Lecture 5

Question 1

Poly-ubiquitin chains?

Answer 1

Mark proteins for degradation and are covalently linked to lysine side chains

Question 2

How is ubiquitination different from post translation modifications?

Answer 2

Add an entire protein instead of just a chemical group

Question 3

Why are there very few genes encoding E1?

Answer 3

Only binds Ub

Question 4

Why are there more genes encoding E2?

Answer 4

Must bind E3

Question 5

Why are there the most genes encoding E3?

Answer 5

E3 provides the specificity for the substrate

Question 6

How does E1 attach the Ub to itself?

Answer 6

Via a thioester bond on a cysteine side chain

Question 7

Why E2 the conjugating enzyme?

Answer 7

Transfers Ub from the E1 to itself to its own cysteine forming a thioester bond

Question 8

E3 ?

Answer 8

E3 ligase transfers the Ub from E2 onto a lysine side chain on the protein that is headed for degration to the same proteasome

Question 9

Isopeptide bond of ubquitination?

Answer 9

C-terminus of ubquitin form a isopeptide bond with the lysine

Question 10

Which amino acids are ubiquitinated?

Answer 10

Lysines and Cysteines

Question 11

Ubiquitin provides another N-terminal to the protein , how many in a monoubiquitnated protein?

Answer 11

Normal protein: one N-terminal , one C-terminal
Monoubiquitinated: two N-terminals, one C-terminal

Question 12

Which lysines can the Ubquitin be added to?

Answer 12

Lys 63, Lys 48 or Lys 11

Question 13

Which lysine needs to be ubiquitinated in order for the protein to be targeted for degradation?

Answer 13

Lysine 48, the others change function of the protein

Question 14

What determines degradation of a substrate?

Answer 14

Specificity of the E3 ligase

Question 15

Three mechanisms for E3 to recognize the substrate for degradation?

Answer 15

1. Quality control degradation of a misfolded protein
2. Constitutive degradation of a native protein to control its level
3. Degradation of a native protein in response to a signal

Question 16

Misfolded protein degradation is regulated by which co-chaperone ?

Answer 16

CHIP(TPR domains for HSC70 and HSP90, and has an E3 ligase domain which binds E2)

Question 17

T/F: Substrates bound to chaperones(Ex. HSP90 ) for a long time are more likely to form a complex with CHIP and get ubiquitinated?

Answer 17

True

Question 18

N-end rule?

Answer 18

Certain residues at the N-terminus are bound by E3 ligases and the protein is ubiquitnated

Question 19

What residues make up the N-end rule ?

Answer 19

If the second amino acid is: Arg, Lys, His, Phe, Trp, Tyr, Leu, IIe (Basic a.a. or large hydrophobic)

Question 20

N-end rule modifications(1) ?

Answer 20

If the second amino acid is Asp or Glu and the cell adds an arginine to the N-terminus then these will also be targeted for degradation

Question 21

N-end rule modifications (2)?

Answer 21

If the seconda amino acid is Asn or Gln and then they are deaminated into Asp and Glu and then Arg is added then these proteins will also be degraded

Question 22

F-box ?

Answer 22

substrate binding protein part of the E3 ligase complex that binds phosphrylated peptide sequences which are going to be degraded

Question 23

SCF ligases?

Answer 23

A type of E3 ligase that recognizes cell signals and degrades functional native proteins

Question 24

Proteasome Structure?

Answer 24

-20S core
-Two 19S regulatory particles(caps)
-Responsible for protein degradation in the cytosol and nucleus and ER (proteins brought to cytosol for degradation)

Question 25

Describe the 20S core of the proteasome?

Answer 25

2 outer rings made up of alpha subunits and 2 inner rings made up of beta subunits. 3 inner beta subunits have protease activity

Question 26

Three functions of the 19s cap of the proteasome?

Answer 26

1. Bring ubiquitinated protein to the core 2. Cut the ubquitins and bring them back to the cytosol 3. ATPase subunits break the tertiary structure of the protein so that it fits in the core

Question 27

What type of activity does the 19S cap with ATPase have?

Answer 27

It is a AAA-family ATPase that can unfold the protein out of its tertiary structure

Question 28

What is the role of the subunits that don't have ATPase activity in the cap?

Answer 28

These subunits remove the Ubs from the protein with a deubiquitinase and the cap has ubiquitin receptors that recognize the Ubs on the protein and bring the protein to the core

Question 29

Ub receptors in the cap role?

Answer 29

-Recognize only K48 Ubs -Protect against premature pre DUB

Question 30

WHat are the intrinsic Ub receptors in the cap of the proteasome?

Answer 30

Rpn10 and Rpn13 bind poly-Ub

Question 31

What are the extrinsic Ub receptors in the cap of the proteasome?

Answer 31

Receptors found in the cytosol (separate from the proteasome). Bind to poly-Ub through Ub associated domain(UBA) and then bind to the cap via a Ub-like domain(UBL)

Question 32

What do the three active beta subuntis in the protein core do?

Answer 32

- One cuts basic AA -One cuts acidic AA -One cuts hydrophobic AA

Question 33

What happens to polar amino acids if the proteasome?

Answer 33

They do not get degraded instead they are released into the cytosol where they will be further degraded into individual AA by proteases in the cytosol