Regulation of Myd88 Dependent Signalling Flashcards
1
Q
Name 2 UBP which mutations in are linked to immune disease
A
NEMO
Optineurin (OPTN)
2
Q
Name an E3 ligase which when mutated is linked to immune disease
A
LUBAC
3
Q
Name 2 protein kinases which when mutated is linked to immune disease
A
IRAK4
TBK1
4
Q
How does Myd88 stimulate NF-kappaB and MAPK signalling?
A
Binds to TLR using Mal IRAK4 is recruited IRAK1 and 2 is recruited Traf6 causes the activation of Tak1 NF-kappaB and MAPK signalling
5
Q
How does Trif cause IRF3 signalling?
A
Trif binds to TLR using TRAM -> Traf3 -> TBK1 -> IRF3
6
Q
What 2 receptor families does Myd88 bind to?
A
IL-1 receptor family
TLR
7
Q
What does IL-1 receptor bind to?
A
IL-1 family members:
- IL-1
- IL-18
- IL-33
8
Q
What do TLRs bind to?
A
PAMPs
9
Q
What are the 2 domains in TLRs?
A
Leucine rich repeat domain
TIR domain
10
Q
What TLRs couple to Myd88?
A
All except TLR3
11
Q
What TLR2 and TLR4 require to recruit Myd88?
A
2nd adaptor, Mal
12
Q
What can TLR3 and TLR4 couple with?
A
Trif
13
Q
How is Trif recruitment to TLR4 mediated?
A
Using Tram
14
Q
What TLRs are expressed on the cell surface?
A
TLR5 TLR1 TLR2 TLR6 TLR4
15
Q
What TLRs are expressed on endosomes?
A
TLR3
TLR7
TLR8
TLR9
16
Q
What is the ligand for TLR5?
A
Flagellin
17
Q
What is the ligand for TLR1/2?
A
Triacylated Lipopeptides
18
Q
What is the ligand for TLR2/6?
A
Diacyclated lipopeptides
19
Q
What is the ligand for TLR4?
A
LPS
20
Q
What is the ligand for TLR3?
A
dsRNA
21
Q
What is the ligand for TLR7/8?
A
ssRNA
22
Q
What is the ligand for TLR9?
A
CpG DNA
23
Q
How is Mal recruited to the TLR?
A
Mal has a TIR domain which binds to TLR TIR domain
24
Q
How does Myd88 bind to Mal?
A
Through the TIR domains
25
How does IRAK4 bind to Mys88?
Through the death domains
26
How does IRAK4 recruit IRAK1/2?
Through its death domain
27
In what form does Myd88 bind to Mal?
As an oligomer
28
What are IRAK1 and 2?
Pseudosubstrates
29
How is TAK1 activated?
by the addition of K63 linked chains
30
What are TRAF proteins?
Tumour necrosis factor receptor (TNFR)-associated factors
31
how many isoforms of TRAF are there?
6
32
Apart from TRAF1, what domain do all of the TRAFs have?
A RING domain
33
What is the RING domain important for?
Function:
- Some are E3 Ub ligases
- In some cases may be for scaffolding
34
What is the function of TRAF5?
Negative regulator:
| - inhibits TLR and IL-6`
35
What is the function of TRAF6?
IL-1/ TLR signalling
36
What is the function of TRAF1 and 2?
Act downstream of the TNR receptor
37
What is the function of TRAF3?
important downstream of CD30
38
What is the function of TRAF4?
involved in neurotophin signalling in the brain
39
What causes the addition of the K63 linked chains to the Myd88osome?
E2 ligase Ubc13 and E3 ligases Pelino 1 and 2 and Traf6
40
What is the Myd88osome?
TLR + Myd88 + IRAK4 + IRAK1/2
41
Where do the K63 linkages attach?
IRAK1/2
| Traf6
42
How do you remove downstream signalling of Myd88?
Have to remove both Pelino 1 and 2 and Traf6
43
What is the Tak1 complex?
It binds to Tab2/3 (only one at a time) and Tab1
44
What do Tab2/3 bind to?
The C-terminal NFZ domain binds to K63 linked PolyUb chains
45
What is the N-terminal domain of Tak1?
Protein kinase domain
46
How is Tak1 activated?
by binding to Tab1
47
What is the N terminal domain of Tab1?
N-terminal pseudophosphate domain
48
What part of Tab1 binds to Tak1?
The C terminal domain
49
What is the function of Tak1?
Can be a MAP3K for p38 and JNK MAPK pathways
| Actives IKK which can activate NF-kappaB and ERK1/2 MAPK cascade
50
How does Enteropathogenic E. Coli target Tab2 and 3 to suppress the immune system?
They inject NIeE into cells via a Type III secretion system -> Methylates Tab2 and 3 -> disrupts NFZ domain and prevents Ub binding -> inhibition of NF-kappaB
51
How do Enteropathogenic E.coli methylate Tab2 and 3?
NIeE encodes a cysteine methylase
52
What polyUb chain binds onto the K63 linked chain?
M1 chain
53
What causes the formation of M1 chain?
Potentially E2 ligase UbcH7
| E3 ligase Lubac
54
What are the 3 protein in LUBAC?
Sharpin
HOIL
HOIP
55
Where is the E3 catalytic site in LUBAC?
In the Rcat RING domain of HOIP
56
What does mutations in the Rcat RING domain of HOIP cause?
Blocks M1 formation downstream of Myd88
57
How does LUBAC interact with the PolyUb chain?
Through the NFZ domains
58
What do mutations in HOIL cause in humans?
Increases susceptibility to bacterial infection and autoinflammatory disorders
59
What do mutations in Sharpin cause in humans?
Chronic proliferative dermatitis
60
Where does Nemo bind?
to the M1 PolyUb chain
61
How is IKK recruited to the complex?
Via the NEMO subunit
62
What 2 subunits of IKK bind to Nemo?
IKKalpha and beta
63
How does Tak1 induce MAPK and NF-kappaB signalling?
Through phosphorylating and activating IKKbeta
64
Does do NEMO bind to M1 PolyUb chains?
Through its UBAN domain
65
What does NF-kappaB function as?
A homo or heterodimer
66
What is NF-kappaB?
A transcription factor
67
How does NF-kappaB function as a transcription factor?
Through the RHD domains
68
What are inhibitors of NF-kappaB?
IkappaBalpha and IkappaBbeta
69
How do IkappaB proteins act as inhibitors?
Through their Rel subunits to keep NF-kappaB in the cytoplasm
70
What family mediates the activation of NF-kappaB?
The IKK family:
- NEMO
- IKKalpha
- IKKbeta
71
What pathways can be used to activate NF-kappaB?
Canonical
Non-canonical
Atypical
IKK independent
72
What does the canonical pathway work downstream of?
Myd88
73
How does the IKK complex activate NF-kappaB?
It phosphorylates IkappaB which is bound to NF-kappaB dimer in the cytoplasm -> NF-kappaB moves to the nucleus and promotes transcription
74
How is IkappaBalpha targeted for degradation?
It is phosphorylated on two serine residues on the D-S-G-X-X-S motif -> recognised by F-box protein b-TcRP -> recruitment of SCF complex -> IkappaBalpha ubiquitylation on K21 and K22
75
What happens if the 2 serine residues in D-S-G-X-X-S in IkappaBalpha are mutated?
Prevents degradation of IkappaBalpha
76
What happens if K21 and K22 of IkappaBalpha are mutated?
stabilises IkappaBalpha
77
What are the components of the SCF complex?
```
Substrate
beta-TcRP
Skp1
Cul1
Rbx1
E2
```
78
What is beta-TcRP?
A receptor
79
What is Skp1?
An adaptor
80
What is Cul1?
A scaffold
81
What is Rbx1?
A RING domain
82
What is the SCF complex?
A multi component E2 ligase complex
83
How is the SCF complex activated?
By the addition of Nedd8 to Cul1 -> conformational change -> frees the E2 and moves it into position to interact with the substrate
84
What is the E2 in the case of SCF binding to IkappaB?
UbcH5
85
What Ub linkage does Cdc34 induce on IkappaB?
K48
86
What is the role of E2 ligase UbcH5?
To add the first Ub onto IkappaB
87
What happens after the first Ub has been added?
UbcH5 is removed and Cdc34 (E2) is added and forms the polyUb chain
88
What does the K48 polyUb chain on IkappaB induce?
IkappaB to undergo proteasomal degradation
89
How do TLRs and IL-1Rs induce MAPK signalling?
Instead of using Raf, they use Tpl2
90
In unstimulated cells, what does Tpl2 exist as?
An inactive complex with Abin2 and p105
91
How does IL-1Rs induce Tpl2 activation?
Tak1 phosphorylates and activates IKKbeta -> phosphorylates p105 -> promotes K48 linked polyUb to p105 -> targeted for degradation -> Tpl2/Abin1/p105 complex dissociates -> Tpl2 is released -> ERK1/2 activation
92
Why does Tpl2 exist as an inactive complex?
It stabilises it and maintains its expression in the cell
93
What does Abin1 KO prevent?
Prevents ERK1/2 activation since Tpl2 is destabilised and not present in the cells
94
What domain does Abin1 have?
A UBAN domain which can bind K63 linked chains and M1 linked chains
95
Is Abin1 binding to K63 and M1 linked chains essential for ERK1/2 signalling?
No
96
What are upstream of JNK?
MKK4 and 7
97
What activates MKK4 and 7?
Tak1
98
What is upstream of p38?
MKK3, 6 and 4
99
what activates MKK3 and 6?
Tak1 and Tpl2
100
How do you prove the requirement for Tak1?
Overexpression studies
Tak1 inhibitors
Loss of function studies
101
what is the issue with Tak1 inhibitors?
they often inhibit other kinases too
102
What happens in you KO Tab2/3 OR Tak1 OR Tab1?
Embryonic lethality
103
Due to embryonic lethality, how is this studied?
Fibroblasts can be isolated from mouse embryos
104
What happens in you KO Tak1?
Prevents IL-1 induced JNK activation
105
What happens in you KO Tab1?
Does not prevent IL-1 induced JNK activation
106
What happens in you KO Tab2?
Does not prevent IL-1 induced JNK activation
107
Why may KO of Tab2 induce this phenotype?
Tab3 could compensate
108
What happens if you KO Tak1 in bone marrow derived macrophages?
They will die during M-CSF induced differentiation
109
What happens if you KO Tak1 in myeloid cells?
Increased levels of ROS
110
What is Ask1?
A member of the MAP3K family
111
How does ROS induce p38 MAPK activation?
TLR4 activation -> ROS increase -> Ask1 activation -> activate p38 MAPK
112
Is Tak1 involved in peritoneal macrophages?
No
113
How does Tak1 KO effect B cells?
Stops TLR induced MAPK activation in B cells
114
Is Tak2 and 3 required in macrophages?
No
115
Is Tak2 and 3 required in B cells?
Yes
116
What is needed to completely block Tak1 signalling?
Loss of Tab1, 2 and 3
