Apoptosis

Question 1

How does apoptosis proceed

Answer 1

Initiates a program, at the end of the program the cell in engulfed by a macrophage.

Question 2

Steps in apoptosis

Answer 2

1. DNA is condensed
2. Shrinkage of the cytoplasm
3. Membrane blebbing
4. DNA cleavage
5. Phosphotidyleserine is transported to the outer membrane (macrophage eat me signal)
6. Tight regulaiton
7. No inflammation
8. Tight control
9. Energy is required

Question 3

Steps in necrosis

Answer 3

1. Cytolysis: membrane destruction
2. Inflammation
3. Random DNA cleavage

Question 4

Two types of apoptosis

What is similar and different about them

Answer 4

Extrinsic and intrinsic, the only difference is where do you get the signal from.

In both of the mechanism the cells activate caspases which are enzymes that eat proteins. There is also MOMP (mitochondria outer membrane permealization) which permealizes the outer membrane of the mitochondria

Question 5

Explain extrinsic apoptosis

Answer 5

When a particular ligand is secreted, our cells have what we call the death receptor. The death domain on the death receptor recruits scaffolding proteins which then recruit caspase 8 from the cytosol. Caspase 8 is then activated (self activated), releases from the receptor and activates MOMP.

There are precaspases and effector caspases. Initiator caspases are the ones that are recruited to the receptor

Question 6

What is a zymogen

Answer 6

An inactivated protein

Question 7

Zymogen version of a caspase

Answer 7

Procaspase

Question 8

How are procaspases activated

Answer 8

They have an alpha subunit and a beta subunit. They are first sequestered around the death receptor (alot of Caspase 8 is sequestered) and then they are cut. They form dimers in their mature form and then they go on to mature effector caspases.

Question 9

Biochemical basis of cutting the caspases

Answer 9

They have dimerized structures and floppy loops coming out of them. These loops are need to be cut to activate the caspases

They are always cut at the aspartate residue

Question 10

Common things between all caspases

Answer 10

They have a common cystiene residue at the active site.

Question 11

What is done to regulate the caspases in a cell

Answer 11

There are proteins called the Inhibitor Apoptosis Protein (IAP). What they do is that when they see a caspase, either active or inactive, they bind to it and close the active site

Question 12

How is apoptosis initiated in the presence of caspase regulation

Answer 12

IAP mediated inhibition is abrogated by smac/DIABLO during apoptosis

Question 13

What are over expressed in cancers

Answer 13

Death receptors

Question 14

What happens downstream of Caspase

Answer 14

1. The kinases are cut, now they are more active and they will phosphorylate whatever they can
2. There is deterioration of the cell cytoskeleton
3. PARP destruction, DNA repair mechanism is now impaired. Capase cleaves PARP
4. DNA fragmentation by caspase activated DNAase
5. Nuclear envelope is degraded by the destruction of the lamin in the envelope
6. Finally phosphotidylserine from the inside of the cell membrane is flipped to be exposed outside for the macrophage to phagocytose the cell

Question 15

How is the DNA cut in apoptosis and how this is avoided in a functioning cell

Answer 15

This is avoided by the protein called Inhibitors of Caspase Activated DNAse (ICAD).

During apoptosis Caspase 3 cleaves ICAD and so now the Caspase activated DNA is turned on and cleaves the DNA

Question 16

What does caspase do to the nucleas during apoptosis

Answer 16

It degrades nuclear lamins

Question 17

What are the 2 proteins associated with activating or deactivating eat me signal

Answer 17

Scrambalase flips this signal out, activating englufement whereas flippase puts this signal back in. During apoptosis flippase is cleaved.

Question 18

Explain intrinsic apoptosis

Answer 18

Due to stress there is activation of a transcription factor called p53. This transcription factor leads to the activation of gene transcription, this leads to PUMA. This is a BH3 binding domain.

Also increased stress can lead to activation of the BH3 proteins

Question 19

What are the 2 kinds of BCL-2 protein

How do these proteins work

Answer 19

They are either pro-apoptopic or anti-apoptopic

So basically we have anti-apoptopic family members that are bound to the pro-apoptopic family members. When BH3 is activated it binds to the anti-apoptopic family members and takes them away from the pro-apoptopic genes that codes for pro-apoptopic proteins. Now these genes are transcribed and they cause MOMP leading to apoptosis

Question 20

What mechanism is a commonality between the extrinsic and intrinsic apoptosis

Answer 20

The activation of pro-apoptopic BCL 2 proteins

Question 21

How does the pro-apoptopic BCL-2 proteins act

Answer 21

They form a pore and incorporate themselves into the mitochondria forming pores.

Question 22

Know what MOMP causes death of mitochondria

Answer 22

Proton gradient is not formed, there are more ROS.

Cytochrome c leaks out in the cytoplasm, it activates caspases, it is a super activator, t forms an apoptosome

Question 23

What process is involved in ubiquitinilation

Answer 23

E1, E2 and E3 proteins are involved (3 is most in number and most specific, followed by E2 and then E1). E1 is activating enzyme, E2 is conjugating enzyme and E3 is ligaing enzyme (it is the one that trasnfers the ubiquitin on the protein that needs to be degraded).

Question 24

What does p53 do

Answer 24

It binds to DNA (since its a transcription factor) leads to shuttind down the cell cycle or if the signal is very strong, it will lead to cell death.

Question 25

How is p53 regulated

Answer 25

MDM2 is bound to p53. MDM2 is itself an E3 so it facilitates in p53 degradation by proteosome

Question 26

What does cancer cells do regarding death receptor

Answer 26

They over express those (well some of the cancer cells over express them)

Question 27

Important thing to remember about p53 and death receptors

Answer 27

Death receptors cause apoptosis in an p53 independent manner

Question 28

What are the causes of lupus

Answer 28

1. Increases apoptosis of monocytes and keratinocytes mediated by increased p53 activity
2. Increased T cell necrosis
3. Impaired mitochondrial function
4. Loss of apoptopic cell clearence
5. Increase in auto-antigen presentation