor-mediated)
- Intrinsic - Death by default (mitochondrial death pathway)
17
Q
Describe the structure of death receptors
A
- Cysteine-rich extracellular domain
- Transmembrane domain
- Intracellular tail with a death domain (DD)
18
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
19
Q
Describe signalling of apoptosis through Fas
A
- Fas ligand (FasL) binds to Fas receptor (on cytotoxic T lymohocytes) and the Fas receptors undergo trimerisation, which brings the three DDs in the intracellular domain 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 and its own DED
- Binding of procaspase 8 to FADD forms DISC (death-induced signalling complex)
- DISC formation results in cross-activation of procaspase 8 by allowing cleavage
- Active caspase 8 is released and forms an active caspase 8 tetramer, which then activates effector caspases
20
Q
Describe the importance of oligomerisation in the Fas / FasL pathway
A
- Some initiator caspases have intrinsic low catalytic activity
- Oligomerisation brings them close enough together to allow transcleavage which allows activation
- Also, at least 2 procaspases are required to form an active caspase tetramer
21
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 DED domains of FADD
- It can therefore incorporate into receptor-procaspase complexes or in other words in between the procaspases that have bound with FADD and therefore prevent cross-activation of the pro-caspases by interfering with the trans-cleavage
- So you don’t form the active caspase-8 tetramers that would otherwise go on to activate the effector caspases involved in apoptosis
22
Q
As an overview, describe death by default (intrinsic pathway)
A
- Cellular stress causes a change in mitochondrial membrane potential
- This leads to release of cytochrome C from the mitochondrion
- This stimulates formation of the apoptosome complex
23
Q
What does the apoptosome consist of?
A
- APAF-1 (apoptotic activating factor 1)
- Cytochrome C
- ATP
- Procaspase 9
24
Q
Describe the domains found within APAF-1 and briefly state their roles
A
- CARD domain - caspase recruitment domain - attracts procaspase 9
- ATPase domain - cleaves ATP required to provide energy for binding
- WD-40 repeats (protein-protein interactions) - binding site for cytochrome C
25
Explain fully, how the intrinsic (death by default) pathway leads to caspase activation and therefore apoptosis
* The cytochrome C released from the mitochondria bind to the WD-40 repeats of APAF-1 and make it form a heptamer structure (apoptosome)
* This requires ATP, and APAF-1 uses its ATPase domain for this
* 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 APAF-1 molecules and their close contact allows them to cross-cleave each other to generate activate caspase 9
* Active caspase 9 can go on to activate effector caspases which ultimately carry out the apoptotic programme
26
1) What type of caspases are caspase 8 and 9?
2) How do caspase 8 and 9, activated by the death of design and death by default apoptotic pathways result in the actual carrying out of the apoptotic programme?
1)
* Initiator caspases
2)
* They both converge on activating caspase 3 - an effector caspase which is the thing that actually carries out the apoptotic pathway
27
What pro-apoptotic protein links the death by default and death by design pathways? Explain how it works
* 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
28
How can energy levels of a cell show whether a cell is going through apoptosis or necrosis?
* Apoptosis requires energy whereas necrosis does not
29
What is an important family of proteins that act as intrinsic modulators of apoptosis?
* Bcl-2 family
30
There are three main groups of Bcl-2 proteins. What is common to all three groups?
* BH3 domain – this is a dimerisation motif, which allows members of the family to form dimers with each other
31
What are the anti-apoptotic Bcl-2 proteins and where are they found?
* Bcl-2
* Bcl-xL
* They are found localised on the mitochondrial membrane
32
What are the pro-apoptotic Bcl-2 proteins and where are they found?
1. Bid
2. Bad
3. Bax
4. Bak
* These are found in the cytoplasm and in the mitochondrial membrane
33
Other than Ras signalling, what other pathway does growth factor binding to growth factor receptors activate?
* PI3-kinase promoting cell survival
34
What type of molecule is PI3-K?
* Lipid kinase
35
What are the main subunits of PI3-K?
* Adaptor subunit
* Targeting subunit
* Catalytic subunit
36
What is the main action of PI3-K?
* PI3-K converts PIP2 to PIP3
37
Outline the whole pathway that goes from growth factor binding then via PI3-K and how it has its effects in regards cellular survival and apoptosis
* Growth factors bind receptors
* This causes dimerisation of these receptors
* This dimerisation results in cross-phosphorylation of tyrosine kinase domains within the intracellular parts of the receptor
* This phosphorylation causes morphological change allowing binding of adaptor proteins
* An adaptor binds which then recruits PI3-K
* PIP3-K (kinase) carries out PIP2 → PIP3
* 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 (a member of the BCL-2 anti-apoptotic family) and has many other effects
* Therefore it promotes cell survival and proliferation
38
Describe the arrangement of the anti-apoptotic and pro-apoptotic proteins when growth factor signalling and the PI3-K pathway is active
* 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
39
Describe how loss of growth factor signalling can lead to apoptosis
* This means loss of activation of the PI3K pathway
* So less PIP3 produced
* So less activation of PKB/Akt
* Bad is allowed to get 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
40
Summarise the effects of PKB/Akt in promoting cell survival
* Phosphorylates and inactivates Bad
* Phosphorylates and inactivates caspase 9
* Inactivates FOXO transcription factors (FOXOs promotes the expression of apoptosis-promoting genes)
41
Outline regulation of apoptosis by BCL-2 family proteins via BH3 heterodimerisation, and thus go over again (there's another flashcard in here about it) how absence of growth factors and the PI3-K pathway leads to apoptosis
* Pro-apoptotic proteins belonging to the BCl-2 family including Bax and Bak are held in their inactive heterodimers by their BH3 domains by the anti-apoptotic proteins belonging to the BCl-2 family including BCl-2 and BCl-xl
* When growth factors are absent, the PIK-3 pathway is inactive
* So PIP3 is not formed
* So PKB is not activated
* This means that Bad cannot be phosphorylated and held in an inactive heterotrimer
* Therefore it is dephosphorylated in the absence of active PKB
* Thus it is released from its heterotrimer
* Now it can form pores in the mitochondrial membrane, which allows cytochrome C to escape into the cytosol and induce apoptosis - cytochrome C apoptosome thing
42
1) Name two extrinsic regulators of apoptosis (anti-apoptotic) and describe their actions
2) Then mention an intrinsic anti-apoptotic regulator - no need to describe their actions
1)
**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
2)
**Anything in the BCL-2 family**
43
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)
b. PTEN Tumour suppressor gene –inactivation will mean reduced likelihood of apoptosis
c. PKB/Akt Oncogene
Y2 - CANCER
flashcards
Decks in class (14)
# Cards
-
Cellular Pathology of Cancer55
-
Oncogenes and Tumour Suppressor Genes18
-
Cell cycle and its control22
-
Signalling Mechanisms of Growth and Division16
-
DNA Damage28
-
External Factors Controlling Division21
-
Angiogenesis31
-
Apoptosis43
-
Invasion – Regulation of Cell Motility47
-
Biological Basis of Cancer Therapy44
-
Leukaemia32
-
Cancer as a Disease – Skin Cancer45
-
Cancer as a Disease – Breast Cancer35
-
Cancer as a Disease – Colorectal Cancer28
