Lecture 3: Apoptosis

Question 1

What is the difference between apoptosis and necrosis?

Answer 1

Apoptosis is ‘good’ cell death. It is programmed cell death, which means it is more regulated, buds coming off cells, less messy. Vesicles carry away contents of cells in a controlled way so the organism as a whole is protected (not endangered by release of cell contents). There is no inflammation.

Necrosis is ‘bad’ cell death. It is messy. The cell explodes (lysis), releasing the contents. This means a virus could be released. There is often inflammation. Necrosis happens when there is absolutely nothing else the cell could do.

Question 2

What does it look like when a cell undergoes apoptosis?

Answer 2

The cell shrinks, then becomes refracted and shines brightly. Then the cell breaks down into many vesicles which carry away the cell contents.

Question 3

When is apoptosis important?

Answer 3

Apoptosis is very important in development. It is essential in making fingers, making the holes in blood vessels and in removing brain nerve cells that are incorrectly attached.

Apoptosis is also important in preventing disease. If T cells with self-reactivity are recognised, they are removed through apoptosis. Otherwise they would the body as part of a faulty immune system. Apoptosis also removes cells in which mutagens/radiation have damaged the DNA irreparably. This prevents cancer.

Question 4

What is Caenorhabditis elegans?

Answer 4

A 1 mm long nematode worm which feeds on E. coli. A fate map of all of the cells in development has been drawn (the researchers got a Nobel Prize for this work). It turns out C. elegans has 1090 somatic cells, 131 of which die by apoptosis. There are 14 essential genes.

Question 5

What are ced mutants?

Answer 5

Ced stands for cell death. Cell death mutants are mutants which cannot undergo correct apoptosis.

Question 6

What are caspases?

Answer 6

Caspases are proteases. Ced-3 is a typical example in C. elegans. All of the caspases in humans have a conserved structure.
Caspase is short for Cysteine aspartate protease. They cleave the C terminal of target proteins to an aspartate residue.

Question 7

How are caspases activated?

Answer 7

Activation of caspases requires two cleavage events. Before the two cleavages the caspase is in an inactive zymogen form. After activation it is called a procaspase. Caspases must be very well regulated - must safeguard cells against uncontrolled protein cleavage.

A prodomain containing the N terminal is cleaved off by an active caspase. The other cleavage site is nearer the C terminus and the cleaved off domain becomes the small subunit of the activated caspase, with the uncleaved region being the large subunit.

Question 8

What are the two types of apoptotic caspase?

Answer 8

Initiators and Effector/executioner.

Question 9

Are there more initiators or effectors?

Answer 9

More effectors

Question 10

Give an example of an initiator and an executioner.

Answer 10

Initiator example: caspase 9

Executioner example: caspase 3

Question 11

How does one activated initiator lead to rapid apoptosis?

Answer 11

Signal amplification. There is a downstream cascade. The one initiator actives many other caspases, which all in turn activate executioners. This means one active initiator can lead to many activates effectors/executioners, causing rapid apoptosis.

Question 12

What are adaptor proteins?

Answer 12

The bridge between caspase and a pro-apoptotic input signal. When a signal for the cell to undergo apoptosis is received, the adaptor tells the initiator, which begins apoptosis.

Question 13

Give an example of an adaptor protein in humans and C. elegans.

Answer 13

Humans - Apaf-1, FADD
C. elegans - ced-4
They are all structurally similar.

Question 14

What are regulator proteins?

Answer 14

Proteins which can promote or prevent apoptosis.

Question 15

Give an example of a regulator protein in humans and C. elegans.

Answer 15

Humans - Bcl-2

C. elegans - ced-9

Question 16

Comment on the number of regulator proteins and common structures.

Answer 16

There are many different regulator proteins, which means that there can be quite complex regulation of apoptosis.
Many regulator proteins have a transmembrane domain at their C terminus, which means this region is rich in hydrophobic amino acid residues.

Question 17

What are the two main classes of apoptotic trigger?

Answer 17

Intrinsic pathway and extrinsic pathway. The intrinsic pathway is suicide and a trigger could be irreparable DNA damage. The extrinsic pathway is caused by an external signal and could be used to kill a self-reactive T cell (immune cell which acts body instead of pathogens).

Question 18

How can mitochondria play a role in intrinsic triggering?

Answer 18

When cytochrome c leaks out of mitochondria, intrinsic apoptosis is triggered. Cytochrome c is not normally found in the cytoplasm. When cyt c is released from an injured or damaged mitochondria, it binds to Apaf-1 (an adaptor protein). These cyt c-Apaf-1 complexes begin to aggregate into heptamers called apoptosomes and then bind inactive procaspase 9 (an initiator). Once enough procaspases have been brought together they are partially active and can continue the cascade (cleavage) of neighbouring proteins. This causes apoptosis.

Question 19

Which regulator proteins can influence the release of cytochrome c from mitochondria?

Answer 19

Bcl-2 in humans and ced-9 in C. elegans are regulator proteins which can prevent apoptosis by preventing the release of cytochrome c from mitochondria. It is the transmembrane domain of the regulator proteins which is important here.

Question 20

How is the Fas death receptor involved in extrinsic triggering of apoptosis?

Answer 20

The Fas death receptors on the target cell are made up of 3 domains: an extracellular ligand-binding domain, a single transmembrane domain and an intracellular death domain. The Fas ligand is present on the outside of killer lymphocytes.

When activated by the binding of a Fas ligand, the death domains on the cytosolic tails of the Fas death receptors recruit intracellular adaptor proteins, which in turn recruit initiator procaspases (procaspase 8 or 10, or both), forming a death -inducing signalling complex (DISC). Once activated in the DISC, the initiator caspases activate downstream executioner procaspases to induce apoptosis.

Question 21

How can extrinsic apoptosis be blocked?

Answer 21

Competitive inhibition.

• decoy receptors which bind the Fas ligand without triggering apoptosis due to no intracellular death domain.
• FLIP caspase-like proteins, which can bind in the DISC, but do not acts as proteases when activated.

Question 22

What is CAD?

Answer 22

Caspase activated DNAase. It gets rid of genomic DNA during apoptosis.

Question 23

What does an activated caspase do during apoptosis?

Answer 23

• Proteolysis of cellular proteins (removes harmful substances before cell releases them)
• Activation of CAD (caspase activating DNAase, gets rid of genomic DNA during apoptosis)

Question 24

What is characteristic about the agarose gel after electrophoresis of the DNA of a cell undergoing apoptosis?

Answer 24

District bands, making up a ‘DNA ladder’

Question 25

Which regions of DNA are most accessible to cleaving proteins like CAD?

Answer 25

The regions of DNA which are not wound around histones.

Question 26

Learning objectives

Answer 26

• describe the key difference between apoptosis and necrosis
• compare and contrast the extrinsic and intrinsic pathways leading to apoptosis
• describe the regulators and mediators of apoptosis, including, Apaf-1 and Bcl-2 like proteins