Lecture 11 - Ubiquitin I

Question 1

Why do cells require protein degradation system?

(2 Points)

Answer 1

1. Normal Protein Turnover (i.e. regulating levels of individual proteins)
2. Prevent Aggregation of Damaged/Misfolded Proteins

Question 2

Provide 3 Examples of Diseases associated with altered protein turnover

Answer 2

1. Cancer - altered degradation of cell cycle control proteins (e.g., p53)
2. Cystic Fibrosis - abnormal trafficking and degradation of mutant proteins
3. Alzheimers/Parkinson’s - accumulation of misfolded proteins

Question 3

Define Ubiquitin

(2 Points)

Answer 3

• Small Globular Protein (76aa), with a protruding C-terminal glycine that can be covalently added to substrate proteins
• Highly conserved in eukaryotes, and ubiquitously expressed in all tissues

Question 4

Describe Ubiquitination in terms of:
(i) Mechanism
(ii) Linkage type
(iii) Polyubiquitin Chains

Answer 4

(i) Ubiquitin is added to a substrate protein via covalent linkage between a Lysine residue on substrate and C-Terminal Glycine (Gly76) on Ubiquitin
(ii) Isopeptide bond (as bond is formed from epsilon-amine group)
(iii) Polyubiquitin chains can be formed by the covalent linkage of Ubiquitin to one of 7 Lysine (or Methionine) residues on another Ubiquitin monomer

Question 5

State the three types of Enzymes involved in ubiquitination, and their roles.

Answer 5

E1 Ubiquitin-Activating enzyme - uses ATP to activate ubiquitin monomer

E2 Ubiquitin-Conjugating enzyme - accepts activated Ubiquitin from E1, then attaches it to specific substrate

E3 Ubiquitin-Ligase Enzyme - substrate selection (recruits E2 ligase to particular substrate)

Question 6

Compare the Ubiquitination mechanisms of HECT and Ring-finger E3 Ubiquitin Ligases

Answer 6

• HECT domain-containing E3 Ligases - form thioester intermediates with ubiquitin
• RING-finger domain-containing E3 Ligases - ubiquitin is transferred directly from E2 to substrate protein

Question 7

How does Ubiquitin play a role in the ubiquitin-proteasome system (UPS)?

Answer 7

Ubiquitin serves as cofactor for selective degradation of proteins by the 26S proteasome, with a (n≥4) K-48 Linked chain being the principal signal for degradation

Question 8

Define the 26S Proteasome in terms of:
(i) Structure
(ii) Assembly

Answer 8

(i) Consists of:
* 19S Cap/Regulator (17 subunits) - recognise substrate via K48-linked chain, and uses ATP to unfold it
* 20S Core (28 Subunits) - contains Thr protease subunits, which digest substrate into short peptides

(ii) Assembly is an ATP-dependent process

Question 9

Summarise the ERAD Pathway of protein degradation

(3 Points)

Answer 9

• Transmembrane E3 Ligase detects misfolded proteins in ER (integral/soluble) and passes them to translocon
• polypeptide is translocated through the translocon into the cytosol (assisted by p97), where it is polubiquitinated by E3 ligase
• K-48 linked chain signals protein for degradation by 26S proteasome

Question 10

How can Ubiquitin Chains vary to encode different information (“Ubiquitin Code”)?

(4 Points)

Answer 10

1. Chain Length
2. Different Linkage Types (e.g., K48, K63, Linear, Forked)
3. Residues other than Lys (e.g., Ser, Thr) may in rare cases be modified with Ubiquitin (Ester Bonds)
4. Ubiquitin monomers can undergo PTM (e.g., Phosphorylation , Acetylation