4.3 Ubiquitin-like proteins

Question 1

Ubiquitin-like proteins

Once people began to look carefully other protein modifiers were identified

What did they find was shared? what was different?

Answer 1

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

Question 2

The ubiquitin-fold

Sumo and ubiquitin

At the structural level are highly conserved

But they are not that conserved at an amino acid level

Answer 2

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

Question 3

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?

Answer 3

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

Question 4

Some of the substrates are now known

Involved in a wide range of processes

Question 5

SUMO
Small ubiquitin-like modifier probably the best studied Ubl

How similar is the sequence identiy?

What is the main difference in SUMO?

Answer 5

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

Question 6

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?

Answer 6

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

Question 7

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?

Answer 7

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

Question 8

General model for SUMOylation

Mono

Poly

SUMO1 terminating

Question 9

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?

Answer 9

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

Question 10

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?

Answer 10

Exogenous chemicals
UV
Ionizing radiation
Errors during DNA replication

All cells have very stringent DNA damage responses to detect and repair damage

Question 11

DNA repair, ubiquitin and SUMO

Is all DNA damage the same?

Answer 11

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

Question 12

DNA repair, ubiquitin and SUMO

Is there overlap between the enzymes involved in DNA repair?

Answer 12

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

Question 13

DNA repair, ubiquitin and SUMO

Today we are going over these two

1. Post-replicative DNA repair
2. Repair of double strand breaks

And how Ub and SUMO are invovled

Question 14

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?

Answer 14

Stalled replication can led to cataastrophic changes to DNA such as trisomy or chromosome breaks

2 Pathways for repairing DNA

1. Trans-lesion synthesis
2. Template switching pathway

Question 15

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

Answer 15

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

Question 16

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

Answer 16

PCNA acts as homotrimer that binds DNA serves as a binding platform for other repair enzymes

Question 17

Strucute of the PCNA trimer

The DNA strand is in the middle

Clever structure for accumulating things around the site of DNA damage

Question 18

Post-replicative DNA repair

PCNA…

What happens?

Answer 18

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

Question 19

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

Answer 19

If PCNA is K63 polyubiquitylated (i.e forms K63 chains) the template switching pathway is used

Question 20

Post-replicative DNA repair

What does

Answer 20

In yeast SUMOylation of PCNA on K164 inhibits the asembly of repair complexes by recruiting inhibitor Srs2

In mammals a USP performs same function

Question 21

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?

Answer 21

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

Question 22

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?

Answer 22

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

Question 23

BRAC1

Breast Cancer Associated 1

What type of ubiquitin ligase is BRAC1?

What is the substrate?

Which pathway does it function?

Answer 23

BRAC1 is a RING-type ubiqutin ligase

The exact susbtrate remains unknown

Functions in pathway that repairs double strand DNA breaks by homologous recombination

Question 24

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

Question 25

Repair of double strand breaks DSBs

Answer 25

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

Question 26

Question 27

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

Question 28

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