L5 - Role of PTMs in NF-kB signalling pathway

Question 1

what is lysine ubiquitination?

Answer 1

involves addition of 76 amino acid ubiquitin protein to the positively charged epsilon-amino group on lysine

conjugation reaction through the C-terminal glycine of ubiquitin

Question 2

how is ubiquitnation different from other modifications?

Answer 2

other modifications involve the addition of small chemical groups to proteins

ubiquitination is the addition of another protein so needs conjugation

Question 3

what is ubiquitin?

Answer 3

a 76 amino acid protein

has the ability to form a wide variety of covalently linked chains

Question 4

how can ubiquitin form so many different chains?

Answer 4

it contains a number of very highly conserved lysine residues on its own structure

it has 7 different lysine residues which are all capable of being ubiquitinated

Question 5

what is the 8th type of modification of ubiquitin?

Answer 5

modification through the amino terminal methionine residue

it has an amino group that can be conjugated to ubiquitin

M1 or linear linkage

Question 6

modifications of ubiquitin

Answer 6

7 types of modifications through conserved lysine residues

an 8th modification through the amino terminal methionine

Question 7

why was ubiquitination overlooked as a modification for a long time?

Answer 7

deubiquitinases are very active so by the time the protein extracts been made, all the ubiquitin chains would have gone

ubiquitin is treated with a proteasome inhibitor so degradation is blocked through the K48 chains

Question 8

what are the 3 classes of protein that mediate ubiquitination?

Answer 8

E1
E2
E3

Question 9

E1

Answer 9

ubiquitin activating enzyme

Question 10

E2

Answer 10

ubiquitin conjugating enzyme

Question 11

E3

Answer 11

ubiquitin ligase

Question 12

how does ubiquitination occur?

Answer 12

1. in an ATP driven reaction, E1 takes ubiquitin and conjugates it to itself activating the ubiquitin molecule
2. E1 transers ubiquitin to E2
3. E2 have specificity for the type of linkage formed
4. E3 interact with proteins to be ubiquitinated and bring the E2 into proximity with the substrate

Question 13

how many of each enzyme is found in the human body?

E1, E2, E3

Answer 13

2 E1
35 E2
617 E3

there are so many E3 as they are the ones that select which proteins get ubiquitinated

Question 14

what are the different types of E3 ligases?

Answer 14

HECT domain E3 ligases

ring ringer or U-box E3 ligases

CUL based ligases

Question 15

HECT domain E3 ligases

Answer 15

interact with E2 protein and the E2 protein transfers the ubiquitin to the E3

E3 then transfers the ubiquitin to the substrate

E3 is acting as a ubiquitin intermediary - has an enzymatic role

Question 16

U-box E3 ligases

Answer 16

interact with the E2 and the substrate

bring them into close proximity so ubiquitination can occur

Question 17

CUL E3 ligases

Answer 17

function as a complex of proteins instead of an individual E3 (like the U-box)

CUL proteins interact with RBX-1 protein which interacts with E2 bringing it in

still proximity based ubiquitination

Question 18

what are the effects of different ubiquitin chains?

Answer 18

they have different effects on the proteins they’re targeting so have different roles in the cell

lots of chains still have an unclear function

Question 19

what chains mediate proteasomal degradation?

Answer 19

K48 and K11

Question 20

what chains mediate protein interactions and localisation?

Answer 20

mono and multi-mono ubiquitination

Question 21

what chain mediates NF-kB activation, DNA repair and lysosomal targeting?

Answer 21

K63 chains

Question 22

what chains mediate NF-kB activation?

Answer 22

K63 and linear chains

Question 23

what is NF-kB?

Answer 23

a protein complex that controls transcription of DNA, cytokine production and cell survival

Question 24

how is NF-kB activated?

Answer 24

1. transcription factor complex (RelA-p50) sits in inactive form in cytoplasm bound to its inhibitor, IkB
2. when cell receives a stimulus (eg. TNF-alpha) a signalling pathway is activated which leads to the activation of the IkB kinase complex (IKK)
3. IKK phosphorylates IkB and targets it for K48 linked ubiqutination
4. IkB is degraded by proteasome, freeing up NF-kB to go into nucleus to regulate transcription

Question 25

what is NF-kB made up of?

Answer 25

RelA and p50

Question 26

how is the IKK complex activated?

Answer 26

in the classical pathway it is a kinase called TAK-1 in the alternative pathway it is a kinase called NIK these kinases phosphorylate the IKK subunits in their activation loop to activate them for kinase activity

Question 27

how is the signal transmitted from the cell surface receptors to the IKK complex?

Answer 27

for years it was unclear missing link is ubiquitin was missed as ubiquitination is lost quickly

Question 28

what is the IkB complex (IKK) made of?

Answer 28

2 catalytic subunits • IKK-alpha • IKK-beta a regulatory subunit • NEMO / IKK-gamma

Question 29

NEMO

Answer 29

has an IKK binding domain in the N-terminus and 2 ubiquitin binding domains in the C terminus the 2 ubiquitin binding domains have specificity for binding the linear form of ubiquitin chain this is critical for NF-kB activation

Question 30

what is proximity induced activation?

Answer 30

cell creates a scaffold that bring components of signalling pathways together to make activation much more efficient

Question 31

what is the scaffold in the NF-kB pathway?

Answer 31

ubiquitin chains

Question 32

what is RIP-1?

Answer 32

receptor interacting protein 1

Question 33

what protein is ubiutinated to form the scaffold in the TNF pathway?

Answer 33

RIP-1

Question 34

how does RIP-1 form the scaffold?

Answer 34

RIP-1 is modified by 2 chains: 1. formation of linear ubiquitin chain • binds NEMO - has motifs specific for linear chains 2. formation of K63 ubiquitin chains • binds to TAK1 kinase complex through its interaction partners - TAB2 and TAB3 - have domains specific got K63 chains RIP-1 brings TAK-1 and IKK close together for efficient activation

Question 35

what are DUBs?

Answer 35

deubiquitinases 95 in the human genome remove ubiquitin chains have specificities for different proteins and different chain types

Question 36

what are the 2 DUBs important in the role of deubiquitinating RIP1?

Answer 36

A20 and CYLD negative regulators deconstruct the scaffold

Question 37

mutations in CYLD?

Answer 37

lead to a form of benign cancer called cylindromatisis - found in the scalp

Question 38

what does the IkB family consist of?

Answer 38

IkB-alpha IkB-beta IkB-epsilon all have conserved lysine residues that can be ubiquitinated to degrade them

Question 39

what type of E3 ligase mediates ubiquitination of IkB?

Answer 39

CUL-1 E3 ligase (=SCF complex)

Question 40

what is the SCF complex composed of?

Answer 40

Skp1 Cullin1 F-box Rbx1

Question 41

what is the F-box?

Answer 41

protein responsible for substrate recognition

Question 42

what is the F-box for IkB-alpha?

Answer 42

beta-TrCP

Question 43

what does beta-TrCP do?

Answer 43

has a WD40 motif which is a reader motif that allows it to interact with phosphorylated substrates can recognise a range of proteins when they become phosphorylated and induce their K48 ubiquitination and degradation

Question 44

regulation of NF-kB function?

Answer 44

once in the nucleus, NF-kB can be subject to lots of types of modifications that help determine its function

Question 45

what can NF-kB regulate?

Answer 45

* nuclear import * stability * DNA binding * transcriptional activity * target gene specificity * interaction with other proteins

Question 46

modifications of the RelA NF-kB subunit?

Answer 46

subjected to lysine acetylation at residues 122/123. 218, 221 and 310 some of these lysines can also be methylated modifications compete with each other modifications at the 218 and 221 sites are particularly important

Question 47

evolutionary conservation of RelA PTM sites

Answer 47

important modifications are conserved • Cys38 - oxidation • K218/221 - acetyl & phospho • Ser276 - phospho are key mechanisms of regulating NF-kB activity in the cell

Question 48

cysteine modifications

Answer 48

cysteines have the ability to form disulphide binds between proteins so they can have a very important structural role in proteins can also be chemically modified can be hydroxylated and sulfhydrated

Question 49

hydroxylation of cysteine

Answer 49

where an agent such as hydrogen peroxide can hydroxylate the cysteine if the cysteine in NF-kB subunits is hydroxylated, its DNA binding ability is totally inhibited potential mechanism by which proteins respond to the redox state of the cell - inhibit NF-kB function under certain cellular conditions

Question 50

sulfhydration of cysteine

Answer 50

addition of a sulfydryl (SH/thiol) group to the cysteine residue TNF-alpha stimulates transcription of the enzyme CSE which generates H2S H2S sulfhydrates the RelA subunit at Cys-38 • increases its ability to bind the co-factor RPS3 (ribosomal protein S3) • stimulates transcriptional activity of NF-kB • stimulates transcription of anti-apoptotic genes