Module 3- Getting Things Where They Need For Signalling?

Question 1

Different types of receptors and examples

Answer 1

Ion channel eg calcium, GABA
GPCR eg rhodopsin
Receptor tyrosine kinase eg TrkA
Enzyme associated eg TLR4
Intracellular receptors eg ER

Question 2

Different types of ligands- how are they different

Answer 2

Endogenous/ exogenous eg drugs
Autocrine, juxtacrine, paracrine, endocrine
Type: peptides, proteins, carbs, lipids, ions

Question 3

Features of toll-like receptors and what they recognise

Answer 3

All share intracellular TIR domain
Same family as IL-1 receptors and MyD88 family
Differ extracellularly to allow different ligand recognition
Act as homodimers or heterodimers
Most at membrane, some at endosome eg 3, 7, 9 or 4 at both membranes
Recognise PAMPs and DAMPs

Question 4

TLR-4: what it recognises and what else is needed for activation. What happens for activation

Answer 4

Recognises LPS from gram negative bacteria and viruses
Requires co-receptors CD14 (membrane bound and soluble) and MD-2
Requires LBP (LPS binding protein)
LPS + LBP + TLR4= dimerisation of TLR4/MD-2 monomers and conformational change in TIRs allowing adapter binding- no intrinsic enzyme activity

Question 5

TLR-4 adapters and features of both

Answer 5

MyD88 and TRIF/TRAM
MyD88 recruited to plasma membrane- used during early immune response
TRIF/TRAM binds to TRAF3 in endosome- used during late immune response
Both bind TIR domain of TLR4
Different downstream effects

Question 6

How does the early immune response with TLR4 work (MyD88 dependent)

Answer 6

MyD88 TIR region interact with dimerised TLR4 TIR regions intracellularly.MyD88 at membrane from interaction with MAL/TIRAP TIR domain, held in membrane by PIP2 domains. MyD88 death domain interacts with IRAK1/4 death domain, leading to IRAK autoP
IRAK-P activates TRAF6 (ub ligase) which poly-ub itself at K63 to form a scaffold
TAK1 is P by IRAK1/4 and activated which then P IKKB which is ub at K48 and degraded, releasing NFkB (in complex with), which is then exposed to its NLS and allows transcription of TNFa, IL-8 and IL-12

Question 7

How does the late immune response with TLR4 work (MyD88 independent)

Answer 7

TRAM interacts with dimerised TLR4 TIR domain and held at membrane by myristoylated lipid modification
TRAM interacts with TRIF, which is activated and by endocytrosis is able to interact with TRAF3/6 on an endosome membrane.
TRAF6 activates NFkB as before
TRAF3 (ub ligase) is ubiquitinated which then interacts with TBk1 which is activated, then P and activates IRF3
IRF3-P moves to nucleus and causes expression of IFN3
Needs endocytosis for interaction to occur and therefore, needed for late immune signalling

Question 8

Dynamin and dynasore in endocytosis in late signalling. Experiment to show this

Answer 8

Formation of vesicle dependent on dynamin
Dynamin inhibited by dynasore
Flow cytometry: compared TLR-4 at membrane with and without dynasore- saw with dynasore there is more at membrane
Western blot: no P of IRF3 with dynasore due to no internalisation
RT-PCR: no IL-6 with dynasore and no IFN-B
ELISA: no IL-6 and no RANTIES with dynasore

Question 9

Roles of HMGB1

Answer 9

Nucleus- binds to and bends DNA to improve access for TF binding to DNA- allows other TFs to bind= regulation
Cytoplasm- binds beclin-1 to induced autophagy
Extracellular- acts as DAMP when cells die or from cell release

Question 10

Structure of HMGB1 and things associated with it

Answer 10

A-box (DNA binding, anti-inflam), B-box (DNA binding, pro-inflam), acidic tail (transcription stimulatory domain)
NLS and NES, interactions with different receptors (TLR4 and RAGE)
Extracellular and intracellular partners: LPS (extracell), beclin 1, peroxiredoxin
Different regions can function differently together or on their own. Different areas can have different signals and interactions also

Question 11

Different states and functions of HMGB1

Answer 11

Release and oxidative state are linked: necrotic release is fully reduced, active secretion is partially reduced, apoptotic release is fully oxidised
The state it is in affects what receptor it acts on and therefore leads to different responses
Passive release or active release (where PTMs also regulate localisation and only occurs in immune cells and neurons)

Question 12

Mediation on HMGB1 oxidation state

Answer 12

Dependent on peroxyredoxin and redoxases. Is enzyme mediated and depends on oxidation state of the cell

Question 13

Features of parkinsons disease

Answer 13

Symptoms develop from ~60yrs- stooped posture, masked face, back rigidity
Unknown causes some possible risk factors are pesticides and timber treatments but genetic and enviro factors also contribute
From a loss of ~70% of dopanamergic neuronal cells (release dopamine) in substantia nigra

Question 14

HMGB1 in parkinsons

Answer 14

Released from dying neurons or active glial cells
Leads to neurodegeneration, neuroinflammation, mitochondrial deformation, apoptosis, autophagy, BBB disruption, effects on TH expression (tyrosine hydroxylase which makes dopamine) and gene transcription

Question 15

Effect of LPS on DA neuronal cells on glia

Answer 15

When treated with LPS and then washed away after different periods- see that the longer treatments cause DA neuronal cell death
Also see same microglia activation if using mimic of parkinsons pathology or LPS

Question 16

Secretion of HMGB1 results on western blotting

Answer 16

Addition of LPS, rotenone and MPP can see HMGB1 secretion after 24 hours in neuron-glia cells
In just glia cultures see secretion after 24 hours then disappears- shows could be early glia response with HMGB1 release and late neuronal response secreting HMGB1

Question 17

effect of HMGB1 on cell death seen with staining and what A-box does to this

Answer 17

HMGB1 alone can activate TH neuronal cell death
When incubate A-box and HMGB1 can block the cell death- A-box potential drug

Question 18

What is the lysosome

Answer 18

Acidic vesicle (pH 4.5-5.5) which has a single membrane and contains transporters and channels to import things for degradation
Has recycling enzymes active at low pH to degrade things
End point of autophagy and endocytosis
Activity increased during starvation
Mutation in any part can cause disease

Question 19

Interactions the lysosome has with other things

Answer 19

Organelle contacts with mitochondria and ER
Fusion with endosomes and autophagosomes
Pro-growth signalling through mTOR on membrane
Anti-growth regulation with TFEB

Question 20

How does TFEB control lysosome synthesis

Answer 20

TF localised to lysosome and P under control conditions
P by many things at different Tyr residues
DeP to cause activation- done by calcineurin and PP2A
DeP exposes NLS and translocates to nucleus
Binds CLEAR (coordinated lysosome expression and regulation) and element in promoters of lysosome and autophagy genes

Question 21

How do enzymes get to the lysosome

Answer 21

Proteins synthesised in RER and trafficked through ER
Motifs or domains recognised by different receptors- LIMP2 in ER, sortilin in golgi and M6P in golgi
M6P needs glycosylation tag for recognition
Taken into endosome by receptors, fuses with lysosome where low pH causes release of enzymes, receptors are recycled back to the ER and golgi through retrograde transport (buds back off)

Question 22

mannose-6 phosphate glycosylation (M6P)

Answer 22

Covalent attachments or carbs to N residues in specific sequence motifs
Initiation- co-translationally occurs in ER, addition of monosaccharide by oligosaccharide transferase OST
Elongation- addition of more complex sugars in golgi and exposure of phosphorylated mannose- recognised by M6P receptor

Question 23

M6P linked protein trafficking from golgi

Answer 23

Captured by M6P receptor or IGF2 receptor in golgi
Then buds from golgi in clatrin coated vesicle, transferred to lysosome, retrograde transport of receptor back to golgi
If enzyme doesnt bind to receptor, escapes through secretory pathway to bind to M6P receptors at membrane and then recycled to lysosome by endocytosis

Question 24

Lysosomal enzymes- features on activity

Answer 24

Activated in acidic environments
Often activated by proteases cleaving pro-peptide sequences
Loss of acidity in disease causes loss of activity
Rupture of lysosomes doesnt cause whole cell degradation due to the loss in activity in cytoplasm

Question 25

Four methods of autophagy lysosome pathway

Answer 25

Macroautophagy: phagophore-> autophagosome, fuses with lysosome
Chaperone-mediated: cargo with specific seq which is exposed when needed to be degraded, binds to heat shock protein (HSPA8), then binds to LAMP2 channel and is taken up
Microautophagy: lysosome invaginates
Endosomal microautophagy: late endosome binding to chaperone mediated molecule, then late endosome fuses with lysosome

Question 26

How does lysosome movement occur

Answer 26

Move along microtubules towards positive pole on cell periphery away from perineuclear membrane
Microtubules bind to cortical actin- mediate the movement
Arl8B interacts with lysosome to allow movement from nucleus to periphery
Arl8B degraded when RNF168 E3 ligase increases as it ub Arl8B, causing lysosomes to not be able to move closer to periphery. Can move back to perinuclear membrane

Question 27

How was it shown experimentally that RNF167 is involved in lysosomal movement

Answer 27

When in a cell with neutral pH, lysosomes stay by nucleus
When in a cell with pH 6.5 they move to periphery
When RNF167 (tagged with HA) is added, then lysosomes move back to nucleus, when it is mutated so it cant do its ubiquitination anymore, the lysosomes move back to periphery
Then quantified it- amount compared to control to show same thing

Question 28

How was it shown experimentally that RNF167 interacts with Arl8B for lysosomal movement

Answer 28

BioID
Fuse with BirA protein which biotinylates anything in cloxe proximity (promiscuous)
Avidin added, binds to biotin, put on beads and pull out with immunoprecipitation. Do mass spec to see which proteins bind to RNF167
Saw Arl8B and VAMP3 bind

Question 29

How was it shown experimentally that RNF167 is involved in lysosomal movement directly with Arl8B- already know it binds

Answer 29

Proving Arl8B is substrate
Fuse RNF167 with FLAG tag and Arl8B with HA tag
Add increasing amounts of RNF-167 and see that Arl8B is decreased in added amounts
When RNF167 mutated so cant ub anymore, dont see a decrease in Arl8B which shows it has a direct effect on it

Question 30

Where could things go wrong in lysosome regulation leading to disease

Answer 30

Change in acidity
Missing enzyme- mutations and variants that change the function
TFEB mysregulation
Mysregulation of trafficking proteins
Lysosome trafficking impaired

Question 31

Importance of lysosome function in neurons

Answer 31

Neurons are long and lysosomes need to travel to control synaptic activity
Require trafficking on microtubules and regulate synaptic activity
When lysosome faults in normal cells it can be diluted out with replication but neurons dont divide so when there is an issue with lysosomes it tends to stay in the neurons= dysfunctional brain disease- most lysosome disorders cause brain dysfunction

Question 32

Features of Batten disease

Answer 32

Child neurodegenerative disease- different symptoms with different onsets between 6 months to 8 years old
Blindness, motor problems, learning difficulties, seizure, hallucinations, inability to walk, talk and feed
All forms fatal
Combination of epilepsy, alzheimers disease, parkinsons disease and blindness in children

Question 33

Biochemistry to identify the cause of CLN2 Batten disease

Answer 33

Run on 2D gel, each spot is a different protein, get better resolution. See a certain protein is missing in late infantile NCL (LINCL)
See that this missing protein causes LINCL to be sensitive to pepstatin with specific activity on haemoglobin substrate
First indication it was a protease (pepstatin insensitive expartile protease)

Question 34

Lysosomal enzyme in CLN2 Batten Disease

Answer 34

Causes by mutations all throughout gene in CLN2 which encodes lysosomal enzyme tripeptidyl peptidase 1 (TPP1)
Regulated by TFEB and known that it cleaves tripeptides from N terminal end of proteins in acidic environment of lysosomes

Question 35

TPP1 and glycosylation and function

Answer 35

Pre-peptide for ER localisation, pro-peptide is cleaved (autocatalytic cleavage)
Need acidity for autocatalytic cleavage, need glycosylation for folding and activity and trafficking
Full enzyme activity with acidity and glycosylation
Seen with western blot in different conditions

Question 36

Alternative trafficking pathway importance for batten disease therapy

Answer 36

Allows proenzymes to be taken up by other cells into lysosomes
Cross-connection allowing treatment of cells from the outside as they can be taken up in endosomes to lysosomes

Question 37

TTP1 enzyme therapy for CLN2 batten disease

Answer 37

Protein too large to get from systemic system through the BBB
Need direct infusion- 4 hour infusion through cannula into the brain once every 2 weeks
Successfully delays disease onset but has risks
Now treated pre-symptomatically
Future= enzyme replacement in eye and gene therapy for one shot treatment