Apoptosis

Question 0

Necrosis

Answer 0

Cell swelling 
Organelle disruption
Plasma membrane rupture
Inflammatory
Externally caused damage-> trauma and disease

Question 1

Apoptosis definition

Answer 1

Programmed cell death
Non inflammatory
Often physiological-> extension of limbs, formation of digits! counter selection of immature B/T cells, ultimate response to DNA damage
Ultimate response to DNA damage

Question 2

Morphological changes in apoptosis

Answer 2

1) onset-> shrinkage of cell-> condensation of cytoplasm
-> shrinkage of nucleus-> nuclear chromatin condensation in to delineated masses against nuclear membranes
2) nucleus fragments (karyorrhexis), cell detaches from surrounding tissue, outlines become convoluted and form extensions
3) blebbing-> extensions separate and plasma membrane seals to form separate membrane around detached solid cellular material-> apoptotic bodies
4) phagocytosis of apoptotic bodies
If not phagocytosed -> undergo degradation-> secondary necrosis

Question 3

Intrinsic apoptosis pathway

Answer 3

Activated by withdrawal of survival signals, often as a consequence of cell damage -> events that effect mitochondrial integrity
Recruited during extrinsic pathway activation by Bid
Leads to cytochrome C realise from mitochondria, a necessary protein component of mitochondrial electron transport chain

Question 4

Stimuli of intrinsic apoptosis

Answer 4

O2 deprivation 
DNA damage
Viral infection 
Hypoxia
Oncogene activation 
Growth factor withdrawal
Cell injury 
Certain steroids
Inability to maintain low Ca levels

Question 5

Intrinsic pathway apoptosis, factors released from mitochondria

Answer 5

Outer mitochondrial membrane becomes permeable-> relase of apoptogenic factors

• Cytochrome C-> binds Apaf (pro apoptotic protease activating factor)-> complex then binds caspase 9 (initiator)-> forms an apoptosome-> activates execution caspase by zymogen cleavage
• SMAC/DIABLO
• pro-caspases
• AIF
• endo G

Question 6

External apoptosis pathway

Answer 6

Stimulation of death receptors by ligands
-> TNFa and deprivation of growth factors
Or fas ligand

Question 7

Death receptor pathway

Answer 7

Death receptors-> subset of TNF-1 receptors -> 3 cysteine rich domains and homologous intracellular domain
Fas/CD95, TNF-R1 and DR3 form a trimer-> binds TNF-1 or other death ligand on the external domain and adaptor proteins internally
Activated TNF-R complex forms the scaffolding for binding two molecules of pro caspase 8

Question 8

Death by fas in the death receptor pathway

Answer 8

1) fas L acts as a ligand for fas-> digomerisation of its R on binding-> associated with clustering of the death domains and binding of FADD
2) FADD binds pro caspase 8 via its death effector domain
3) forms a complex of fas, FADD, and procaspase 8-> death inducing signalling complex
4) self cleavage of pro caspase 8-> caspase 8
5) caspase 8 activates effector caspases

Question 9

Caspas 8 effects

Answer 9

An initiator caspase
Activates execution caspases 3,6,7
Also cleaves Bcl-2(Bid)-> activates the mitochondrial integritity (intrinsic) pathway-> interacts with BAX-> cytochrome C relase-> binds APAF with dATP and pro caspase 9-> cleaves pro caspase 3 and 7
-> stress pathway

Question 10

Fas

Answer 10

=CD95=APO-1
Type 1 membrane protein
Monoclonal antibodies cytoxic for human cell lines isolated-> recognised cell surface proteins Fas
PC60 T cell lymphoma-> specifically kills cells expressing fas

Question 11

Death by TNFR-1 in the death receptor pathway

Answer 11

Various other death domain containing proteins can form once the receptor is activated
TNF-R is activated by TNFa-> binds either TRAF or TRADD
TRAF expression is controlled by survial signals (NFuB)-> binding signals stress mediated inflammatory signals->activation of MAPKK and JNKS-> RIP dependent activation of IKK and NFaB
Loss of TRAF expression favours TRADD binding-> recruits FADD for caspase 8

Question 12

Caspases

Answer 12

Cysteine proteases that cleave peptide bonds next to an Aspartate residue
Present as pro caspases-> activated by proteolytic cleavage
Initiator caspases-> cleave other caspases
Execution caspases-> cleave cellular protiens involved in maintianing cellular integrity and endonucleases CAD

Question 13

Caspase substrates

Answer 13

Cytoplasmic-> beta catenin, IP3R
Nuclear-> lamins, SRF
Protein kinases-> PKC isoforms PRK2
Other signalling protiens-> pro-interleukin, Ras, GAP
Cell cycle-> Rb, p21, p27
Apoptotic mediators-> pro caspases

Question 14

BCL-2 family

Answer 14

Decision makers that integrate prodeath and anti death signals
Originally identified in follicular lymphoma
Led-9 homologue
Extends cell survival
> 15 members

Question 15

Anti apoptotic Bcl

Answer 15

Bcl-2
Bcl-X-> regulates outer mitochondrial membrane integrity
Bcl-w
Mol-1
Localise predominantly in outer mitochondrial membrane
Complex with APAF-> inhibit apoptosome formation
Heterodimerase with BAK and BAX-> block pore formation
Sequester and inhibit BH-3 only pro apoptotic factors
Viral homologues-> EBV, BHRF-1

Question 16

Proapoptoic Bcl

Answer 16

Activated by conformational change or multimerisation
Channel forming-> BAX, BAK, BOK-> associate with VDACS-> release of apoptogenic factors
BH3-> activated by dephosphorlyation, cleavage,due novo expression-> activation of multi domain pro apoptotic factors-> dimerise-> form and ion channel that promotes cyto c release
-> inactivate anti apoptotic factors
Bid-> activates BAX and binds Bcl-2-> frees APAF

Question 17

Inhibitors of apoptosis

Answer 17

Eight human protiens 
Expression up regulated by NFaB
Inhibit/ubiquylating caspases 
Down regulated by phosphorylation
IAP kinase stimulated by activated fas R

Question 18

PI3K dependent survival pathway

Answer 18

Activated by binding of growth factors
-> activates AKT-> results in phosphorylation of the pro apoptotic BAD-> inactivated.
Fork head phosphorylation-> deactivation of PI3K-> normally causes production of fas ligand

Question 19

Clearance of apoptotic cells

Answer 19

By phagocytes
Macrophages recognise apoptotic cells-> phosphatidylserine, calrecticulin, thrombospondin
Ingest apoptotic cells by phagocytosis
Release anti inflam-> TGFb1-> resolves inflammation and promotes tissue repair

Question 20

Too much apoptosis

Answer 20

Neurodegeneration
Atherosclerosis
Multiple sclerosis
AIDS-> HIV infects CD4+ lymphocytes-> increased susceptibility to apoptosis-> progressive loss of CD4 T cells

Question 21

Too little apoptosis

Answer 21

Autoimmunity
Chronic inflammation
Cancer-> Bcl-2 over expressed
-> B cell lymphomas, breast cancers, prostate, colorectal