The diverse members of the mammalian HSP70 machine show distinct chaperone-like activities Jurre Hageman, Maria van Waarde-Verhagen, Alicja Zylicz, Dawid Walerych, Harm H. Kampinga
To cite this version:
Jurre Hageman, Maria van Waarde-Verhagen, Alicja Zylicz, Dawid Walerych, Harm H. Kampinga. The diverse members of the mammalian HSP70 machine show distinct chaperone-like activities. Bio- chemical Journal, Portland Press, 2011, 435 (1), pp.127-142. 10.1042/BJ20101247. hal-00576982
HAL Id: hal-00576982 https://hal.archives-ouvertes.fr/hal-00576982 Submitted on 16 Mar 2011
HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Biochemical Journal Immediate Publication. Published on 14 Jan 2011 as manuscript BJ20101247
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Figure 1
A
prokaryotes unicellular eukaryotes multicellular eukaryotes
compartmentalization multi cellularity multi-domain proteins cellular specialization development
B DNAK/HSPA C DNAJ
20 100
15 80
60 10 40 5 Number of genes Number of genes 20
0 0 A. thaliana A. thaliana T. kodakaraensis M. jannaschii T. acidophilum M. mazei S. aureus M. pneumoniae E. coli Y. pestis angola S. cerevisiae C. elegans D. melanogaster H. sapiens T. kodakaraensis M. jannaschii T. acidophilum M. mazei S. aureus M. pneumoniae E. coli Y. pestis angola S. cerevisiae C. elegans D. melanogaster H. sapiens
Archaea Eubacteria Eukaryota Archaea Eubacteria Eukaryota THIS IS NOT THE VERSION OF RECORD - see doi:10.1042/BJ20101247 Accepted Manuscript
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Figure 2
A HSPA and HSPH Phylogram Predicted/Known localization Experimental localization
HSPH4 ER
HSPH1
1000 HSPH2 Cytosol 743 HSPH3 HSPH1 HSPH2 HSPH3 HSPA14 Cytosol 756 HSPA13 ER Trichotomy
581 HSPA9 Mitochondria HSPA14 1000 HSPA5 ER
HSPA6 1000 HSPA1L 1000 517 1000 HSPA1A HSPA1A HSPA1L HSPA2 1000 Cytosol
945 HSPA1B
HSPA2 1000 HSPA8 HSPA6 HSPA8 HSPA12A Mitochondria? 1000 HSPA12B ? 0.1
B DNAJA DNAJA2 Cytosol
DNAJA2
DNAJA3 Mitochondria
Trichotomy
DNAJA3
DNAJA1 Cytosol/Membrane
1000 DNAJA1
DNAJA4 Cytosol/Membrane 0.1
DNAJA4 C DNAJB
DNAJB2a Cytosol/Nucleus 1000 DNAJB2b ER DNAJB8 Cytosol/Nucleus DNAJB2a 436 DNAJB7 Nucleus 848 DNAJB6a 1000 Cytosol/Nucleus THIS IS NOT THE VERSION OF RECORD - see doi:10.1042/BJ20101247 DNAJB6b
DNAJB9 DNAJB6b DNAJB8 DNAJB9 Trichotomy ER 598 DNAJB11 994 DNAJB13 1000 DNAJB5 998 Cytosol 1000 DNAJB4 731 DNAJB1 AcceptedDNAJB1 ManuscriptDNAJB4 DNAJB5 DNAJB12a 1000 Nucleus DNAJB12b 1000 DNAJB14a 1000 Cytosol 0.1 DNAJB14b
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Figure 3
ABHek-293 Inducibility Hek-293 Abundance for HSPH/HSPA -HS
GAPD = 36.3% Gene control 1 hour 3 hours 6 hours HSPH3 = 1.7% HSPH2 = 1.0% HSPH1 1,0 1,9 8,6 10,0 HSPH1 = 2.2% HSPH2 1,0 1,5 2,6 1,8 HSPH HSPA14 = 0.8% HSPH3 1,0 0,5 1,2 1,4 HSPA9 = 8.2%
HSPA1A = 0.0% Gene control 1 hour 3 hours 6 hours HSPA1A 1,0 495,7 3121,0 401,7 HSPA1B 1,0 3,0 8,0 1,1 HSPA1B = 15.8% HSPA1L 1,0 13,5 112,0 8,0 HSPA2 1,0 1,0 1,1 1,4 HSPA1L = 0.0% HSPA2 = 0.0% HSPA5 1,0 2,7 10,2 7,0 HSPA HSPA5 = 0.2% +HS HSPA6 1,0 832,2 4588,5 157,7 HSPA6 = 0.0% HSPA8 1,0 1,1 1,7 0,8 HSPA8 = 33.9% HSPA9B 1,0 0,8 0,9 0,9 HSPA8 = 16.1% HSPA14 1,0 0,8 0,8 0,9 HS HSPA9 = 2.0% HSPA14 = 0.2% Gene control 1 hour 3 hours 6 hours HSPH1 = 5.3% HSPH2 = 0.7% DNAJA1 1,0 1,2 2,1 3,0 DNAJA2 1,0 1,4 1,1 1,3 HSPH3 = 0.6% DNAJA3 1,0 1,4 0,7 1,2 DNAJA4 1,0 1,2 1,1 0,8 DNAJB1 1,0 30,5 79,6 4,8 GAPD = 10.3% DNAJB2 1,0 1,3 0,8 0,9 DNAJB4 1,0 2,4 6,5 2,3 DNAJ DNAJB5 1,0 1,4 1,1 1,4 DNAJB6 1,0 1,8 2,9 2,4 DNAJB7 1,0 1,4 0,8 1,0 HSPA6 = 22.1% DNAJB8 1,1 1,4 1,6 5,3 HSPA1A = 5.0% DNAJB9 1,0 1,5 3,9 1,4 HSPA5 = 0.6% DNAJB11 1,0 1,5 2,4 2,5 DNAJB12 1,0 1,3 0,6 1,2 HSPA2 = 0.0%
HSPA1L = 0.0%
C Hek-293 Abundance for DNAJ
-HS HSPA1B = 37.1%
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% THIS IS NOT THE VERSION OF RECORD - see doi:10.1042/BJ20101247
D Various Cell-lines/Tissues ABCB1 HYOU1 IGF1R IGF2R DNAJA1 DNAJA2 DNAJA3 DNAJA4 DNAJB1 DNAJB11 DNAJB2 DNAJB4 DNAJB5 DNAJB9 DNAJC4 DNAJC5 DNAJC7 DNAJC8 HSPA1A HSPA1L HSPA2 HSPH2 HSPA6 HSPA8 HSPA9 HSF1 HSPH1 RPS27A RPS27A Hek-293 Hek-293 A549 Accepted ManuscriptA549 HepG2 HepG2 Hela Hela SHSY-5Y SHSY-5Y VH-25 VH-25 Liver Liver Brain Brain Mixed cell-lines Mixed cell-lines
Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited THIS IS NOT THE VERSION OF RECORD - see doi:10.1042/BJ20101247 © 2011 The Authors Journal compilation © 2011Portland Press Limited Licenced copy.Copying isnotpermitted, except withpriorpermission and asallowedby law. Biochemical JournalImmediatePublication.Publishedon14Jan2011asmanuscriptBJ20101247 D Figure 4 C B A Luciferase 37°C Luciferase 45°C Luciferase CHAPS HD74Q HD74Q GAPD
% luciferase activity Hek-293 Chaps GAPD 10 20 30 40 50 60 0 Tet
mRFP mRFP
HSPH HSPH1 HSPH1 - Accepted+ NO HSP ManuscriptHSPH2 HSPH2 -+-+-+ HSPAH3 HSPH3 GFP HSPA1A HSPA1A
HSPH1 HSPH HSPA1L HSPA1L HSPH2 HSPA2 HSPA2 HSPA -
+-+ HSPA6 HSPA6 HSPH3 HSPA8 HSPA8 HSPA1A HSPA14 HSPA14 SSA1 SSA1
- DNAK DNAK
+ HSPA1L
-+-+-+ DNAJA1 DNAJA1 HSPA2 DNAJA2 DNAJA2 HSPA6 HSPA DNAJA3 DNAJA3 HSPA8 DNAJA4 DNAJA4 - DNAJ
- - +-+ HSPA14 DNAJB1 DNAJB1 DNAJB2a SSA1 DNAJB2a DNAJB4 DNAJB4 DNAJB5 DNAJB5
+ DNAK DNAJB6b DNAJB6b -+-+-+ DNAJB8 DNAJB8 DANAJA1 D D D D D DNAJA2 GFP GFP GAPD V5 GFP
DNAJA3 -
- - +-+ DNAJA4
DNAJB1 DNAJ
+ DNAJB2 -+-+-+ DNAJB4
DNAJB5
DNAJB6 -
+-+ DNAJB8
DNAJB9 D D D D GAPD GFP GFP V5 THIS IS NOT THE VERSION OF RECORD - see doi:10.1042/BJ20101247 Figure 5 B A © 2011 The Authors Journal compilation © 2011Portland Press Limited Licenced copy.Copying isnotpermitted, except withpriorpermission and asallowedby law. Biochemical JournalImmediatePublication.Publishedon14Jan2011asmanuscriptBJ20101247 D HD74Q GFP % luciferase activity 10 20 30 40 50 60 0 O23
mRFP mRFP
HSPH HSPH1 HSPH1 HSPH2 HSPH2 HSPH3 HSPH3 HSPA1A HSPA1A Accepted ManuscriptHSPA1L HSPA1L HSPA2 HSPA2 HSPA HSPA6 HSPA6 HSPA8 HSPA8 HSPA14 HSPA14 SSA1 SSA1 DNAK DNAK
DNAJA1 DNAJA1 DNAJA2 DNAJA2 DNAJA3 DNAJA3 DNAJA4 DNAJA4 DNAJ DNAJB1 DNAJB1 DNAJB2a DNAJB2a DNAJB4 DNAJB4 DNAJB5 DNAJB5 DNAJB6b DNAJB6b DNAJB8 DNAJB8 Suppression C aggregation Luciferase of polyQ Folding HSPA14 DNAJB8 DNAJB6b DNAJB5 DNAJB4 DNAJB2a DNAJB1 DNAJA4 DNAJA3 DNAJA2 DNAJA1 DNAK SSA1 HSPA8 HSPA6 HSPA2 HSPA1L HSPA1A HSPH3 HSPH2 HSPH1 mRFP Chap e-9 O23 Hek-293 u u u oy PolyQ PolyQ Luc Luc Luc + + + + + + + ------eodn Agg.Suppression Refolding + + + + + + ------+ + + + + + ------Hek-293 + + + + ------O23 + + + + ------Biochemical Journal Immediate Publication. Published on 14 Jan 2011 as manuscript BJ20101247
Figure 6
A C HSPA1A 1 --MAKAAAIGIDLGTTYSCVGVFQHGKVEIIANDQGNRTTPSYVAFTDTERLIGDAAKNQ 50 HSPA6 1 MQAPRELAVGIDLGTTYSCVGVFQQGRVEILANDQGNRTTPSYVAFTDTERLVGDAAKSQ * *************** * *** ********************* ***** * Tet - Tet + 40 HSPA1A 59 VALNPQNTVFDAKRLIGRKFGDPVVQSDMKHWPFQVINDGDKPKVQVSYKGDTKAFYPEE HSPA6 61 AALNPHNTVFDAKRLIGRKFADTTVQSDMKHWPFRVVSEGGKPKVRVCYRGEDKTFYPEE **** ************** * ********** * * **** * * * * ***** 30
HSPA1A 119 ISSMVLTKMKEIAEAYLGYPVTNAVITVPAYFNDSQRQATKDAGVIAGLNVLRIINEPTA HSPA6 121 ISSMVLSKMKETAEAYLGQPVKHAVITVPAYFNDSQRQATKDAGAIAGLNVLRIINEPTA ****** **** ****** ** ********************* *************** 20
HSPA1A 179 AAIAYGLDRTGKGERNVLIFDLGGGTFDVSILTIDDGIFEVKATAGDTHLGGEDFDNRLV
% luciferase activity 10 ATPase HSPA6 181 AAIAYGLDRRGAGERNVLIFDLGGGTFDVSVLSIDAGVFEVKATAGDTHLGGEDFDNRLV domain ********* * ****************** * ** * **********************
HSPA1A 239 NHFVEEFKRKHKKDISQNKRAVRRLRTACERAKRTLSSSTQASLEIDSLFEGIDFYTSIT 0 HSPA6 241 NHFMEEFRRKHGKDLSGNKRALRRLRTACERAKRTLSSSTQATLEIDSLFEGVDFYTSIT *** *** *** ** * **** ******************** ********* *******
HSPA1A 299 RARFEELCSDLFRSTLEPVEKALRDAKLDKAQIHDLVLVGGSTRIPKVQKLLQDFFNGRD HSPA6 301 RARFEELCSDLFRSTLEPVEKALRDAKLDKAQIHDVVLVGGSTRIPKVQKLLQDFFNGKE *********************************** ********************** HspA6 Control HspA1A LNKSINPDEAVAYGAAVQAAILMGDKSENVQDLLLLDVAPLSLGLETAGGVMTALIKRNS HSPA1A 359 361 LNKSINPDEAVAYGAAVQAAVLMGDKCEKVQDLLLLDVAPLSLGLETAGGVMTTLIQRNA HSPA6 ******************** ***** * ************************ ** ** HspA1A-HspA6 PstI HspA6-HspA1A PstI HspA6-HspA1A HspA1A-HspA6 Van91I Van91I HspA6-HspA1A Tet - +-+-+-+-+-+-+ HSPA1A 419 TIPTKQTQIFTTYSDNQPGVLIQVYEGERAMTKDNNLLGRFELSGIPPAPRGVPQIEVTF TIPTKQTQTFTTYSDNQPGVFIQVYEGERAMTKDNNLLGRFELSGIPPAPRGVPQIEVTF DV5 HSPA6 421 Substrate ******** *********** *************************************** binding domain DGAPD
HSPA1A 479 DIDANGILNVTATDKSTGKANKITITNDKGRLSKEEIERMVQEAEKYKAEDEVQRERVSA DIDANGILSVTATDRSTGKANKITITNDKGRLSKEEVERMVHEAEQYKAEDEAQRDRVAA HSPA6 481 ******** ***** ********************* **** *** ****** ** ** * D
HSPA1A 539 KNALESYAFNMKSAVEDEGLKGKISEADKKKVLDKCQEVISWLDANTLAEKDEFEHKRKE KNSLEAHVFHVKGSLQEESLRDKIPEEDRRKMQDKCREVLAWLEHNQLAEKEEYEHQKRE HSPA6 541 ** ** * * * * ** * * * *** ** ** * **** * ** * C-terminal DHSPA1A domain HSPA1A 599 LEQVCNPIISGLYQGAGGPGPGGFGAQGPKGGSGSGPTIEEVD DHSPA8 HSPA6 601 LEQICRPIFSRLYGGPGVPGGSSCGTQARQGDPSTGPIIEEVD *** * ** * ** * * ** * * * ** ***** 30 DGAPD Control shRNA 25 Van91I 241 PSTI 351 B 20 HSPA1A ATPase Substrate binding C-terminal 1 385 537 641 15 Van91I 243 PSTI 353 10 HSPA6 ATPase Substrate binding C-terminal 1 385 537 643 % luciferase activity 5
0 Vector HSPA6 HSPA1A HSPA6-HSPA1a PSTI HSPA6-HSPA1a HSPA1A-HSPA6 PSTI HSPA1A-HSPA6 HSPA6-HSPA1a Van91I HSPA6-HSPA1a E OverexpressionF RNAi Van91I HSPA1A-HSPA6 100 70
80 60 RNAi: 50 Vector 60 HSPA1A 40 HSPA6 40 30 20 % luciferase activity
% luciferase activity 20 10
THIS IS NOT THE VERSION OF RECORD - see doi:10.1042/BJ20101247 0 0 Expression: Expression: Vector Vector Vector Vector HSPA6 HSPA6 HSPA6 HSPA1A HSPA1A HSPA1A DV5 DHSPA1A DHSPA6 DHSPA6 DGAPD
DHSPA1A RNAi: Mock Mock Mock Mock HSPA6 HSPA6 HSPA6 HSPA6 HSPA1A DAcceptedGAPD ManuscriptHSPA1A HSPA1A HSPA1A non-thermotolerant thermo- Control Thermotolerant tolerant
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Control DNAJA1 DNAJA2 DNAJA4
nu NiNtabeads DNAJB1 Input DNAJB2a Accepted Manuscript DNAJB4 DNAJB5 DNAJB6b DNAJB7 DNAJB8 TRI TRT
Control DNAJA1 DNAJA2 DNAJA4 DNAJB1 DNAJB2a DNAJB4 DNAJB5 DNAJB6b DNAJB7 DNAJB8 TRI TRT Biochemical Journal Immediate Publication. Published on 14 Jan 2011 as manuscript BJ20101247
Figure 8
AB 100 + ATP + DNAJB1 HSPA6 100 HSPA1A + DNAJB1 Control 80 80 60 60 HSPA1A 40 40 HSPA6 + DNAJB1 HSPA6
Light scattering (RLU) 20 20 HSPA1A Luciferase activity (RLU) DNAJB1 0 Control 0 020406080 0 5 10 15 20 Time (min) Time (min)
C D RT 40 degrees Celcius 20 min
50 HSPA6 + DNAJB1 HSPA6
40 Control Control HSPA1A HSPA1A + DNAJB1 HSPA1A + DNAJA1 HSPA6 HSPA6 + DNAJB1 HSPA6 + DNAJA1 HSPA1A + DNAJB1 30 HSPA1A 20 P53 tetramer bound to p21 promoter % ATP Hydrolyzed 10 DNAJB1 sequence Control 0 0 20 40 60 80 100 120 Time (min) THIS IS NOT THE VERSION OF RECORD - see doi:10.1042/BJ20101247 Accepted Manuscript
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Mock Control HSPA1A mRFP HSPA6
Mock tolerant Thermo- HSPA1A
HSPA6 Overlay
Tet + Tet - Tet + Tet -
% Survival % Survival 1e-5 1e-4 1e-3 1e-2 1e-1 1e+0 1e+1 1e+2 1e-4 1e-3 1e-2 1e-1 1e+0 1e+1 1e+2 04 08 0 120 100 80 60 40 20 0 04 08 0 120 100 80 60 40 20 0 Time at45 Time at45 °C (min) °C (min) HspA6 Control HspA1A HspA1A TT HspA6 TT TT Control HspA1A HspA6 Control HspA1A TT HspA6 TT TT Control