Ubiquitin Signalling (1)

Question 1

What happens if mutated proteins are degraded?

Answer 1

Get accumulation of the mutated proteins leading to toxic protein aggregates

Question 2

What are the two main cellular pathway which result in the disposal of damaged or misfolded proteins?

Answer 2

Autophagy pathway

Proteasomal pathway

Question 3

Out of 22k genes which are encoded in human cells, how many are part of the ubiquitin system?

Answer 3

~1,000

Question 4

How many amino acids are in a ubiquitin molecule?

Answer 4

76

Question 5

How does ubiquitin bind to proteins?

Answer 5

Post translationally via lysine residues

Question 6

What are isopeptides?

Answer 6

A bond between the C-terminus of ubiquitin and the epsilon minor groups of the side chain of the lysine residue

Question 7

What do isopeptide bonds provide?

Answer 7

Flexibility

Question 8

How does the addition of ubiquitin alter the proteins function?

Answer 8

Target it for degradation
Changes its subcellular location
Bind to downstream effector

Question 9

How many different lysine residues can ubiquitin bind to?

Answer 9

7 different lysine residues

Question 10

As well as lysine, what other site can ubiquitin bind to? And what type of bond does it form?

Answer 10

N-terminal Methionine

Peptide bond

Question 11

What is monoubiquitylation?

Answer 11

Addition of a single ubiquitin onto a substrate

Question 12

What is multiple ubiquitylation?

Answer 12

Multiple lysine residues on a single substrate are modified with Ub

Question 13

What is polyubiquitylation?

Answer 13

Addition of Ub onto more Ub

Question 14

What is heterotypic polyubiquitylation?

Answer 14

Polyubiquitin chains with different linkages

Question 15

What are the two types of heterotypic polyubiquitylation?

Answer 15

Mixed

Branched

Question 16

What is mixed heterotypic polyubiquitylation?

Answer 16

Can start off with Ubs being via one link and then changes to a different linkage e.g. K63 to begin with and then K48

Question 17

What is branched heterotypic polyubiquitylation?

Answer 17

You have a polyubiquitylated chain, then one of the Ub have 2 linkages and forms a branch

Question 18

What post translational modifications can happen to Ub?

Answer 18

Phosphorylated
Acetylated
Addition of other ubiquitin like modifiers

Question 19

Name 2 examples of Ub like modifiers

Answer 19

SUMO

NedA

Question 20

How does ubiqutylation regulate downstream cellular processes?

Answer 20

Proteins with ubiquitin binding domains (UBDs) will recognises the Ub signals and couple to downstream responses

Question 21

How is Ub controlled and regulated?

Answer 21

By deubiquitinases (DUBs)

Question 22

What do DUBs do to Ub?

Answer 22

They cleave isopeptide bond between the Ub and its substrate or between Ub molecules -> reverse the protein signal e.g. stop it being degraded by the proteasome
Antagonise the function of E3

Question 23

How many DUBs are there?

Answer 23

~100

Question 24

How many UBDs are there?

Answer 24

~20

Question 25

How many proteins contain UBDs?

Answer 25

~250

Question 26

How many E1 are there?

Answer 26

~2

Question 27

How many E2 are there?

Answer 27

~40

Question 28

How many E3 are there?

Answer 28

~600

Question 29

How many proteins are regulated by Ub?

Answer 29

~7k

Question 30

What is Lys48 involved in?

Answer 30

Proteasomal degradation

Question 31

What is Lys63 involved in?

Answer 31

NF-kappaB signalling (PAK1 is activated by Lys63 polyUb -> NF-kappaB)

Question 32

What is Met1 involved in?

Answer 32

TNFR signalling (by activating IKK)

Question 33

What is Lys6 involved in?

Answer 33

DNA damage response and dispose of damaged mitochondria by autophagy

Question 34

What are the 4 Ub patches?

Answer 34

Ile44 patch Phe4 patch Ile36 patch Asp58

Question 35

What is the function of the patches?

Answer 35

To recognise different chains

Question 36

Give the structure of Lys6-linked diubiquitin

Answer 36

One of the Ubs has a Ile44 patch and the other had a Ile36 patch at the interface (where they join) Closed conformation

Question 37

Give the structure of Lys11-linked diubiquitin

Answer 37

Has two Ile36 patches at the interface | Closed conformation

Question 38

Give the structure of Lys48-linked diubiquitin

Answer 38

Has two Ile44 patches at the interface | Closed conformation

Question 39

Give the structure of Met1 and Lys63-linked diubiquitins

Answer 39

Open conformation

Question 40

Name the UBDs which recognise Ile44 patches in monoUb

Answer 40

``` Ub interaction motif (UIM) MIU (Motif interaction Ub) - reverse of UIM CUE GAT UBA ```

Question 41

Name a UBD which doesn't always bind to Ile44

Answer 41

zinc finger UBDs

Question 42

What are the types of Zinc finger UBDs? And what do they bind

Answer 42

UVZ - recognises Ile44 patch | IsoT - recognises Ile36 and the C-terminal tail of Ub

Question 43

What is special about IsoT?

Answer 43

Since it can bind to the C-terminal tail of Ub, it can bind to free Lys76 - important in Ub chains which have been cleaved and released and need to be broken down

Question 44

Name a UBD which recognises polyUb chains

Answer 44

Rap80

Question 45

Describe Rap80

Answer 45

it has 2 UIM motifs which occur in tandem Each recognise Ile44 patches of one Ub It only binds to polyUb which are K63 linked

Question 46

Why does Rap80 only recognised K63 linked polyUb?

Answer 46

Due to the linker which seperates the 2 UIM

Question 47

Name 2 single UBDs which can recognise 2 Ubs

Answer 47

TAB2 ZNF | hHR23 UBA

Question 48

Describe TAB2 ZNF

Answer 48

Found on the TAB2 protein Binds K63 linked polyUb Has two interfaces which both recognise a Ile44 patches

Question 49

What is TAB2?

Answer 49

Part of the PAK1 complex involved in NF-kappaB signalling

Question 50

Describe hHR23 UBA

Answer 50

Found on hHR23 Recognises K48 chains - targets them for degradation by the proteasome Has 2 interfaces

Question 51

How is ZNF able to bind to K63?

Answer 51

Change it to a closed conformation from an open one

Question 52

Name a UBD which recognises PolyUb Met1

Answer 52

NEMO

Question 53

What are the 3 classes of Ub ligase?

Answer 53

RING HECT E3 ligase RER

Question 54

What is a RING Ub ligase?

Answer 54

A substrate and a charged E2 | Gets a direct transfer of Ub onto the substrate

Question 55

What is a HECT E3 ligase?

Answer 55

Accept a charged Ub from a charged E2 ligase and passes it onto the lysine of the substrate

Question 56

What is an RER Ub ligase?

Answer 56

Hybrid of a RING and HECT E3 ligase

Question 57

How does HECT E3 ligases work?

Answer 57

A charged E2 entraps the HECT E3 ligase -> Ub is transferred onto a cysteine-kappa-cysteine of the HECT -> Ub is transferred onto a Lys of the substrate

Question 58

How are proteins targeted for degradation?

Answer 58

UBE2C will add a single Ub onto the substrate (priming) and then goes away -> UBE2S will then add more Ub onto the initial substrate (elongation) - causes branched chains

Question 59

Which chains are better for degradation?

Answer 59

Branched (K48 linked with more than 4 Ub)

Question 60

What are homotypic chains better for?

Answer 60

Signalling

Question 61

Why is protein degradation important?

Answer 61

Protein aggregates are a marker of neurodegenerative diseases such as Huntington's, Parkinson's and Alzheimers

Question 62

What are the 2 subunits of the proteasome?

Answer 62

a 19S regulatory particle and a 20S co-particle

Question 63

What is the function of the co-particle

Answer 63

To chop up the proteins by the 3 different proteolytic sites it contains

Question 64

Why are large protein aggregates not targeted by the proteasome?

Answer 64

Since they have large globular heads

Question 65

How does the proteasome work?

Answer 65

RP is the lid and has receptors which bind to the Ub chains -> Proteins enter as folded proteins -> ATPase pulls it through -> it unfolds as it enters the CP