L5 chaperones and HSR in cytosol (PF3)

• Heat shock proteins (HSPs) are found in the cytosol, endoplasmic reticulum (ER), and other organelles like mitochondria and ribosomes

  
• HSP90 binds to client proteins in an open conformation, closes around them to trap them in a stable folded state, then releases the client proteins and begins the cycle again

  
• The functional cycle of HSP90 is regulated by ATP binding and hydrolysis: ATP binding causes HSP90 to close around the client protein, and ATP hydrolysis causes HSP90 to release the client protein
• The GroEL cycle involves a protein folding process where GroEL, a chaperonin, provides a cage for proteins to fold by themselves

  
• The functional cycle of HSP90 involves binding to client proteins in an open conformation, closing around them to trap them in a stable folded state, then releasing them after ATP hydrolysis

  
• HSP70 assists in bringing the client protein to HSP90 along with Hop, creating a multi-chaperone complex
• Hop, a co-chaperone, recognizes both HSP70 and HSP90 through a motif called EEVD
• When a client is bound, HSP90 binds to ATP, and p23 stabilizes the binding of the client to HSP90, changing HSP90 to a closed conformation
• HSP90 recognizes hydrophobic patches and polar residues, interacting with kinases, receptors, and transcription factors
• Inhibiting HSP90 activity has been used as an anti-cancer therapy, affecting proteins like cellular-src kinase involved in signaling cell growth
• HSP60, like GroEL in E. coli, provides a cage for proteins to fold by themselves, with a timer and space for proper folding
• The GroEL functional cycle involves binding to ATP, changing conformation to allow substrate entry, providing time and space for protein folding, then releasing the protein after ATP hydrolysis
• all heat shock proteins are chaperones, but not all chaperones are heat shock proteins
• • constitutive heat shock proteins are found in the cell all the time
  • inducible heat shock proteins are found only in response to stress
• the heat shock reponse is activated by unfolded cytosolic proteins under heat stress, oxidative damage, and proteasome inhibition.

  • the heat shock response mainly referes to the transcription of heat shock proteins being upregulated and the transcription of other genes being down regulated.
  • the response continues after stress is removed, meaning the HSP proteins are very stable and present for a while, as they need time to refold all of the misfolded proteins.
• heat shock factor 1 mediates the heat shock reponse. it has a DNA binding domain, a regulatory domain and a transcription activation domain. the DNA sequence the HSF1 identifies is called the heat shock element.
• activation of HSF1:

  • HSF1 stays a monomer because it’s recognized by HSP90 and is held as a monomer. HSF1 has no compeition against HSP90
  • when we increase the amount of unfolded proteins, HSF1 is liberated (because it has more compeition for HSP90) and trimerizes
  • then HSF1 will bind to the promoter of the HS genes, this will increase number of HSP to balance the amount of unfolded proteins in the cell
  • then HSP90 then becomes able to bind to HSF1 done (less compeition) and HSF1 becomes a monomer again (inactive)
• HSP70 works as a monomer. cytosilic version is HSC70 and HSP70, in the ER it's called BiP, it is also found in the mitochondria and ribosomes
• when HSP70 is bound to ATP, it cannot bind to the substrate. will only bind to substrate when HSP70 is bound to ADP. it needs the help of co-chaperones (HSP40s called DNAJs or nucleotide exchange factors that promote substrate release)
• HSP40 recognizes client protein and brings it to HSP70
• ATPase activity of HSP70 is induced, leading to HSP70 becoming bound to ADP, promoting a conformational change allowing HSP70 to bind to the substrate
• Once the substrate is bound, HSP70 is bound to ADP, and the NEF liberates the ADP and binds itself to HSP70
• After the client is released, the protein will fold by itself into the native state or HSP70 gives the misfolded protein to another chaperone for proper folding
• types of DNAJs action: some DNAJs have dimerization domain, others have substrate binding domain. some transfer the substrate, while others allow the substrate to fold by itself (those that hold are ATP independent chaperones). HSP40 can also bring HSP70 to protein complex instead of bringing substrate to HSP70.
• HSP40s (DNAJs) have a J domain which binds transiently to HSP70 which hydrolyzes ATP to allow HSP 70 to bind to the substrate (ATPase activity induced).
• what are nucleotide exchange factors?

  • NEF remove the ADP from HSP70
  • ATP binds to HSP70 only when NEF dissassociates
• HSP90 works as a homodimer, it is constitutive and ATP controls the opening and closing of the dimer. recognizes hydrophobic patches. co-chaperone is p23. the c-terminus has dimerization domain, middle region recognizes client N terminus is ATP binding domain
• how does Hop recognize HSP70 and HSP90

  • because both the HSP70 and HSP90 have a motif, EEVD that can be recognized by Hop domain called the TPR domain
  • TPR domain can be specific for HSP70, 90 or both of them
  • TPR co-chaperones often have other domains that interact with the substrate directly
  • HOP has domains which bind to both HSP70 and HSP90
  • p52 (FKB52) has only the HSP90 domain, and has the peptidyl-prolyl domain (chaperone to specific prolines)
  • CHIP binds either HSP70 or HSP90 (not both), and has a ubiquitin ligase domain to degrade proteins
• functional cycle of HSP70

  1. HSP70 with the protein is bringing the client to the HSP90 along with Hop. create a multi-chaperone complex
  2. Hop is a co-chaperone that can recognize the HSP70 and HSP90.
  3. when client is bound, HSP90 binds to the ATP, and p23 stabilizes binding of client to HSP90 which changes the HSP90 to a closed conformation
  4. p52 stabilizes the dimer as well
  5. the ATP is hydrolyzed, p23, client and p52 are all released, now ADP is in the ATP binding domain
  6. ADP is released and now the HSP90 can bind to other client proteins
• HSP60 is a constitutive protein, provide a cage to allow the protein to fold by itself. GroEL is cyllinder (7 subunits) and GroES has cap (7 subunits). depending on the binding of the ATP, the size of the GroEL will change, changing which substrates can enter the cage
• • when GroEL is not binding to the nucleotide, they are smaller. when HSP60 is not bound to nucleotides, it is smaller and has a wall of hydrophobic residues. so this will bring a misfolded protein to it.
  • when the GroEL is bound to ATP, HSP60 gets taller. there are more hydrophilic residues in this region. if we have a hydrophobic residue inside, this cage provides the space to allow the protein to binds by itself.
• GroEL functional cycle

  1. the HSP60 is in its normal state with the hydrophobic residues towards the center of the cage.
  2. then it binds to ATP making the cage bigger and the hydrophobic residues no longer face the middle, promoting the binding of the GroES component
  3. it gives the protein 7 seconds and space to allow the protein to fix its folding by itself.
  4. when the GroEL is bound to ATP, that liberates the GroES, liberates the protein and the ADP