LEC 8: Ubiquitination

Question 1

Ubiquitination

Answer 1

Proteins are marked for Degradation by the addition of Ubiquitin Molecules

Question 2

The Proteosome

Answer 2

A cylindrical protein that functions to degrade all types of proteins
- Degrades via chemical reaction of proteolysis, breaks peptide bonds

Question 3

Ubiquitin Conjugation

Answer 3

E1: Ubiquitin-activating enzyme
E2: Ubiquitin-conjugating enzyme
E3: Ubiquitin Ligase

Question 4

The Ubiquitination Enzymes: E1

Answer 4

E1 activates ubiquitin through a high energy thioester linkage to a Cys side chain on the E1 protein

Question 5

The Ubiquitination Enzymes: E2

Answer 5

Specific E2’s function in specific pathways

Question 6

The Ubiquitination Enzymes: E3

Answer 6

E3 (ubiquitin Ligase) transfers ubiquitin to the target molecule.
- Different E3 enzymes recognise different destruction signals in proteins.

Question 7

Activation of Ubiquitin Ligase

Answer 7

1. Phosphorylation
2. Ligand-binding induced conformational change
3. Accessory protein binding induces conformational change

Question 8

Ubiquitin and the 4 Lysines

Answer 8

There are 4 lysines (K) in ubiquitin that can form bonds with other ubiquitins.

• The type of bond determines the fate of the target protein.
• A single target protein can be modified at a number of different lysines with either a single ubiquitin, or a polyubiquitin chain or a combination of both.

Question 9

The N-End Rule

Answer 9

The susceptibility of a protein to ubiquitin-mediated degradation is dictated by the N-terminal amino acid.

• There are a number of destabilising amino acids at the N-end (Arg, Lys, His, Phe, Leu, Tyr, Trp, Ile, Asp, and Gln). This is known as the N-end rule
• Degradation initiates with the binding of a ubiquitin to the N-end

Question 10

Activation of a Destruction Signal

Answer 10

1. Phosphorylation by a protein kinase
2. Unmasking by protein dissociation
3. Creation of a new terminus by proteolytic cleavage

Question 11

Regulation of Cellular Processes

Answer 11

E.g. Epidermal Growth Factor Receptor Tyrosine Kinase (EGFR Tyrosine Kinase)

• The addition of ubiquitin to a protein is tightly regulated by a series of accessory factors that modulate the activity of the E3.
• Regulation of Epidermal Growth Factor Receptor Tyrosine kinase signal transduction is an example of how E3 regulates cellular processes

Question 12

Non-Degradative Ubiquitination

Answer 12

Ubiquitin addition does not always lead to degradation of the target protein, can change the susceptibility of the protein to other modifications such as:

• Methylation, Phosphorylation
• Invading pathogens are recognised by PRR (Pathogen Recognition Receptors)
• Toll like receptors are PRRs. They sense pathogens and communicate a signal through to the NF-kB transcriptional activator

Question 13

TLR Signal Transduction Pathway

Answer 13

1. Activation of the receptor kinase leads to ubiquitination of TRAF.
2. The ubiquitinated TRAF increases the susceptibility of the NF-kB/IkB complex to phosphorylation.
3. Phosphorylation causes release of the NF-kB transcription activator allowing it to enter the nucleus
4. Where it activates the expression of target genes

Question 14

Control of Transcription

Answer 14

• The transcriptional activator recruits RNAPII complex to the promoter. Part of the complex is an E3 ligase.
• Once transcription initiates the E3 ligase ubiquitinates the activator rendering it inactive
• This prevents multiple rounds of transcription initiation and maintains strict control over promoter activity

Question 15

Addition of ubiquitin can affect susceptibility to other modifications

Answer 15

Addition of ubiquitin to H3 and or H2B affects the susceptibility of these proteins to methylation.