Protein Folding (Madura) Flashcards
Ubiquitin
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
Ubiquitination
hydrophobic stripe promotes int’xn with proteosome:
- UbCH: cleaves pep bonds b/w pro-Ub to activate
- E1: Ub activating: activates Ub w ATP (thioester)
- E2: Ub conjugating: mediates transfer of Ub from E1 to E3 or substrate (thioester)
- 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*
Immunoproteosome
- 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
Inflammatory response
- proteasome processes NFκ (TF) precursor
- NFκ inhibited by IκBα (until inflamm occurs)
- Stress induces E1-3 & Ub to target & activate Iκ Kinase complex, which phosphorylates IκBα
- P-IκBα degraded by E1-3 & Ub
- NFκ now activated TF → stress response
Inflammatory pathway & Ub/proteasome
- NFKB partial hydrolysis
- IK kinase inactivation
- IKBα degradation
Proteosome regulation
- zymogen protease; pro seq removal coupled to assembly
- multiUb protein target
- ATP dependent
- Gated channel; sequestered hydrolysis
GroEL/Folding
Key features:
- Large exposed surface cavity
- hydrophobic res coat (unfolded proteins expose hydrophobic core) - convert chemical energy to mechanical
- flip hydrophobic residue to polar
Pauling/Anfinsen limitations
- observed small protein refolding spontaneously
- limited to small proteins with simple/single structure domain
- larger proteins w multiple domains, distant interacting regions
Molten Globule features:
- near final state collection of related intermediate structures (all 2o)
- driven by elimination of water from hydrophobic core
Hif1 function
- Activates stress responsive (hypoxia induced) genes
- promote angiogenesis, anaerobic metabolism, resist apoptosis (cancer)
Compare and Contrast Normoxia and Hypoxia
(degradation, transcriptional, translational)
Ub activating enzyme (E1)
Activates Ub
Binds 2 Ubs as a ubiquitin-adenylate, and via a thioester bond
ATP-dependent stop
Ub-conjugating enzyme (E2)
Mediates the transfer of Ub from E1 to either E3 or substrate
Ub-protein ligase (E3)
Attaches the Ub to substrate via an isopeptide bond
(Parkin is an E3 enzyme, and mutations are associated with Parkinsons Disease)
Parkin
E2 ligase that causes early onset Parkinson’s Disease
Parking might bind and translocate E2 enzymes to the proteosome
E6
HPV high risk variant binds E6 (E3 ligase)
E6AP boudn to E6 no longer degrades desginated substrate but instead degrades p53
Hif-1
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
When is energy needed?
1) Activation of Ubiquitin
2) Protein unfolding when degraded by proteosome
What non-covalent interactions are important?
Multi-Ub chain forms HYDROPHOBIC a interaction with the proteosome
When Ub chain collapses, the interaxions btwn Ubs are hydrophobic as chain compresses
