8: Necrosis and Apoptosis 3

Question 1

extrinsic pathway over view: what happens

Answer 1

death ligand binds to death receptors - adaptors, disc formation, caspase 8 activation, and then caspase 3 execution pathway.

Question 2

intrinsic pathway overview: what happens

Answer 2

radiation, toxins, hypoxia, etc. cause mitochondrial changes. formation of apoptosome. caspase 9 activation, then caspase 3 activation.

Question 3

caspase independent pathway: through what? normally located?

Answer 3

AIF: apoptosis inducing factor. AIF normally located in intermembrane space of mitochondria

Question 4

caspase independent pathway: overview of events

Answer 4

cell receives signal to die, for ex. ROS. AIF release from mit. migrates to cell nucleus, binds to and triggers destruction of DNA and cell death.

Question 5

misfolded proteins produced when? they trigger?

Answer 5

constantly. trigger a protective stress response aka UPR: unfolded protein response.

Question 6

prolonged ER stress and UPR activation result in? misfolded proteins can also?

Answer 6

result in activation of cell death pathways. misfolded proteins also aggregate and interact with chaperones and transcription factors.

Question 7

what proteins play a key role in communication between ER and mitochondria? which specific ones?

Answer 7

BCl2 family: Bcl2, Bcl XL, BAX, BAK, B1-1 and BIK

Question 8

other ER stress proteins

Answer 8

BAP31, p53 dependent products NOXA and PUMA

Question 9

ER stress is coupled to?

Answer 9

specific independent pathways, and intrinsic and extrinsic apoptotic pathways

Question 10

caspases stand for? how many types?

Answer 10

cysteine dependent aspartate specific proteases. 14 different caspases in mammals.

Question 11

caspases: synthesized as? activated by?

Answer 11

inactiv procaspases. all (except the initial caspase in a cascade) are activated by proteolytic cleavage by another caspase

Question 12

sequence of events in caspase activation

Answer 12

prodomain cleaved off, capsase divided into small and large subunits. cleaved subunits associated to form active capsase

Question 13

which caspases asoociated with inflamation? apoptosis?

Answer 13

inflam: 1, 4, 5, 11 - 14. apoptosis: 3, 6, 7 are executioners. 2, 8, 9, 10 are initiators

Question 14

6 caspase substrates

Answer 14

other capsases. DNase. regulators of survival pathways. regulators of apoptosis like Bcl2, bclXL, XIAP, Bid. kinases like AKT, Raf1. structural proteins like laminin and actin.

Question 15

apoptosis vs. necrosis: dislocated how? represent?

Answer 15

two processea re temporally dislocated, likely represent two extremes of a continuum.

Question 16

necrosis process can start only and exclusively when?

Answer 16

the cell dies, and is an irreversible process = no return

Question 17

A vs. N: cell size and membrane?

Answer 17

A: shrinkage and fragmentation, membrane is blebbed by integrity maintained. N: cell swells, smoothing and lysis of membrane.

Question 18

A vs. N: mitochondria?

Answer 18

A: increased membrane permeability but structurally ok. N: swells and disordered structure

Question 19

A vs. N: nuclei and DNA degradation?

Answer 19

A: chromatin clumped and fragmented, DNA degraded regularly every 180 bp, DNA appears in cytosol. N: nuclei swells, membrane disrupted, diffuse and random degradation of DNA

Question 20

A vs. N: process?

Answer 20

A: DNA-programmed cascade that requires protein synthesis, RNA transcription and ATP. N: no protein synthesis, RNA transcription, is energy independent and see ATP depletion

Question 21

A vs. N: inducing stimuli?

Answer 21

A: developmental programs and disease processes. N: disease processes

Question 22

5 assays for apoptosis

Answer 22

cytomorphological changes. DNA fragmentation. detection of caspases, cleaved substrates, regulators and inhibitors. membrane alterations. mitochondrial assays.

Question 23

tunnel staining: stands for? does what? problem?

Answer 23

terminal deoxynucleotidyl transferase mediated dUTP nick end labeling. labels fragmented ends of DNA. detects apoptosis, but DNA will break in necrosis too.

Question 24

how to measure DNA fragmentation

Answer 24

gel electophoresis: compare with DNA ladder to see it if breaking every 180 bp

Question 25

gel electrophoresis: what do you see in A vs. N

Answer 25

A: “laddering”: while N you just see a diffuse band near the top (larger size) - no trace of laddering and overall smaller DNA signal

Question 26

diseases associated with inhibition of apoptosis? increase apoptosis?

Answer 26

inhibition: cancer, autoimmune disorders, viral infections. increased: neurodegenerative disorders, ischemic injuries, AIDS, toxin induced liver disease (alcohol)