III. Signal transduction and cell cycle | 55. Function and activation of apoptotic caspases, role of granzyme B

Question 1

I. Apoptosis
1. What is Apoptosis?

Answer 1

Apoptosis is a form of programmed cell death, and is important when it comes to organ sculpting (differentiation of fingers), removal of unnecessary structures and destruction of damaged/harmful cells (DNA damage, viral infection).

Question 2

I. Apoptosis
6A. What are the Events of apoptosis?

Answer 2

1. Cell will shrink and turn round
2. Chromatin condenses and fragments together with the nucleus
3. Cell membrane blebs and protrudes (protrusions =
   apoptopodia)
4. Protrusions will fragment (separate) and form apoptotic bodies
5. Apoptotic bodies are eaten by other cells

Question 3

I. Apoptosis
2. Can you name other forms of cell death except apoptosis?

Answer 3

necroptosis, pyroptosis etc.

Question 4

I. Apoptosis
3. What is the main difference between apoptosis and other forms of cell death (necroptosis, pyroptosis etc.)?

Answer 4

The main difference between apoptosis and other forms of cell death (necroptosis, pyroptosis etc.) is the induction of inflammation
1. Apoptosis: cell shrinks without lysis (PM does not break) and does not spread its content
=> no inflammation is induced
- Other forms of cell death: cell swelling with lysis
-> emitting the intracellular content
-> induces inflammation

Question 5

I. Apoptosis
4. What is Necrosis?

Answer 5

Necrosis is a non-programmed way of cell death.
- Happens when the cell is so damaged that it cannot undergo programmed cell death.
- Cell swelling will cause bursting and induce inflammation.
- The inflammation will cause a lot of cells to die
=> Therefore, necrosis = death of many cells; apoptosis = death of one cell at a time

Question 6

I. Apoptosis
5. What are major causes of apoptosis?

Answer 6

• Lack of survival signals
• Stress (oxidative stress, ER stress)
• Oncogenic insult
• DNA damages (radiation/chemicals)
• Viral infection

Question 7

I. Apoptosis
6B. What happen after apoptosis?

Answer 7

After apoptosis, phagocytosis occurs:
- Attraction of phagocyte via ‘’eat me’’ signals
- ‘’eat me’’ signals are exposed phosphatidylserines and other phospholipids, which will be translocated to the outer bilayer of the PM and recognized by a phagocyte

Question 8

II. Caspases
1. What are Caspases?

Answer 8

Caspases are a family of (aspartate-specific cysteine) protease enzymes which induce apoptosis.

Question 9

II. Caspases
2. What is the mechanism of caspases?

Answer 9

Caspases are a family of (aspartate-specific cysteine) protease enzymes which induce apoptosis.
- They will cleave specific sequences in proteins located inside the cell, causing changes that lead to cell death.
-These proteases have cysteine at their active site and cleave their target proteins at specific aspartic acids => therefore, they’re called caspases (c for cysteine, asp for aspartate).

Question 10

III. Caspase activation
1. How are caspases synthesized and activated?

Answer 10

Caspases are synthesized in the cell as inactive precursors (procaspases) and are activated only during apoptosis.

Question 11

III. Caspase activation
2. What are the 2 classes of apoptotic caspases?

Answer 11

initiator caspases and effector caspases

Question 12

III. Caspase activation - Initiator caspases (2, 8, 9, 10)
3A. What are the features of Initiator caspases (2, 8, 9, 10)?

Answer 12

• The caspases which initiate the apoptotic process
• Normally exist as pro-caspases in the cytosol
• The major function of initiator caspases is to activate effector caspases

Question 13

III. Caspase activation - Initiator caspases (2, 8, 9, 10)
3B. What is the mechanism of Initiator caspases (2, 8, 9, 10)?

Answer 13

• An apoptotic signal will trigger the assembly of multiple initiator caspases, where
  pairs of caspases go together to form a dimer, resulting in protease activation
• Each caspase dimer then cleaves its partner at a specific site in the protease domain,
  which will stabilize the active complex and is required for their proper enzymatic
  activity (= self-activation)

Question 14

III. Caspase activation - Initiator caspases (2, 8, 9, 10)
3C. What is the major function of Initiator caspases (2, 8, 9, 10)?

Answer 14

The major function of initiator caspases is to activate effector caspases

Question 15

III. Caspase activation - Effector caspases (3, 6, 7)
4A. What are the feature(s) of Effector caspases (3, 6, 7)?

Answer 15

Normally exist as inactive dimers

Question 16

III. Caspase activation - Effector caspases (3, 6, 7)
4B. What are the feature(s) of Effector caspases (3, 6, 7)?

Answer 16

• When they are cleaved by initiator caspases at a site in the protease domain, they change to their active conformation
• Once activated, effector caspases will catalyze the protein cleavage events that kill the cell
• One inhibitor caspase complex can activate many effect caspases, resulting in an amplification of the proteolytic cascade
• Effector caspases can also be activated by molecules that are not caspases, such as granzyme B or calpain (Ca2+)

Question 17

IV. Caspase cascade
1. What are the features of Caspase cascade?

Answer 17

• The caspase cascade is destructive, self-amplifying and irreversible.
• The effector caspase will cleave various proteins, which will be known as ‘’death substrates’’.

Question 18

IV. Caspase cascade
2. What are the examples of “death” substrates?

Answer 18

1. Cell adhesion proteins => cleavage of these leads to detachment of the cell
2. Lamin filaments => Nuclear fragmentation
3. Cytoskeletal proteins => shrinking, blebbing and protrusion of the cell
4. Golgi matrix proteins => fragmentation of the Golgi

Question 19

IV. Caspase cascade
3A. Make a schematic diagram of caspase cascade?

Answer 19

Question 20

IV. Caspase cascade
3B. What are the 5 factors that lead to activation of initiator caspases?

Answer 20

1. GF withdrawal
2. DNA damage
3. Stress
4. Death ligand
5. Oncogene insult

Question 21

IV. Caspase cascade
3C. What are the 5 consequences of cleavage of death substrates?

Answer 21

1. DNA fragmentation
2. Alteration of cell shape
3. Organelle fragmentation
4. Activation of phagocytotic cells

Question 22

IV. Caspase cascade
3D. What are 1, 2, 3?

Answer 22

1. Activation of initiator caspases
2. Activation of effector caspases
3. Cleavage of death substrates

Question 23

IV. Structure of caspases
1. What is the structure of initiator caspase?

Answer 23

Initiator caspases contain special domains:
- DED = death effector domain
- CARD = caspase-recruitment domain
=> These domains are responsible for the auto-activation.
=> They are not present in effector caspases, which explains why effector caspases cannot perform auto- activation

Question 24

V. Inhibitors of apoptosis (IAPs)
1. Why do we need Inhibitors of apoptosis (IAPs)?

Answer 24

Since the activation of the caspase cascade leads to an irreversible cell death, the cell must have mechanisms ensuring that these caspases are only activated when appropriate.

Question 25

V. Inhibitors of apoptosis (IAPs)
2. What is the role of ‘’inhibitor of apoptosis’’?

Answer 25

• Since the activation of the caspase cascade leads to an irreversible cell death, the cell must have mechanisms ensuring that these caspases are only activated when appropriate.
• This mechanism is done by proteins of the ‘’inhibitor of apoptosis’’ family, better known as IAP proteins.

Question 26

V. Inhibitors of apoptosis (IAPs)
3. What. is the mechanism of Inhibitors of apoptosis (IAPs)?

Answer 26

• IAP proteins are able to bind and inhibit the activation of caspases. In order for the apoptosis to happen, the inhibition of the IAP proteins must occur.
• They will be inhibited by proteins released from the mitochondria.