L5 - Role of PTMs in NF-kB signalling pathway Flashcards
what is lysine ubiquitination?
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
how is ubiquitnation different from other modifications?
other modifications involve the addition of small chemical groups to proteins
ubiquitination is the addition of another protein so needs conjugation
what is ubiquitin?
a 76 amino acid protein
has the ability to form a wide variety of covalently linked chains
how can ubiquitin form so many different chains?
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
what is the 8th type of modification of ubiquitin?
modification through the amino terminal methionine residue
it has an amino group that can be conjugated to ubiquitin
M1 or linear linkage
modifications of ubiquitin
7 types of modifications through conserved lysine residues
an 8th modification through the amino terminal methionine
why was ubiquitination overlooked as a modification for a long time?
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
what are the 3 classes of protein that mediate ubiquitination?
E1
E2
E3
E1
ubiquitin activating enzyme
E2
ubiquitin conjugating enzyme
E3
ubiquitin ligase
how does ubiquitination occur?
- in an ATP driven reaction, E1 takes ubiquitin and conjugates it to itself activating the ubiquitin molecule
- E1 transers ubiquitin to E2
- E2 have specificity for the type of linkage formed
- E3 interact with proteins to be ubiquitinated and bring the E2 into proximity with the substrate
how many of each enzyme is found in the human body?
E1, E2, E3
2 E1
35 E2
617 E3
there are so many E3 as they are the ones that select which proteins get ubiquitinated
what are the different types of E3 ligases?
HECT domain E3 ligases
ring ringer or U-box E3 ligases
CUL based ligases
HECT domain E3 ligases
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
U-box E3 ligases
interact with the E2 and the substrate
bring them into close proximity so ubiquitination can occur
CUL E3 ligases
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
what are the effects of different ubiquitin chains?
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
what chains mediate proteasomal degradation?
K48 and K11
what chains mediate protein interactions and localisation?
mono and multi-mono ubiquitination
what chain mediates NF-kB activation, DNA repair and lysosomal targeting?
K63 chains
what chains mediate NF-kB activation?
K63 and linear chains
what is NF-kB?
a protein complex that controls transcription of DNA, cytokine production and cell survival
how is NF-kB activated?
- transcription factor complex (RelA-p50) sits in inactive form in cytoplasm bound to its inhibitor, IkB
- when cell receives a stimulus (eg. TNF-alpha) a signalling pathway is activated which leads to the activation of the IkB kinase complex (IKK)
- IKK phosphorylates IkB and targets it for K48 linked ubiqutination
- IkB is degraded by proteasome, freeing up NF-kB to go into nucleus to regulate transcription
what is NF-kB made up of?
RelA and p50
how is the IKK complex activated?
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
how is the signal transmitted from the cell surface receptors to the IKK complex?
for years it was unclear
missing link is ubiquitin
was missed as ubiquitination is lost quickly
what is the IkB complex (IKK) made of?
2 catalytic subunits
• IKK-alpha
• IKK-beta
a regulatory subunit
• NEMO / IKK-gamma
NEMO
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
what is proximity induced activation?
cell creates a scaffold that bring components of signalling pathways together to make activation much more efficient
what is the scaffold in the NF-kB pathway?
ubiquitin chains
what is RIP-1?
receptor interacting protein 1
what protein is ubiutinated to form the scaffold in the TNF pathway?
RIP-1
how does RIP-1 form the scaffold?
RIP-1 is modified by 2 chains:
- formation of linear ubiquitin chain
• binds NEMO - has motifs specific for linear chains - 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
what are DUBs?
deubiquitinases
95 in the human genome
remove ubiquitin chains
have specificities for different proteins and different chain types
what are the 2 DUBs important in the role of deubiquitinating RIP1?
A20 and CYLD
negative regulators
deconstruct the scaffold
mutations in CYLD?
lead to a form of benign cancer called cylindromatisis - found in the scalp
what does the IkB family consist of?
IkB-alpha
IkB-beta
IkB-epsilon
all have conserved lysine residues that can be ubiquitinated to degrade them
what type of E3 ligase mediates ubiquitination of IkB?
CUL-1 E3 ligase (=SCF complex)
what is the SCF complex composed of?
Skp1
Cullin1
F-box
Rbx1
what is the F-box?
protein responsible for substrate recognition
what is the F-box for IkB-alpha?
beta-TrCP
what does beta-TrCP do?
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
regulation of NF-kB function?
once in the nucleus, NF-kB can be subject to lots of types of modifications that help determine its function
what can NF-kB regulate?
- nuclear import
- stability
- DNA binding
- transcriptional activity
- target gene specificity
- interaction with other proteins
modifications of the RelA NF-kB subunit?
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
evolutionary conservation of RelA PTM sites
important modifications are conserved
• Cys38 - oxidation
• K218/221 - acetyl & phospho
• Ser276 - phospho
are key mechanisms of regulating NF-kB activity in the cell
cysteine modifications
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
hydroxylation of cysteine
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
sulfhydration of cysteine
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
