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Flashcards in TNF receptor family Deck (50)
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1

What is the role of apoptosis

Maintains tissue homeostasis by balancing cellular life and death

2

What is the result of cell death overtaking proliferation

Diseases such as neurodegeneration, immunodeficiency and infertility can take hold

3

What is the result of cell proliferation being greater than cell death

Cancers and autoimmune disorders become likely.

4

What are three characteristics of apoptosis

Membrane blebbing, chromatin condensation and cellular shrinkage

5

what family of receptors are responsible for inhibiting apoptosis and how were they discovered

TrkA,B and C. Exp - Limb bud removal reduced motor neurons found on the ipsilateral side of the spinal cord. Ectopic limb bud addition resulted in additional motor neurons on the ipsilateral side - Targets of innervation secrete limiting amounts of survival factors to generate a balance between the size of the target organ and the number of innervating neurons - The neurotrophic hypothesis.

6

How can the NGF signal be transduced at the tips of growing neuronal processes

Sympathetic neurons placed in a TC system that allowed the somas and neurites to be bathed in different media - anti-NGF added to the soma, NGF added to the growing neurites. When TrkA receptors at the neurite terminal bind NGF they are internalised. They can then signal locally and induce growth or are retrogradely transported back to the soma, the dimer signals in the soma and activates MAPKs - ERK moves into the nucleus and induces gene expression and differentiation.

7

At what rate are the Trk dimers transported at back to the soma

2-20mm per hour (Very slowly)

8

How is the P75LNTR different to the Trk receptors for NGF

P75LNTR is not a tyrosine kinase - it is a TNF receptor

9

What does LNTR mean

low-affinity neurotrophic receptor

10

What is similar between all TNF receptor extracellular domains

Cysteine repeats

11

How many times does a TNFR pass through the plasma membrane

once

12

What is surprising about the TNF receptor ligands

They are single pass, membrane bound proteins, although some can be shedded and become soluble

13

How is receptor activation by the ligand regulated

Antibodies for TNFR ligands sequester the ligand and prevent receptor binding and activation. Or to occupy the receptor itself

14

What are the ligands of P75LNTR and what is its role

NGF, BDNF, NT3/4 - role in regulating apoptosis in the nervous system

15

What is the structure of the death domain

composed entirely of alpha helices - protein to protein interactions

16

What is the Fas-receptor otherwise known as and what is its ligand

Known as CD-95 and ligand = Fas

17

What is the result of a mutation in the Fas gene

Causes autoimmune lymphoproliferative syndrome, with a size increase in lymph nodes and spleen

18

How is Fas receptor and ligand complex regulated

By PTPN13 - a large protein tyrosine phosphatase which binds to the C-terminal tail of the FasR. This regulates autophosphorylation and prevents the FasR from reaching the cells surface.

19

How does PTPN13 prevent the FasR from reaching the plasma membrane

PTPN13 interacts with SDCCAG3 which regulates the transport of the FasR to either the PM or the lysosome

20

How does PTPN13 expression differ in cancerous cells compared to normal ones

Far more expression in cancer cells so less FasR at the cell surface so less activation/less likelihood of induced apoptosis.

21

What is the structure of TNF-Alpha

A trimer - The TNF-Alpha ligand has a swiss roll structure which forms anti-parallel beta sheets

22

What are the two models for ligand induced activation of TNF receptors

1. Individual TNF receptors are brought together by the soluble TNF ligand in their extracellular domains, this brings the death domains together on the intracellular
2. There is a pre-ligand associated trimer, after ligand binding the trimer undergoes a conformational change to bring together the death domains. More likely to be true, efficient one step process.

23

what are the 11 steps of the extrinsic apoptotic pathway

1. Trimeric ligand induces trimerisation of the FasR
2. This triggers interaction of death domains
3. They undergo a conformational change
4. Leads to the recruitment of FADD
5. FADD also contains a death domain (the death domains interact together)
6. The death domains are recruited to the membrane and undergo a conformational change
7. FADD is composed of another domain - DED
8. DED interacts with pro caspase 8 (interacts by its DED domain)
9. Pro caspases activate themselves by cutting off the DED domain (proteolytic activation step)
10. The active caspase 8, activates further downstream effector caspases
11. They cut a number of proteins in the cell which are crucial for cell survival

24

What are caspases 8,9 and 10

Initiator caspases

25

What are caspases 3,5 and 7

Effector caspases

26

What is the difference between a procaspase and a caspase (only for initiator caspases)

A pro caspase is inactive due to it still having its DED domain intact

27

What is the structure of Caspases

They are beta and alpha domains

28

What is the reason for there being multiple steps in activating caspases

More positions for regulation. Also the upregulation of signalling (an amplification cascade of proteases)

29

What changes are made to the structure of the procaspase molecules

They are one polypeptide chain initially but after binding of DED to DED on FADD
1. Removal of DED domain
2. Further cleavage through the beta and alpha domains, leaving two small domains of each.
3. The active caspase is composed of beta and alpha subunits
4. This active caspase will diffuse into the cytosol and find the effector procaspase and cleave its prodomain at the N-terminus and activate it.

30

What is the difference between activation of initiator caspases and effector caspases

To activate initiator caspases the prodomain containing the DED domain is cleaved off, in effector caspases there is no DED domain so only the prodomain is cleaved

31

What cell types undergo intrinsic apoptosis

Type 2 cells

32

What level of caspase 8 activation induces intrinsic apoptosis

When activated at low levels

33

What is the name of the protein that caspase-8 cleaves

Bid- an inhibitor of apoptosis, it produces tBid (truncated Bid)

34

What is the role of tBid

Facilitates the association and integration of Bax into the outer mitochondrial membrane

35

What is the role of Bax

It forms pores within the outer membrane, this allows for the release of cytochrome C from into the cytoplasm

36

What is the role of cytochrom C

It binds to Apaf-1 and induces the formation of an apoptosome

37

What is Apaf-1

Protein made up of CARD, CED-4 and WD40 domains

38

What is an apoptosome

A macromolecule
, multisubunit complex of active Apaf-1
CARD domain in the centre
CED-4 as the stalk
WD40 at the periphery and binds cytochrome C

39

What is the role of the apoptosome

Activates procaspase 9 by promoting the release of the CARD domain

40

What is the role of caspase 9

Activates further downstream effector caspases

41

What step acts as an irreversible step in mitochondrial targeting of tBid

Posttranslational N-myristoylation of the pro-apoptotic molecule BID

42

What other pathways are activated by Fas binding

JNK, MAPK, NF-kB, P38

43

What is the role of cFLIP

Possible that this molecule is also recruited to the FADD receptor and/or caspase 8/10. cFLIP has an inhibitory role and is a way to regulate apoptosis on the cytosolic side. Important due to the number of other pathways activated by activated Fas/TNFR

44

How has cFLIP been targeted for cancer therapies

siRNAs that specifically knock down the expression of cFLIP in diverse human cancer cell lines augmented TRAIL-induced DISC recruitment and increased the efficacy of chemotherapeutic agents.

45

How can we measure apoptosis

FACS analysis - E.g. if there are a mixture of cells and only a fraction are expressing a certain apoptotic protein, use an antibody that recognises the ECD and sort the cells based on the intensity of their fluorescence

46

What marker is used in FACS for early apoptotic cells and why

In a living cell phosphatidyl serine is only found on the intracellular side of the plasma membrane. Early apoptotic cells will present phosphatidyl serine on the outer leaflet of the plasma membrane. AnnexinV has an affinity for it and can be coupled to a dye called FITC.

47

What marker is used in facts for late stage apoptosis and why

Usually Pi and 7AAD cannot enter the cell. At later stages of apoptosis/necrosis the cells become leaky and allow dyes such as Pi and 7AAD to enter the cell and stain the nucleus

48

How are the apoptotic and late stage/necrotic cell FACS data shown

x axis = ANNEXIN
y axis = Pi/ 7AAD
cells high in both are likely to be necrotic/ late stage apoptotic, cells high in annexin are likely to be apoptotic and cells low in both are likely to be viable cells.

49

What players are responsible for the TNF receptor to become activated

TNF trimer
TNF receptor
TNFR1- associated death domain protein (TRAAD) - binds FADD - apoptosis
TNFR1- associated factor 2 (TRAF) - binds NFkB - Transcription factor for preinflammatory genes (involved in chronic inflammation diseases)

50

What is required for the activation of FADD after TNF receptor activation

Dependent on internalisation of the TNF receptor - maturation to complex II