3- MoD; Cell Damage

Question 1

what is necrosis?

Answer 1

death to a group of cells affected by an injurious agent/ event - often too little blood flowing to tissue, damaging cells which need to be removed

acute, deliberate inflammation to clear cell debris via phagocytosis

Question 2

causes of necrosis

Answer 2

injury (to numerous cells within a tissue)
infection
infarction
inflammation
cancer = reduced blood supply to cells within a tumour mass

Question 3

describe the process of necrosis

Answer 3

infectious event/ agent damages a group of cells - affects blood supply to the area

decreased blood supply = decreased oxygen = decreased ATP production

decreased activity of ATP dependent ion channels/ pumps - induces ionic imbalance

causes changes in cell osmolarity, and an increase in volume as water is drawn in = adds pressure on organelle membranes

pressure causes lysosomal membrane rupture and the release of lysosomal enzymes which degrade cell contents

cell continues to swell in this time - it gets so big the cell membrane ruptures and releases cell debris into the extracellular space

cell debris cleared by phagocytosis

Question 4

differentiate and describe reversible and irreversible swelling

Answer 4

reversible swelling - before lysosomal cell rupture, all the events before are reversible

if the cell is suddenly reoxygenated - ATP production increases, increases activity of ATP dependent pumps which restores ionic balance and cell osmolarity
= decreases swelling

irreversible swelling - following lysosomal membrane rupture, events are irreversible as there’s too much damage and the cell is no longer capable of ATP production

Question 5

describe the microscopic nuclear changes within a cell undergoing necrosis

Answer 5

chromatin condenses and shrinks, and is degraded by DNAses

nucleus fragments

Question 6

describe the microscopic biochemical changes within a cell undergoing necrosis

Answer 6

intracellular enzymes and proteins are released into extracellular space - e.g. creatine kinase, lactate dehydrogenase

these are detectable in blood and urine - can be used to measure the extent of tissue damage and necrosis

Question 7

describe the microscopic cytoplasmic changes within a cell undergoing necrosis

Answer 7

loss of cellular structures as materials are digested by enzymes

proteins denature and aggregate - cytoplasm turns white and opaque instead of transparent

Question 8

what is apoptosis?

Answer 8

selective, programmed cell death in one or fewer cells - without an inflammatory response

Question 9

causes of apoptosis

Answer 9

normal healthy physiological and pathological reaction

healthy:
- occurs in embryogenesis to form individual fingers
- endocrine-dependent tissue atrophy
- normal tissue turnover

pathological:
- lack of growth factor inducing neuronal death
- DNA damage-mediated apoptosis
- cell death in tumours in the middle of the mass, have the most limited blood supply
- immune response induced by CD8+ T cells, NK cells

Question 10

compare apoptosis and necrosis

Answer 10

apoptosis = selective, programmed cell death in one or fewer cells
- all events are IRREVERSIBLE and ATP dependent
- cell SHRINKS as cytoskeleton is disassembled
- cell contents are ORDERLY packaged in membrane-bound vesicles which express new molecules and are phagocytosed without an inflammatory response
- no/ limited leakage of cytosolic components

necrosis = death to a group of cells affected by an injurious agent/ event
- events are reversible up to a point, all ATP dependent
- cell SWELLS from water influx following changes in cell osmolarity
- lysosomal rupture releases enzymes which degrade cell material HAPHAZARDLY
- lots of leakage of cytosolic components and cell debris released into extrac. space
- phagocytosis of cell debris is an acute inflammatory reaction

Question 11

describe the microscopic cytoplasmic changes within a cell undergoing apoptosis

Answer 11

cell shrinkage from cytoskeleton disassembly and packaging organelles into membrane-bound vesicles

cell fragmentation as vesicles bud off - phagocytosed by macrophages

limited leakage compared to necrosis - less inflammation triggered

Question 12

describe the microscopic nuclear changes within a cell undergoing apoptosis

Answer 12

nuclear chromatin condenses on nuclear membrane

DNA cleaved within nucleus

Question 13

describe the microscopic biochemical changes within a cell undergoing apoptosis

Answer 13

expression of charged sugar molecules on the outer surface of cell membranes = recognised by macrophages to enhance phagocytosis and reduce inflammation

protein cleavage by proteases and caspases

Question 14

describe the differences in DNA digestion between apoptosis and necrosis, as shown on a chromosomal DNA run on agarose gel

Answer 14

normally there’s a single high molecular weight band of unaffected DNA

apoptosis = smaller band produced as DNA is digested - orderly

necrosis = non-specific DNA digestion causes a DNA smear

Question 15

what signals promote cell survival?

Answer 15

cell-cell or cell-matrix contacts
growth factors
cytokines

Question 16

what signals promote cell apoptosis?

Answer 16

disruption of cell-cell/ cell-matrix contacts = important in cancers as cell move throughout body for metastasis

lack of growth factors
DNA damaging agents
death domain ligands

Question 17

what are the two types of apoptosis? give examples for the types

Answer 17

relative to the cell - intrinsic and extrinsic

intrinsic - e.g. DNA damage, interrupted cell cycle or protein synthesis, viral infection

extrinsic - e.g. withdrawing survival factors like mitogens, TNF extracellular signal, T or NK cells

Question 18

what are caspases?

Answer 18

cysteine aspartate-specific proteases - aspartate is the specific immuno-acid motif it binds to

act as a point of convergence for intrinsic and extrinsic apoptotic mechanisms

Question 19

describe the caspase cascade, and how it relates to apoptosis

Answer 19

caspase cascade is a pyramidal caspase - involves signal amplification, starts with one initiator caspase, activates many molecules further downstream

starts with an initiator caspase - convergence point for intrinsic and extrinsic apoptotic mechanisms

many initiator caspases within a cell at high concs - brought together in close proximity and cleave themselves

initiator caspase X cleaves procaspase Y at two sites - first the N terminal, then the C terminal, before rearranging small and large subunit of procaspase Y into an active configuration

initiator caspase activates downstream caspases - activate further effector downstream caspases

effector caspases have additional substrates - activate more caspases and other proteins = e.g. lamin for nuclear envelope, signalling, DNA replication or repair proteins

activated caspases and proteins contribute to cellular changes in apoptosis - e.g. chromatin condensing and shrinking, nucleus fragmenting, plasma membrane blebbing

Question 20

what are the initiator caspases in humans?

Answer 20

caspase 8 and 9

Question 21

what are the effector/ downstream caspases in humans?

Answer 21

1, 3, 6, 7

Question 22

describe the proteolytic mechanism for procaspases to activate

Answer 22

initiator caspases initiate autoproteolysis OR an initiator caspase will cleave a downstream caspase at 2 sites:
- first the N terminal, then the C terminal

then procaspase will rearrange small and large subunit into an active configuration

Question 23

what is the main overall trigger for intrinsic apoptosis?

Answer 23

intrinsic apoptosis is induced by cytochrome C release from the mitochondria

cytochrome C release occurs from anything that changes mitochondrial membrane permeability making it more permeable - e.g. oxidative stress, lack of growth factors

Question 24

components for cytochrome C-induced apoptosis?

Answer 24

cytochrome C - release from mitochondria

APAF-1/ apoptotic protease activating factor protein = has a cytochrome C binding site, APAF domain and CARD domain

procaspase-9 = has CARD and protease domains

procaspase 9 and APAF-1 share a CARD domain

Question 25

describe the mechanism for cytochrome C-induced apoptosis

Answer 25

cytochrome C is released from mitochondria under oxidative stress - binds to cytochrome C binding site on APAF-1

induces conformational change = favours dimerization of APAF-1 proteins

creates a microenvironment with a high conc. of CARD domains - recruits procaspase-9 to complex due to shared CARD domains

other proteins are also recruited to complex - creates a big multimeric protein complex called an apoptosome

protease domains of recruited procaspase-9 in close proximity induce autoproteolysis/ self-cleavage and activation into active caspase 9

caspase 9 goes from membrane to cytoplasm - triggers caspase cascade by cleaving downstream caspases

Question 26

what molecule regulates cytochrome C release?

Answer 26

BCL-2 family proteins = regulate permeability of mitochondrial outer membrane pores

Question 27

describe the regulation of cytochrome C release

Answer 27

BCL-2 family proteins regulate the permeability of mitochondrial outer membrane pores through a balance of pro-apoptotic and anti-apoptotic BCL-2 proteins

anti-apoptotic proteins - e.g. BCL-2, BCL-XL
pro-apoptotic proteins - e.g. Bax, Bad, Bid

not all BCL-2 proteins are involved in mitochondrial membrane permeability BUT they do all share a BH3 domain - makes dimerization/ multimerization easier

Question 28

list pro-apoptotic BCL-2 family proteins

Answer 28

Bad
Bid
Bax

Question 29

list anti-apoptotic BCL-2 family proteins

Answer 29

BCL-2
BCL-XL

Question 30

describe how the balance of apoptotic BCL-2 factors governs cytochrome C release and cell death - interactions between Bax, BCL-2 and Bad

Answer 30

1. Bax binds to mitochondrial pore as a pro-apoptotic protein = allows cytochrome C release = allows apoptosis
2. BCL-2 binds to Bax as a anti-apoptotic protein = closes pore = prevents cytochrome C release = prevents apoptosis
3. Bad binds to BCL-2 more strongly than BCL-2 binds to Bax =displaces BCL-2 from pore as a pro-apoptotic signal = allows cytochrome C release and apoptosis

Question 31

what regulates BCL-2 proteins?

Answer 31

depends on:
1. gene expression
2. type of post-translational modifications
3. if the cell is in an environment with many growth factors/ survival signals

e.g. expression of TP53 following DNA damage, or phosphorylation as a post-translational modification

Question 32

how does TP53 regulate BCL-2 family proteins and apoptosis?

Answer 32

DNA damage occurs - activates expression of TP53 gene = TP53 TF = increase expression of pro-apoptotic Bax

Bax inserts itself into membrane, creates new pores, increases mitochondrial membrane permeability

not enough anti-apoptotic BCL-2 to block its effects = causes cytochrome C release

Question 33

how does phosphorylation as a post-transcriptional modification regulate BCL-2 family proteins and apoptosis?

Answer 33

if cell is in a growth factor rich environment - act as survival signals

growth factors activate AKT/PKB (protein kinase B)

PKB phosphorylates Bad - prevents it from binding and displacing BCL-2 = prevents pores opening

prevents cytochrome C release and apoptosis

(removing growth factors as survival signals would prevent this phosphorylation)

Question 34

what are the components of extrinsic apoptosis?

Answer 34

transmembrane receptor - has a ligand binding domain and death domain

death adaptor protein - has a death domain and death effector domain

procaspase-8 - has a death effector domain and protease domain

Question 35

describe the mechanism for extrinsic apoptosis with TNF as the ligand

Answer 35

TNF binds TNF- transmembrane receptor at the ligand binding domain = induces a conformational change for the recruitment of more TNF receptors

creates an environment with a high concentration of death domains - promoters dimerization/ multimerization and the recruitment of FAAD (death adaptor protein)

recruiting FADD proteins creates a high conc. environment of death effector proteins = promotes recruiting procaspase-8 due to shared death effector domain

creates a multimeric DISC protein complex - high conc of death effector domains promotes autoproteolysis of procaspase 8 into active caspase 8

active caspase 8 goes from membrane to cytoplasm - activates downstream caspases and other substrates = triggers apoptotic cellular changes