4.3 Ubiquitin-like proteins Flashcards
Ubiquitin-like proteins
Once people began to look carefully other protein modifiers were identified
What did they find was shared? what was different?
They found proteins that shared structural conservation with ubiquitin but are not similar in the amino acid sequnece, named it ubiquitin-like proteins (Ubls)
Comserved 3D structure called ubiquitin-fold
The ubiquitin-fold
Sumo and ubiquitin
At the structural level are highly conserved
But they are not that conserved at an amino acid level
From a review in 2005
Taking the structures of
Ubiquitin
and Ubiquitin like proteins
- Sumo
- Nedd8
Superimposed them and you can see the conserved 3D structure
They look like ubiquitin but are not
UBL’s (ubiquitin like proteins)
What do they all share?
In some cases what is thought to be not required?
How diverse are the functions of UBLs?
Cross talk betwen Ub and Ubl?
All Ubls share similar but distinct attachment machinery to ubiquitin
In some cases the E3 equivalent is not always required
The functions of Ubl proteins is as diverse as that of Ub
Corss talk between Ub and Ubl attachment machinery
Ub and Ubl are distinct but are very much interlinked
Some of the substrates are now known
Involved in a wide range of processes
SUMO
Small ubiquitin-like modifier probably the best studied Ubl
How similar is the sequence identiy?
What is the main difference in SUMO?
Shows about 18% sequence identifiy with ubiquitn but very similar structurally
Main difference is N-terminal extension that makes SUMO ~11kDa
N-terminal extension
Sumo is longer at the N-terminal end
SUMOylation
SUMO is much more divergent across evolution than ubiquitin
How many genes does yeast have the encode SUMO? How many do humans have? of which how many are identical?
The attachment pathway uses E1, E2 and E3 analogues - how many of them are there?
Yeas has one gene (Smt3)
Humans have 3 (SUMO1-3) plus a probable pseudogene SUMO4.
SUMO 2 and 3 are almost identical in sequence and are considered to be functionally redundant
E1 is a dimer
E2 has only 1
E3 there is only 10
SUMOylation
Where is SUMO attached?
What is different about SUMO in comparison to ubiquitin?
What is the consensus site?
Is polySUMOylation possible?
Does SUMO1 have consensus lysine?
Like Ub, SUMO is covalently attached to lysine residues via C-terminal glycine
Unlike ubiquitin a consensus site for modification cab be indentified:
4KxE/D
Where 4 = hydrophobic residue
x = anything
polySUMOylation is possible
SUMO1 doesnt have consensus lysine so is considered to be for monoSUMOylation or acts as chain terminator
Like Ub, SUMOylation is dynamic with SENPs removing SUMO
General model for SUMOylation
Mono
Poly
SUMO1 terminating
SUMOylation
What does SUMO do? is it all linkd to Ub?
However
When SUMO and Ub are both involved what happens?
Is SUMO related to proteasome?
Huge range of roles for SUMO not all linked to Ub
However in many cases SUMOylation antagonizes ubiquitylation
Sumoylation also indirectly linked to proteasome degradation by SUMO-targeted ubiquitin ligases - STUbLs
Ubiquitin ligases that bind to SUMOylated proteins and add K48-linked Ub chains
One example of one system where SUMOylation is very important
The role of SUMO in maintaing genome integrity
DNA genome is under constant attack - such as?
Exogenous chemicals
UV
Ionizing radiation
Errors during DNA replication
All cells have very stringent DNA damage responses to detect and repair damage
DNA repair, ubiquitin and SUMO
Is all DNA damage the same?
All DNA damage is not the same - there is not ONE DNA repair pathway becasue different things damage DNA differently
There are multiple types of DNA damaged repaired by distinct systems
When DNA damage doesnt work you get mutations and disease, most frequently cancers
DNA repair, ubiquitin and SUMO
Is there overlap between the enzymes involved in DNA repair?
There is overlap between different repair pathways
- Common enzymes
- If one pathway doesnt repair damage another will have a go
Huge range of roles for both Ub and SUMO in repair systems
Proteasomal and non-proteasomal processes are both involved
DNA repair, ubiquitin and SUMO
Today we are going over these two
- Post-replicative DNA repair
- Repair of double strand breaks
And how Ub and SUMO are invovled
Post-replicative DNA repair
DNA damage can cause stalling of replication forks
What can stalled replication lead to?
What are the 2 pathways for repairing DNA to prevent stalling?
Stalled replication can led to cataastrophic changes to DNA such as trisomy or chromosome breaks
2 Pathways for repairing DNA
- Trans-lesion synthesis
- Template switching pathway
Post-replicative DNA repair
What does the trans-lesion synthesis pathway involve?
What do these polymerases do?
Template switching pathway uses sister chromadtid as template for repair - is this prone to errors
Trans-lesion synthesis involves specialized DNA polymerase (n, I, k, REV1 and E) to randomly introduce bases opposite legion –> these are error prone but this is still better than a stall
Template switching pathway is error free
Post-replicative DNA repair
Both of these pathways involve the proliferating cell Nuclear Antigen protein (PCNA)
What does PCNA do?
The ubiquitylation status of PCNA determines which of the two repairing pathways is used
PCNA acts as homotrimer that binds DNA serves as a binding platform for other repair enzymes
Strucute of the PCNA trimer
The DNA strand is in the middle
Clever structure for accumulating things around the site of DNA damage
Post-replicative DNA repair
PCNA…
What happens?
Replication fork
PCNA is travelling along
When the replication fork encounters damage you get ubiquitination
Rad6-and Rad18 stick Ub on the PCNA
The PCNA then recruits Poln
Which then randomly introduces bases
On encountering damage PCNA is monoubiquitylated on K164
Monoubiquitylation recruits polymerases such as Poln, which randomy introduces bases
Post-replicative DNA repair
How do you get switching between the trans-lesion and template switching
PCNA is initially ubiquinatied - the chain is made longer by another E3 enzyme in this case it is called Rad5 with Ubc13 and mms2
It is polyubiqunaited
If PCNA is K63 polyubiquitylated (i.e forms K63 chains) the template switching pathway is used
Post-replicative DNA repair
What does
In yeast SUMOylation of PCNA on K164 inhibits the asembly of repair complexes by recruiting inhibitor Srs2
In mammals a USP performs same function
Xeroderma Pigmentosum XP
What type of this disease is?
What are patients unable to repair?
What does sunlight exposure lead to?
What mutations cause XP?
XP variant 5 is caused by?
What are current treatments for XP?
What is the survival percentage?
Autsomal recessive disease
Patients unable to repair UV induced DNA damage
Minimal exposure to sunlight leads to metastatic malignant melanoma and carcinoma
XP variants 5 caused by mutations in Poln, trans-lesion pathway no longer funcational
Current treatment for all XP variants is to minimize UV exposure and treat cancers as they appear
Only 40% of XP patients survive past 20 - some manage to survive into 40s
BRAC1
Breast Cancer Associated 1
What are BRAC1 linked to?
If a woman has an abnormal BRAC1 gene what is her chance of getting breast cancer? and ovarian cancer?
What is the treatment for BRAC1?
BRAC1 mutations in BRAC1 known to cause massive risk in incidence of breast and ovarian cancer
Women with abnormal BRAC1 gene have 80% chance of getting breast cancer in their life-time
Also have 55% increased risk of getting ovarian cancer
Treatment - Double breast vasectomy - Angelina Jolie
BRAC1
Breast Cancer Associated 1
What type of ubiquitin ligase is BRAC1?
What is the substrate?
Which pathway does it function?
BRAC1 is a RING-type ubiqutin ligase
The exact susbtrate remains unknown
Functions in pathway that repairs double strand DNA breaks by homologous recombination
Repair of double strand breaks - DSBs
Is the most dangerougs type of DNA damage as all DNA related process are affected
Repair involves complex series of PTM and subsequent recruitment of repair proteins
Depending on what proteins are recruited damage is either repaired by homologus recobination HR or non-homologous end joining NHEJ pathways
Repair of double strand breaks DSBs
Initial event is phosphorylation of H2AX which then recruits the DNA damage check point protein MDC1 which is then in turn phosphorylated by the kinase ATM
This phosphorylaition leads to the recruitment of several Ub ligases that ultimately promote the Ub of H2A and other chromatin proteins
This Ub cascade then leads to the recruitment of BRAC1/ and or 53BP1
BRAC1 initiates the HR pathway - 53BP1 initiates NHEJ
Repair of double strand breaks DSBs
SUMOylation is also heavily involved in DSB repair
Both BRAC1 and 53BP1 are positively regulated by SUMOylation (BRAC1 is SRUbl)
SUMOylation of MDC1 promotes is Ub/proteasome mediated turnover by recruitment of STUBl
Conclusion
Ubiquitin and Ubiqutin-like proteins are some of the most important post translational modifications that exist in Eukaryotic cells
Ubiquitin is not just for degradation despite what you might read in text books
