Tumor Necrosis Factor - Dr Erdman

Question 1

what happen when homeostasis is disrupted?

Answer 1

> proliferation = cancer, autoimmune disease

> cell death = neurdegeneration, immunodeficiency

Question 2

What is apoptosis

Answer 2

• programmed cell death
• maintain tissue homeostasis by balancing cellular life and death
• when disregulated, cause premature cell loss

Question 3

Features of apoptotic cells

Answer 3

• membrane blebbing

* cellular shrinkage*condensation of chromatin

Question 4

Explain the experiment that study the cell survival

Answer 4

FIRST EXPERIMENT
* victor hamburger
* chick embryo’s spinal cord ( motor neurons)
* remove limb bud
*number of motor neuron decrease dramatically in the limb of missing limb bud
* * conclusion **
limb bud is necessary for survival of sensory and motor neurons in the spinal cord

SECOND EXPERIMENT

• extra limb bud transplanted
• increase in the number of motor neurons

> > The neurotrophic hypothesis derived
- Target of innervation ( limb bud) secretes limiting survival factors (neurotrophic factor)- to generate a balance between the 1) size of organ, 2) the number of innervation

Question 5

What is the neurotrophic hypothesis

Answer 5

• Target of innervation ( limb bud) secretes limiting survival factors (neurotrophic factor)- to generate a balance between the 1) size of organ, 2) the number of innervation

Question 6

How do we find out about the molecular basis of the neurotrophic factors?

Answer 6

experiment by cohen and montalcini

assay

1. sympathetic ganglion extracted
2. ganglion concentrated with cell bodies
3. put some NGF
4. observed axon growth
5. later work to purify NGF

54: neurite outgrowth assay
60: NGF purified
69: NGF purified to homogeneity

Question 7

What are Trk Receptor

Answer 7

• Receptor tyrosine kinase
• TrkA,B,C
• Trk A binds to NGF
• Trk B binds to BDNF
• Trk C binds to NT-3

– this work shows that receptor tyrosine kinase not only regulate proliferation but also SURVIVAL (of neu=rite outgrowth )

Question 8

Describe the experiment that proves Trk receptor can signal locally and retrogradely

Answer 8

1. sympathetic neurons placed in petri dish with tight seal ( not allowing substance from outised to enter the soma area)
2. in petri dish (PD)1 , put anti-NGF in soma… result = neurite failed to survived
3. in PD 2 , put anti NGF in soma area. then put NGF in the neurite area. Result = neurite survived eventhough the soma received anti NGF
4. suggestive that there is a retrograde transport mechanism of delivering NGF to the soma

CONCLUSION of the experiment

1. Receptor can be endocytosed
2. retrograde transport of the endosome ( containing the activated receptors)
3. endosome receptor complex find other binding partner ( cell-body signalling speciic signalling components) to promote cell survival
4. called signalling endosome

Question 9

Explain signalling endosome

Answer 9

• can be found in neurons
• Ligand (NGF) bound to receptor (Trk A) in the –NEURITE– and become activated
• the activated receptor in endosomed and known as signallling endosome
• signalling endosome is retrogradely transported to the cell bodies
• then binds to other signalling molecules ( such as cell-body specific signalling molecules)
• promote cell survival

Question 10

What the hell is p75

Answer 10

p75 LNTR / p75 LNGFR

• p75 (L)ow affinity (N)euro(T)rophic (Re)eceptor
• is NOT a receptor tyrosine kinase
• founding member of the NGFR
• contain the DEATH DOMAIN
• Basically it is just historically important because people first discovered DEATH domain from p75. it is still a mystery~

Question 11

TNF problem usually arise what problem

Answer 11

autoimmune disease

inflammatory disease

Question 12

what structures are related to death domain?

Answer 12

• Death DOmain
• Death Effector DOmain (DED)
• CARD domain

Question 13

what will happen if you have FAS gene mutation

Answer 13

• autoimmune disease
• phenotype =
  a) increased proliferation of lymphocytes
  b) size increase in lymphoid organs like lymph nodes or spleen. non-malignant

Question 14

what is a PTPN13?

Answer 14

Regulate membrane trafficking of FAS Receptor. so FAS R is now present at the CELL SURFACE

FAS R+ PTPN13 -> more FAS R at cell surface

overexpressed PTPN -> reduced FAS R in cell surface, cancerous cells ( no apoptosis )

Question 15

FAS Rcan only exert it action when

Answer 15

it is in the cell surface ( regulated by PTPN13)

Question 16

what happen when you have over expression of PTPN13

Answer 16

less FAS R on surface, become cancerous

Question 17

what is the structure of TNF alpha Ligand

Answer 17

Fas Ligand aka CD95 L is a transmembrane protein

classed as TNF -a

TRIMERIC

3 identical subunit

have JELLY ROLL STRUCTURE

Question 18

Describe the activation of the TNF receptor

Answer 18

the trimeric TNF-a causes TRIMERISATION of the FAS ligand

Question 19

What is the difference between the oligomerization of the RTK and TNFR?

Answer 19

the trimeric TNF-a (or FASL) will cause trimerisation of the TNFR (or FAS R)

the RTK will dimerize
-ligand will bind to each RTK (aka 1 ligand per RTK as oppose to 1 trimeric ligand per TNFR/ FASR)

Question 20

compare the RTK and TNFR/FASR clustering and intracellular domain changes

Answer 20

both will cluster (RTK dimerize, TNFR/FASR trimerize)

both oligomerization transduces signal by conformational change

in RTK, clustering of the Tyrosine Kinase Domain TKD cause transautophosphorylation, thus creating dosking site for SH2 and PLC gamma domain

in TNFR/FASR, trimerization causes clustering of the death domain, causing conformational change , thus creating a docking site for downstream signalling

Question 21

pre-ligand association domain

Answer 21

a theory

• TNFR is a preformed trimer
• like insulin
• upon ligand binding, will have conformational change

Question 22

Describe the 2 model of activation of TNFR

Answer 22

a) TNFR have ligand binding domain, trimerisation is caused by trimeric ligand binding. not preformed
b) PLAD - pre ligand association domain, describe the idea that the PLAD domain assist in the formation of PREFORMED TNFR TRIMER. the proformed trimer is not active. can only be activated upon ligand binding . idea similar to insulin receptor

Question 23

p75 NTR binds to what

Answer 23

all (pro)-neurotrophin

Question 24

what effect does pro-neurotrophin and mature neurotrophin have on p75 NTR

Answer 24

proneurotrophin - cell death and degenration

neurotrophin - survival and neurite growth

Question 25

TNF super family are all membrane bound?

Answer 25

No. all of them are membrane bound but SOME of them have splice variant of soluble version

Question 26

TNFR superfamily

Answer 26

- is much more complicated in terms of structure than RTKs - extracellular ligand binding domain are cycteine-rich - 2 subfamily ( with DEATH DOMAIN and without DD) - ones with DD are able to induce apoptosis

Question 27

Does phosphorylation takes place in TNF R?

Answer 27

NO in Death domain --unlike in RTKs who will phosphorylate in the TKD upon TKD clustering and recpetor dimerization--

Question 28

Explain the extrinsic pathway of apoptosis

Answer 28

1. (JUXTACRINE SIGNALLING) Fas L is membrane bound to other cell, usually immune cells such as T cells. But we also have splice variant of soluble TNF-a molecule 2. TNFSF are trimeric, have jelly roll structures of beta sheets. Binds to corresponding TNFRSF. 3. 2 ideas of TNFRSF activation, a) conventional. no ligand, no trimeric receptor. b) PLAD. Preformed Ligand Association Domain theory describe that the receptors are actually preformed trimers. But only activated upon ligand binding 4. ligand binding > Receptor activation > conformational change in the DEATH DOMAIN 5. death domain clustering upon receptor trimerisation, conformation change creates docking site for downstream signalling 6. INSERT PICTURE HERE 7. death domain in the FASR will bind to DeathDomain of FAS Associated Death Domain (FADD). 8. FADD has DD and Death Effector Domain (DED) 9. FASR DD binds to FADD DD 10. FADD DED binds to DED on caspase 8/10 prodomain 11 INITIATOR caspase 8/10 have a) DED in the prodomainb) large a subunit c) small b subunit. EEFECTOR caspase only have Large a subunit and s b subunit. does NOT have DED doamain in the prodomain. Prodomain will be cleaved by active caspase 12) 2 x L and 2 X S subunit of the same type of caspase becomes the active caspase 13. DED of initiator caspase will be cleaved. active initiator caspase allows for more effector caspase production by cleaving the prodomain of the effector caspase ( no DED ) 14. a lot of caspase --->> APOPTOSIS 15. effector caspase is NOT membrane bound/ FADD bound and does NOT have a DED in its prodomain

Question 29

Why do we have a lot of stages in a pathway

Answer 29

1. amplifaction of signal (such as in RAS RAF MEK ERK ) | 2. more possible level of regulation

Question 30

How do we measure the activation of the caspase?

Answer 30

western blot > look out for new band showing up ( caspase/protease) > greater or lesser intensity

Question 31

type 1 cells vs type 2 cells in TNFR?

Answer 31

Type 1 cell >> extrinsic only | Type 2 cell >> extrinsic PLUS INTRINSIC pathway

Question 32

Describe type 2 cell in TNF R

Answer 32

1. Caspase 8 cleaved Bid into truncated Bid (tBid) 1. 2 tBid undergo myristoylation of N-terminal glycine by NMT and translocated to the mitochondria 2. myristoylated tBido on the mitochondria facilitate recruitment of Bak, the protein that will form the pores that releases cytochrome c 3. activation of a channel within the mitochondria so that cytochrome c can be released from the mitochondria 4. Cytochrome c activate Apaf. Apaf have CARD domain. binding of cytochrome c to apaf will assemble a few Apaf into APOPTOSOME 5. Apoptosome = molecular machinary to activate initiator caspase (procasoase 9) 6. Apoptosome will cleave CARD domain on m the procaspase 9 7. active caspase 9 activate procaspase 3

Question 33

What is the significance of truncated Bid

Answer 33

Bid -> tBid via cleavage by caspase 8 >>> creating new N terminus and C terminus (ye la kan dah cleave) tBid translocate to the mitochondria and undergo myristoylation, the lipid modification where the N-terminal glycine is added with myristic acid by NMT N-myristoyltransferase. this facilitate the recruitment of Bak, the protein that will form pores that release cytochrome c from the mitochondria *New N terminal GLYCINE will be added Myristic acid by NMT (N- myristoyltransferase) tBid undergo Myristoylation , a type of lipid modification.

Question 34

What is an apoptosome

Answer 34

1. a structure formed by several Apaf with bound cytochrome c 2. the CARD domain of the Apaf form the middle portion 3. Apoptosome will cleave CARD domain from the procaspase 9

Question 35

What is other pathway of apoptosis by the FAS receptor?

Answer 35

NF-KB FAS are activating the extrinsic, intrinsic and NFKB pathways

Question 36

How can we measure apoptosis

Answer 36

1. Phosphatidyl serine (component of plasma membrane) > in healthy cell, PS found only in the inner leaflet > PS will flip from inner leaflet to outer leaflet in dying/ apoptotic cells 2. Annexin5-FITC (annexin5 with fluoroform) added to the culture, annexin 5 will bind to PS. >> the annexin5-FITC is not permeable, so cannot visualise those in the inner leaflet of healthy cells 3. in healthy cell, no PS staining.