Apoptosis Flashcards
1
Q
What is apoptosis also known as?
A
Programmed cell death
2
Q
Why do we have programmed cell death?
A
To remove:
- Harmful cells
- e.g. Cells with viral infection, DNA damage
- Developmentally defective cells
- e.g. B lymphocytes expressing antibodies against self-antigens
- Excess/unnecessary cells - this happens during embryonic development e.g:
- Brain to eliminate excess neurons
- Sculpting of digits and organs
- Sculpting of digits prevents webbing of hands (fingers being joined together)
- Liver regeneration
- Apoptotic cells releasing growth signals that stimulates the proliferation of progenitor cells
- Obsolete organs
- e.g. Mammary epithelium at the end of lactation
We can also exploit apoptosis → chemotherapeutic killing of cells
3
Q
Define apoptosis.
A
- Regulated cell death
- Controlled disassembly of cellular contents without disruption
- NO inflammatory response
4
Q
Define necrosis.
A
- Unregulated cell death
- Associated with:
- Trauma
- Cellular disruption
- Inflammatory response
5
Q
Describe the process of necrosis.
A
- Plasma membrane becomes permeable
- Plasma membrane damaged due to trauma so it becomes more permeable
- There is cell swelling and rupture of cellular membranes
- More water can enter the cell due to increased permeability → rupture
- Proteases are released → autodigestion and dissolution of the cell
- Lysosomes rupture → lysosomal enzymes (including proteases) released into cyctoplasm → autodigestion
- Dissolution - i.e. cell dissolves in ECF
- Localised inflammation
- Cellular contents released into ECF
- Once cellular contents ae no longer contained in the cell, they are perceived as DAMPs → triggers inflammatory response
6
Q
Describe the process of apoptosis.
A
Apoptosis has two phases:
- Latent phase
- Execution phase
Latent phase
- Death pathways are activated
- BUT cells appear morphologically (structurally) the same
Execution phase
- Loss of microvilli and intercellular junctions
- Cell shrinkage
- Loss of plasma membrane asymmetry
- Phosphatidylserine (phospholipid) is normally in the inner leaflet
- However, during apoptosis it appears in the outer leaflet
- This allows the apoptotic cell to be recognised and removed by phagocytes
- Chromatin and nuclear condensation
- DNA fragmentation
- Formation of membrane blebs
- Bleb = bulge of plasma membrane
- Fragmentation into membrane-enclosed apoptotic bodies
- Therefore, no inflammation
7
Q
Why is there no inflammation in apoptosis?
A
Because the plasma membrane remins intact
- This is unlike necrosis where the plamsa membrane ruptures and cellular contents is released into the ECF
-
Once cellular contents ae no longer contained in the cell, they are perceived as DAMPs → triggers inflammatory response
- DAMP = damage associated molecular pattern
8
Q
How is DNA modified in apoptosis and how can you tell this experimentally?
A
DNA modificaton - DNA fragmentation
Experimental evidence:
- DNA ladders on agarose gel
- Lots of DNA fragments of different sizes forms a ladder-like appearance during gel electrophoresis
- More DNA ‘ends’
- These are labelled by adding an extra fluorescently-tagged base
- This method for detecting DNA fragmentation is known as a TUNEL assay
9
Q
What are apoptotic bodies removed by?
A
Apoptotic bodies are phagocytosed by surrounding cells which can include:
- Non-professional phagocytes - e.g. epithelial cells
- Macrophages
10
Q
Give 4 types of cell death? Define the last 2.
A
- Apoptosis (PCD)
- Necrosis
- Apoptosis-like PCD
- Necrosis-like PCD
PCD = programmed cell death
Apoptosis-like PCD
- Some, but not all, features of apoptosis
- Example of a difference:
- In apoptosis-like PCD, you have display of phagocytic recognition molecules before plasma membrane lysis
Necrosis-like PCD
- Variable features of apoptosis before cell lysis
- You don’t get chromatin condensation
-
But you get varying degrees of other apoptosis-like features
- e.g. Externalisation of phosphatidylserine before lysis
- ‘Aborted apoptosis’
- A standard apoptosis programme is initiated
- But is blocked at the level of caspase activation, so takes alternative, caspase-independent routes
REMEMBER:
- There are more than just these 4 types of cell death
- Essentially there is a graded response of cell death
- Cell death can be apoptosis, necrosis, or anywhere in between the two
11
Q
What are caspases?
A
Caspases = cysteine-dependent aspartate-directed proteases
Caspases are:
- Executioners of (i.e. execute) apoptosis
- Activated by a proteolysis cascade
How they work:
- They have a cysteine residue in their active site that is required for their activity
- They cleave proteins just after their aspartate residue
NOTE: Residue = monomer
12
Q
What are the classes of caspases? Desribe each class.
A
INITIATOR CASPASES
These are caspases: 2, 9, 8, 10
FUNCTION:
- Trigger apoptosis by cleaving and activating the effector caspases
STRUCTURE:
- They have 2 subunits: p20 and p10
- Subunit = separate polypeptide chain
- But p20 and p10 are initially joined (part of the same polypeptide) on the procaspase protein
- The procaspases have an prodomains
-
Caspase 2 and 9: CARD
- Caspase recruitment domain
- Caspase 8 and 10: DED (x2)
- Death effector domain
-
Caspase 2 and 9: CARD
- These pro-domains allow homotypic (within the same type) protein-protein interactions
EFFECTOR CASPASES
These are caspases: 3, 6, 7
FUNCTION:
- Carry out the apoptotic programme
STRUCTURE:
- They have 2 subunits: p20 and p10
13
Q
How do procaspases mature to become active caspases?
A
- Caspases activated by proteolytic cleavage to form a large subunit (LS) and small subunit (SS)
- Initiator caspases also have a prodomain which is cleaved and removed
- 2LS + 2SS → active heteroteramer (i.e. active caspase)
EXPLANATION (extra):
Initiator caspases - e.g. caspase 8
- Initiator procaspases have a prodomain (e.g. DED)
- This allows recruitment of other initiator procaspases of the same type → oligomerisation
- e.g. Oligomerisation between procaspase 8 molecules
- Oligomerisation allows the procaspases to cleave each other → transcleavage
- This forms a:
- Large subunit (LS) - p20
- Small subunit (SS) - p10
- Prodomain - removed
- LS and SS from each procaspase (so 2LS and 2SS) fold to form a heterotetramer
Effector caspases - e.g. caspase 3
- Initiator caspases cleave the effector procaspases to form a:
- Large subunit (LS) - p20
- Small subunit (SS) - p10
- LS and SS from each pro-caspase (so 2LS and 2SS) fold to form a heterotetramer
14
Q
What is the main purpose of the caspase cascade?
A
- Amplification
- Divergent responses
- Regulation
REMEMBER: Initiator caspases trigger apoptosis by cleaving and activating effector caspases which carry out the apoptotic programme
NOTE:
- Caspase 2 is an initiator caspase as it can undergo transcleavage
- But it can also be cleaved by other caspases which makes it like an effector caspase
15
Q
How do effector caspases execute the apoptotic programme?
A
Effector caspases carry out their function by cleavage
They can:
-
Inactivate proteins or complexes
- e.g. Cleavage of nuclear lamins → nuclear breakdown
-
Activate enzymes by:
- Direct cleavage of pro-enzyme (zymogen)**
- Cleavage of inhibitory molecules, releasing the active enzyme
- e.g. Caspase-activated DNase (CAD)
- Types of enzymes activated:
- Protein kinase
- Nuclease → DNA fragmentation (e.g. CAD)
16
Q
What are the two mechanisms of caspase activation?
A
Death by design – receptor-mediated (extrinsic) pathways
Death by default – mitochondrial (intrinsic) death pathway
17
Q
Describe the structure of death receptors and their ligands.
A
All cells have death receptors on their surface
They have a:
- Extracellular cysteine-rich domain
- Transmembrane domain
- Intracelllular cytoplasmic tail which contains the death domain (DD)
Death receptor ligands:
- Are trimers → receptors trimerise
- Can be:
- Secreted (from other cells)
- Transmembrane - present on the plasma membrane of another cell
18
Q
In receptor-mediated apoptosis, what do the death receptors interact with?
A
Death receptors interact with adaptor proteins - there are two:
- FADD
- FLIP
FADD
- Activates apoptosis
- Structure - two main domains:
- DED (death effector domain)
- DD (death domain)
FLIP
- Inhibits apoptosis
- Structure:
- 2 DED domains
- The long form (FLIPL) can also have a caspase-like domain
DD and DED domains bind to similar domains on other proteins
19
Q
Describe the signalling process through death receptors (extrinsic pathway).
A
EXAMPLE:
- Death receptor = Fas
- Fas is upregulated on infected cells
- Ligand = Fas ligand (FasL)
- Fas L - trimeric transmembrane protein expressed on lymphocytes
Process:
- Trimeric ligand binds to death receptor → receptor trimerisation
- The receptor then recruits the adaptor protein FADD
- This is done via interactions between the DD domain of the receptors and the DD domain of FADD
- The FADD molecules can then recruit pro-caspase 8 molecules → procaspase 8 oligomerisation
- This is done via interaction between the DED domain of FAD and the DED pro-domain of procaspase 8
- All of this results in formation of DISC (death-inducing signalling complex) - composed of:
- Death receptor
- FADD
- Procaspase 8
- Oligomerisation of procaspase 8 (DISC formation) results in cleavage and activation
20
Q
Describe how oligomerisation of procaspase 8 results in their activation.
A
- Oligomerisation of procaspase 8 molecules brings them into close contact
- This allows transcleavage of procaspase 8
- One procaspase in the oligomer cleaves the other
- Cleavage → large and small subunit (gets rid of prodomain)
- Transcleavage can happen because:
- Some initiator procaspases have intrinsic low catalytic activity
- Oligomerisation triggers a conformational change in some initiator caspases, activating them
- Due to transcleavage, you need at least 2 procaspases to form an active tetramer
- Because one procaspase has to cleave the other
21
Q
How does FLIP inhibit procaspase 8 activation?
A
- FLIP has 2 DED domains at its N-terminus, same as procaspase 8
- i.e. They are homologous in terms of their DED domains
- This means that FLIP can also interact with FAD via their DED domains
- i.e. Interaction between the DED domain of FAD and the DED domain of FLIP
- This means that FLIP competes with procaspase 8 for binding to FADD
- However, FLIP has no proteolytic activity
- So it incorporates into the receptor-procaspase complexes and prevents procaspase activation by interfering with transcleavage
22
Q
What does caspase 8 do once activated?
A
Caspase 8 activates downstream effector caspases (in a proteolytic cleavage cascade)
- The effector caspases carry out the apoptotic programme
23
Q
Describe the mitochondrial regulation of apoptosis (intrinsic pathway).
A
- Cellular stresses
- Loss of mitochondrial membrane potential
- Release of cytochrome c and other apoptosis inducing factors from mitochondria
- Formation of the apoptosome complex
NOTE: Each step leads to the next
24
Q
Give examples of cellular stresses which can lead to loss of the mitochondrial membrane potential.
A
- Lack of or overstimulation by growth factors
- p53 activation due to DNA damage
- Reactive oxygen species (ROS)
NOTE: Mitochondrial membrane potential refers to the inner membrane
25
Describe the formation of the apoptosome.
The **apoptosome** *('wheel of death')* is made up of:
* APAF-1 *(apoptotic activating factor-1)*
* Cyctochrome c
* ATP
* Procaspase 9
APAF-1 structure
* CARD domain *(at its N-terminus)*
* CARD = caspase recruiting domain
* ATPase domain
* WD-40 repeats
* *Made up of repeats of a sequence of approx. 40 AAs*
* Mediate protein-protein interactions
Apoptosome formation:
* When cytochrome c binds to the WD-40 repeats of APAF-1 , the APAF-1 proteins come together to form a **heptamer**
* ****Also requires ATP *(binding to the APAF-1)*
* The CARD domains of the heptamer can then interact with **procaspase 9** molecules via their CARD domains
* So each Apaf-1 in the heptamer can potentially bind a procaspase 9
* Interaction between the APAF-1 heptamer and procaspase 9 molecules → procaspase 9 oligomerisation
* Oligomerisation → transcleavage of procaspase 9 → active tertramer released *(2LS + 2SS)*
26
What does caspase 9 do once activated?
Initiate a caspase cascade *(proteolytic cleavage cascade)*
27
What determines whether a cell will undergo apoptosis or necrosis?
**Energy levels** in the cell determines whether it will undergo apoptosis OR necrosis
* Apoptosis requires energy because the **apoptosome requires ATP**
28
What is the role of Bid?
* Bid links the receptor-mediated and mitochondrial death pathways
* Caspase 8 from the receptor-mediated pathway cleaves Bid, activating it
* BID enhances release of mitochondrial proteins (including **cytochrome c**), thus engaging the intrinsic pathway
* The major difference between the intrinsic and extrinsic is that the **intrinsic pathway requires ATP**
* So, whichever pathway is stimulated initially, the intrinsic pathway is involved in apoptosis
* Therefore, **apoptosis** is an **active process -** *requires ATP*
29
What are the Bcl-2 family proteins?
These are **intrinsic modulators of apoptosis** *(i.e. modulate the intrinsic pathway)*
There are 3 main groups of proteins in the Bsl-2 family
* They **all** contain a **BH3** domain
* BH3 is a **dimerisation motif**
* Allows proteins in the Bcl-2 family to associate and dimerise with each other
* Some of the proteins contain other domains including a transmembrane domain
* The transmembrane domain attaches these proteins to the outer mitochondrial membrane
* Group 3 is the BH3 only family
* *Can have a transmembrane domain but no other BH domains*
30
What two categories are the Bcl-2 family proteins divided into?
**Anti-apoptotic**
* These include:
* Bcl-2
* Bcl-xL
* They _inhibit_ apoptosis
* They are localised to the mitochondria
* *Specifically the outer mitochondrial membrane*
**Pro-apoptotic**
* These include:
* Bid
* Bad
* Bax
* Bak
* They _promote_ apoptosis
* They move between the cytosol and mitochondria
* *Specifically the outer mitochondrial membrane*
31
Describe the two pathways which can occur when a growth factor binds to a receptor.
**Pathway 1**
* Growth factor *(ligand)* binds to growth factor receptor *(tyrosine kinase receptor)*
* This leads to receptor dimerisation → cross-phosphorylation → docking of Grb2 *(adaptor protein)* → activates Ras → ERK cascade → cell growth (and proliferation)
**Pathway 2**
* Growth factor *(ligand)* binds to growth factor receptor *(tyrosine kinase receptor)*
* This leads to receptor dimerisation → cross-phosphorylation → docking of a **different** protein: **PI3-K** → activates PI3-K pathway → cell survival *(i.e. anti-apoptotic effects)* and proliferation **
32
Describe the PI3-K signalling pathway.
PI3-K = phosphatidylinositol 3-kinase
* A **lipid** kinase (NOT a protein kinase)
* *i.e. phosphorylates lipids not proteins*
* Involved in:
* Control of cell growth
* Cell survival
Pathway:
* PI3-K binds to the receptor → PI3-K activated
* Once activated PI3-K phosphorylates **PIP2 → PIP3**
* PIP2 is a phospholipid present in the plasma membrane
* PIP3 activates **protein kinase B (PKB)**
* ****PKB is also known as **Akt**
* PKB is **anti-apoptotic**
IMPORTANT:
* PI3-K has 2 subunits
* p85 - adaptor
* p110 - kinase *(i.e. catalytic)*
* PI3-K binds to **phosphorylated tyrosines** on the tyrosine kinase receptor via its **adaptor subunit**
* *p85 subunit has SH2 domains which allow this*
* This activates the **kinase subunit** which catalyses **PIP2 → PIP3**
33
How does PKB/Akt **block apoptosis** to induce cell survival?
* Phosphorylates and inactivates **Bad**
* Phosphorylates and inactivates **caspase 9**
* Inactivates **FOXO** transcription factors
* FOXOs promote expression of apoptosis-promoting genes
* *Promotes the intrinsic and extrinsic pathway*
* Other - e.g. stimulates ribosome production and protein synthesis
* *Impaired ribosome biogenesis → p53-mediated apoptosis*
* *So stimulating ribosome production prevents this*
* *Also, in general, you need ribsome production to allow protein synthesis*
* *And you need sufficient protein synthesis, so the cell has enough resources (e.g. enzymes) to carry out its normal function and survive*
34
Describe how the Bcl-2 family of proteins regulate apoptosis.
Cell survival *(picture A)*
* The anti-apoptotic proteins *(Bcl-2. Bcl-xL)* form a **heterodimer** with the the pro-apoptotic proteins *(Bax, Bak)*
* Dimerisation via **BH3 domains**
* *Both pro- and anti-apoptotic proteins have BH3 domains*
* **Heterodimer on outer mitochondrial membrane
* **PKB** phosphorylates **Bad** *(pro-apoptotic)*
* This results in Bad forming a heterodimer with the protein 14-3-3
* REMEMBER: This requires presence of a **growth factor** stimulating the PI3-K pathway
* The pro-apoptotic proteins are held **inactive** in their heterodimers
Apoptosis *(picture B)*
* Growth factor **absent →** no PI3-K pathway → PKB does **not** phosphorylate Bad → **Bad is active**
* Bad binds to Bcl-2 and Bcl-xL, displacing the pro-apoptotic proteins (Bax, Bak)
* *i.e. Bax and Bak released from their heterodimer → **active***
* Bax and Bak then form a pore in the outer mitochondrial membrane → cytochrome C released from mitochondria
* *Bax and Bak oligomerise to form a pore complex*
* **Once cytochrome C is in the cytosol, it can stimulate the formation of the apoptosome → apoptosis
35
What is the role of PTEN?
* It's a lipid kinase which dephosphorylates PIP3 back to PIP2
* This turns off activation of PKB and the PI3-K pathway
* This pathway promotes cell survival and protein synthesis *(and proliferation)*
* So by turning off this pathway, it **promotes apoptosis**
* By turning off a pathway that promotes cell survival and proliferation, PTEN acts as a **tumour suppressor**
* *You need this **negative regulation** to prevent the cell from dividing uncontrollably*
36
What do IAPs do?
IAP = **I**nhibitor of **A**poptosis **P**roteins
IAPs inhibit *(regulate)* the **extrinsic** pathway of apoptosis by:
* Binding to procaspases and preventing activation
* Binding to active caspases and inhibiting their activity
37
Summarise how the intrinsic and extrinsic pathways of apoptosis are inhibited.
NOTE: Inhibiting the apoptotic pathways is **cytoprotective** because it protects the cell from death
**Intrinsic pathway inhibition:**
* Bcl-2
* Bcl-xL
**Extrinsic pathway inhibition:**
* FLIP
* IAPs
**Both pathways:**
* Growth factor pathways via PI3’-K and PKB/Akt
38
State some proto-oncogenes and tumour suppressors associated with apoptosis.
**Proto-oncogenes:**
* Bcl-2
* PKG/Akt
EXPLANATION (extra):
* These are anti-apoptotic genes → promote cell survival
* So, if these genes mutated, you get cell survival even when apoptosis should occur *(e.g. DNA is damaged)*
* Cell survival allows cell proliferation, which could lead to tumour formation
**Tumour suppressor:**
* PTEN
* *It inhibits the PI3-K pathway which promotes cell survival and proliferation*
* *This negative regulation prevents the cell from dividing uncontrollably*
39
State some therapeutic uses of apoptosis.
* Stimulating apoptosis in harmful cells
* e.g. Cells with viral infection or DNA damage
* These cells can be oncogenic *(can potentially cause cancer)*
* Chemotherapeutic killing of tumour cells
* e.g. Dexamethasone stimulates DNA cleavage
* *DNA fragmentation is a key feature of apoptosis*
