Lecture 16: Protein turnover and protein ubiquitination

Question 1

What are the main turnover pathways in eukaryotes?

Answer 1

1. Endosome-lysosome pathway:
   degrades extracellular and cell-
   surface proteins
2. Ubiquitin-proteasome pathway:
   degrades proteins from cytoplasm,
   nucleus and ER
3. Mitochondria (and chloroplasts): have
   their own proteolytic system of
   bacterial origin

Question 2

Why is protein turnover regulated?

Answer 2

1. Metabolic flexibility
   a. Peptide hormones
   b. Key regulatory enzymes
   c. Receptor molecules
2. Physiological control and regulation
   a. T-cell activation; antigen presentation
   b. Cell cycle control: cyclins
3. Removal of abnormal proteins
   a. Protein oxidation; e.g., Met and Cys
   sidechains
   b. Denature or misfolding

Question 3

What is the molecular mechanism for protein turnover?

Answer 3

1. Ubiquitination:
   a. Covalent attachment of ubiquitin to
   intracellular proteins (v. specialised
   form of post-translational
   modification)
2. Ubiquitin:
   a. 76AA protein
   b. highly conserved
   c. “ubiquitously” expressed, but only in
   eukaryotes
   d. C-terminal Gly-Gly dipeptide
   e. “ubiquitous” = expressed in all cell
   types (only in eukaryotes)

Question 4

How does ubiquitin attach?

Answer 4

1. Via glycine-76 at C-terminus to beta-

amino group of lysine

Question 5

What is poly-ubiquitination? Why can it occur?

Answer 5

1. Target proteins often ubiquitinated at
   more than one lysine residue
2. Ubiquitin can be ‘ubiquitinated’ up to
   around 10x - called “polyubiquitination”
3. Why?
   Ubiquitin itself has 6 different Lysine’s for attachment of others. Specifically Lys48 (Lys48 polyubiquitination - only these proteins go to the proteasome for lysis once reach ~10 ubiquitin’s)

Question 6

What are the roles of monoubiquitylation and different polyubiquitinations?

Answer 6

1. Monoubiquitylation at one site:
   a. Histone regulation
   b. Endocytosis
   c. Viral budding
2. Monoubiquitylation at many sites:
   a. Histone regulation
   b. Endocytosis
   c. Viral budding
3. Polyubiquitylation of Lys48:
   a. Proteolysis
4. Polyubiquitylation of Lys48, Lys29, Lys63:
   a. Proteolysis

Question 7

What are the different steps/enzymes involved in ubiquitination?

Answer 7

1. “Activated” by E1 enzyme
2. “Conjugated” by E2 enzyme
3. “Ligated” by E3 to target protein
4. “Shredded” in proteasome complex

Question 8

Elaborate on the E1 / Ubiquitin Activating Enzyme

Answer 8

1. Binding sites for:
   a. Ubiquitin
   b. Nucleotide
2. Only one E1 enzyme in genome

Step 1: E1 + Ubiquitin -> E1 : Ub complex
(non covalent)
Step 2: E1-Ub + ATP -> E1 : Ub-AMP + PPi
(pyrophosphate)
Step 3: E1-Ub-AMP -> E1-S-C-O-Ub
(covalent attachment of “activated”
Ub to E1 via thioester linkage (to
Cys 626 of E1)

E1 NOW ACTIVE

Question 9

Elaborate on E2 / Ubiquitin Conjugating Enzyme

Answer 9

1. Ubiquitin transferred from E1 to E2
   a. via thioester linkage
   b. Approx 35 types of E2 in genome

Step 1: E1-S-C-O-Ub + E2 -> E2-S-C-O-Ub +
E1

E2 enzyme still cant transfer ubiquitin to target proteins (now needs E3)

Question 10

Elaborate on E3 / Ubiquitin Liagases

Answer 10

1. Directly targets “E2-S-C-O-Ub” to proteins destined for degredation
2. Wide spread + v. numerous
3. E3 helps E2 transfer ubiquitin onto lysine sidechains (‘isopeptide bond’)

Step 1: E2-S-C-O-Ub + E3 + target protein-K -> Target protein-K-NH-C-O-Ub + E2 + E3

Question 11

What are all 5 steps of ubiquitination?

Answer 11

Step 1: E1 + Ub -> E1 : Ub complex
Step 2: E1-Ub + ATP -> E1 : Ub-AMP + PPi
Step 3: E1-Ub-AMP - E1-S-C-O-Ub + AMP
Step 4: E1-S-C-O-Ub + E2 -> E2-S-C-O-Ub
+ E1
Step 5: E2-S-C-O-Ub + E3 +
target protein-K -> Target
protein-K-NH-C-O-Ub + E2 + E3

Question 12

What is the 20S proteasome?

Answer 12

1. Deals with Lys-48 poly-Ub substrates
2. V abundant (~1% of cellular protein)
3. Contains 20S core particle with:
   a. 4 rings of 7 subunits each (28 subunits)
   b. 2 outer rings of alpha-subunits
   c. 2 inner rings of beta-subunits

Question 13

What are the structural features of the 20S proteasome?

Answer 13

1. Has a 19S cap
   a. one at each end
   b. top cap recognises polyUb-proteins
   c. 20S + 2x19S = “26S PROTEASOME”
   d. 6 subunits (~20 proteins)
   e. Each subunit has ATPase activity

Question 14

Elaborate on the 19S cap / “unfoldase”

Answer 14

1. Recognise poly-Ub proteins
2. ATP hydrolysis coupled to
   conformational changed in target protein
   (reverse chaperone)
3. Partially unfolded protein pulled into ring
   a. pulled deeper by more ATP hydrolysis
   b. Often target protein released and
   unaltered
4. 19S also has “de-Ubiquitinating” activity
   a. “Shaving” of proximal ubiquitin
   b. “trimming” of distal ubiquitin

Question 15

What proteases are found within the proteasome?

Answer 15

1. One catalytic beta-subunit has a
   “chymotrypsin-like” activity with
   preference for tyrosine or
   phenylalanine at the P1 (Peptide
   carbonyl) position
2. One has “trypsin-like” activity with
   preference for arginine or lysine at
   the P1 position
3. One has “post-glutamyl/caspase”
   activity with preference for glutamate
   or other acidic residue at the P1 position

THREE CATALYTIC SUBUNITS WITH DIFFERENT SUBSTRATE SPECIFICITY

Question 16

What are DUBs?

Answer 16

1. “Deubiquitinating Enzymes”
2. ~120 in genome
3. Ubiquitin-specific processing protease
   (UBP)
   a. Removes large polyUb side chains
4. Ubiquitin C-terminal hydrolases (UCH)
   a. removes monoubiquitin
   b. ubiquitin from the c-terminus of polyUb
   sidechains
5. DUBs are cysteine proteases

Question 17

What can go wrong with the ubiquitin proteasome system

Answer 17

1. Accelerated or decreased degeneration

Question 18

What is the therapeutic potential for proteasome inhibition?

Answer 18

1. Velcade (bortezomib)
   a. small molecule inhibitor
   b. Inhibits “chymotrypsin-like” activity
   c. Only approved proteasome inhibitor
   d. 1.5 billion $ drug
   e. used in multiple myeloma
   f. sensitises cell to apoptosis