Tumour Necrosis Factor (TNF)/NGF receptors

Question 1

1. what can too much cell death lead to? (3)
2. what can too little cell death lead to? (2)
3. give a few examples of dieases associated with TNF signalling.
4. give 3 ways in which T cells become resistant to apoptosis in autoimmune diseases.

Answer 1

1. immunodeficiency, neurodegeneration and infertility
2. cancer and autoimmune disease
3. sepsis, asthma, IBD, diabetes, myocarditis, RA
4. IL-1 and TGF-beta decrease fas expression inhibit apoptosisBcl-1 expression increases the threshold for apoptosissFas inhibits the fas mediated apoptotic pathway

Question 2

1. describe the characteristics of apoptosis
2. describe the characteristics of necrosis
3. how can apoptosis be measured using annexin V, FITC, propodium iodine and FACS machine?

Answer 2

1. cell shrinks and chromatin condenses. cell blebbing. blebs are phagocytosed. it is a single celled event that doesnt disrupt the surrounding environment. Requires ATP. membranes remain intact
2. the influx of water leads to cell swelling and eventually cell lysis. IC components are released into the EC enviro. Large areas of tissue are affected. Inflammation is elicited. ATP is deleted.
3. Annexin V binds to phosphatidyl serine. Phosphatidyl serine is only found in the extracellular leaflet of membranes during apoptosis, thus Annexin V can only bind to membranes of cells that are undergoing apoptosis. FITC is a flurophore which is used to label annexin V. propidium iodide is used to label cells undergoing late phase apoptosis, as during this stage, membranes are leaky so propidium iodine can enter cells. FACS machines are used to sort fluorescent cells (apoptotic cells) from non-fluorescent cells.

Question 3

1. what were the results of removing the limb bud?
2. what were the results of transplanting an extra limb bud?
3. what is the neurotrophic hypothesis?
4. what was the first neurotrophic factor to be identified?
5. what was the first neurotrophic factor receptor to be identified?
6. what are the effects of ligand binding to this receptor?
7. name 3 other NTF receptors.

Answer 3

1. a reduction in the number of neurons in the spinal cord
2. an increase in the number of neurons in the spinal cord
3. innervation targets secrete survival factors to generate a balance between the size of the target organ and the number of innervating neurons
4. NGF
5. p75^LNTR (low affinity receptor)
6. couldn’t elicit the neurite cell growth as seen when NGF is added
7. TrkA, TrkB and TrkC

Question 4

1. what does p75 bind to?
2. what binds to a) TrkA, b) TrkB c) TrkC?
3. what does activation of Trk lead to?
4. what does activation of p75 lead to?

Answer 4

1. all neurotrophins
2. a) NGF b) BDNF and NT-4 c) NT-3
3. neurite growth and cell survival (via Akt)
4. apoptosis

Question 5

1. what type of motifs are found in the extracellular domain of TNF receptors, and what do they mediate?
2. what type of domains are found in the cytosolic domains of receptors? How were these domains identified? what are these domains made up of
3. name 2 other domains similar to 2 that are found on downstream effectors.
4. how many transmembrane domains do receptors posess?
5. what is the structure of the ligands? What does this mean? how are the different types of ligand produced?

Answer 5

1. cysteine rich repeats that facilitate ligand binding
2. death domains, that consisit of alpha helices. They were identified by sequence allignment of Fas and TNF-R1
3. death effector domains (DED) and caspace recruitment domains (CARD)
4. 1
5. posess an EC domain, cytosolic domain and TM domain, therefore ligands ca also act as receptors (receptor-ligand interraction can evoke downstream signalling in the ligand posessing cell) - membrane bound or cytosolic forms of the receptor are formed by alternative splicing.

Question 6

1. how are receptors activated?
2. name the 2 models that describe how this process (1) is acheived
3. why is p75 activation unusual?

Answer 6

1. trimerisation
2. receptors are present as monomers, and TNF ligand promotes trimerisationreceptors are present as trimers, (PLAD domain enables pre-ligand association). Ligand binding induces a conformational changes to bring the death domains together.

Question 7

1. where is complex 1 formed and what does it consist of?
2. where is complex 2 formed and what does it consist of?
3. which complex promotes apoptosis. Describe the process.
4. how is the signal amplified?

Answer 7

1. at the plasma membrane. consists of , TRADDTRAF2 and RIP
2. following internalisation. consists of TRADD, FADD and RIP
3. complex 2. DD of receptor interracts with the DD of RIP. DD of RIP and TRADD (which are no longer associated with the DD of the receptor) interract with the DD of FADD. FADD recruits pro-caspase 8 via its DED domains.
4. many caspases can be recruited to and activated by complex. each caspase can cleave many downstream proteins.

Question 8

1. what are caspases synthesised as?
2. name the 3 initiator caspases?
3. how are initiator procaspases activated?
4. how can the cleavage of caspases be seen using western blotting?

Answer 8

1. procaspases
2. 8, 9 and 10
3. they bind to FADD via their DED domains. FADD cleaves the procaspase, removing the DED domains and bringing the alpha and beta domains together to form a catalytically active caspase. As the DED domain is removed, the caspase dissociates from the complex and cleaves effector procaspases
4. application of a Fas ligand produces an additional band.

Question 9

describe 2 ways in which there is crosstalk with the intrinisc apoptotic pathway

Answer 9

1. nomally, BID prevents the binding of BAX and BAX to the MT; BAX and BAK binding promotes the release of cytochrome C. Caspase 8, as activated by TNF complex 2 breaks down BID, thus relieves the inhibition of BAX and BAK binding
2. the release of cytochrome C activates APAF, which promotes the formation of the apoptosome. the apoptosome can interract with the CARD domain of pro-caspase 9 which facilitates its cleavage.

Question 10

1. why can TRAF molecules act as E3 ubiquitin ligases?
2. what are TRAFs signal transducers for? What are their signalling outcomes?
3. how have TRAFs been implicated in cancer?

Answer 10

1. they contain a RING finger domain
2. toll like receptors. They activate NF-kB and MAPK promoting cell survival
3. they act as tumour supressor genes and inhibit B cell survival. Deletion/inactivation mutations are frequently found in B cell neoplasms. SNPs of TRAF3 are associated with altered risk of multiple myeloma.

Question 11

1. name 2 reasons why neurotrophic factors and their receptors are ideal candidates for therapeutic intervention in neurodegenerative disease?
2. what is the biological paradox associated with p75?
3. how can this information be used therapeutically?

Answer 11

1. because neurotrophic factors show potent neurotrophic and regenerative functions in models of neurodegenerative disease, and p75 is upregulated in models of MND/in response to peripheral nerve crush
2. despite being upreguated in models of neurodegenerative disease, p75 plays a part in neuronal cell death. Why does the NS induce a molecule aid the demise of neurons in response to injury?
3. develop molecules to inhibit p75 in cases of neural injury and degeneration.

Question 12

1. name 2 factors that are essential for programmed necrosis
2. how was one of these factors identified?
3. when is the complex of these 2 factors assembled?

Answer 12

1. RIP1 and RIP3
2. RIP3 was identified by an RNAi screen
3. RIP3 is only recruited to RIP1 following internalisation (Complex 2). The RIP1/RIP3 complex is only formed during programmed necrosis and not apoptosis.

Question 13

1. how was the Trk oncogene identified as the NGF receptor? What was the control?
2. how was the affinity of the NGF binding determined?
3. how did scientists investigate whether the low affinity binding sites in the study corresponded to the LNTF?
4. what were the findings of each of the experiments?

Answer 13

1. TrkB expressing cell lines were incubated with ¹²⁵I-NGF, and compared binding to PC12 cells which were known to express a high affinity NGFR
2. scatchard plot analysis of ¹²⁵I-NGF binding to TrkB cell lines and PC12 cell lines
3. cell extracts from PC12 cell lines and the TrkB cell lines were immunoprecipitated using anti-p75^LNTR antibodies
4. cell lines expressing TrkB bound to NGF efficiently. PC12 cells also bound to NGF efficientlythe affinity of NGF binding in TrkB cells was comparable to that of high affinity receptors in PC12 cellsantibodies immunoprecipitated LNGFR from PC12 cells but nor from the TrkB cell lines.

Question 14

1. how were the kinetics of inactivation followed?
2. how did researchers determine the compartments that receptors were internalised to?
3. how did researchers investigate the association of TNFR with DISC proteins?
4. what was the purpose of creating point mutations in the TRID motif?
5. what were the results?

Answer 14

1. fluorescence microscopy, whereby TNFR was labelled with biotin
2. analysed receptorsomes (vesicles containing receptors) by westerm blotting for markers of Intracellular compartments
3. immunopreciptation
4. to examine the effects on internalisation, DISC recruitment and apoptosis
5. at 4^oC, biotin was seen at the surface, whilst at 37^oC was found in endocytic vesicles, then MVBs, then lysosomes

isolated receptorsomes early on contain EE markers. Later they contain LE markers (LAMP1)

follwing internalisation, RIP1, FADD, TRADD and caspase 8 are recruited to receptorsomes. RIP1, TRADD, FADD and procaspase 8 co-immunoprecipitated with TNFR1

deletion of TRID by point muations inhibited internalisation, DISC recruitment and apoptosis.