Protein Folding (Madura)

Question 1

Ubiquitin

Answer 1

Energy usage:

• Ub activation (E1)
• proteasome assembly
• substrate unfolding

Ubiquitin features:

• Highly Reactive carboxy terminus
• Small hydrophobic patches →stripe
• Surface lysine residues
  *different chains mediate diff effects

Question 2

Ubiquitination

Answer 2

hydrophobic stripe promotes int’xn with proteosome:

1. UbCH: cleaves pep bonds b/w pro-Ub to activate
2. E1: Ub activating: activates Ub w ATP (thioester)
3. E2: Ub conjugating: mediates transfer of Ub from E1 to E3 or substrate (thioester)
4. E3: Ub protein ligase: attaches Ub to substrate w isopep bond 5. UbP: cleave isopeptide bonds b/w Ub & substrates
   * *Parkin is E3, associated to Parkinson’s*

Question 3

Immunoproteosome

Answer 3

• specifically cleaved proteins sent to cell surface to facilitate antigen presentation on MHCs

generates peptides from viral, etc agents, 9-12 NTs long, designed well to be expressed on cleft of MHCs

NFκ a TF for Hif1

Question 4

Inflammatory response

Answer 4

1. proteasome processes NFκ (TF) precursor
2. NFκ inhibited by IκBα (until inflamm occurs)
3. Stress induces E1-3 & Ub to target & activate Iκ Kinase complex, which phosphorylates IκBα
4. P-IκBα degraded by E1-3 & Ub
5. NFκ now activated TF → stress response

Question 5

Inflammatory pathway & Ub/proteasome

Answer 5

• • NFKB partial hydrolysis
• • IK kinase inactivation
• • IKBα degradation

Question 6

Proteosome regulation

Answer 6

• zymogen protease; pro seq removal coupled to assembly
• multiUb protein target
• ATP dependent
• Gated channel; sequestered hydrolysis

Question 7

GroEL/Folding

Answer 7

Key features:

• Large exposed surface cavity
• hydrophobic res coat (unfolded proteins expose hydrophobic core) - convert chemical energy to mechanical
• flip hydrophobic residue to polar

Question 8

Pauling/Anfinsen limitations

Answer 8

• observed small protein refolding spontaneously
• limited to small proteins with simple/single structure domain
• larger proteins w multiple domains, distant interacting regions

Question 9

Molten Globule features:

Answer 9

• near final state collection of related intermediate structures (all 2^o)
• driven by elimination of water from hydrophobic core

Question 10

Hif1 function

Answer 10

• Activates stress responsive (hypoxia induced) genes
• promote angiogenesis, anaerobic metabolism, resist apoptosis (cancer)

Question 11

Compare and Contrast Normoxia and Hypoxia

(degradation, transcriptional, translational)

Answer 11

Question 12

Ub activating enzyme (E1)

Answer 12

Activates Ub

Binds 2 Ubs as a ubiquitin-adenylate, and via a thioester bond

ATP-dependent stop

Question 13

Ub-conjugating enzyme (E2)

Answer 13

Mediates the transfer of Ub from E1 to either E3 or substrate

Question 14

Ub-protein ligase (E3)

Answer 14

Attaches the Ub to substrate via an isopeptide bond

(Parkin is an E3 enzyme, and mutations are associated with Parkinsons Disease)

Question 15

Parkin

Answer 15

E2 ligase that causes early onset Parkinson’s Disease

Parking might bind and translocate E2 enzymes to the proteosome

Question 16

E6

Answer 16

HPV high risk variant binds E6 (E3 ligase)

E6AP boudn to E6 no longer degrades desginated substrate but instead degrades p53

Question 17

Hif-1

Answer 17

Hypoxia Inducible Factor

Normal circumstances: Hif-1 is made and destroyed (bc don’t usually have hypoxia)

Hypoxia conditions: Hif-1 is stabilized

Mutiations: (Von Hippel-Lindau); Mutant VHL cannot recognize Hif-1; Hif-1 stabilized –> goes to nucleus to turn on Hypoxia indiced proteins –>increase vascularization of region

What is happening here: E3 ligase (VHL) is unable to recognize substrate (Hif-1)

Also could have mut Hif-1 that cannot recognize wt VHL

Question 18

When is energy needed?

Answer 18

1) Activation of Ubiquitin
2) Protein unfolding when degraded by proteosome

Question 19

What non-covalent interactions are important?

Answer 19

Multi-Ub chain forms HYDROPHOBIC a interaction with the proteosome

When Ub chain collapses, the interaxions btwn Ubs are hydrophobic as chain compresses