Protein Folding, Hsp70, Hsp90, co-chaperones (Karagoz)

Question 1

What is the Anfinsen’s dogma? Explain the structures of proteins.

Answer 1

• Primary: AA seq.
• Secondary: alpha-helix / beta-sheet
• Tertiary: interaction of the secondary structures (disulphide bridges, hydrogen bonds, van der Waals interactions…)
• Quartery: multiple subunit (peptide chains) interaction

Primary defines tertiaty, tertiary defines specific native, functioning fold - how was this proven:
Native protein + urea (chaotropic agent) -> protein reduced (diluphide bridges -S-S- go to -SH HS-)
1.1) Remove urea and then oxidize -> protein can go back to its native form (tertiary structure restored) and is resistant towards oxidative agents
1.2) Oxidize and then remove urea -> protein couldn’t go back to its native state (tertiary structure not restored) and got oxidized

Question 2

Briefly describe the homeostasis of the proteome. What cellular processes have to be in equilibrium in order for the homeostasis to be maintained?

Answer 2

• protein synthesis
• protein degradation
• protein folding and sorting
• UPR, ISR and HSR

Folding rates and synthesis rates have to be in equilibrium

Question 3

What force predominantly drives the folding of proteins? Name other interactions/structures formed. What causes protein to aggregate? Why do they aggregate, from an energetical point of view?

Answer 3

Main force: hydrophobic collapse to obtain lowest intramolecular E state.
Other forces: disulphide bridge formation, hydrogen bonds, coloumbic interactions (- and + charges), van der Waals

Aggregates formation:
- DNA mut.
- limited tRNA
- damaged mRNA
- translation errors
- high temperatures
- heavy metals
- oxidative stress
Aggregates have even lower intermolecular E than the natively folded proteins.

Question 4

What are the roles of chaperones?

Answer 4

ATP-hydrolysis driven:
- folding / unfolding / refolding / degradation of proteins
- clathrin coat destabilization
- assembly/dissassembly of complexes
- protein translocation
- apoptosis
- stress response actors
ATP-hydrolysis driven self assembly and remodeling for aforementioned functions

Question 5

Name classes of chaperones.

Answer 5

• Hsp90 (constitutively expressed = Hsp90beta, stress induced = Hsp90alpha, mitochondrial = Grp75 / mtHsp75, ER =TRAP1 / Grp95)
• Hsp70 (constitutively expressed = Hsc70, stress induced = Hsp70, mitochondrial = Grp75 / mtHsp75, ER = Bip / Grp78)
• Hsp60
• Hsp40
  …

Question 6

What are chaperonins? Name examples and describe them.

Answer 6

Type of chaperones: HSP60 family almost practically across all ORG, large, double-ring complex that entrap substrates, mostly stress induced.
TYPE 1
- GroEl and its GroES co-chaperonin (bacterial)
- Hsp60 and its Hsp10 co-chapronin (mammalian)
- 2 7-membered rings
TYPE 2
- TRIC and its CCT subunits (mammalian) and prefoldin co-chaperone that forms a cage-like structure
- 2 8-membered rings

CCT1-8:
- apical dom.: substrate binding
- intermediate dom.: folding
- equatorial dom.: ATPase
- prefoldin cage goes on top of substrate
- hydrophobic ring on the inside

Spatial constrains: cannot fit alot of substrates into the cage
Advantages: substrate cannot aggregate, no kinetically entrapped substrates

Question 7

What are co-chaperones? Name examples.

Answer 7

Chaperones that modify chaperones:
- NEFs (GrpE, Armadillo, Hsp110)
- AAA proteins
- JDP
- TPR dom.-containing proteins (HOP, CHIP)

Question 8

Name the subunits of Hsp70 and its mechanism of action. What is the importande of the J domain? What is entropic pulling? What are the functions of Hsp70?

Answer 8

Structure:
- NBD (ATP or ADP binding)
- flexible linker for allosteric interactions
- SBD beta binds substrate
- SBD alpha is an alpha-helical lid

Mechanism:
1) ATP binds NBD -> SBD beta-alpha open up and prevent hydrolysis of ATP
2) Non-native substrate binds -> SBD alpha lid moves and enables partial ATP hydrolysis because substrate pushed the linker and prevented full hydrolysis
- JDP (a partner of Hsp70 binding to J-dom.) overcomes this and enables full hydrolysis
- JDP also remodels the Hsp70 itself and substrate for better compatibility and higher binding affinity
3) ATP -> ADP -> SBD alpha lid closes on the substrate-binding cleft and prevents substrate from escaping
4) NEF removes ADP and adds ATP
5) Native substrate is released

Entropic pulling: swinging and pulling of the translocating protein OR unfolding and refolding of the protein using ATP hydrolysis (2nd option found online)

Functions:
Housekeeping-related:
- de-novo protein synthesis folding
- translocation
- assembly and disassembly of compexes
- protein activity regulation
- protein handover
- proteolysis protection
Stress-related:
-aggregation prevetion
- dissaggregation of aggregates
- protein refolding
- degradation

Question 9

Name the subunits of Hsp90 and its mechanism of action. What are the functions of Hsp90?

Answer 9

Late folding events downstream of Hsp70. Chaperones for aggregation-prone proteins and unstable regulatory proteins. Wide variety of protein targets.
- ER Hsp90: Grp94
- Mitochondrial Hsp90: Hsp75

Hsp70 binds more hydrophobic substrates, Hsp90 binds less hydrophobic substrates because they have already been (partially folded) and have less hydrophobic residues outside the substrate. HOP co-chaperone facilitates transport.

Structure: shaped like a V, homodimer, top-down description
- N-term. dom. with a lid: binds ATP and promotes lid closing
- middle dom.: binds substrate, also charged interactions
- C-term. dom.: dimerization dom
- MEEV motif

Mechanism:
1) ADP bound to N-term: open conformation
2) Substrate binds to middle dom. -> ATP bidns -> lid closes and trapps the substrate
Closed state regulated by co-chaperones:
- HOP: negatively influence the entry to closed state
- CDC37: negatively influence the entry to closed state
- AHA1: positively influence the entry to closed state
3) ATP -> ADP -> open state conformation and refolded substrate release
Open state regulated by co-chaperones:
-p23: negatively influence the entry to open state