Cell Biology - Apoptosis

Front 1

What organism was apoptosis originally observed in?

Back 1

C. elegans

Front 2

What is the difference between apoptosis and necrosis?

Back 2

Apoptosis:
- characteristic morphological changes
- surface produces a signal to trigger the process
- does not cause any inflammatory response

Necrosis
- cell swells and eventually bursts
- causes inflammatory response

Front 3

What are the four main roles of apoptosis during development?

Back 3

1. Unwanted cell elimination (ex. in growth and definition of digits)
2. Unwanted structures eliminated (ex. tail in development of frog)
3. Regulates cell numbers (ex. half of nerve cells undergo apoptosis because there are not enough survival factors for them all)
4. Quality control

Front 4

What four characteristics can we look for to determine if a cell has undergone apoptosis?

Back 4

- DNA has been cleaved
- phosphatidyl serine on the extracellular side of the cell
- mitochondria lost electron potential across membrane so can no longer transport proteins
- cytochrome C in the cytosol

Front 5

What is the TUNEL technique?

Back 5

TdT-mediated dUTP Nick End Labeling
- labels cells that died due to apoptosis
- enzyme terminal deoxynucleotidyl transferace (TdT) adds chains of labeled deoxynucleotide (dUTP) to the 3'OH ends of DNA fragments

Front 6

What do caspases do and how do they do it?

Back 6

- have cysteine at their active site and cleave their target proteins at specific aspartic acids
- synthesized in the cell as inactive precursors (procaspases) which are activated by cleavage by another caspase
- procaspase is split into a large and small subunit that form a heterodimer and 2 of these assemble to form the active tetramer
- once activated caspases cleave and thereby activate other procaspases resulting in an amplifying proteolytic cascade
- caspases then in turn cleave target proteins

Front 7

What happens in the extrinsic pathway?

Back 7

- Fas ligand on the surface of the killer lymphocyte activates Fas death receptors on the surface of the target cell
- Cytosolic tail of Fas receptor then recruits FADD adaptor protein via the death domain on each FADD (FADD = Fas-associated death domain)
- each FADD then recruits a procaspase-8 or procaspase-10 or both and FADD and the procaspase form DISC (Death Inducing Signal Complex)
- DISC then brings procaspase molecules close together which activates them causing them to cleave one another and become a caspase
- Activated caspase-8 and caspace-10 then cleave executioner procaspases producing a cascade of caspases leading to apoptosis

Front 8

What are the two inhibitory proteins that inhibit apoptosis?

Back 8

1. Decoy receptors - have ligand binding domain but not a death domain
2. FLIP - intracellular blocking protein, resembles procaspase-8 and procaspase-10 and competes with them but doesn't actually have a proteolytic domain to cleave caspases

Front 9

What happens in the intrinsic pathway?

Back 9

- cytochrome C is released from the mitochondria and activates Apaf1 to hydrolyze its bound dATP to dADP
- this induces the complexes of Apaf1 and cytochrome C to aggregate and form a large, heptameric apoptosome
- this then recruits procaspase-9 through a caspase recruitment domain (CARD) in each protein
- the procaspase-9 molecules are activated within the apoptosome and are now able to cleave and activate downstream executioner procaspases which leads to a caspase cascade

Front 10

What do Bcl2 proteins do broadly?

Back 10

regulate the intrinsic pathway by controlling the release of cytochrome C into the cytosol

Front 11

What are the anti-apoptotic Bcl2 proteins?

Back 11

ex. Bcl2 and Bcl-XL
- share four distinctive Bcl2 homology (BH) domains (BH1, BH2, BH3 and BH4)

Front 12

What are the 2 subfamilies of the pro-apoptotic Bcl2 proteins?

Back 12

- BH123 proteins (Bax and Bak) - structurally similar to Bcl2 but lack BH4 domain
- BH3-only proteins (Bad, Bim, Bid, Puma and Noxa) - share sequence homology with Bcl2 in only the BH3 domain

Front 13

What happens in the absence of an intrinsic apoptotic stimulus?

Back 13

- anti-apopototic Bcl2 proteins bind to and inhibit the BH123 proteins on the mitochondrial outer membrane
- prevent innapropriate release of cytochrome C

Front 14

What happens in the presence of an intrinsic apoptotic stimulus? (Two things)

Back 14

- the BH123 proteins aggregate on the outer mitochondrial membrane in a tunnel and release cytochrome C from the intermembrane space into the cytosol
- BH3-only proteins are activated and bind to the anti-apoptotic Bcl2 proteins so that they can no longer inhibit the BH123 protein

Front 15

Where are Bak and Bax located?

Back 15

Bak - tightly bound to the membrane at all times
Bax - in cytosol, only moves to membrane when there is a signal

Front 16

What does DNA damage cause?

Back 16

- leads to activation of p53 which leads to transcription of genes encoding BH3-only proteins Puma and Noxa which assist in causing apoptosis

Front 17

What did the study by Ren et al find?

Back 17

- triple knockout of Bid, Bim and Puma cuased rate of apoptosis to dramatically decrease (BH3-only proteins are therefore very important for inducing apoptosis)
- apoptosis is also triggered due to lack of nutrients like low potassium
- essential role of Bid, Bim and Puma in activating Bax and Bak and are required for Bax and Bak to form oligomers

Front 18

What do IAPs do?

Back 18

- in the absence of an apoptotic stimulus, Inhibitors of Apoptosis prevent accidental apoptosis caused by the spontaneous activation of procaspases
- IAPs are lcoated in the cytosol and bind to and inhibit any spontaneously activated caspases
- some IAPs also ubuiquitylate the caspases they bind to

Front 19

What do anti-IAP proteins do?

Back 19

- when an apoptotic stimulus activates the intrinsic pathway, among the proteins that are released from the mitochondrial intermembrane space are the anti-IAP proteins, which bind to and block the inhibitory activity of the IAPs
- allow for apoptosis to resume

Front 20

What are survival factors?

Back 20

- extracellular signal molecules that inhibit apoptosis

Front 21

What are three examples of survival factors?

Back 21

1. increased production of anti-apoptotic Bcl2 protein
2. inactivation of pro-apoptotic BH3-only Bcl2 protein
3. Inactivation of anti-IAPs