Lecture 6- Apoptosis

Question 1

give an example of cell death being important in normal processes

Answer 1

• apoptosis of cells joining digits (fingers and toes)- no webbed hands/feet
• tubes of cells formed by central cells undergoing apoptosis

Question 2

what do dying cells release?

Answer 2

signals into their surroundings

Question 3

what are the 3 main ways cells can die?

Answer 3

• necrosis
• apoptosis
• autophagy

Question 4

what happens during necrosis?

Answer 4

• cell swells
• plasma membrane loses integrity
• components of cell released into surrounding tissue (detected as DAMPs)

Question 5

what happens during apoptosis?

Answer 5

• cell shrinks
• plasma membrane retains integrity
• cell phagocytosed by macrophages

Question 6

what happens during autophagy?

Answer 6

• plasma membranes maintain integrity
• organelles are broken down and used as nutrients- meaning cell doesn’t always die from autophagy
• cell is basically eating itself from inside out using lysosomes

Question 7

what is apoptosis a response to?

Answer 7

cellular stress. e.g. infection or damage

Question 8

what does apoptosis minimise (as opposed to necrosis)?

Answer 8

collateral damage

Question 9

give the 6 main steps of how apoptosis occurs

Answer 9

• cell shrinkage
• cytoskeleton collapse
• loss of nuclear membrane
• chromatin condenses and DNA cleaved into fragments
• membrane blebs and breaks off into apoptotic bodies
• cell surface alters to attract phagocytes

Question 10

give a flow diagram showing a general (non-specific) cell pathway showing controlled cell response to stimuli

Answer 10

signal –> activates receptor –> series of enzymes –> biological effects

Question 11

during a normal signaling pathway, what enzyme is usually used?

Answer 11

protein kinases

Question 12

what enzymes are used in the apoptotic signaling pathway?

Answer 12

proteinases

Question 13

which proteinases are used in apoptosis?

Answer 13

caspases

Question 14

what type of peptidase are caspases?

Answer 14

endopeptidases

Question 15

what does a endopeptidase do?

Answer 15

cleaves specifically within a protein - DOES NOT DEGRADE CELL. Done to turn protein substrate on

Question 16

what does the active site of caspase contain?

Answer 16

cysteine amino acid

Question 17

where do caspase cleave substrate?

Answer 17

after aspartate residue

Question 18

are all caspases involved in apoptosis?

Answer 18

no

Question 19

generally, in what 2 ways do caspases kill cells?

Answer 19

• cuts up DNA

- allows apoptotic cell to be recognised by phagocytes

Question 20

how do caspases cut up DNA?

Answer 20

CAD (Caspase Activated DNAse) is usually bound to an inhibitor (ICAD)
ICAD is cleaved at one site by caspase to remove the Inhibitor
the DNAse then dimerises, enters nucleus and cuts DNA between histones

Question 21

how do caspases signal an apoptotic cell for phagocyte recognition?

Answer 21

• lipids in plasma membrane aren’t evenly distributed between inner and outer leaflet - phosphatidylcholine and phosphatidylserine are normally on inner leaflet
• iPLA2 is cleaved by caspase, activating it to cut phosphatidylcholine to release lysophosphatidylcholine that diffuses as a signal to phagocytes to attract to sell
• one type of flippase is turned off by caspase, catalysing phosphatidylserine to switch to be on the outerleaflet to signal to macrophages

Question 22

what are the 2 main groups of caspase?

Answer 22

• initiator caspase

- executioner caspase

Question 23

do initiator caspase have large or small pro-domain?

Answer 23

large

Question 24

do executioner caspase have large or small pro-domain?

Answer 24

small

Question 25

how are executioner caspases activated?

Answer 25

they’re proteolytically cleaved by initiator caspases

Question 26

what is a caspase in an inactive form called?

Answer 26

zymogen

Question 27

draw a flow diagram showing the caspase flow from signal to biological substrate

Answer 27

signal –> receptor –> initiator caspase (casp.9)–> executioner caspase (casp. 7 + 3) –> biological substrate

Question 28

how must executioner zymogers become active caspases?

Answer 28

start as dimers and be cleaved to monomers . active monomer caspase.

Question 29

how must initation zymogens become active caspases?

Answer 29

start as monomers- induced dimerisation. active dimer caspase

Question 30

how specifically is caspase 9 activated?

Answer 30

• it’s activated by the apoptosome
• apoptosome is formed when APAF-1 is oligermerised by cytochrome C to a heptamer
  the heptameric APAF-1 complex exposes its CARD domain which binds to the CARD domain of caspase 9
• this recruits multiple molecules of caspase 9 to the apoptosome- giving an active dimer
• the active caspase 9 cleaves itself from the apoptosome and can go on to activate caspase 3 and 7

Question 31

how is apoptosome formation controlled?

Answer 31

• by the mitochondria which sequesters cytochrome C

Question 32

how is cytochrome C involved in life and death of cell?

Answer 32

life- has an important role in the electron transport chain in respiration
death- allows caspase activation for apoptosis

Question 33

what controls cytochrome release from mitochondria?

Answer 33

BCL-2 protein family

Question 34

what does BAX do?

Answer 34

promotes cell death by antagonising BCL-2 expression

Question 35

What does BCL-2 alone do for cell?

Answer 35

keeps it alive

Question 36

what domain structure do all members of the BCL-2 family contain?

Answer 36

BH3

Question 37

What does BCL-2 set for apoptosis to occur?

Answer 37

a threshold- only once enough BCL-2 is released from cell can BAX be activated in order to activate cytochrome c etc.

Question 38

what are BH3 only proteins regulated by? (2)

Answer 38

GF signalling

DNA damage

Question 39

What does DNA damage regulate?

Answer 39

PUMA via p53 (tumour suppressor)

Question 40

what does chemotherapy try to do?

Answer 40

activate BH3 to cause cell death

Question 41

what can suppress apoptosis (2 gene types)?

Answer 41

• oncogenes

- tumour supressor genes