TNF receptor family Flashcards
What is the role of apoptosis
Maintains tissue homeostasis by balancing cellular life and death
What is the result of cell death overtaking proliferation
Diseases such as neurodegeneration, immunodeficiency and infertility can take hold
What is the result of cell proliferation being greater than cell death
Cancers and autoimmune disorders become likely.
What are three characteristics of apoptosis
Membrane blebbing, chromatin condensation and cellular shrinkage
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.
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.
At what rate are the Trk dimers transported at back to the soma
2-20mm per hour (Very slowly)
How is the P75LNTR different to the Trk receptors for NGF
P75LNTR is not a tyrosine kinase - it is a TNF receptor
What does LNTR mean
low-affinity neurotrophic receptor
What is similar between all TNF receptor extracellular domains
Cysteine repeats
How many times does a TNFR pass through the plasma membrane
once
What is surprising about the TNF receptor ligands
They are single pass, membrane bound proteins, although some can be shedded and become soluble
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
What are the ligands of P75LNTR and what is its role
NGF, BDNF, NT3/4 - role in regulating apoptosis in the nervous system
What is the structure of the death domain
composed entirely of alpha helices - protein to protein interactions
What is the Fas-receptor otherwise known as and what is its ligand
Known as CD-95 and ligand = Fas
What is the result of a mutation in the Fas gene
Causes autoimmune lymphoproliferative syndrome, with a size increase in lymph nodes and spleen
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.
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
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.
What is the structure of TNF-Alpha
A trimer - The TNF-Alpha ligand has a swiss roll structure which forms anti-parallel beta sheets
What are the two models for ligand induced activation of TNF receptors
- Individual TNF receptors are brought together by the soluble TNF ligand in their extracellular domains, this brings the death domains together on the intracellular
- 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.
what are the 11 steps of the extrinsic apoptotic pathway
- Trimeric ligand induces trimerisation of the FasR
- This triggers interaction of death domains
- They undergo a conformational change
- Leads to the recruitment of FADD
- FADD also contains a death domain (the death domains interact together)
- The death domains are recruited to the membrane and undergo a conformational change
- FADD is composed of another domain - DED
- DED interacts with pro caspase 8 (interacts by its DED domain)
- Pro caspases activate themselves by cutting off the DED domain (proteolytic activation step)
- The active caspase 8, activates further downstream effector caspases
- They cut a number of proteins in the cell which are crucial for cell survival
What are caspases 8,9 and 10
Initiator caspases
What are caspases 3,5 and 7
Effector caspases
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
What is the structure of Caspases
They are beta and alpha domains
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)
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
- Removal of DED domain
- Further cleavage through the beta and alpha domains, leaving two small domains of each.
- The active caspase is composed of beta and alpha subunits
- This active caspase will diffuse into the cytosol and find the effector procaspase and cleave its prodomain at the N-terminus and activate it.
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
What cell types undergo intrinsic apoptosis
Type 2 cells
What level of caspase 8 activation induces intrinsic apoptosis
When activated at low levels
What is the name of the protein that caspase-8 cleaves
Bid- an inhibitor of apoptosis, it produces tBid (truncated Bid)
What is the role of tBid
Facilitates the association and integration of Bax into the outer mitochondrial membrane
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
What is the role of cytochrom C
It binds to Apaf-1 and induces the formation of an apoptosome
What is Apaf-1
Protein made up of CARD, CED-4 and WD40 domains
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
What is the role of the apoptosome
Activates procaspase 9 by promoting the release of the CARD domain
What is the role of caspase 9
Activates further downstream effector caspases
What step acts as an irreversible step in mitochondrial targeting of tBid
Posttranslational N-myristoylation of the pro-apoptotic molecule BID
What other pathways are activated by Fas binding
JNK, MAPK, NF-kB, P38
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
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.
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
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.
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
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.
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)
What is required for the activation of FADD after TNF receptor activation
Dependent on internalisation of the TNF receptor - maturation to complex II
