Ubiquitination (Versteeg)

Question 1

Briefly explain the role of PTMs and name its types.

Answer 1

Quicker than de-novo synthesis, variability (smaller genome but alot of PTMs bring variability, complex formation, localization, activation/inactivation, degradation)
PTMs are either: written, read or errased

Chemical/complex molecules or proteins:
- methylation
- phosphorylation
- acetylation
- glycosylation
- isoprenylation
- ubi
- sumoylation

Question 2

What is the role of ubi based on its pattern?

Answer 2

• proteosomal degradation
• autophagy
• protein localization
• endocytosis
• protein activity regulation
• cell cycle regulation (time sensitive)
• transcription
• DNA repair

Question 3

Where on a protein is ubi attached? Describe the bond.

Answer 3

Any lysine (K) of a protein can be ubi. Attached via isopeptide bond (on a side chain) with two glycines (GG) on the C-term LRLRGG (G76) motif.

Question 4

Where in the call can a protein be ubi?

Answer 4

• nucleus
• cytoplasm
• not in ER, Golgi and mitochondria (need to be transported from lumen to cytosol)

Question 5

What other bonds can ubi make except isopeptide bond?

Answer 5

• peptide bond (to N-term)
• thioester bond (SH on Cys)
• oxyester bond (OH on Ser/Thr

Question 6

Describe the structure and functions of a proteosome

Answer 6

Top-down:
- ubi substrate binding dom.
- regulatory ATPase dom.
- catalytic dom. for degradation (qield: 2-25 peptide residues of AA or antigens)
- regulatory dom. with ubi receptors

Question 7

What patterns can ubi make?

Answer 7

Substrates can be:
- monoubi (one ubi)
- polymonoubi (multiple unchained ubi)
- polyubi
homo: ubi always binds on the same K of previous ubi
heterotypic: ubi binds on different K of previous ubi

8 functionally different ubi PTM: mono, polymono, Kn (like K48, most common), linear chain (Met ubi attachment)

Question 8

Describe the E1-2-3 ligase cascade.

Answer 8

Attachment of ubi on a substrate: pyramid effect
1) E1 (activating enzyme): ATP -> AMP consumption, conjugates ubi to itself via thioester bond
2) E2 (conjugating enzyme): conjugates ubi to itself via thioester and dispaces E1
3) E3 (ligating enzyme): ligates substrate to ubi and displaces E2
—
4) Deubiquitinating enzyme can deubiquinate substrate

When Es get displaced: back to E-SH form (thioester bond reduced)

Question 9

Distinguish E2s based on the type of substrate they interact with. What are the two ways how ubi chains can be elongated?

Answer 9

Transferring ubi onto:
- substrate
- other ubi
- both

Two ways how ubi chains can be elongated:
- sequentially
- en-block

Question 10

Describe the structure of E2 conjugators and how they interact with E3 ligases and E1 activators. Why are they interacting together?

Answer 10

Conserved catalytic fold (UBC) on E2:
α-helix 1, loop 7, loop 4 -> E3 interaction via Zn ring loops

Interaction with E3:
- α-helix 1 + loop 7 + loop 4 — hydrogen bonds + van der Waals — ZIA + α1/ZIB + ZIIB
- puts E2 in a locked position and prepares it for a nucleophilic attack
- linch pin on E3 RING: position the C-term of ubi for nucleophillic attack and locks it, otherwise it moves too much, E2 usually loaded with ubi

Interaction with E1:
- reloading of ubi upon ubi transfer to substrate
- E2 binds E1 -> ubi reloading from E1 -> E2 dissociation

E2 regulated by: accessory E3 helix, E2 or ubi (alosteric, +/-)

Question 11

What type of E3 ligases are there? Where do they discharge? Which ones have catalytic cys?

Answer 11

HECT:
- thioester bond
- have cat. cys
- E2 discharges on cys
- mechanism: ubi replaces E2 -> ubi translocated to substrate

RING:
- no bond
- E2 dock and discharge from Zn finger
- mechanism: E2 binds ubi -> ubi is translocated to substrate -> E2 is displaced

RBR:
-like RING, has one more ring
- thioester bond
- have cat. cys
- mechanism: ubi binds E2 -> ubi translocated directly to E3 ring -> ubi translocated to substrate

Question 12

How can RING E3 ligases assemble?

Answer 12

RING-based dimerization:
- dimerize on RINGS (via C-termini)

N+C-based dimerization: cullin RING formation
- 4 helix bundle dimerization
- only 7 cullins but have many adaptors so can bind many substrates

• partially enclosed RING with proteins:
  Substrate -> F-box protein -> Skp1 -> Cul1 -> R(ing)-box protein -> E2 with ubi that is translocated to substrate

Question 13

How can E3 ligases be regulated? Name three examples.

Answer 13

• E3 binding inhibitor: Cand1 binds Cullin -> sub cannot bind
• Allosteric activation: dipeptide binds Ubr1 (Cullin like sub blocker) -> sub binds
• Small molecular co-substrate: Auxin helps sub to bind SCF (Cullin like)
• Preudosubstrate inhibitor: **Acm1 (like-sub without K) binds **APC (Cullin like) -> sub cannot bind

Question 14

How is the poly-ubi topology determined?

Answer 14

Determined by the last enzyme bound with ubi: RBR E3, HECT E3, E2 (RING E3 has no contact with ubi)

Question 15

Describe the role of ubi in normoxia and hypoxia. Wat TF is involved in it? What does the TF induce?

Answer 15

HIF-1α: TF
- angiogenesis
- metastasis, invasion
- metabolic reprogramming
- cancer stemness
Because: VEGF, GLUT1, EPO go up

Normoxia:
1) HIF-1α stays in cytosol -> hydroxylated on prolines by PHD
2) E3 RING ligase with Von Hippel-Lindau (VHL) adaptor attaches ubi and recognized hydroxyprolil 564
3) Degradation

Hypoxia:
1) HIF-1α translocated to nucleus
2) HIF-1α with other subunits initiate transcription