Old C > Apoptosis > Flashcards

Apoptosis Flashcards

(53 cards)

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1
Q

key difference between apoptosis and Necroptis/necrosis

A

apoptosis is “tidy” and regulated

necrosis/necroptosis is “messy” and inflammatory.

= No inflammation as its just a normal thing in development

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2
Q

describe apoptosis vs necroptosis

A

apoptosis - programmed cell death:
controlled proscess –> no leakage of cell contents

= no inflammation

Necroptosis - reglated necrosis:
triggered by severe stress –> cell contents spill out = inflammation

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3
Q

role of apoptosis in development

A

shapes tissues = removes webbing in embryos

controls neuron number in brain –> cells that dont make connections removed

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4
Q

what are the 2 main pathways of apoptosis

A

Extrinsic - death receptor:
Fas/FasL –> FADD + Pro-caspase-8 via death domains and death effector –> BID to tBID –> MOMP

Intrinsic - mitochondrial:
internal stress –> DNA damage,growth factor loss

= all create apoptosome via Cytochrome-c and Apaf1

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5
Q

what are caspases

A

cysteine proteases that cleave proteins at specific aspartic acid residues

exist as pro-caspases

initiator (8,9) activate effector (3,6,7)

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6
Q

which caspases are effector and which are initiator

A
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7
Q

describe the structure of caspases and what must happen to activate in initiator vs effector caspases

A

N-terminal Pro-domain bound to a large and small subunit

Inititiator caspases have LONG pro-domains that contain CARD/DEs

= clustering/dimersation of caspases in DISC/apoptososome/inflamasome activates caspase
= autocleavage may happen LATER but is not required for activation

Effector caspases already exist as inactive dimers

= initiator caspases cut at the linker between large and small subunits
= cleavage causes comformational change of active site -> activated via proteolytic cleavage

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8
Q

describe some effects of activating effector caspases

A

cleave hundreds of proteins –> structural and regulatory proteins inactivated

= CAD and ROCK1

cause rapid cellular destruction:
- anti-apoptic proteins destroyed
- cell-cell and cell-matrix contacts
- nuclear structure destroyed
- DNA destroyed/fragmented

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9
Q

what is DNA degraded by

A

CAD
Caspase activated deoxynuclease

ICAD is the precursor –> cleaved by effector caspase-3 –> cleaves DNA between nucleosome

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10
Q

how does CAD cleave DNA

A

cuts between nucleosomes –> 180bps apart

= looking at agarose gel seperation with CAD = ladder at 180 shows cleaved DNA

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11
Q

what happens to phosphatidylserine in apoptic cells and describe the enzymes involved

A

lipid usaully on inner leaflet of bilayer in normal cell –> moves to outer in apoptic via Scramblase

= Scramblase activated by caspases –> allows macrophages to engulf

= Flippase ‘flips lipid back’ to inner if movement was accidental BUT is inhibited by caspases

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12
Q

what is membrane blebbing

A

sign of apoptosis

effector caspases cleave ROCK1 –> controls contraction of cells via actomyosin

ROCK1 stuck ‘active’ = contract forcefully and uncontrollably = blebs

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13
Q

what type of proteins decide fate of cell

A

Bcl-2 - 3 types

  1. Pro-survival –> Bcl-2,Bcl-XL and Mcl-1

= protect mitochondrial membrane

  1. Pro-apoptic –> Bax,Bak

= creates pores in membrane

  1. BH-3 only –> sensors –> Bid,PUMA,Bad

= actiavte Bax/Bak

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14
Q

where is cytochrome-c found

A

inner membrane space of the mitocondria

usally used in oxidative phsophorylation –> loss –> elctron transport chain fail

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15
Q

Bcl-2 protein family structure in each group and why its like this

A

3 groups within Bcl-2 family proteins each with specific set of conserved Bcl-2 homology domains (BH)

Pro-surivival - Bcl-2/Mcl-1:

all 4 BH domains and C-terminal Transmembrane domain (TM) for insertion into outer mitochondrial membrane

= bind and sequester BH-3 onlt proteins and Bax/bak preventing MOMP

Apoptic - Bax/Bak:

BH1-3 domains NOT BH4 and TM domain for membrane insertion

= Bax/Bak oligomerise to form pores in MOM

BH3-only - Bid/bad/bim/PUMA:

only contain BH3 and some have no TM = just sensors/activators -> do not require mitochondrial membrane insertion

= either activate Bax/bak directly or neutralise Bcl-2 survival proteins to ‘sensitise’ the MOM

= ‘Sentinels’ for stress, oncogene activation, DNA damage

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16
Q

side by side of Bim vs Bad

A

bid cleaved by Fas/pro-caspase-8

Bad acted on by kinase (Erk) and phosphtase (calcineurin) for control

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17
Q

diagram of simple apoptosis from induction of Bax into membrane of mitochondria

A
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18
Q

describe the relationship between Bcl-2 and Bax on the mitochondrial membrane

A

Bax binds to membrane and Bcl-2 –> ‘titrates’ out the Bcl-2 protecting the membrane

Bax homo-ogliomerises and inserts into outer membrane

= Bax/Bak (already in membrane) form pores

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19
Q

describe how the apoptosome forms

A

Cytochrome c causes Apaf-1 monomer to bind dATP and/or ATP

dATP binding triggers oligomerisation

Exposes CARD domain = Caspase recognition domain –> recruits pro-caspase 9

Pro-caspase activated by associating with other pro-caspase 9 monomers

= dimerisation activates ‘activator’ caspases (8,9)

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20
Q

diagram of Bcl-2 family and apoptosis seesaw

A
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21
Q

what are the 2 ways that BH3 only proteins cause apoptosis

A

Indirectly - ‘sensitisers’:
bind to pro-surival proteins

“neutralize the brakes” = prevent pro-survival proteins inhibiting Bax and Bak

= Bad

Directly - ‘activators’:
small subset (tBID/Bim) directly bind and activate Bax/bak

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22
Q

what does the drug Venetoclax do against cancers

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A

blocks Bcl-2 pro-survival protein

= restores apoptosis in B-cell lymphomas

HOWEVER does not inhibit Mcl-1

= apoptosis still inhibited

23
Q

challenges of using venetoclax as a drug to block Bcl-2 and restore apoptosis in cancers

Study These Flashcards
A

some cancers rely on other pro-surival proteins like Mcl-1

= resistant to drug as apoptosis not restored and Bax/Bak still blocked

= combo therapy used like Mcl-1 inhibitors

24
Q

what is venetoclax and exmple of

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A

BH3 mimics

= bind and inhibit pro-survival Bcl-2 family proteins

25
diagram for overall idea of BH3 mimics
26
what do chemotherapeutic drugs rely on
apoptosis --> by damaging DNA or mitotic damage = sensed (p53) --> signalling pathway --> apoptic machinery
27
describe a simple summary of the extrincisc death receptor pathway
death ligand bind to death receptor (Fas/FasL) Fas recruits FADD via death domains = FADD has death AND death effector domains (DED) FADD recruits initiator caspases (8) via DED (death effector domains) domains 2 downstream pathways: 1. initiator caspase activate effector caspases (3) --> cleavage of cellular compoents = ICAD --> CAD = DNA (180bps)= extrinsic pathway = mitochondrial independant 2. amplification of mitochondrial: cleavage of Bid --> tBid = MOMP = apoptosome form
28
what is DISC
death-inducing-signalling-complex = FADD (adaptor) AND pro-caspase-8 FADD has death domains which are recruited to death domains of Fas AND death effector domains (DED) which recruit pro-caspase-8 = clustering/dimersation actiavates caspase-8
29
what is the Fas ligand and Fas receptor structure like
trimer ligand causes clustering of Fas receptors intracellular domain has death domains adaptor proteins have death domains that are recruited (FADD)
30
describe what happens when the trimer FasL bind to Fas
FasL is a trimer --> needed as Fas requires 'clustering' to function FasL binds causing Trimerisation of 3x Fas reptors intracellular domain of receptor has death domain (DD) = Trimerisation of Fads reveals Death domain FADD adaptor protein has DD and DED = FADD binds to one of the clustered death domains and recruits pro-caspase-8 = DISC DISC clustering due to trimerastion of Fas receptors means DISCs in high conc = dimersation activates initiator pro-caspase 8 pro-caspase-8 cleaves Pro-caspase-3 and Bid = direct extrinsic pathway or indirect amplification of mitochondrial intrinsic pathway
31
what happenes in autoimmune lymphoproliferative syndrome (ALPS)
mutations in death domain or truncated FasR = defective apoptosis = build up of lymphocytes and higher risk of lymphomas
32
what percentage of collecteral cancers have reduced or lost Fas expression
50% = resistant to apoptosis at the top of the microvili
33
what is DcR3
Decoy death receptor contains death domain BUT no Death effector domain (DED) = binds and mops up FasL BUT has no downstream signalling pathway
34
what is FLIP (flip between apoptosis and not)
FLIP binds to FADD and OUTCOMPETES pro-caspase-8 on death effector domains no inititiator caspases recruited to begin casade = prevents DISC formation
35
what is TRAIL and why does it present a therapeutic approach to some cancers compared to Fas
TRAIL (TNF-related apoptosis-inducing ligand) = cytokine that binds to DR4/5 death receptors adding excess TRAIL overwhelms decoy receptors on cancers and activates the upregulated DR4/DR5 on tumor cells = DISC forms and triggers apoptosis Normal tissues are protected because they express higher numbers of decoy receptors = Fas has broad expression on normal cells aswell as cancer and causes toxicity/off-target effects
36
Name 4 ways that cancers can evade externally stimulated apoptosis
1. Fas mutation/downregulation 2. Expression of decoy receptors (DcR3) 3. FLIP overexpression 4. Caspase-8 mutations
37
BH3-only proteins activate Bax/Bak and promote apoptosis - how is Bid activated
Cleavage by Caspase-8 in the DISC Bid is cytosolic --> cleavage into active tBid = moves into nucleus and directly activates Bax/Bak to ogliomerise and form pore/MOMP
38
overall diagram of Bcl-2 family
39
Describe the action of the BH3 only protein Bad and how its controlled (activated/inactivated)
BH3-only proteins are sensors = either inhibit pro-survival Bcl-2 proteins OR can directly activate Bax/Bak Bad is a 'sensitiser' -> inhibits pro-survival Bcl-2 family proteins = Bcl-2 (NOT Mcl-1 = bid) Allows/frees Bax/Bak to induce MOMP via pore formation Inactive Bad: phosphorylated by survival signalling (Akt) --> bound by 14-3-3 proteins --> cytosolic --> no apoptosis Active Bad: dephosphorylated by overactive calcineurin/lack of survival signalling from Akt = naturally dephosphorylates --> moves to mitochondria --> binds Bcl-2/Bcl-xl --> Bax/Bak activated indirectly
40
how can survival signalling prevent apoptosis via Bad
Akt phosphoylates Bad = phospho-Bad is bound by 14-3-3 protein and kept inactive Bad cant titrate Bcl-2 proteins out of mitochondira for apoptosis = if survival signalling drops --> Bad is de-phosphoylated/activated
41
describe the relationship between the Ras pathway and Bim (BH3-only)
Erk phosporylates Bim = phosphorylated Bim ubiquitinated and destroyed Bim usaully binds and inhibits pro-surival proteins on mitonchodria = Bcl-2 and Mcl-1 Loss of Bim = no removal of the 'protecting' proteins = Bax/Bak cant form pore for apoptosis
42
what 2 things are released from damaged mitochonria in MOMP leading to apoptosis
1.Cytochrome-c = Apaf-1 --> caspase-3/7 --> apoptosis 2. SMAC - neutralises IAPs such as XIAP = IAPs are caspase inhibitors so SMAC is needed to STOP IAPs inhibiting caspases/apoptosis
43
what are IAPs (XIAP is an exmaple)
Caspase inhibitors (3,7.9) family of proteins with BIR1/2/3 domains --> for different caspase binding inhibit and target caspases for degredation = SMAC inhibits IAP action and allows apotosis to continue as normal = IAPs are commonly overexpressed in cancer to block cell death
44
how does SMAC bind to XIAP and what has this led to in drug development
SMAC binds to BIR2 domains of XIAP that would usaully inhibit effector caspases synthethic 'SMAC mimetics' mimic this peptide and allow apoptosis to continue in cancer cells Done in combo therapy with TRAIL agonists --> TRAIL alone may not trigger apotpsosis due to upregulation of IAPs = shown to have tumour eradication in mice
45
summary of regulators and therapies in apoptosis 2 lecture
46
describe the difference between activation of Initiator vs Executioner caspases
1. Initiators (9,8,10) dimerise to become active monomers/pro-caspases are inactive and are brought together by adaptor complexes = apoptososome or DISC Dimerisation causes activation --> autocleavage may come after to stabilse BUT dimersation is key activation step 2. Executioners (3,6,7) stored ALREADY as inactive dimers cleavage by initiator caspases cause structural rearrangement and activation = dimers BEFORE and AFTER activation
47
how is Bad activated/inactivated
Inactive Bad: phosphorylated by survival signalling (Akt) = bound by 14-3-3 proteins and sequestered in cytosol --> no apoptosis Active Bad: dephosphorylated by overactive calcineurin from stress signals/lack of growth signalling (Akt) = Bad not phosphorylated --> can move to mitochondria --> binds and neutralises Bcl-2/Bcl-xl = apoptosis = Bax/Bak activated indirectly
48
SMAc vs XIAP summary
49
what is PUMA and its role and activation
p53-upregulated modulator of apoptosis = BH3-only pro-apoptic protein directly activates Bax/Bak for MOMP AND neutralises pro-survival Bcl-2 proteins Severe DNA damge (apoptosis not arrest path) -> ATM -> Mdm2 phosphorylation and release of p53 -> p53 stabilise and bind PUMA promoter = ATM Ser46 phosphorylation allowing K120 acetylation by HATs = key switch between p53s cell cycle 'arrest' (p21) and 'apoptosis' pathway (PUMA
50
what decides p53s acetylation which then promotes 'arrest' or 'apoptosis' - 2 points
Different phosphorylation marks on p53 create “landing pads” for specific HATs = Different DNA damage strengths recruit different HATs
51
describe the action of Bcl-2 proteins vs Bax
Bax is a cytosolic protein partially activated by stress signals reveals BH3 domain = ogliomerisation domain to form pores in mitochondrial membrane Bcl-2 pro-surivival proteins bind BH3 domains inhibiting and sequestering protein = cannot form pores for MOMP and is now stuck neutralisation of Bcl-2s by BH3-only proteins like tBid,Bad and Bim (some also directly activate Bax/Bak) = release of Bax and inhibiting the inhbitors 'sensitises' membrane fore MOMP = more succeptible
52
overall diagram of apoptic proteins and describe what is meant by sensitisers vs activator BH3 only proteins
sensitisers: Neutralise/titrate out protecting Bcl-2s = 'sensitise' the membrane = make it more vulrable by removing its protection = Bad activators: direvtly activate Bax/Bak to ogliomerise and form pores = Bid and Bim
53
explain how dimerisation of initiator caspases causes activation
Initiator caspases have a 'split active site' = substrate binding pocvket are floppy and disorganised = catalytic cysteine cannot cleave dimerisation rearranges active site of each monomer = substrate binding groove formed correctly = cleavage at aspartic acid residue After dimerisation they cleave themselves stabilizing the active form AND allowing regulation by IAPs
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