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BGM2002 CS > L5 - Role of PTMs in NF-kB signalling pathway > Flashcards

L5 - Role of PTMs in NF-kB signalling pathway Flashcards

1
Q

what is lysine ubiquitination?

A

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

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2
Q

how is ubiquitnation different from other modifications?

A

other modifications involve the addition of small chemical groups to proteins

ubiquitination is the addition of another protein so needs conjugation

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3
Q

what is ubiquitin?

A

a 76 amino acid protein

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

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4
Q

how can ubiquitin form so many different chains?

A

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

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5
Q

what is the 8th type of modification of ubiquitin?

A

modification through the amino terminal methionine residue

it has an amino group that can be conjugated to ubiquitin

M1 or linear linkage

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6
Q

modifications of ubiquitin

A

7 types of modifications through conserved lysine residues

an 8th modification through the amino terminal methionine

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7
Q

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

A

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

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8
Q

what are the 3 classes of protein that mediate ubiquitination?

A

E1
E2
E3

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9
Q

E1

A

ubiquitin activating enzyme

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10
Q

E2

A

ubiquitin conjugating enzyme

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11
Q

E3

A

ubiquitin ligase

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12
Q

how does ubiquitination occur?

A
  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
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13
Q

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

E1, E2, E3

A

2 E1
35 E2
617 E3

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

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14
Q

what are the different types of E3 ligases?

A

HECT domain E3 ligases

ring ringer or U-box E3 ligases

CUL based ligases

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15
Q

HECT domain E3 ligases

A

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

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16
Q

U-box E3 ligases

A

interact with the E2 and the substrate

bring them into close proximity so ubiquitination can occur

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17
Q

CUL E3 ligases

A

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

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18
Q

what are the effects of different ubiquitin chains?

A

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

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19
Q

what chains mediate proteasomal degradation?

A

K48 and K11

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20
Q

what chains mediate protein interactions and localisation?

A

mono and multi-mono ubiquitination

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21
Q

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

A

K63 chains

22
Q

what chains mediate NF-kB activation?

A

K63 and linear chains

23
Q

what is NF-kB?

A

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

24
Q

how is NF-kB activated?

A
  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
25
Q

what is NF-kB made up of?

A

RelA and p50

26
Q

how is the IKK complex activated?

A

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

27
Q

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

A

for years it was unclear

missing link is ubiquitin

was missed as ubiquitination is lost quickly

28
Q

what is the IkB complex (IKK) made of?

A

2 catalytic subunits
• IKK-alpha
• IKK-beta

a regulatory subunit
• NEMO / IKK-gamma

29
Q

NEMO

A

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

30
Q

what is proximity induced activation?

A

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

31
Q

what is the scaffold in the NF-kB pathway?

A

ubiquitin chains

32
Q

what is RIP-1?

A

receptor interacting protein 1

33
Q

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

A

RIP-1

34
Q

how does RIP-1 form the scaffold?

A

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

35
Q

what are DUBs?

A

deubiquitinases

95 in the human genome

remove ubiquitin chains

have specificities for different proteins and different chain types

36
Q

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

A

A20 and CYLD

negative regulators

deconstruct the scaffold

37
Q

mutations in CYLD?

A

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

38
Q

what does the IkB family consist of?

A

IkB-alpha
IkB-beta
IkB-epsilon

all have conserved lysine residues that can be ubiquitinated to degrade them

39
Q

what type of E3 ligase mediates ubiquitination of IkB?

A

CUL-1 E3 ligase (=SCF complex)

40
Q

what is the SCF complex composed of?

A

Skp1
Cullin1
F-box
Rbx1

41
Q

what is the F-box?

A

protein responsible for substrate recognition

42
Q

what is the F-box for IkB-alpha?

A

beta-TrCP

43
Q

what does beta-TrCP do?

A

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

44
Q

regulation of NF-kB function?

A

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

45
Q

what can NF-kB regulate?

A
  • nuclear import
  • stability
  • DNA binding
  • transcriptional activity
  • target gene specificity
  • interaction with other proteins
46
Q

modifications of the RelA NF-kB subunit?

A

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

47
Q

evolutionary conservation of RelA PTM sites

A

important modifications are conserved
• Cys38 - oxidation
• K218/221 - acetyl & phospho
• Ser276 - phospho

are key mechanisms of regulating NF-kB activity in the cell

48
Q

cysteine modifications

A

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

49
Q

hydroxylation of cysteine

A

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

50
Q

sulfhydration of cysteine

A

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

BGM2002 CS (14 decks)

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