TRAIL-TRAIL

Question 1

What are the two types of regulated necrosis

Answer 1

Necroptosis and pyroptosis

Question 2

How does necrosis differ to apoptosis>

Answer 2

Necrosis: There is an opening in the plasma membrane of the cell, followed by the release of cytoplasmic proteins into the extracellular space. Apoptosis: Cell dies, becomes packaged into apoptotic bodies which can be taken up by neighboring cells or phagocytes

Question 3

Which type of cell death gives off an alarm signal to the immune system

Answer 3

Necrosis

Question 4

What are the orderly succession of morphological changes in apoptosis

Answer 4

Shrinkage, Nuclear fragmentation, chromatin condensation, apoptotic bodies segregate, DNA cleavage and phagocytosis

Question 5

what is secondary necrosis>

Answer 5

If no macrophages or other surrounding cells are available to the apoptotic cell, secondary necrosis occurs which is the release of intracellular contents into the extracellular space

Question 6

What is TNF?

Answer 6

Tumour necrosis factor

Question 7

What is TRAIL?

Answer 7

An apoptosis-inducing member of the TNF family.

Question 8

What is the difference between TRAIL receptors 1,2,3 and 4?

Answer 8

TRAIL receptors 1 and 2 (DR4/5) have a full, intracellular death domain. TRAIL -R3 has no intracellular death domain at all. TRAIL-R4 has a truncated death domain. Therefore TRAIL-R3/4 are considered to be apoptosis inhibiting receptors

Question 9

In what structure does TRAIL exist? How does it activate TRAIL-Rs?

Answer 9

As a homotrimer which crosslinks three TRAIL receptors.

Question 10

What happens following crosslinking of TRAIL-R1/2?

Answer 10

The death domains of the receptors adopt a conformation that allows for the recruitment of FADD via its death domain. Via its death effector domain; FADD recruits pro-caspase 8. Procaspase 8 homodimerisation within the DISC leads to cleavage and release of active caspase-8 from the DISC.

Question 11

What structural domains does FADD have?

Answer 11

It has a death domain and a death effector domain

Question 12

What molecule prevents the homodimerisation and subsequent activation of procaspase-8 (long form)

Answer 12

c-FLIP long - Very similar to procaspase-8 structure so dimerises with procaspase-8. Cleavage of a single procaspase and c-FLIP long then occurs

Question 13

What is the method of procaspase-8 inhibition exhibited by c-FLIP short

Answer 13

The caspase domain of procaspase-8 is unable to dimerise with anything so there is a complete inhibition of procaspase-8 at the DISC

Question 14

How do you distinguish which c-FLIP structure is present

Answer 14

By looking at the fragments of caspase-8 at the DISC when immunoprecipitated from a cell. If it’s full length then c-FLIP short is responsible. If it is p43 of caspase 8 it is due to c-FLIP long.

Question 15

What does active caspase 8 do to induce death.

Answer 15

It cleaves the molecules Bid and (pro)caspase-3.

Question 16

What subsequent changes have to occur to procaspase-3 following caspase-8 mediated cleavage to induce its full activity?

Answer 16

A maturation step which requires the activity of caspase-3 itself.

Question 17

What molecule blocks the maturation step of procaspase-3

Answer 17

XIAP - XIAP binds to procaspase-3 and prevents its intrinsic cleavage. It also inhibits the activity of caspase-9 in the apoptosome.

Question 18

What is Bid? What is tBid?

Answer 18

A proapoptotic member of the Bcl-2 family. tBid is the active form of Bid following cleavage by caspase-8. It activates to Bax and Bak at the mitochondria which form pores in the mitochondrial membrane so proteins in the intermembranal space of the mitochondria are released.

Question 19

What are two important compunds released from the intermembranal space in mitochondria during apoptosis?

Answer 19

Cytochrome c and Smac/DIABLO

Question 20

What is the role of cytochrome c in apoptosis?

Answer 20

It forms the apoptosome with caspase-9 (requires Apaf-1). The apoptosome then induces the pre-activation of procaspase-3

Question 21

What is the role of Smac/DIABLO in apoptosis?

Answer 21

It binds to the surface of XIAP which removes the inhibition of procaspase-3 and caspase-9, inducing apoptosis.

Question 22

How does a cancer cell overcome apoptosis following chemo/radiotherapy?

Answer 22

Upregulation of Bcl-2/Bcl-xL - antiapoptotic members of the Bcl-2 family of proteins.

Question 23

What are BH3 mimetics?

Answer 23

They activate Bax/Bak directly or inactivate Bcl-2/Bcl-xL (antiapoptotic Bcl-2 family members) which induces the permeabilisation of the mitochondrial membrane.

Question 24

How do Smac mimetics work?

Answer 24

Either leads to autodegredation of XIAP or the removal of the inhibitory action of XIAP by releasing the caspase-3/9

Question 25

What three methods are there to induce apoptosis therapeutically>

Answer 25

1. Targeting the death receptor (antibodies or TRAIL)
2. Targeting XIAP via Smac mimetics
3. BH3 mimetics which either activate Bax/Bak or inactivate Bcl-2/Bcl-xL/Mcl-1

Question 26

What is the role of Cdk9

Answer 26

Forms part of the transcription elongation factor b. it phosphorylates RNA pol II allowing for elongation. Blocking of Cdk9 therefore stops transcription.

Question 27

How does Cdk9 inhibition sensitize cancer cells to TRAIL induced apoptosis?

Answer 27

Loss of Cdk9 inhibits the expression of cFLIP and Mcl-1. (Mcl-1 is another antiapoptotic Bcl-2 family member)

Question 28

Why does global Cdk9 inhibition only effect Mcl-1 and cFLIP)?

Answer 28

Because they have a high turnover, so transcriptional repression significantly inhibits their production compared to other proteins.