Chinese Journal of Biochemistry and Molecular Biology. (diamide, H 2 N NH 2 ) (DTT) HSF1 HSF1, C 153 S C S. HSF1 C 36 T C 103 Y
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1 ISSN CN ΠQ Chinese Journal of Biochemistry and Molecular Biology 21 (3) : HSF1 3 ( ) 1 (heat shock transcription factor l HSF1) HSF1 HSF1 5 1 (diamide H 2 N NH 2 ) (DTT) HSF1 DNA HSF1 DNA ; C 153 S C S HSF1 HSF1 C 36 T C 103 Y HSF1 (ox2 HSF1) C 153 S C S C 373 S C 378 S ox2 HSF1 HSF1 C 153 C 373 C 378 ; C 153 C 373 C 378 HSF1 1 Q256 Q71 LIN Zheng 3 HSF1 may have Integrated Redox Dependent Regulation of Cysteine Residues into Its Function Response HUANG Fan LUO Lan ZHANG Shi2Hong MA Zhong2Fu WU Xing2Gang XU Kang ( The First Hospital Affiliated to Sun Yat2Sen University Guangzhou China) Abstract To evaluate the fundamentally important role of redox status of human heat shock transcription factor 1 ( HSF1) in biological regulation cysteine site2specific mutants of HSF1 were constructed and used to determine which of the five cysteine residues may be engaged in disulfide cross2link Analysis of the in vitro transcribed and translated HSF1 proteins showed that while mutation of C 36 and C 103 had no effect on the redox sensitivity of HSF1 the mutation of C 153 or double mutation of C 373 and C 378 to serine rendered HSF1 insensitive to oxidizing reagent diamide and prevented its conversion to ox2 HSF1 HSF1 with a single cysteine to serine mutation at either the C 373 or C 378 position obtained different ox2 HSF1 conformers in the presence of diamide The results suggest that cysteines residues of HSF1 particularly C 153 and C 373 C 378 are necessary and sufficient to form an intramolecular disulfide cross2link under an oxidizing condition and likely provide an off2switch triggered by oxidation to prevent the activation of HSF1 Key words heat shock transcription factor1 cysteine mutant redox : : (No ) (No 31695) 3 Tel : E2mail : com Received : July ;Accepted :October Supported by grants from the National Natural Science Foundation of China (No ) and Guangdong Natural Science Foundation (No 31695) 3 Corresponding author Tel : E2mail : com
2 HeLa 10 % (HSF) DMEM 5 % CO d HSF1 1 [1 ] HSF min [5 ] DNA Bradford Protein Assay (Bio2Rad) 113 HSF1 HSF1 529 C 36 C 103 C 153 C 373 C 378 PCR ( PCR2Mediated Cysteine Specific [2 ] mutagenesis) HSF1 4 2 [3 ] 2 1 PCR DNA [4 ] HFS1 2 DNA 2 2 PCR C 153 C 373 HSF1 5 Nco (CCATGG) 1 ;C 373 Xho 369 (ACC) (AGC) HSF1 ( HSF1 ) :C 36 T C 103 Y C 153 S HSF1 DNA ( PflM ) 5 2TTCCTGACCAAGCTGTGGA23 ( Nco ) 5 2GGGGGAGGCCATGG HSF1 3 C 153 C 373 C 378 GCT23 ;C 373 S C 378 S ( Nco ) 5 2 TCCTGCCAGCCCCATGGCCT23 ( Bgl ) 5 2GTTTATAGATCTCTGCCT23 ( ) :C 36 T 1 (Cys36 Thr36) 5 2ACGCGCTCATCACC 111 New England Biolabs Inc ; ECL Western [ 2 32 P]dATP (S A CiΠmmol) [ 35 S] (S A CiΠmmol) TGGAGCCCGA23 ; 5 2TCGGGCTCCAGGT GATGAGCGCGT23 ;C 103 Y (Cys103 Tyr103) 5 2AGCACCCATACTTCC TGCGT23 5 2ACGCAGGAAGTATGGG TGCT23 ; C 153 S Amersam ; Sigma (Cys153 Ser153) 5 2GGAAGCAGGAGTCC ;TNT Promega ATGGACTCCA23 5 2TGGAGTCCATGGACT ; cdna pjc20 (HSF1) Rutgers CCTGCTT23 ; C 373 S (Cys373 Ser 373) Dr Liu ; 5 2ACCTCGAGCCCTGAAAAGTCCCTCAGCGTA23 ; ( EMSA) HSF12DNA 5 2TACGCTGAGGGACTTTTCAGGGCTCGA HSE 5 2GCCTCGAATGTTCGCGAAGTTT GGT23 ; C S ( Cys373ΠCys378 CG23 ( ) PCR Ser373ΠSer378 ) 5 2TGAAAAGTTCCCT GeneAmp PCR system 2400( Perkin2Elmer) CAGCGTAGCCTCCCTGGACAAGAAT23 5 2
3 3 :HSF1 343 TGTCCAGGGAGGCTACGCTGAGGGAACTTTTCA23 ; C 378 S (Cys378 Ser378) C S IgG ECL Western HSF1 PCR HSF1 DNA 116 ( EMSA) [5 ] pjc20 HSF1 DNA HSF1 cpm C 36 T C 103 Y PflM Nco P 4 2 pjc20 HS F1 ( PflM ΠNco ) ;C 153 S PCR PflM Sph pjc20 HS F1 ( PflM ΠSph ) Nco ; min 4 % C 373 S C S Nco Bgl HSF1 DNA min ; pjc20 HS F1 ( Nco ΠBgl ) C 378 S Bpu10 Bgl pjc20 C S C 378 S pjc20 HS F1 Bpu10 ΠBgl ( Eppendorf 2510 ) DNA DH5 DNA (QIAfilter Midi QIAGEN) DNA DNA ( ) kd ngaan2 HSE 500 g poly (diπdc) mmolπl 5 min 25 ; DTT 5 mmolπl 5 min HSF1 6 C 36 T C 103 Y C 153 S C 373 S C 378 S ; C S DNA ABI310 ( Pirken2Elmer) (TCA) 114 Promerga TNT g [ 35 S ] T ( l cpm ) T7 EcoR 212 HSF1 DNA cdna TNT RNA T7 ( Fig11 ) [ 35 S] TNT 25 l 30 HSF1 120 min 1 L DNA (TCA) [ 35 S] 1 25 ; 2 SDS (42 60 min) ; 3 (2 mmolπl 5 min 25 ) 115 Western ; 4 DTT (5 mmolπl 5 min 25 ) SDS (WT) C 36 T C 103 Y C 153 S C 373 S HSF1 C 378 S DNA ( 2) ; ( 2mercaptoethanol) DTT ( 3) DTT (2 mmolπl 5 min 25 ) ( 4) C 153 S C S DNA ( 5 mmolπl DDT 3) HSF1 15 min 1 mmolπl DDT SD2Dry 213 HSF1 HSF1
4 Fig 1 Effects of diamide on the in vitro activation of wild type and cysteine mutant of HSF1 EMSA of HSE2binding activity The wild type and mutants of HSF1 were transcribed and translated in vitro HSE2binding of the wild type and cysteine mutants of HSF1 was determined under control (25 ; lane 1 if each construct) in vitro heat activated (42 60 min ; lane 2) diamide treated (2 mmolπl 5 min at 25 ) and then heat activated (lane 3) and diamide2 and DTT2 treated (5 mmolπl 5 min at 25 ) and then heat activated (lane 4) conditions For the comparison aliquots of HeLa cell extract trated under identical conditions WT: wild type ; WCE: Whole Cell Extract Fig12 HSF1 [ 35 S] Fig 3 Western (2 mmolπ HSF1 L 5 min 25 ) HSF1 C 36 T C 103 Y [ 35 S] HSF1 (ox2hsf1) ( ) C 153 S C S ( 8 10 ) HSF1 Fig 1 HSF1 C 153 C 373 C 378 ox2hsf1 ox2hsf1 WT yes yes C 36 Y yes yes C 105 Y yes yes C 153 S no no C 373 S no yes C 378 S yes no Fig3 Resolution and detection of redox conformers of HSF1 by Western blotting The transcription in vitro and translated HSF1 protein without and with diamide treatment (2 mmolπl 5 min at 25 ) was subjected Fig 2 Redox conformers of HSF1 wild type and cysteine mutants In vitro translation was carry out in the presence of [ 35 S ] methionine Diamide (2 mmolπl) was the added to the translation mixture Samples were subjected to native gel electrophoresis and the presence of 35 S2labeled proteins was detected by autoradipgraghy WT: wild type to native gel electrophoresis and immuno2western blot Whole cell extract from HeLa cell was included in lane 1 and lane 2 as control The position of the disulfide cross2linked conformers of HSF1 ox2 HSF1 and as indicated Chart of comparing results of conformational shift patterns indicating apparent presence or absence of two diamide2induced HSF1 bands (ox2hsf1 and ox2hsf1 ) WT: wild type ; WCE: whole cell extract
5 3 :HSF1 345 [9 (WCE) HSF1 DNA ] ; C 36 T C 103 Y HSF1 (ox2hsf1) ( 4 6 8) C 153 S ( 10) C 373 S C 378 S 2 RsrA SigR ox2hsf1 C 373 S ox2 R [12 ] ; HSF1 ( 12) ; C 378 S ox2hsf1 ( 14) C 153 ox2 [13 14 ] HSF1 C 153 C 373 C 378 C 153 C 373 ox2 HSF1 ; C 153 C 378 ox2hsf1 ( Fig 3 ) WCE HSF1 5 ox2hsf1 ( 2) C 153 C 373 HSF1 ox2hsf1 C 378 (LZ 4) HSF1 HSF1 3 OxyR [10 11 ] ;RsrA Sigma [15 16 ] HSF1 C 153 C 373 C 378 ( Fig 4) HSF1 [17 ] [6 8 ] HSF1 N C C Ref C 373 C 378 (Cys 62 [7 ) NF2 B ] ; (TTF1) HSF1 (Cys 58 ) TTF1 HSF1 mutants Residues position Relevance to functional domains Cys21 Thr P36 2sheet ;2SH points away DBD Cys22 Tyr P103 Proximity to 2helix3 of DBD Cys23 Ser P153 Leu2zipper outside of trimer domain Cys24 Ser p373 Just 5 of Leu2zipper # 3 Cys25 Ser p378 Just 5 of Leu2zipper # 3 Cys4 5 SerΠSer p374π379 Fig 4 Illustration of cysteine locations on functional domains of primary structure relative to HSF1 cdna clone HSF1 5 C 153 S C S HSF1 ox2hsf1 HSF1 C 36 T C 103 Y HSF1 C 373 S C 378 S HSF ox2hsf1 ox2hsf1 HSF1 C 153
6 C 373 C HSF1 DNA HSF1 DNA 9 Tell G Pines A Paron I D C 153 C 373 C 378 HSF1 Ahn [18 ] 2 HSF1 HSF1 C 35 C 105 HSF1 DNA HSF1 HSF1 5 [ ] 5 HSF1 DNA HSF1 ( References) 1 Zhong M Orosz A Wu C Direct sensing of heat and oxidation by Drosophila heat shock transcription factor Mol Cell (1) : Moran L K Gutteridge J M Quinlan G J Thiols in cellular redox signaling and control Curr Med Chem (7) : Deneke S M Thiol2based antioxidants Curr Top Cell Regul 2000 Hai2Meng Progress on the study of coiled coils in protein structures 36 : Chin J Biochem Mol Biol) (5) : Sen C K Cellular thiols and redox2regulated signal transduction Curr 18 Ahn S G Thiele D J Redox regulation of mammalian heat shock factor Top Cell Regul : Choi H S Lin Z Li B Liu A Y Age2dependent decrease in the heat2 1 is essential for Hsp gene activation and protection from stress Dev (4) : Genes inducible DNA sequence2specific binding activity in human diploid fibroblasts J Biol Chem (29) : Wu C Heat shock transcription factors : structure and regulation Annu Rev Cell Dev Biol : MEF2 20 Pirkkala L Nykanen P Sistonen L Roles of the heat shock ( Zhang Wen2Wei Xu Lie2Ming Regulation of transcriptional factor MEF2 to multiple signal conduction pathways and biological functions Biol) (4) : Chin J Biochem Mol 7 Georgiou G How to flip the (redox) switch Cell (5) : (Li Zai2Quan Zhou Ai2Ru Tang Zhao2Shu Molecular mechanism on endoplasmic reticulum stress responses Chin J Biochem Mol Biol) (3) : Elia A Bisca A Kelley M R Manzini G Damante G Redox effector factor21 regulates the activity of thyroid transcription factor 1 by controlling the redox state of the N transcriptional activation domain J Biol Chem ( 17 ) : Zheng M Aslund F Storz G Activation of the OxyR transcription factor by reversible disulfide bond formation Science (5357) : Aslund F Zheng M Beckwith J Storz G Regulation of the OxyR transcription factor by hydrogen peroxide and the cellular thiol2disulfide status Proc Natl Acad Sci USA (11) : Kang J G Paget M S B Seok YJ Hahn M Y Bae J B Hahn J S Kleanthous C Buttner M J Roe J H regulated by redox change RsrA an anti2sigma factor EMBO J (15) : Stamler J S Hausladen A Oxidative modifications in nitrosative stress Nat Struct Biol ( 4) : Cooper C E Patel R P Brookes P S Darley2Usmar V M Nanotransducers in cellular redox signaling : Modification of thiols by reactive oxygen and nitrogen species Trends Biochem Sci (10) : Zou J R Baler R Dahl G Voellmy R Activation of the DNA2binding ability of human heat shock transcription factor 1 may involve the transition from an intramolecular to an intermolecular triple2stranded coiled2coil structure Mol Cell Biol (11) : Liu A Y I (Lin Zheng Huang Fan Ma Zhong2Fu Xu Kang Liu Alice Y Redox2 dependent changes in structure and function of hhsf1 Acta Biochim Biophys Sin) (6) : (Wei Xiang Zeng Xian2Gang Zhou transcription factors in regulation of the heat shock response and beyond FAS EB J (7) : Zhong M Orosz A Wu C Direct sensing of heat and oxidation by Drosophila heat shock transcription factor Mol Cell (1) :
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