Lecture 4 Flashcards
HSP70 Chaperones (Cytosol, ER)
Cytosol
Inducible: HSP70
Constitutive: HSC70
ER
BiP
HSP90 Chaperones (Cytosol, ER)
Cytosol:
Inducible + Constitutive : HSP90 alpha and beta
ER:
GRP94
Chaperonin (Cytosol and ER)
Cytosol:
Constituted + inducible : TRiC
ER:
-None
Heat Shock Response proteins?
HSP70, HSP90 alpha + beta, HSP60
Unfolded Response Protein?
BiP and GRP94
HSP70
-70kDa monomer
-ATPase domain controls substrate binding
- ATP bound : not bound to peptide
-ADP bound : bound to peptide(open -confomation)
-Binds hydrophobic regions
HSP70 Co-chaperones?
-DNAJ (HSP40) promotes HSP70 substrate binding
-NEF promotes substrate release
HSP40 J domain role?
J domain:
- Binds transiently to HSP70 causes it to hydrolyze ATP to ADP (active form) and bind the polypeptide
-Does not bind the substrate
HSP40 Specific domain role?
Determines specificty of HSP40
-Can be substrate binding domains
Substrate binding domains of DNAJ?
Bind hydrophobic regions of the susbtrate and transfer the substrate to HSP70 during ATP hydrolysis
DNAJ that do not bind the substrate?
Complex binds the substrate then DNAJ recruits HSP70s to the complex
NEF?
Remove ADP from HSP70 and allow ATP to bind
-Opens the HSP70 ATPase domain and weakens interactions with nucleotide
-WHen NEF dissociates ATP binds
How does HSP70 support folding?
-Binds hydrphobic regions of folding intermediates and rpevents aggregation
-Release of polypeptide provides chance for the protein to fold to native state
HSP90
-Homodimers(2 identical subunits joined at the C-termni) (180kDa)
-Dimer can open and closed based on ATPase cycle
-Substrates bind along the sides of HSP90
Co-chaperone of HSP90?
p23, stabalizes the closed form of HSP90
When do HSP90 bind to substrates?
Late stages of folding binds to hydrophobic and polar surrfaces
HOP
Co-chaperone that transfers an almost folded susbtrate form the HSP70 cycle to the HSP90 cycle. HSP70 dissociates from the protein when HSP90 binds ATP
How can HOP bind both HSP90 and HSP70?
Cystolic HSP90 and HSP70 have similar C-terminal sequence motifs (EEVD) which TPR domains from HOP recognizes
Three types of TPR Co-Chaperones?
- HOP: has TPR domains that bind HSP70 and HSP90
- FKBp52: has an HSP90 TPR domain and PPlase domains
- CHIP: has TPR domains for both HSP90 and HSP70 and a ubiquitin ligase domain
PPlase domain?
Peptidyl-prolyl isomerase: chaperone that is specific to prolines since prolines are the only amino acid that can be in both cis/trans conformation in the polypeptide chain (The PPlase helps move between the two conformations)
What types of proteins does HSP90 fold?
Kinases, receptors, transcription factors (responsible for cell signaling pathways)
-Kinases don’t require HSC70 to help folding
Mutation in a kinase?
Misfolded kinases can cause cancer
Normal kinases vs Viral kinases ?
C-src(normal): normal kinases are involved in signalling cell growth and have autoregulatory domains that are not always active, requires HSP90 to be folded and active
V-src: Viral kinases are always active and induce cells to become cancerous
How are HSP90s used as a target to develop anti-cancer drugs?
HSP90s are needed to create functional and properly folded kinases. If we treat cells that are viral if HSP90 inhibitor they will no longer be able to create functional kinases. However now HSF1 will all be active which will increase HSPs and refold the mutated kinases(this treatment didn’t work)
E. Coli HSP60?
GroEL: middle of the cage2 rings made up of 7 identical subunits
GroES: cap 7 identical subunits
Homologous to HSP60 in humans
GroEL domains?
- ATPase domain
- Substrate binding domain
GroES ATP-bound?
Unbinds the substrate(protein that needs folding), protein is released into the polar cavity and GroES cap binds to the GroEL. GroEL increases in size due to conformational change in the substrate binding domain
GroES not bound to ATP?
Binds to the substrate and the susbtrate binding domain is in a down conformation leading to a smaller cage size
How does GroEL promoter folding?
- Substrate is in a polar cavity (hydrophiic will not interact with the protein)
-Provides space to fold
-Confinement promotes folding for more compact conformations
T/F: Mitochondrial HSP60 acts like GroEL?
True
Human chaperonin in cytosol?
-No cap
-Has a TRiC ring complex
-long substrate binding domains form the cavity
