Lecture 7 (apoptosis)

Question 1

Types of cell death mechanisms

Answer 1

-Apoptosis
-Autophagy
-Pyroptosis
-Necroptoisis
-Necrosis

Question 2

Key morphological changes that apoptotic cells undergo:

Answer 2

-cell shrinkage
-blebbing
-nuclear fragmentation
-formation of apoptotic bodies

Question 3

Morphological steps of apoptosis:

Answer 3

1. Apoptotic signal
2. Cell shrinkage and chromatin condensation
3. Nuclear fragmentation and membrane blebbing
4. Apoptotic body formation

Question 4

Triggers of apoptosis

Answer 4

-Radiation
-Cytotoxic agents
-Toxins
-Growth factor/nutrient/oxygen deprivation

Question 5

Caspases involvement in apoptosis:

Answer 5

Acts as a enzymatic mediator of apoptosis.
-Expressed as larger inactive pro-enzymes (pro-caspases).

Question 6

Function and structure of caspase

Answer 6

-It is a cysteine protease that cleaves after aspartic acid residues.
-It is composed of a large and small subunit.

Question 7

Two types of Caspases

Answer 7

1. Initiator caspase
2. Executioner caspase

Question 8

Types of initiator caspases and their domains

Answer 8

-Caspase 8, 9, and 10
-Prodomain has DED in procaspase 8 and 10 and CARD in procaspase 9.
-These domains allow for homophilic interactions with adaptor proteins

Question 9

Types of executioner caspases and how they are activated:

Answer 9

-Important in both intrinsic and extrinsic pathways
-Caspase 3 and 7
-Activation by proteolytic cleavage, mediated by initiator caspases
-Procaspase cleavage leads to tetramer of small and large heterodimers

Question 10

3 ways executioner caspases kill cells?

Answer 10

-It disassembles the cell structures by degradation of nuclear lamina.
-It inactivates apoptotic inhibitors e.g i-CAD
-It deregulates other protein activity e.g gelsolin to actin depolymerisation

Question 11

The Intrinsic Pathway of Apoptosis:

Answer 11

-Also known as mitochondrial pathway
-Activated by stresses signals being relayed to the mitochondria. Leading to mitochondrial outer membrane permeabilization (MOMP), which releases apoptotic proteins into the cytoplasm

Question 12

Pro-apoptotic proteins released in the intrinsic pathway:

Answer 12

1. Cytochrome C which associates with other proteins such as APaf-1 to allow for the formation of apoptosome.
2. Smac (Diablo) inactivates a group of anti-apoptotic proteins called IAPs (inhibitors of apoptosis)

Question 13

Cytochrome c fuction:

Answer 13

-Cytochome c + Apaf-1 + procaspase 9 = activation of caspase 9
-Caspase9 leads to activation of
effector caspases

Question 14

What is APAF-1:

Answer 14

-It is a scaffold protein and stands for apoptosis protease activating factor 1.
-Together with cytochrome c and procaspase 9 it activates caspase 9.
-It also oligomerizes in response to cytochrome c release and forms a large complex known as apoptosome.

Question 15

Apoptotic proteins:

Answer 15

-Proapoptotic (BH3 only): BIM, BAD, NOXA, PUMA, BID which activate Bax/Bak
-Proapoptotic (BAX subfamily): BAX, BOK and BAK which are essential for MOMP
-Anti-apoptotic: Bcl-2, Bcl-XL, MCL-1, Bcl-w are necessary for cell survival

Question 16

BH3 proteins:

Answer 16

-Activate BAX/BAK either through direct binding and/or indirectly by binding to their repressors, prosurvival BCL2 protein

Question 17

How are BH3 proteins regulated and activated and give examples of some:

Answer 17

-BH3 proteins are regulated at the transcriptional and post-translational level
and activated in response to given stimuli:
Examples:
-UV activates Bim
-Genotoxic damage activates Noxa, PUMA.
-Cytokine deprivation activates Bad
-Death receptors activates tBid

Question 18

What determines cytochrome c release

Answer 18

-The relative levels of pro and anti-apoptotic proteins within each channel determine whether cytochrome c will be released from the mitochondrion.

Question 19

Inhibitors of apoptosis (IAPs):

Answer 19

-Family of 8 proteins including c-IAP1, c-IAP2, XIAP.
-X-IAP inhibits caspase-3 and -9
-cIAP1 ubiquitinates caspase-3 and -7

Question 20

Apoptosis in cancer cells:

Answer 20

-Upregulation of anti-apoptotic proteins is common in cancer (e.g. Bcl-2 and Mcl-1).
-Inactivation of pro-apoptotic proteins (e.g. Bax is inactivated in the majority of colon cancers)

Question 21

Signaling regulation of the intrinsic pathway is controlled by:

Answer 21

-AKT
-P53

Question 22

AKT role in intrinsic pathway:

Answer 22

AKT is a kinase activated by numerous growth factor receptor signals.
-AKT is anti apoptosis and does this by:
-Inactivating Bad and indirectly downregulating BIM and PUMA expression through the transcription factor, FOXO3a.
-It also prevents cleavage of Bid from extrinsic pathway.

Question 23

P53 role in intrinsic pathway:

Answer 23

-P53 is proapoptotic and induces apoptosis by inducing expression of BAX, PUMA and NOXA (pro-apoptotic) and inactivating Bcl-2 (anti-apoptotic).

Question 24

Extrinsic pathway of apoptosis:

Answer 24

-Also known as death receptor pathway
-Induced by external signalling caused by ligand binding to a specific membrane receptor

Question 25

Two ways to activate death receptor pathway signalling:

Answer 25

1. Increase ligand expression
2. Increase receptor expression

Question 26

Ligands and their Death receptor:

Answer 26

-Death receptors/ligands are members of the TNF family of proteins
TRAIL - DR4 and DR5
TNF - TNFR1
FasL - Fas (APO-1/CD95)

Question 27

Key steps in the extrinsic pathway:

Answer 27

1.Ligand-receptor binding
2. Receptor trimerization
3. Recruitment and formation of Death
Inducing Signalling Complex (DISC)
4. DISC recruits and activates the initiator
caspase. DISC triggers self cleavage, thereby activating, caspases.
5. Active initiator caspases then activates
executioner caspases

Question 28

What happens when the death receptors timerisation:

Answer 28

It brings the 3 Death Domains (DD) together which allows for recruitment of FADD (Fas associated death domain), which forms the Death Inducing Signalling Complex (DISC).

Question 29

Active Caspase 8:

Answer 29

-Also known as initiator caspase as it can lead to direct activation of caspase 3 and activation of BID which promotes MOP

Question 30

What is anoikis?

Answer 30

-Greek for homeless. In biology it means anchorage independent.
-It is a unique mode of apoptosis induced after loss of cell adhesion to ECM (through integrin).
-This results inactivation of pro-survival signaling (PI3K-Akt pathway or NK—κB) and caspase 8 activation.

Question 31

Directly killing through the extrinsic pathway:

Answer 31

-Done by FasL
-Fas expression can be increased due to p53 activation

Question 32

FasL:

Answer 32

-Can be membrane bound on nearby cell or truncated-soluble form.
-FasL (ligand) on cytotoxic T cell binds to Fas (receptor) on tumour cell
-Stimulates extrinsic pathway in tumour cell and perforin and granzyme release from cytotoxic T cell.

Question 33

How does perforin and granzyme B cause apoptosis:

Answer 33

They are proteins that break holes in the cell membrane to release its contents.

Question 34

How does the body prevent CD8+ cells from attacking any cells containing FasL?

Answer 34

When DNA damage occurs then MHC I will display a new antigen that will show CD8+ to attack it.

Question 35

How do phagocytic cells clean up destroyed apoptotic cells.

Answer 35

-They recognize phosphatidylserine on the apoptotic cell surface and then engulf them.
-As a result the PCs release anti-inflammatory cytokines.