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Apoptosis Flashcards

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AP® Biology flashcards Biology 101 flashcards
1
Q

why is programmed cell death good?

A

unlike necrotic cell death, it presents leakage of inflammatory cell content that could result in autoimmune diseases

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2
Q

what are 5 key changes that occur within the cell during apoptosis?

A
  • the nucleus and cytoplasm shrink
  • chromatin condenses and nuclei become fragmented
  • blabbing of the cell membrane occurs
  • apoptotic cells are phagocytes by their neighbours or by phagocytes
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3
Q

generally, when does apoptosis occur? (5)

A
  • during development for sculpting of digits
  • deleting structures not needed
  • adjusting cell number during neural development
  • tissue homeostasis in self renewing tissues
  • removing harmful cells
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4
Q

what model was apoptotic pathways first discovered in and why?

A

c.elegans, the hermaphrodite has invariable development in which 131 cells of 1090 are apoptosed. the cell lineages have also all been traced. this allowed people to look for mutants with changes to this.

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5
Q

what mutants revealed which different components of the apoptosis pathway?

A
  • CED-3: no apoptosis
  • CED4: no apoptosis
  • CED9: more that 131 cell deaths
  • EGL-1: no cell death
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6
Q

what is the homologue to CED-3 in mammals?

A

caspases

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7
Q

what is the homologue of CED-4 in mammals?

A

apaf-1

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8
Q

what is the simple apoptotic pathway in c-elegans?

A
  • CED-9 is on the OMM and binds to CED-4 to prevent its action.
  • EGF-1 binds to CED-9, releasing CED4 from binding CED9.
  • CED-4 forms a dimer called the apoptosome and CED-3 is recruited to the apoptosome which goes on to trigger cell death.
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9
Q

what evidence is there that CED-3 (procaspases) are required for apoptosis in c.elegans?

A

in CED-3 mutants there is no apoptosis

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10
Q

what type of protein are caspases?

A

proteases

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11
Q

what is the cleavage target for caspases?

A
  • the cleavage site is always after an aspartic residue
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12
Q

what is substrate specificity of capsizes determined by? give an example

A

4 residues that are n-terminal to the cleavage site. E.g caspase 3 prefers DEVD

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13
Q

what does the mature caspase look like?

A

a heterotetromer with two active sites

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14
Q

what are the two ways in which caspases can be activated?

A

by dimerisation or by cleavage of the linker between the small and large subunits.

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15
Q

what are the two types of caspases?

A

initiator caspases and effector caspases

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16
Q

how are initiator caspases activated?

A

by dimerisation which is mediated by the pro-domain which interacts with adaptor proteins FADD or apaf-1

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17
Q

how are effector caspases activated?

A

by cleavage between the small and large subunit

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18
Q

list 3 effector caspsases and name their distinguishing features.

A

-3, -6, -7, have a short prodomain

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19
Q

list 2 initiator caspases

A

8 and 9

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20
Q

which initiator caspases plays a key role in the death receptor signalling pathway ? how does this work?

A

caspase 8.
-Fas is a cell surface integral membrane protein containing extracellular cysteine-rich repeat domains, a transmembrane domain and an intracellular region with a death domain.
- Binding of Fas ligand to Fas leads to trimerization of Fas and formation of a death-inducing signalling complex (DISC) and ultimately activation of the cell death programme.
- The Fas death domain recruits an adaptor protein, FADD, which contains a death domain and death effector domain (DED).
- The DED of FADD then recruits procaspase-8 via a DED-DED interaction (the long prodomain of caspase-8 contains a DED).
capsase * then cleaves capsize 3- the effector

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21
Q

explain how the initiator capsize 9 is activated

A

Caspase-9 is an initiator caspase that plays a key role in the mitochondrial pathway of apoptosis.
Caspase-9 is activated by associating with a protein cofactor, Apaf-1. Cytochrome c, released from the mitochondria during apoptosis, is also required for caspase-9 activation. The three proteins form a large complex known as the apoptosome.
Cytochrome c and ATP-dependent oligomerization of Apaf-1 allows recruitment of procaspase-9 into the apoptosome complex. The CARD motif in the prodomain of caspase-9 interacts with Apaf-1. Activation of caspase-9 is mediated by dimerization in the apoptosome which activates it.
- caspase 9 then activates capspace 3 by cleavage

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22
Q

name 5 caspase targets and their roles

A
  1. ICAD- inhibit the caspase activated DNAse that cuts genomic DNA (CAD)- its cleavage releases CAD
  2. cleavage of nuclear laming for nuclear shrinkage and budding
  3. BID- a pro-apoptotic member of the BCL-2. tBID induced mitochondrial changes that lead to cytochrome C release
  4. PAK2- allows blebbing
  5. fodrin- leads to loss of cell shape
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23
Q

how can the death receptor and mitochondrial pathway of apoptosis be linked?

A

caspase 8 cleaves bid to form bid which is a bh3-only domain

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24
Q

is apaf-1 in the cytosol?

A

yes

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25
Q

what are three groups of BCL-2 protein family and what is either of their roles and components?

A

group 1: antiapoptotic and have four BH domains (BCL-2, BCL-XL, MCL1 and BCL-W)
group 2: proapoptotic (Bax and Bak and BOK)
group 3: proapoptotic and their only shared homology is a BH3 domain. They are called BH-3 only proteins. (BIM, PUMA, BAD)

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26
Q

what are the roles of BAX and BAK?

A

they are essential of inserting into the outer mitochondrial membrane and permeabilising it, release cytochrome C

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27
Q

explain how BAX functions

A

BAX translocates fro the cytoplasm to the mitochondria where it oligomerises and inserts into the outer membrane, forming a pore.

  • tBID facilitates BAX translocation.
  • BH3 only proteins (BIM and PUMA) can also induce a conformational change in BAX which facilitates its translocation to the membrane
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28
Q

generally what is the role of BH-3 only protiens?

A

to inhibit BCL-2 and BCL-xl (which function to inhibit cytochrome C release) proteins and also to activate Bax

29
Q

how do BCL-2 and BCL-xl inhibit cytochrome C release?

A

binding to it and prevent oligomerisation and membrane insertion

30
Q

what are IAPs? give an example

A

they are direct inhibitors of caspases. XIAP inhibits caspases 2, 7 and 9

31
Q

how do IAPs inhibit caspase function?

A
  • they have a BIR domain and a c-terminal RING domain
  • the BIRs bind to the surface of the capsizes
  • the sequences between the BIRS block the catalytic grooves of the target enzymes
  • the ring domain can as a ubiquitin ligase, facilitating ubiquintaitona nd degradation of the bound capsizes
32
Q

what is the inhibitor of IAPs? how does this work?

A

SMAC/Diablo - SMAC is released from mitochondria during apoptosis and binds directly to certain IAP proteins (via an N-terminal A V P I motif) and prevents them from binding to caspases.
- Smac/ Diablo is release from mito which inhibits IAPs and prevents them from inhibiting caspaases. So when mito release occurs, caspases are allowed to be active and apoptosis procudes

33
Q

how can DNA damage activate the apoptotic pathway?

A

p53 activates BAX and also PUMA and NOXA

34
Q

in which cell type of the Fas death pathway used?

A

type 1 such as thymocytes

35
Q

in what type of cell in the mitochondrial pwtah important

A

type 2 like liver cells

36
Q

why are there two types of pathways in different cells?

A

type 2 cells have high levels of XIAP compared to type 1 cells
in these type 2 cells, permabilisation of the mito is important to release Smac which inhibtis XIAP which allows apoptosis ot proceed

37
Q

name two ways in which apoptosis is inhibited by survival factors

A
  1. NGF/ IGF binds to trkA on the surface of neurons which sends a signal which inhibits the caspases so if you take away this isgnal, you get apoptosis.
    IGF-1 and NGF activate the PI3K pathway
    IGFR activates IRS-1 and RAS which activate PI3K which activates AKT (PKB)
    one of PKB’s substrates is bad which is a BH-3 only protein whcih triggers apoptosis by binding ot BCL-XL, when Akt is actiave it phos Bad which allows it to be reocgnised by 14-3-3 which sequesters phos bad in the cytoplas so that it cant interatc with BCL-xl and so cant trigger aopotosis
  2. FKHRL1 (Foxo3a) which binds to the promotes of the Fas ligand or BIM (Bh3-nly) activating their transcription - activating apoptosis. But activation go AKT via NGF or IGF e.g. results in the phos of FOXO3a and theen it binds 14-3-3- and stays in the cytoplasm so can’t worm
38
Q

what inhibits bcl-2 and bcl-ml normally

A

??

39
Q

give two examples of systems in the body that need apoptosis to regulate cell proliferation

A

the haematopoeitc system and the intestinal epithelium

40
Q

describe an experiment which shows that BCL- 2inhibits apoptosis

A

enforced expression of BCL-2 rendered haematopoietic cells refractr=ory to deaht induced by cytokine deprivation and promoted lymphocyte accumulation in mice.

41
Q

what sets the mitochondrial apoptotic threshold?

A

the various levels of the BCL-2 protein family- BCL-2 antiapoptic predominately.
when enough BH3 only proteins have been stimulated in response to various cytotoci stressed to exceed the apoptoci threshold, bad and bak begin to form oligomers that permeabilise the mitochondrial outer membrane

42
Q

what is the general structure of BAX, BAK, BOK and also the BCL-2 and BCLxl

A

globular, a helical bundle surrounding a central hydrophobic core- this can bind to the amphiphatic helix of BH3 only proteins

43
Q

how are bh3-only proteins activated?

A

BH3-only proteins, which are induced transcriptionally or post transcriptionally by cytotoxic stress signals

44
Q

what do the pro-survival proteins seem to bind?

A

bax

45
Q

what is BAK likely inhibited by (2)

A

ACL1 and BCL-Xl

46
Q

what is the difference between the localisation of BAK and BAX?

A
  • BAK resides on the mitochondria, with its transmembrane domain spanning the outer membrane
  • BAX is regulalry cycled to the membrane of the mito in healthy cells but is translocated back, possibly by interactions with pro-survival proteinns such as BCl-2
47
Q

how does the activation of BAX allow it to insert into the membrane and what activates this?

A

BID or BIM can activate it:
- two distinct activation sites have been proposed: the canonical hydrophoboc groove, which is homologous to that of the pro-survival proteins, and an alternative site on the opposite side of BAX.
- the rear activation site is thought to be a trigger for extruding the BAX transmembrane domain from the groove and thereby accelerating the basal shuttling of BAX between the cytosol (inert BAX) and the outer mitochondrial membrane (activated BAX).
the groove activation site may then drive subsequent transitions. However, the failure of mutations of the rear activation site to abolish apoptosis induction indicates that it might not be essential for the activation of BAX.

48
Q

explain the entire step of BAX activation by a BH3 only protein

A
  1. BAX is translocated to the OMM by binding BH3-only protein. The transmembrane released from the BAX surface groove then inserts into the OMM
  2. the binding promotes the release of the latch domain from the core domain of the BAX groove. This may act to dissociate the BH3-only protein
  3. In step 5, protrusion of the BAX BH3 domain allows two such molecules to form the BAX BH3‐in‐groove dimer
  4. oligomers form- it is not known how
  5. it is thought the large oligomers may induce tension to the OMM, inducing permabilisation
  6. cytochrome is released
49
Q

how can perturbartion to the apoptotic pathway be linked to tumorgenesis?

A

loss of the pro-aoptotic BCL-2 family members such as BIM, PUMA, BAD and BAX also accelerates tumorigenesis that it driven by such oncogenic lesions
blockin apoptosis presumably promotes tumorigenes by keeping cells alive long enough to acquire oncogenic mutations that drive the neoplastic progression, as well as my countering apoptotosis that oncogenes such as MYC drive under certain conditions of cellular stres

50
Q

how is p53 mutation linked to apoptosis-driven therapy

A

for example, p53 mutation, which is prevelant in diverse types of tumour, impairs the induction of PUMA and NOXA in response to DNA damage-inducing chemotherapeutics, and many lymphoid malignancies have increased levels of BCL-2

51
Q

how has huntingtin disease been linked to apoptosis?

A
  • Huntington’s Disease appears to result from an aber- rant expansion of CAG repeats encoding a polyglutamine repeat in the Huntingtin protein. The mutant protein pathogenesis involves caspase-8 activation
  • which is thought to occur because of an altered complex formed by mutant Huntingtin protein
  • One model holds that active caspase-3 cleaves Huntingtin into self-aggregating fragments that prove toxic to the affected neurons.
52
Q

what is the evidence that caspase-mediated apoptotiss is not the only mechanism for cell death in development?

A
  1. inactivation of mouse cell death genes leads to only relativley minor developmental defects and can often survive embryonic development. one reason may be the considerable redundancy within the caspase family and the existence of multiple mechanisms for caspase inactivation. for example, some effector caspases can be activated in the absence of apaf1 function.
  2. but there is evidence that there is a back up mechanism: Although apoptosis is the major cell death mechanism in developing limbs, inactivation of proapoptotic genes in the mouse only partially prevents the removal of the interdigital tissue, suggesting that backup mechanisms exist when apo- ptosis fails
53
Q

how many neurons die during development?

A

50%

54
Q

what is the neurotrophic theory and what does it ensure?

A

Neuronal survival is regulated by the availability of limiting amounts of target-derived neurotrophic factors.
there growth factors are produced by the target tissues. It ensures that:
1. neurons that project to the wrong targets are eliminated because these targets dont produce the right factors
2. all target cells become innervated
3. the number of innervating neurons is correctly matched to the number of target cells.

55
Q

what type of neurons have been close studied in terms of growth factor response?

A

sympathetic

56
Q

have has the dependence of sympathetic neurons on growth factors been demonstrated?

A

if you culture sympathetic neurons in a dish and remove NGF the cells die via apoptosis

57
Q

how did a group first identify the signalling pathway involved in the death programme in response to NGF removal in sympathetic neurons?

A

they looked for changes in expression of genes just after the withdrawal of NGF and they looked at a family of transcription factors: c-jun, jun B and Jun D. These form heterodimers with the Fos proteins to bind to the DNA binding site called the ATF binding site.

58
Q

once they had identified the Jun family how did they identify the exact target?

A
  • they obtained antibodies against each member and used them in immunohistochemistry and blotting experiments and they found that just c-jun protein changed. just after the removal of NGF is removed, c-jun protein increases and is activated via phosphorylation- via JNK
59
Q

how did they test the role of c-jun in apoptosis in sympathetic neurons? (3)

A
  • they made an expression vector for a dominant negative protein and when they exressed this, it inhibits cell death after NGF withdrawal- so c-jun is required for apoptossi following removal of growth factors
  • they also used an expresison vector called JIP-1 which inhibits -cjun phos, also when you do this and withdraw NGF you dont get apoptosis
  • if you make a KO of the c-jun gene in symp neurons and this also stops cell death
60
Q

what is the c-jun pathay

A

Rac1/cdc42 is thought to activate MLKs which activates JNKK which activate JNK which activates c-jun which upregulates pro apoptotic genes and also itself

61
Q

how was it shown that symp neurons rely on the mito pathway for apoptosis? what is upregulated after withdrawal

A

if you KO cas9 cas3 or apaf1 you dont get apop in NGF withdarwl
the release of cytochrome C is regulated by bax after NGf withdarwl, and bcl-2 and bcl-x inhibit this

62
Q

how did they show that c-jun was involved cytochrome C release?

A

they removed NGF and the cell expressing b-gal in the presence of hoechst you see cells have apoptotic nucleus and the cytochome C has a diffused pattern across the cell- using staining
but in neurons infected with lenti express DN c-jun, when you withdarw, there is normal nuclei and cytochrome c does not have a diffuse localisation
this shows that c-jun is requried for the release of cytochrome c

63
Q

how did they find out how c-jun acts to induce cytochrome C release?

A
  • they showed in immunoblotting experiments that symp neurons express several bax, bad, bak, bid and bim
  • but after NG deprivation, only one changed in level and this was the Bim protein, which increases after NGF withdrawal
    -This increase in the BIM protein is due to an increase in BIM mrna as measured by micro analysis
  • this increase requires the atcivity of c-jun, DN reduced the increase in mRNA
  • they carried out gain and loss of BIM
    overexpression of BIM in the presence of NGF
  • they found this induces cytochrome C release and apoptosis
  • they made AONs- rescue half the neurons that wouldve died after NGF withdrawal
  • also a group of BIM KO mice in symp- the BIM KO symp die more slowly after NGF withdrawl
64
Q

what was surprising about the KO of BIM in sum neurons with NGF removal?
what did they do about this?

A
  • it delayed death but didn’t completely protect
  • they found that only 2 other BH3 only proteins were upregulate after NGF withdrawal: DP5, PUMA,
  • they did KOs of these and found that KO BIM partially protected neurons from apoptosis, Dp5 has a minor protective effect, Puma has the same effect as BIM
65
Q

what is the overlap theory about NGF withdrawal and apoptosis triggering

A
  • it upregulates c-jun which upregulates Puman, DP5 and BIM, this inhibits BCL2 and BCL-xl and activates BAX- leading to mitochondrial apop
  • FOXO is also upregulated which activates Bim
66
Q

how is retinal degeneration linked to apoptosis?

A

Photoreceptor neurons die by apoptosis in mouse models of retinal degeneration. Expression of the p35 caspase inhibitor in the eye blocks retinal degeneration in Drosophila

67
Q

what is the homologue of EGL-1?

A

BH-3 only proteins

68
Q

what is the homologye of caspase 9?

A

bcl-2

69
Q

how does the removal of NGF trigger the upregulation of c-jun?

A

NGF acts to inhibit cdc42- cdc42 acts to activate c-jun via JNKK and JNK - so when NGF is removed, cdc42 can activate c-jun

Advanced molecular cell biology (6 decks)

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