Module 3- Getting Things Where They Need For Signalling? Flashcards
Different types of receptors and examples
Ion channel eg calcium, GABA
GPCR eg rhodopsin
Receptor tyrosine kinase eg TrkA
Enzyme associated eg TLR4
Intracellular receptors eg ER
Different types of ligands- how are they different
Endogenous/ exogenous eg drugs
Autocrine, juxtacrine, paracrine, endocrine
Type: peptides, proteins, carbs, lipids, ions
Features of toll-like receptors and what they recognise
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
TLR-4: what it recognises and what else is needed for activation. What happens for activation
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
TLR-4 adapters and features of both
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
How does the early immune response with TLR4 work (MyD88 dependent)
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
How does the late immune response with TLR4 work (MyD88 independent)
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
Dynamin and dynasore in endocytosis in late signalling. Experiment to show this
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
Roles of HMGB1
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
Structure of HMGB1 and things associated with it
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
Different states and functions of HMGB1
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)
Mediation on HMGB1 oxidation state
Dependent on peroxyredoxin and redoxases. Is enzyme mediated and depends on oxidation state of the cell
Features of parkinsons disease
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
HMGB1 in parkinsons
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
Effect of LPS on DA neuronal cells on glia
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
Secretion of HMGB1 results on western blotting
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
effect of HMGB1 on cell death seen with staining and what A-box does to this
HMGB1 alone can activate TH neuronal cell death
When incubate A-box and HMGB1 can block the cell death- A-box potential drug
What is the lysosome
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
Interactions the lysosome has with other things
Organelle contacts with mitochondria and ER
Fusion with endosomes and autophagosomes
Pro-growth signalling through mTOR on membrane
Anti-growth regulation with TFEB
How does TFEB control lysosome synthesis
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
How do enzymes get to the lysosome
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)
mannose-6 phosphate glycosylation (M6P)
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
M6P linked protein trafficking from golgi
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
Lysosomal enzymes- features on activity
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
