1-2: Cell Death

Question 1

Explain the importance of cell signalling during cell death

Answer 1

Cell signalling can induce cell death and determine what form of cell death occurs, but also…

Signalling by the dying cell determines the response to it (for example, apoptotic cells release anti-inflammatory signals, and other signals to prevent further damage and maintain tissue integrity)

Question 2

What are the morphological differences between apoptosis and necrosis?

Answer 2

In necrosis, cell contents can spill out, causing inflammation and tissue damage…

whereas apoptosis is more carefully controlled and results in no observable inflammation - but DOES show cell shrinkage, NEBD, DNA cleavage, and other changes requiring ATP

Question 3

Describe the process of Casp9 activation and its subsequent effects

Answer 3

• Cytosolic Casp9 is an Inactive Monomer (as [Casp9] = 9-20nM, Kd is 50µM)
• When cytC is released from mitochondria, it binds to APAF1, inducing unfolding and oligomerisation and exposing the CARD domain
• CARD homeotypically binds Casp9 CARD, recruiting Casp9’s to the apoptosome (shifting the dissociation constants to allow FORMATION OF ACTIVE DIMERS)
• Active Casp9 dimers cleave their own CARD domains and are released to activate ECs (e.g., 3 and 7)

Question 4

Describe how Casp3 changes the plasma membrane, and the significance of this

Answer 4

Casp3 cleaves iPLA2, which in turn cleaves PhosphoCholine to form LYSOPHOSPHATIDYLCHOLINE (“find me” signal)

Casp3 also cleaves Xkr8, which then flips PhosphatidylSerine to the outer leaflet (“eat me” signal)

Question 5

Name ~8 of the best understood cleavage events carried out by Caspases

Answer 5

1. ICAD -> CAD -> DNA fragmentation
2. Acinus + Helicard (a DNA helicase) -> Chromatin Condensation
3. Kinases (e.g., ROCK-1, PAK2 -> membrane blebbing)
4. Gelsolin -> Depolymerises F-actin (may facilitate membrane blebbing)
5. IFs e.g., Cytokeratin-18 and Vimentin [AND] IF organisation proteins e.g., Gas2 and Plectin -> breaks down cytoskeleton architecture
6. Proteins involved in cell adhesion or communication at Desmosomes, Gap Junctions, Adherens Junctions (e.g., ß-cadherins, E-cadherin)
7. ER and Golgi proteins (e.g., golgin-160, Bap31)
8. Nuclear matrix proteins -> Lamina Disassembles, Nuclear Pore Transport Disrupted

Question 6

Describe the significance of PARP in apoptosis

Answer 6

Casp3+7 inactivate PARP (a DNA repair enzyme) to save ATP for apoptotic processes

This is important as PARP would use even more ATP under oxidative stress and DNA damage

Question 7

What are the two main categories of apoptosis-derived signals?

Answer 7

1. Those that direct immune cells to clear debris and inhibit inflammation and fibrosis
2. Those that directly signal neighbouring cells to prevent tissue damage

Question 8

Describe how Bcl2 was discovered

Answer 8

A mutation causing Follicular Lymphoma in B Cells (hence Bcl2), involving a reciprocal translocation between chromosomes 14 and 18, resulting in increased Bcl-2 production and greater cell survival

Question 9

Describe the different types of Bcl-2 family proteins

Answer 9

Anti-apoptotic Bcl2s share BH1-4 domains and a TransMembrane domain, and block OMM permeabilisation

Pro-apoptotic Bcl2s lack BH4 and form holes in OMM

BH3-only proteins lack all except BH3, and regulate the other two groups

Question 10

Name the Bcl2 proteins in each group

Answer 10

AA: Bcl2, BclX, BclW, Mcl1, BFL-1/A1

PA: Bax, Bak, Bok

BH3-only: PUMA, Bik, Bim, Bad, Bid, BMF, HRK, NOXA

Question 11

Describe the interactions between the different groups of Bcl-2 proteins

Answer 11

• Activator BH3s (BID/BIM) bind to PAs (BAK/BAX) to activate them
• AAs (2, X, MCL-1) bind to, and sequester, both PAs and BH3s ^
• Sensitiser BH3s (BAD/NOXA) sequester AAs