Chaperones

• Chaperones are proteins that help fold proteins into their correct 3D shape
• Chaperones bind to exposed hydrophobic regions of other misfolded proteins preventing their aggregation and assisting their correct native state folding
• Chaperones are important in preventing the accumulation of toxic aggregates
• Heat shock proteins are highly conserved proteins that maintain cellular proteostasis, protecting cells from stress
• Stress conditions that damage proteins and upregulate heat shock proteins:

  • Extreme temperatures
  • Oxidative conditions
  • Expression of foreign proteins e.g. viral
  • Chemical e.g. heavy metals
• Stress activates heat shock transcription factors e.g. HSF1
• Heat shock protein are classified into families based on their size in kD - hsp100, hsp90, hsp70, hsp60, and small hsps
• General properties of chaperones:

  • Assist folding
  • Recognise exposed hydrophobic residues
  • ATPase activity
  • Compartment specific
  • Proteostasis mechanism
• Common eukaryotic chaperones:

  • Hsp70 - clamp
  • Hsp60 - barrel / isolation chamber
  • Hsp90 - mechanical force
• Hsp70 is involved in:

  • Transmembrane translocation
  • Folding
  • Disaggregation
  • Endocytosis and exocytosis
  • Protein degradation
• Hsp70 structure involves 2 domains joined by a linker:

  • ATP binding / hydrolysis (ATPase)
  • Substrate binding
• Hsp70 binds to extended hydrophobic regions of polypeptides
• ADP bound Hsp70 - linker extends the lid and substrate binding domain holds the substrate
• ATP bound Hsp70 - lid and substrate binding domain bind ATPase opening the substrate binding domain - substrate is released
• Action of Hsp70

  1. Unfolded / partially folded substrates are presented by co chaperones e.g. hsp40 recruits hsp70
  2. ATP hydrolysis is promoted by hsp40 - closes cleft on hsp70 (stabilised by hsp70 interacting protein)
  3. Exchange of ADP for ATP promoted by a nucleotide exchange factor
  4. Polypeptide either folds or re binds
  5. Cycles of binding and release prevent aggregation and promote folding
• Nucleotide exchange factors are proteins that stimulate the exchange of nucleoside diphosphates for nucleoside triphosphates bound to other proteins
• Hsp40 target hsp70 to different sites/functions - helical J domain activates hsp70 ATPase activity by binding near ATPase substrate binding domain linker
• Hsp40 have an elongated structure with a V shaped dimer containing 2 tandem beta sub domains for substrate binding
• identification of hsp70 substrates:

  1. Pulse label cell 5 minutes with radioactive amino acids
  2. Hsp70 associated with newly synthesised protein
  3. Lyse cell and centrifuge to remove membranes
  4. Immunoprecipitate with antibody to hsp70 - traps associated polypeptide