Harm Kampinga - cssi-educational porto alegre - chaperone concepts

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Harm Kampinga - cssi-educational porto alegre - chaperone concepts

  1. 1. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  Kampinga Cell  Stress  and  Chaperones: basic  concepts Harm H. Kampinga Dept. of Cell Biology, UMC Groningen, The Netherlands 1
  2. 2. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  KampingaOriginally,   heat   shock   genes   (Ritossa,   1962)   and   later   heat   shock   proteins  (Tissieres  et  al  1974),  were  described  to  be  genes/proteins   of  which  the  expression  increased  aBer  an  heat  shock.   Ritossa,  1962 Tissieres  et  al  .,  1974 2
  3. 3. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  KampingaOriginally,   heat   shock   genes   (Ritossa,   1962)   and   later   heat   shock   proteins  (Tissieres  et  al  1974),  were  described  to  be  genes/proteins   of  which  the  expression  increased  aBer  an  heat  shock.  Heat  shock  proteins  (HSP)  were  defined  as  proteins  encoded  by  genes  that  contain  so-­‐called  “heat  shock  elements   (HSE)  in  their  genes  that   are  regulated  by  the  “heat  shock  transcripNon  factor-­‐1  (HSF-­‐1)” 3
  4. 4. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  KampingaOriginally,   heat   shock   genes   (Ritossa,   1962)   and   later   heat   shock   proteins  (Tissieres  et  al  1974),  were  described  to  be  genes/proteins   of  which  the  expression  increased  aBer  an  heat  shock.  Heat  shock  proteins  (HSP)  were  defined  as  proteins  encoded  by  genes  that  contain  so-­‐called  “heat  shock  elements   (HSE)  in  their  genes  that   are  regulated  by  the  “heat  shock  transcripNon  factor-­‐1  (HSF-­‐1)” Akerfelt  et  al.,  2011 3
  5. 5. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  Kampinga Originally,   heat   shock   genes   (Ritossa,   1962)   and   later   heat   shock   proteins   (Tissieres  et  al  1974),  were  described  to  be  genes/proteins   of  which  the  expression   increased  aBer  an  heat  shock.  Heat  shock  proteins  (HSP)  were  defined  as  proteins  encoded  by  genes  that  contain  so-­‐called  “heat  shock  elements   (HSE)  in  their  genes  that   are  regulated  by  the  “heat  shock  transcripNon  factor-­‐1  (HSF-­‐1)”Nowadays,   we   know   that   many   situaNons,   both   physiologically   and   stressful  condiNons  can  acNvate  HSF-­‐1 Morimoto  1998 4
  6. 6. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  Kampinga Originally,   heat   shock   genes   (Ritossa,   1962)   and   later   heat   shock   proteins   (Tissieres  et  al  1974),  were  described  to  be  genes/proteins   of  which  the  expression   increased  aBer  an  heat  shock.  Heat  shock  proteins  (HSP)  were  defined  as  proteins  encoded  by  genes  that  contain  so-­‐called  “heat  shock  elements   (HSE)  in  their  genes  that   are  regulated  by  the  “heat  shock  transcripNon  factor-­‐1  (HSF-­‐1)”Nowadays,   we   know   that   many   situaNons,   both   physiologically   and   stressful  condiNons  can  acNvate  HSF-­‐1There  are  many  members  in  at  least  5  different  heat  shock  proteins  families  (HSP90,  HSP70,  HSP40,  chaperonins,  and  small  HSP). sHsp Hsp40 Hsp90 Chaperonins(HspB: 10 members) (DnaJ: 50 members) (HspC: 5 members) (HspD/E+ CCT: 2+12 members) Hsp70 (HspA+H: 13+4 members) Kampinga  et  al.,    Cell  Stress  Chaperones.  (2009)   5
  7. 7. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  KampingaHSP  belong  to  the  group  of  Molecular  Chaperones 6
  8. 8. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  KampingaHSP  belong  to  the  group  of  Molecular  Chaperones A  chaperone   is  an  adult  or  typically  older  person  who  accompanies  or  supervises  one  or  more   young,  unmarried  men  or  women  during  social  occasions  usually  with  the  specific   intent  of  prevenNng  inappropriate  social  or  sexual  interacNons  or  illegal  behavior   (e.g.,  underage  drinking,  drug  use).   6
  9. 9. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  KampingaHSP  belong  to  the  group  of  Molecular  Chaperones A  chaperone   is  an  adult  or  typically  older  person  who  accompanies  or  supervises  one  or  more   young,  unmarried  men  or  women  during  social  occasions  usually  with  the  specific   intent  of  prevenNng  inappropriate  social  or  sexual  interacNons  or  illegal  behavior   (e.g.,  underage  drinking,  drug  use).   6
  10. 10. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  KampingaHSP  belong  to  the  group  of  Molecular  Chaperones A  chaperone   is  an  adult  or  typically  older  person  who  accompanies  or  supervises  one  or  more   young,  unmarried  men  or  women  during  social  occasions  usually  with  the  specific   intent  of  prevenNng  inappropriate  social  or  sexual  interacNons  or  illegal  behavior   (e.g.,  underage  drinking,  drug  use).   6
  11. 11. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  KampingaHSP  belong  to  the  group  of  Molecular  Chaperones A  chaperone   is  an  adult  or  typically  older  person  who  accompanies  or  supervises  one  or  more   young,  unmarried  men  or  women  during  social  occasions  usually  with  the  specific   intent  of  prevenNng  inappropriate  social  or  sexual  interacNons  or  illegal  behavior   (e.g.,  underage  drinking,  drug  use).   6
  12. 12. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  KampingaHSP  belong  to  the  group  of  Molecular  ChaperonesDefiniNon:   any  protein  (HSP  being   the  largest  group),  which  interacts,  stabilizes   or  helps  a  non-­‐naNve  protein  to  acquire   its  na?ve   conforma?on  but  is  not  present  in  the  final  funcNonal  structure 7
  13. 13. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  KampingaHSP  belong  to  the  group  of  Molecular  ChaperonesDefiniNon:   any  protein  (HSP  being   the  largest  group),  which  interacts,  stabilizes   or  helps  a  non-­‐naNve  protein  to  acquire   its  na?ve   conforma?on  but  is  not  present  in  the  final  funcNonal  structure 7
  14. 14. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  KampingaHSP  belong  to  the  group  of  Molecular  ChaperonesDefiniNon:   any  protein  (HSP  being   the  largest  group),  which  interacts,  stabilizes   or  helps  a  non-­‐naNve  protein  to  acquire   its  na?ve   conforma?on  but  is  not  present  in  the  final  funcNonal  structure 7
  15. 15. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  KampingaHSP  belong  to  the  group  of  Molecular  ChaperonesDefiniNon:   any  protein  (HSP  being   the  largest  group),  which  interacts,  stabilizes   or  helps  a  non-­‐naNve  protein  to  acquire   its  na?ve   conforma?on  but  is  not  present  in  the  final  funcNonal  structure 7
  16. 16. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  KampingaHSP  belong  to  the  group  of  Molecular  ChaperonesDefiniNon:   any  protein  (HSP  being   the  largest  group),  which  interacts,  stabilizes   or  helps  a  non-­‐naNve  protein  to  acquire   its  na?ve   conforma?on  but  is  not  present  in  the  final  funcNonal  structure 8
  17. 17. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  KampingaHSP  belong  to  the  group  of  Molecular  ChaperonesDefiniNon:   any  protein  (HSP  being   the  largest  group),  which  interacts,  stabilizes   or  helps  a  non-­‐naNve  protein  to  acquire   its  na?ve   conforma?on  but  is  not  present  in  the  final  funcNonal  structure 8
  18. 18. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  KampingaHSP  belong  to  the  group  of  Molecular  ChaperonesDefiniNon:   any  protein  (HSP  being   the  largest  group),  which  interacts,  stabilizes   or  helps  a  non-­‐naNve  protein  to  acquire   its  na?ve   conforma?on  but  is  not  present  in  the  final  funcNonal  structureThe   basic   chaperone   acNon   comprise   an   iteraNve   cycles   of   client   binding   to   and  release   from   HSPs   in  which   clients   may   fold   or   may   be   degraded  if   folding   is   not  successful   Kampinga & Craig - Nature Reviews | Molecular Cell Biology 9
  19. 19. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  KampingaHSP  belong  to  the  group  of  Molecular  ChaperonesDefiniNon:   any  protein  (HSP  being   the  largest  group),  which  interacts,  stabilizes   or  helps  a  non-­‐naNve  protein  to  acquire   its  na?ve   conforma?on  but  is  not  present  in  the  final  funcNonal  structureThe   basic   chaperone   acNon   comprise   an   iteraNve   cycles   of   client   binding   to   and  release   from   HSPs   in  which   clients   may   fold   or   may   be   degraded  if   folding   is   not  successful  Such   acNon   is   required   for   assisted   folding   of   nascent   proteins   and   for   protein  translocaNon   (consNtuNvely   expressed   HSP)   and   for   refolding   of   stress-­‐unfolded  proteins  (consNtuNve  and  heat-­‐inducible  HSP). Kampinga & Craig - Nature Reviews | Molecular Cell Biology 9
  20. 20. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  Kampinga HSP  belong  to  the  group  of  Molecular  Chaperones DefiniNon:   any  protein  (HSP  being   the  largest  group),  which  interacts,  stabilizes   or   helps  a  non-­‐naNve  protein  to  acquire   its  na?ve   conforma?on  but  is  not  present  in   the  final  funcNonal  structure The   basic   chaperone   acNon   comprise   an   iteraNve   cycles   of   client   binding   to   and   release   from   HSPs   in  which   clients   may   fold   or   may   be   degraded  if   folding   is   not   successful   Such   acNon   is   required   for   assisted   folding   of   nascent   proteins   and   for   protein   translocaNon   (consNtuNvely   expressed   HSP)   and   for   refolding   of   stress-­‐unfolded   proteins  (consNtuNve  and  heat-­‐inducible  HSP).nascent Kampinga & Craig - Nature Reviews | Molecular Cell Biology 9
  21. 21. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  Kampinga HSP  belong  to  the  group  of  Molecular  Chaperones DefiniNon:   any  protein  (HSP  being   the  largest  group),  which  interacts,  stabilizes   or   helps  a  non-­‐naNve  protein  to  acquire   its  na?ve   conforma?on  but  is  not  present  in   the  final  funcNonal  structure The   basic   chaperone   acNon   comprise   an   iteraNve   cycles   of   client   binding   to   and   release   from   HSPs   in  which   clients   may   fold   or   may   be   degraded  if   folding   is   not   successful   Such   acNon   is   required   for   assisted   folding   of   nascent   proteins   and   for   protein   translocaNon   (consNtuNvely   expressed   HSP)   and   for   refolding   of   stress-­‐unfolded   proteins  (consNtuNve  and  heat-­‐inducible  HSP).Native stressnascent Kampinga & Craig - Nature Reviews | Molecular Cell Biology 9
  22. 22. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  KampingaHSP  belong  to  the  group  of  Molecular  ChaperonesDefiniNon:   any  protein  (HSP  being   the  largest  group),  which  interacts,  stabilizes   or  helps  a  non-­‐naNve  protein  to  acquire   its  na?ve   conforma?on  but  is  not  present  in  the  final  funcNonal  structureThe   basic   chaperone   acNon   comprise   an   iteraNve   cycles   of   client   binding   to   and  release   from   HSPs   in  which   clients   may   fold   or   may   be   degraded  if   folding   is   not  successful  Such   acNon   is   required   for   assisted   folding   of   nascent   proteins   and   for   protein  translocaNon   (consNtuNvely   expressed   HSP)   and   for   refolding   of   stress-­‐unfolded  proteins  (consNtuNve  and  heat-­‐inducible  HSP).Heat-­‐induced   HSP  up-­‐regulaNon  (e.g.   Hsp70)  thus  protects  cells  against   the  toxicity  of  subsequent  heaNng  (thermotolerance). 10
  23. 23. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  KampingaHSP  belong  to  the  group  of  Molecular  ChaperonesDefiniNon:   any  protein  (HSP  being   the  largest  group),  which  interacts,  stabilizes   or  helps  a  non-­‐naNve  protein  to  acquire   its  na?ve   conforma?on  but  is  not  present  in  the  final  funcNonal  structureThe   basic   chaperone   acNon   comprise   an   iteraNve   cycles   of   client   binding   to   and  release   from   HSPs   in  which   clients   may   fold   or   may   be   degraded  if   folding   is   not  successful  Such   acNon   is   required   for   assisted   folding   of   nascent   proteins   and   for   protein  translocaNon   (consNtuNvely   expressed   HSP)   and   for   refolding   of   stress-­‐unfolded  proteins  (consNtuNve  and  heat-­‐inducible  HSP).Heat-­‐induced   HSP  up-­‐regulaNon  (e.g.   Hsp70)  thus  protects  cells  against   the  toxicity  of   subsequent   heaNng   (thermotolerance)   and   this   correlates   with   increased  refolding  capacity. Luciferase 100 % luciferase activity + HSP 90 60 40 - HSP 20 0 HS 0 1 2 hrs after HS 11
  24. 24. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  KampingaHSP  belong  to  the  group  of  Molecular  ChaperonesDefiniNon:   any  protein  (HSP  being   the  largest  group),  which  interacts,  stabilizes   or  helps  a  non-­‐naNve  protein  to  acquire   its  na?ve   conforma?on  but  is  not  present  in  the  final  funcNonal  structureThe   basic   chaperone   acNon   comprise   an   iteraNve   cycles   of   client   binding   to   and  release   from   HSPs   in  which   clients   may   fold   or   may   be   degraded  if   folding   is   not  successful  Such   acNon   is   required   for   assisted   folding   of   nascent   proteins   and   for   protein  translocaNon   (consNtuNvely   expressed   HSP)   and   for   refolding   of   stress-­‐unfolded  proteins  (consNtuNve  and  heat-­‐inducible  HSP).Heat-­‐induced   HSP  up-­‐regulaNon  (e.g.   Hsp70)  thus  protects  cells  against   the  toxicity  of   subsequent   heaNng   (thermotolerance)   and   this   correlates   with   increased  refolding  capacity. toxicity  protec*on chaperone  ac*vity HSP70 HSP70 Nollen et al | Mol Cell Biol 1999 12
  25. 25. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  Kampinga CHAPERONE  ACTIVITY in  vitro  assays 13
  26. 26. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  Kampinga CHAPERONE  ACTIVITY in  vitro  assays 1.  heat  substrate  +/-­‐  HSP2.  measure  substrate  aggrega*on 13
  27. 27. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  Kampinga CHAPERONE  ACTIVITY in  vitro  assays 1.  heat  substrate  +/-­‐  HSP2.  measure  substrate  aggrega*on light  sca<ering client:  HSP 1:0 1:1 1:2 1:5 13
  28. 28. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  Kampinga CHAPERONE  ACTIVITY in  vitro  assays 1.  heat  substrate  +/-­‐  HSP 1.  heat  substrate  +/-­‐  HSP2.  measure  substrate  aggrega*on 2.  reincubate  at  37oC  +/-­‐  HSP  and  +/-­‐  ATP 3.  measure  ac*vity light  sca<ering client:  HSP 1:0 1:1 1:2 1:5 13
  29. 29. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  Kampinga CHAPERONE  ACTIVITY in  vitro  assays 1.  heat  substrate  +/-­‐  HSP 1.  heat  substrate  +/-­‐  HSP2.  measure  substrate  aggrega*on 2.  reincubate  at  37oC  +/-­‐  HSP  and  +/-­‐  ATP 3.  measure  ac*vity light  sca<ering ac=vity  assays client:  HSP  /  ATP client:  HSP 1:0 1:5  /  with  ATP 1:2  /  with  ATP 1:1 1:1  /  with  ATP 1:2 1:5  /  no  ATP 1:5 1:0  /  no  ATP 13
  30. 30. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  Kampinga CHAPERONE  ACTIVITY in  vitro  assays 1.  heat  substrate  +/-­‐  HSP 1.  heat  substrate  +/-­‐  HSP 2.  measure  substrate  aggrega*on 2.  reincubate  at  37oC  +/-­‐  HSP  and  +/-­‐  ATP 3.  measure  ac*vity light  sca<ering ac=vity  assays client:  HSP  /  ATP client:  HSP 1:0 1:5  /  with  ATP 1:2  /  with  ATP 1:1 1:1  /  with  ATP 1:2 1:5  /  no  ATP 1:5 1:0  /  no  ATPaggrega?on  preven?on (“holdase”) 13
  31. 31. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  Kampinga CHAPERONE  ACTIVITY in  vitro  assays 1.  heat  substrate  +/-­‐  HSP 1.  heat  substrate  +/-­‐  HSP 2.  measure  substrate  aggrega*on 2.  reincubate  at  37oC  +/-­‐  HSP  and  +/-­‐  ATP 3.  measure  ac*vity light  sca<ering ac=vity  assays client:  HSP  /  ATP client:  HSP 1:0 1:5  /  with  ATP 1:2  /  with  ATP 1:1 1:1  /  with  ATP 1:2 1:5  /  no  ATP 1:5 1:0  /  no  ATPaggrega?on  preven?on suppor?ng  refolding (“holdase”) (“foldase”) 13
  32. 32. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  Kampinga CHAPERONE  ACTIVITYsHsp Hsp40 Hsp60/10 Hsp90(HspB) (DnaJ) (HspD/E) (HspC) Hsp70 (HspA) 14
  33. 33. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  Kampinga CHAPERONE  ACTIVITY ATP-independent ‘holders’sHsp Hsp40 Hsp60/10 Hsp90(HspB) (DnaJ) (HspD/E) (HspC) Hsp70 (HspA) 14
  34. 34. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  Kampinga CHAPERONE  ACTIVITY ATP-independent ‘holders’sHsp Hsp40 Hsp60/10 Hsp90(HspB) (DnaJ) (HspD/E) (HspC) ATP-dependent ‘holders & folders’ Hsp70 (HspA) 14
  35. 35. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  Kampinga CHAPERONE  ACTIVITY ATP-independent ‘holders’sHsp Hsp40 Hsp60/10 Hsp90(HspB) (DnaJ) (HspD/E) (HspC) ATP-dependent ‘holders & folders’ Hsp70 (HspA) The Hsp70 machine 14
  36. 36. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  Kampinga CHAPERONE  ACTIVITY The  Hsp70  machine 15
  37. 37. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  Kampinga CHAPERONE  ACTIVITY The  Hsp70  machine Kampinga & Craig - Nature Reviews | Molecular Cell Biology 16
  38. 38. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  Kampinga CHAPERONE  ACTIVITY Hsp90 Mayer  et  et  al.  2002 17
  39. 39. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  Kampinga CHAPERONE  ACTIVITY Hsp90 Mayer  et  et  al.  2002 18
  40. 40. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  Kampinga CHAPERONE  ACTIVITY GroEL/ES  (Hsp60/10) Ranson  et  al.  2001   Ranford  et  al.  2000   19
  41. 41. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  Kampinga CHAPERONE  ACTIVITY GroEL/ES  (Hsp60/10) Tang  et  al.  2006   20
  42. 42. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  Kampinga CHAPERONE  ACTIVITY small  Hsp Kim  et  al  1998 Jaya  et  al.  20069 21
  43. 43. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  Kampinga CHAPERONE  ACTIVITY small  Hsp Teydmer  et  al  2011 22
  44. 44. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  Kampinga CHAPERONE  NETWORK HSPC Kampinga & Craig - Nature Reviews | Molecular Cell Biology 23
  45. 45. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  Kampinga CHAPERONE  NETWORK HSPCHSPD/E Kampinga & Craig - Nature Reviews | Molecular Cell Biology 24
  46. 46. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  Kampinga CHAPERONE  NETWORK HSPCHSPD/E HSPB Kampinga & Craig - Nature Reviews | Molecular Cell Biology 25
  47. 47. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  Kampinga Originally,   heat   shock   genes   (Ritossa,   1962)   and   later   heat   shock   proteins   (Tissieres  et  al  1974),  were  described  to  be  genes/proteins   of  which  the  expression   increased  aBer  an  heat  shock.  Heat  shock  proteins  (HSP)  were  defined  as  proteins  encoded  by  genes  that  contain  so-­‐called  “heat  shock  elements   (HSE)  in  their  genes  that   are  regulated  by  the  “heat  shock  transcripNon  factor-­‐1  (HSF-­‐1)”Nowadays,   we   know   that   many   situaNon,   both   physiologically   and   stressful  condiNons  can  acNvate  HSF-­‐1There  are  many  members  in  at  least  5  different  heat  shock  proteins  families  (HSP90,  HSP70,  HSP40,  chaperonins,  and  small  HSP)  that  work  in  a  cellular  network. sHsp Hsp40 Hsp90 Chaperonins(HspB: 10 members) (DnaJ: 50 members) (HspC: 5 members) (HspD/E+ CCT: 2+12 members) Hsp70 (HspA+H: 13+4 members) Kampinga  et  al.,    Cell  Stress  Chaperones.  (2009)   26
  48. 48. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  KampingaButOriginally,   heat   shock   genes   (Ritossa,  have so many proteins   why do humans 1962)   and   later   heat   shock   HSPs? (Tissieres  et  al  1974),  were  described  to  be  genes/proteins   of  which  the  expression   increased  aBer  an  heat  shock.   Heat  shock  proteins  (HSP)  were  defined  as  proteins  encoded  by  genes  that  contain   so-­‐called  “heat  shock  elements   (HSE)  in  their  genes  that   are  regulated  by  the  “heat   shock  transcripNon  factor-­‐1  (HSF-­‐1)” Nowadays,   we   know   that   many   situaNon,   both   physiologically   and   stressful   condiNons  can  acNvate  HSF-­‐1 There  are  many  members  in  at  least  5  different  heat  shock  proteins  families  (HSP90,   HSP70,  HSP40,  chaperonins,  and  small  HSP)  that  work  in  a  cellular  network. sHsp Hsp40 Hsp90 Chaperonins (HspB: 10 members) (DnaJ: 50 members) (HspC: 5 members) (HspD/E+ CCT: 2+12 members) Hsp70 (HspA+H: 13+4 members) Kampinga  et  al.,    Cell  Stress  Chaperones.  (2009)   26
  49. 49. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  KampingaButOriginally,   heat   shock   genes   (Ritossa,  have so many proteins   why do humans 1962)   and   later   heat   shock   HSPs? 1. compartmentalization escribed  to  be  genes/proteins   of  which  Per-UPR?) (Tissieres  et  al  1974),  were  d (cyt/nuc-HSR; ER-UPR; mit-UPR; the  expression   increased  aBer  an  heat  shock.   Heat  shock  proteins  (HSP)  were  defined  as  proteins  encoded  by  genes  that  contain   so-­‐called  “heat  shock  elements   (HSE)  in  their  genes  that   are  regulated  by  the  “heat   shock  transcripNon  factor-­‐1  (HSF-­‐1)” Nowadays,   we   know   that   many   situaNon,   both   physiologically   and   stressful   condiNons  can  acNvate  HSF-­‐1 There  are  many  members  in  at  least  5  different  heat  shock  proteins  families  (HSP90,   HSP70,  HSP40,  chaperonins,  and  small  HSP)  that  work  in  a  cellular  network. sHsp Hsp40 Hsp90 Chaperonins (HspB: 10 members) (DnaJ: 50 members) (HspC: 5 members) (HspD/E+ CCT: 2+12 members) Hsp70 (HspA+H: 13+4 members) Kampinga  et  al.,    Cell  Stress  Chaperones.  (2009)   26
  50. 50. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  KampingaButOriginally,   heat   shock   genes   (Ritossa,  have so many proteins   why do humans 1962)   and   later   heat   shock   HSPs? 1. compartmentalization escribed  to  be  genes/proteins   of  which  Per-UPR?) (Tissieres  et  al  1974),  were  d (cyt/nuc-HSR; ER-UPR; mit-UPR; the  expression   increased  aBer  an  heat  shock.   2. Heat  shock  proteins  (HSP)  were  defined  as  proteins  encoded  by  genes  that  contain   differential regulation (e.g., many HSPs are NOT heat-/HSF-1 regulated) so-­‐called  “heat  shock  elements   (HSE)  in  their  genes  that   are  regulated  by  the  “heat   shock  transcripNon  factor-­‐1  (HSF-­‐1)” Nowadays,   we   know   that   many   situaNon,   both   physiologically   and   stressful   condiNons  can  acNvate  HSF-­‐1 There  are  many  members  in  at  least  5  different  heat  shock  proteins  families  (HSP90,   HSP70,  HSP40,  chaperonins,  and  small  HSP)  that  work  in  a  cellular  network. sHsp Hsp40 Hsp90 Chaperonins (HspB: 10 members) (DnaJ: 50 members) (HspC: 5 members) (HspD/E+ CCT: 2+12 members) Hsp70 (HspA+H: 13+4 members) Kampinga  et  al.,    Cell  Stress  Chaperones.  (2009)   26
  51. 51. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  KampingaBut why do humans have so many HSPs? 1. compartmentalization (cyt/nuc-HSR; ER-UPR; mit-UPR; Per-UPR?) 2. differential regulation (e.g., many HSPs are NOT heat-/HSF-1 regulated) -HS DNAJB12 = 0.1% DNAJB11 = 2.0% +HS DNAJB9 = 0.3% DNAJB8 = 0.0% DNAJB7 = 0.1% DNAJB6 = 2.4% HS DNAJB5 = 0.0% DNAJB4 = 0.6% DNAJB2 = 0.0% GAPD = 29.0% GAPD = 82.9% DNAJA1 = 2.5% DNAJA1 = 4.2% DNAJA2 = 0.4% DNAJA3 = 0.4% DNAJA2 = 1.4% DNAJA4 = 0.0% DNAJA3 = 2.3% DNAJA4 = 0.0% DNAJB1 = 2.8% DNAJB2 = 0.0% DNAJB1 = 62.1% DNAJB4 = 0.3% DNAJB5 = 0.1% DNAJB6 = 2.3% DNAJB7 = 0.3% DNAJB8 = 0.0% DNAJB9 = 0.2% DNAJB11 = 2.4% DNAJB12 = 0.7% Hageman  et  al  2011  . 27
  52. 52. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  KampingaBut why do humans have so many HSPs? 1. compartmentalization (cyt/nuc-HSR; ER-UPR; mit-UPR; Per-UPR?) 2. differential regulation (e.g., many HSPs are NOT heat-/HSF-1 regulated) 3. functional specificity (structural differences) sHsp Hsp40 Hsp90 Chaperonins (HspB: 10 members) (DnaJ: 50 members) (HspC: 5 members) (HspD/E+ CCT: 2+12 members) Hsp70 (HspA+H: 13+4 members) Kampinga  et  al.,    Cell  Stress  Chaperones.  (2009)   28
  53. 53. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  KampingaBut why do humans have so many HSPs? 1. compartmentalization (cyt/nuc-HSR; ER-UPR; mit-UPR; Per-UPR?) 2. differential regulation (e.g., many HSPs are NOT heat-/HSF-1 regulated) 3. functional specificity (structural differences) sHsp Hsp40 Hsp90 Chaperonins (HspB: 10 members) (DnaJ: 50 members) (HspC: 5 members) (HspD/E+ CCT: 2+12 members) Hsp70 (HspA+H: 13+4 members) Kampinga  et  al.,    Cell  Stress  Chaperones.  (2009)   28
  54. 54. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  KampingaFUNCTIONAL  SPECIFICITY  OF  THE  HSP70  MACHINE Kampinga & Craig - Nature Reviews | Molecular Cell Biology 29
  55. 55. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  Kampinga FUNCTIONAL  SPECIFICITY  OF  THE  HSP70  MACHINEThe  Hsp70  machine  is  involved  in  many  different  biochemical  and  biological  funcNons  ranging   from  co-­‐translaNonal  folding,  to  protein  translocaNon  across  membranes,  from  protein   remodeling  to  protein  degradaNon. Kampinga & Craig - Nature Reviews | Molecular Cell Biology 29
  56. 56. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  Kampinga FUNCTIONAL  SPECIFICITY  OF  THE  HSP70  MACHINEThe  Hsp70  machine  is  involved  in  many  different  biochemical  and  biological  funcNons  ranging   from  co-­‐translaNonal  folding,  to  protein  translocaNon  across  membranes,  from  protein   remodeling  to  protein  degradaNon. 1.  Role  expansion  HSP70  family Kampinga & Craig - Nature Reviews | Molecular Cell Biology 29
  57. 57. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  Kampinga FUNCTIONAL  SPECIFICITY  OF  THE  HSP70  MACHINEThe  Hsp70  machine  is  involved  in  many  different  biochemical  and  biological  funcNons  ranging   from  co-­‐translaNonal  folding,  to  protein  translocaNon  across  membranes,  from  protein   remodeling  to  protein  degradaNon. 1.  Role  expansion  HSP70  family Hsp70 Yeast Human 0 200 400 600 800 1000 Ssa1-4; Ssb1,2 HSPA1,2,6,7,8 Cytosol Kar2 HSPA5 ER Ssc1;SSq1;Emc10 HSPA9 Mitochondria HSPA12A,B Cytosol Ssz1 HSPA14 Cytosol Kampinga & Craig - Nature Reviews | Molecular Cell Biology 29
  58. 58. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  Kampinga FUNCTIONAL  SPECIFICITY  OF  THE  HSP70  MACHINE The  Hsp70  machine  is  involved  in  many  different  biochemical  and  biological  funcNons  ranging   from  co-­‐translaNonal  folding,  to  protein  translocaNon  across  membranes,  from  protein   remodeling  to  protein  degradaNon. 1.  Role  expansion  HSP70  family Hsp70 Yeast Human 0 200 400 600 800 1000 Ssa1-4; Ssb1,2 HSPA1,2,6,7,8 Cytosol Kar2 HSPA5 ER Ssc1;SSq1;Emc10 HSPA9 Mitochondria HSPA12A,B Cytosol Ssz1 HSPA14 Cytosol Hsp70  are  structural  almost  idenNcal  (same  domain  structure)  Hsp70  show  all  similar  biochemical  acNvity  (an  ATP-­‐dependent  client  protein  binding/release  cycle).   Except  HSPA14  that  lacks  substrate  binding Kampinga & Craig - Nature Reviews | Molecular Cell Biology 29
  59. 59. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  Kampinga FUNCTIONAL  SPECIFICITY  OF  THE  HSP70  MACHINEThe  Hsp70  machine  is  involved  in  many  different  biochemical  and  biological  funcNons  ranging   from  co-­‐translaNonal  folding,  to  protein  translocaNon  across  membranes,  from  protein   remodeling  to  protein  degradaNon. Kampinga & Craig - Nature Reviews | Molecular Cell Biology 30
  60. 60. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  Kampinga FUNCTIONAL  SPECIFICITY  OF  THE  HSP70  MACHINEThe  Hsp70  machine  is  involved  in  many  different  biochemical  and  biological  funcNons  ranging   from  co-­‐translaNonal  folding,  to  protein  translocaNon  across  membranes,  from  protein   remodeling  to  protein  degradaNon. 2.  Role  expansion  DNAJ  protein  family Kampinga & Craig - Nature Reviews | Molecular Cell Biology 30
  61. 61. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  Kampinga FUNCTIONAL  SPECIFICITY  OF  THE  HSP70  MACHINE The  Hsp70  machine  is  involved  in  many  different  biochemical  and  biological  funcNons  ranging   from  co-­‐translaNonal  folding,  to  protein  translocaNon  across  membranes,  from  protein   remodeling  to  protein  degradaNon. 2.  Role  expansion  DNAJ  protein  family Yeast Human 0 100 200 300 400 500 600 Yeast Human 0 100 200 300 400 500 600Promiscous client binding Promiscous client bindingYdj1 DNAJA1 Ydj1 DNAJA1Xdj1 DNAJA2;A4 Xdj1 DNAJA2;A4Apj1 Apj1Scj1 Scj1Mdj1 DNAJA3 Mdj1 DNAJA3Sis1 DNAJB1 Sis1 DNAJB1 DNAJB4;B5 DNAJB4;B5 DNAJB11 DNAJB11 DNAJB9 DNAJB9 DNAJB2a,2b DNAJB2a,2bDjp1 Djp1Caj1 Caj1 DNAJB6a,6b DNAJB6a,6b DNAJB8 DNAJB8 DNAJB7 DNAJB7Erj5 Erj5 DNAJB12a,12b DNAJB12a,12b DNAJB14a,14b DNAJB14a,14b DNAJC18 DNAJC18Selective client binding Selective client bindingJjj1 DNAJC21 590/531 Jjj1 DNAJC21 590/531Jjj3 DNAJC24 Jjj3 DNAJC24 DNAJC5,5b,5g FuncNonal  classificaNon  DNAJ  proteins DNAJC5,5b,5gJac1 DNAJC20 Jac1 DNAJC20Cwc23 DNAJC17 Cwc23 DNAJC17 DNAJC10 DNAJC16 793 782 1:  Client  binding  and  client  delivery  to  Hsp70 DNAJC10 DNAJC16 793 782Swa2 DNAJC6 668 913 Swa2 a)  promiscuous  client  binding DNAJC6 668 913 b)  selecNve  client  binding DNAJC26 1311 DNAJC26 1311 DNAJC27 DNAJC27 DNAJC3 DNAJC3 DNAJC7 2:  No  client  binding,  just  ATPase  sNmulaNon Jem1 DNAJC7Jem1 DNAJC29 4306 4579 a)  tethering  Hsp70  to  a  locaNon/  a  posiNon DNAJC29 DNAJC14 4306 702 4579 DNAJC14 702 DNAJC22Client binding unclear b)  no  tethering DNAJC22 Client binding unclear Kampinga & Craig - Nature Reviews | Molecular Cell Biology DNAJB13 DNAJB13 31
  62. 62. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  Kampinga FUNCTIONAL  SPECIFICITY  OF  THE  HSP70  MACHINE The  Hsp70  machine  is  involved  in  many  different  biochemical  and  biological  funcNons  ranging   from  co-­‐translaNonal  folding,  to  protein  translocaNon  across  membranes,  from  protein   remodeling  to  protein  degradaNon. 2.  Role  expansion  DNAJ  protein  family Yeast Human 0 100 200 300 400 500 600 Yeast Human 0 100 200 300 400 500 600Promiscous client binding Promiscous client bindingYdj1 DNAJA1 Ydj1 DNAJA1Xdj1 DNAJA2;A4 Xdj1 DNAJA2;A4Apj1 Apj1Scj1 Scj1Mdj1 DNAJA3 Mdj1 DNAJA3Sis1 DNAJB1 Sis1 DNAJB1 DNAJB4;B5 DNAJB4;B5 DNAJB11 DNAJB11 DNAJB9 DNAJB9 DNAJB2a,2b DNAJB2a,2bDjp1 Djp1Caj1 Caj1 DNAJB6a,6b DNAJB6a,6b DNAJB8 DNAJB8 DNAJB7 DNAJB7Erj5 Erj5 DNAJB12a,12b DNAJB12a,12b DNAJB14a,14b DNAJB14a,14b DNAJC18 DNAJC18Selective client binding Selective client bindingJjj1 DNAJC21 590/531 Jjj1 DNAJC21 590/531Jjj3 DNAJC24 Jjj3 DNAJC24 DNAJC5,5b,5g FuncNonal  classificaNon  DNAJ  proteins DNAJC5,5b,5gJac1 DNAJC20 Jac1 DNAJC20Cwc23 DNAJC17 Cwc23 DNAJC17 DNAJC10 DNAJC16 793 782 1:  Client  binding  and  client  delivery  to  Hsp70 DNAJC10 DNAJC16 793 782Swa2 DNAJC6 668 913 Swa2 a)  promiscuous  client  binding DNAJC6 668 913 b)  selecNve  client  binding DNAJC26 1311 DNAJC26 1311 DNAJC27 DNAJC27 DNAJC3 DNAJC3 DNAJC7 2:  No  client  binding,  just  ATPase  sNmulaNon Jem1 DNAJC7Jem1 DNAJC29 4306 4579 a)  tethering  Hsp70  to  a  locaNon/  a  posiNon DNAJC29 DNAJC14 4306 702 4579 DNAJC14 702 DNAJC22Client binding unclear b)  no  tethering DNAJC22 Client binding unclear Kampinga & Craig - Nature Reviews | Molecular Cell Biology DNAJB13 DNAJB13 31
  63. 63. Cell  Stress  and  Chaperones:  basic  concepts  -­‐  Harm  H.  Kampinga FUNCTIONAL  SPECIFICITY  OF  THE  HSP70  MACHINE The  Hsp70  machine  is  involved  in  many  different  biochemical  and  biological  funcNons  ranging   from  co-­‐translaNonal  folding,  to  protein  translocaNon  across  membranes,  from  protein   remodeling  to  protein  degradaNon. 2.  Role  expansion  DNAJ  protein  family Yeast Human 0 100 200 300 400 500 600 Yeast Human 0 100 200 300 400 500 600Promiscous client binding Promiscous client bindingYdj1 DNAJA1 Ydj1 DNAJA1Xdj1 DNAJA2;A4 Xdj1 DNAJA2;A4Apj1 Apj1Scj1 Scj1Mdj1 DNAJA3 Mdj1 DNAJA3Sis1 DNAJB1 Sis1 DNAJB1 DNAJB4;B5 DNAJB4;B5 DNAJB11 DNAJB11 DNAJB9 DNAJB9 DNAJB2a,2b DNAJB2a,2bDjp1 Djp1Caj1 Caj1 DNAJB6a,6b DNAJB6a,6b DNAJB8 DNAJB8 DNAJB7 DNAJB7Erj5 Erj5 DNAJB12a,12b DNAJB12a,12b DNAJB14a,14b DNAJB14a,14b DNAJC18 DNAJC18Selective client binding Selective client bindingJjj1 DNAJC21 590/531 Jjj1 DNAJC21 590/531Jjj3 DNAJC24 Jjj3 DNAJC24 DNAJC5,5b,5g FuncNonal  classificaNon  DNAJ  proteins DNAJC5,5b,5gJac1 DNAJC20 Jac1 DNAJC20Cwc23 DNAJC17 Cwc23 DNAJC17 DNAJC10 DNAJC16 793 782 1:  Client  binding  and  client  delivery  to  Hsp70 DNAJC10 DNAJC16 793 782Swa2 DNAJC6 668 913 Swa2 a)  promiscuous  client  binding DNAJC6 668 913 b)  selecNve  client  binding DNAJC26 1311 DNAJC26 1311 DNAJC27 DNAJC27 DNAJC3 DNAJC3 DNAJC7 2:  No  client  binding,  just  ATPase  sNmulaNon Jem1 DNAJC7Jem1 DNAJC29 4306 4579 a)  tethering  Hsp70  to  a  locaNon/  a  posiNon DNAJC29 DNAJC14 4306 702 4579 DNAJC14 702 DNAJC22Client binding unclear b)  no  tethering DNAJC22 Client binding unclear Kampinga & Craig - Nature Reviews | Molecular Cell Biology DNAJB13 DNAJB13 31

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