Protein Ubiquitination

Question 1

Ubiquitin:

Answer 1

Small Globular 76 Amino Acid Protein
8.5 kDa protein
Found in all eukaroytic cells, involved in many cellular processes
Human vs Yeast: 96% Identical
There are 4 human genes that each express a pre-Ub protein with a different C-terminal sequence. These different C-terminal extensions are removed to each generate identical Ub proteins ending in GlyGly.

Question 2

The carboxyl group of the C-terminal Gly of Ub acts as an acceptor to form an isopeptide bond with an amino group in the substrate.
TRUE OR FALSE

Answer 2

FALSE
The carboxyl group of the C-terminal Gly of Ub acts as a DONOR to form an isopeptide bond with an amino group in the substrate.

Question 3

ε-amino group of a lysine residue of the substrate protein (or sometimes the N-terminal NH2 group) acts as an acceptor.
TRUE OR FALSE

Answer 3

TRUE
Ub also has Lys 6, 11, 27, 29, 33, 48, 63 and N-terminal amino group (Met1) that can act as acceptors for reacting with C-termini of further Ub donors, to form chains.

Question 4

Homotypic polyubiquitin chains:

Answer 4

Contain a single linkage type (i.e. K48-K48-K48 etc.)

Question 5

Heterotypic polyubiquitin chains:

Answer 5

Contain different linkage types within the same chain

Question 6

‘Linear’ means that the N-terminal NH2 of Met1 of Ub (NOT Lys) is the acceptor for the C-terminus of the donor Ub. TRUE OR FALSE

Answer 6

TRUE

Question 7

In branched polyubiquitin chains, a single Ub is extended at 2 or more Lys residues.
TRUE OR FALSE

Answer 7

TRUE

Question 8

Extended chain conformation:

Answer 8

Linking the C-terminal carboxyl of the incoming donor Ub (distal to substrate) to Lys63 or Met1 of the acceptor Ub (proximal to the substrate) positions the C-terminal Gly of the donor as far away as possible from the previous Ub-Ub or Ub-substrate linkage made by the acceptor, generating an extended chain conformation.

Question 9

Compact chain conformation:

Answer 9

Linking the donor to Lys48 of the acceptor Ub gives a compact structure, with the donor (distal) Ub positioned on the side of the acceptor Ub.

Question 10

Polyubiquitin chains with different linkages are recognized by different BLANKS

Answer 10

Ubiquitin Binding Domains (e.g. UBA, UIM, UBZ)

• UBDs dock non-covalently onto Ub and Ub chains.
• Some UBDs recognise a particular chain toplogy and some require a particular Ub chain length.
• Others dock onto the substrate as well as the attached Ub
• There are UBDs within a wide variety of regulatory proteins whose functions determine the role and the fate of the ubiquitinated protein e.g. lysosomal degradation

Question 11

Ub is chemically activated in a two-step reaction by the E1, then transferred to an E2 by condensation.
TRUE OR FALSE

Answer 11

FALSE
Ub is chemically activated in a two-step reaction by the E1, then transferred to an E2 by TRANSTHIOLATION.

1. The E1 uses ATP to activate the C-terminal carboxyl group of Ub (Gly 76) to form a mixed anhydride with AMP that binds non-covalently to the E1.
2. The Ub–adenylate is then transferred to a cysteine residue of E1, resulting in a Ub–E1 thiolester and release of AMP.
3. A second Ub is adenylated.
4. The first Ub from Ub-E1 is transferred to an E2

Question 12

E2 Ub-conjugating enzymes:

Answer 12

A ubiquitin-conjugating (UBC) domain on the E2 docks non-covalently with Ub-E1.
The Ub-E1 transfers its first Ub from the Cys of E1 to the E2, resulting in a thiolester bond between C-terminal carboxyl of Ub and the catalytic Cys residue on the E2.

Question 13

E2s can contact E1 and E3 at the same time

TRUE OR FALSE

Answer 13

FALSE
E2s cannot contact E1 and E3 at the same time – the binding sites overlap.
The Ub-E2 must dissociate from the E1, so that it can interact with an E3.

Question 14

There are two mechanistically distinct types of E3 ubiquitin–protein ligases: What are they?

Answer 14

HECT domain E3s

RING E3s

Question 15

How do HECT domain E3s work?

Answer 15

Ub is transferred from the E2 to a thiol group on HECT domain E3s and the Ub is then conjugated to a lysine side chain of the substrate (and as the chain grows, to a K in a Ub attached to the core substrate).

The result is formation of an isopeptide bond between Ub and the substrate.

Question 16

How do RING E3s work?

Answer 16

For RING E3s and U-box E3s, the E3 functions as an adapter or matchmaker, juxtaposing the E2-Ub thiolester and substrate to allow transfer of Ub directly from the E2 to an NH2 group of the substrate (and as the chain grows, to a K in a Ub attached to the core substrate).

The result is formation of an isopeptide bond between
Ub and the substrate.

Question 17

The RING type E3 ubiquitin ligases

Answer 17

• Most E3s contain a RING finger or structurally related U-box, which act as matchmakers – bring substrate and charged E2 together
• In The RING finger domain, a consensus sequence
  containing 8 cysteine and histidine residues coordinate
  two Zn ions in a ‘cross-braced’ fashion.
• RING is the 15th most common domain in human genome
• The RING finger is not inherently catalytic.
• The RING binds to the E2-Ub and brings it close to the substrate acceptor Lys
• The RING allosterically activates the E2

Question 18

As the 3-step process advances, specificity increases:
E1 interacts with all E2s, which interact with a more limited subset of E3s, which in turn each target a limited array of protein substrates.
How many of each E(X) are there in humans, and how is specificity achieved?

Answer 18

2 Human E1s
~38 Human E2s
100s of Human E3s
1000s of Substrates

Specificity:
When working with a RING-type E3, the E2 determines the Ub chain type and the E3 selects the substrate.

Question 19

3 Step ubiquitin sytstem:

Answer 19

1. Ub+ATP+Mg2+ drives E1 > E1-Ub
2. Transfer of Ub from E1 to E2
3. Interaction of E2-Ub with E3 to cause ubiquitination of the substrate

Question 20

The E3 determines the Ub chain linkage and topology

TRUE OR FALSE

Answer 20

FALSE
The E2 determines the Ub chain linkage and topology

The E2 orients the acceptor Ub in a way that exposes only the favoured Lys residue to its active site (charged with the donor Ub), leading to the formation of Ub chains of a specific linkage.
Some E2s can catalyze both initiation (monoubiquitylation of substrate) and chain extension (polyubiquitylation).
Other E2s have specific roles in Ub chain initiation or elongation so a different E2 is required for the priming monoubiquitylation.
Some E3s work with more than one E2 – one for initiation and another for extension

Question 21

How are substrates recognized by the E3s?

Answer 21

The substrate docks onto recognition sites (“recognins”) on the E3s, which are distinct from the HECT or RING domains.

A wide variety of “recognins” exist that dock onto different types of motifs in the substrates that are to be selected for ubiquitylation by the E3.

1. Sequence motif in the substrate
2. Post-translational modification of the substrate:
3. Post-translational modification of the E3:
4. Via adapter proteins

Question 22

Humans contain six cullins that each interact with a variety of different specificity modules or receptors
TRUE OR FALSE

Answer 22

TRUE

e.g. CUL1 is the cullin, Skp1 is its adapter protein, F box is its substrate

Question 23

Some RING E3 ubiquitin ligases are multisubunit Cullin-RING Ub ligases (CRLs), such as SCF in which substrate specificity and E3 activity are performed by different proteins
How is the gap between the substrate bound to the F-box protein and the Ub on the E2 bridged?

Answer 23

SCF is activated by covalent attachment of Nedd8 to the C-terminal region of the Cullin scaffold, which causes a conformational change in the cullin that allows Rbx1 (the RING E3) to swing freely. The unleashed Rbx1 binds more tightly to the Ub-E2 and can ‘reach’ substrates that have docked onto the F-box ‘substrate receptor’.

Question 24

The E3 docks onto target proteins with an exposed degradation signal (a cullin)
TRUE OR FALSE

Answer 24

FALSE

The E3 docks onto target proteins with an exposed degradation signal (a DEGRON)

Question 25

How many ubiquitins are required for recognition by the proteasome and degradation?

Answer 25

> 4 ubiquitins (up to ~50) in a chain are required for recognition by the proteasome and degradation

Question 26

The proteasome constituents:

Answer 26

19S cap x 2

20S core

Question 27

20S core of the proteasome:

Answer 27

Hollow cylinder stack of 4 heptameric rings

Contains 6 proteolytic sites internally:

• 2x Tryptic
• 2x Chymotryptic
• 2x Peptidylglutamyl-peptidase

Linearized protein required. (Linearisation occurs at the cap that the protein enters)

Intact ubiquitin and small peptide products are released during this ATP-dependent process.

Question 28

19S cap x 2 of the proteasome:

Answer 28

Contains ~20 subunits

• The 19S binds to the 20S while hydrolyzing ATP.
• The C-terminal HbYX motif of the 19S AAA ATPase - works like a key-in-a-lock to induce gate opening.
• UBD recognises tetra-Ub (K48) on the substrate target
• DUBs deubiquitinate the substrate target (Ub is recycled)
• Chaperones unfold the substrate target, which is translocated through the open gate into the inner chamber of the 20S.

Question 29

What can go wrong in Ubiquitin systems to cause disorder and disease?

Answer 29

Example 1: Cervical cancer caused by Human Papilloma Virus (HPV)

• E6-AP is a HECT E3 ligase.
• Human Papilloma Virus expresses the E6 oncoprotein inside infected human cells.
• E6 binds to both p53 ‘guardian of the genome’ protein and to E6-AP.
• Via E6-AP, the p53 is polyubiquitylated (K48 chains).
• The p53 that is tagged by K48-linked chains is degraded by the proteasome.
• Without E6, the E6-AP does not normally ubiquitylate p53.

Example 2: Maternally inherited deletion of UBE3A encoding E6-AP causes Angelman syndrome

• Due to loss of UBE3A gene from the maternally-inherited copy of Chromosome 15.
• UBE3A is on maternal and paternal copies of chromosome 15, but differs in the patterns of methylation (imprinting). Paternal silencing of UBE3A occurs in certain brain regions; only the maternal allele is active in the hippocampus and cerebellum.
• In AS, E6-AP protein is not present in hippocampus and cerebellum.