ISSN 100727626 CN 1123870ΠQ 2005 6 Chinese Journal of Biochemistry and Molecular Biology 21 (3) :341 346 HSF1 3 ( 510080) 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 373 378 S HSF1 HSF1 C 36 T C 103 Y HSF1 (ox2 HSF1) C 153 S C 373 378 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 510080 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 :2004207229 :2004210228 (No 30070829) (No 31695) 3 Tel :02028775576628377 E2mail : zhenglin2 @hotmail com Received : July 29 2004 ;Accepted :October 28 2004 Supported by grants from the National Natural Science Foundation of China (No 30070829) and Guangdong Natural Science Foundation (No 31695) 3 Corresponding author Tel :02028775576628377 E2mail : zhenglin2 @hotmail com
342 21 112 HeLa 10 % (HSF) DMEM 5 % CO 2 37 3 d HSF1 1 [1 ] HSF1 42 60 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 1 1 2 5 3 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 3 000 CiΠmmol) [ 35 S] (S A 1 016 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 373 378 S ( Cys373ΠCys378 CG23 ( ) PCR Ser373ΠSer378 ) 5 2TGAAAAGTTCCCT GeneAmp PCR system 2400( Perkin2Elmer) CAGCGTAGCCTCCCTGGACAAGAAT23 5 2
3 :HSF1 343 TGTCCAGGGAGGCTACGCTGAGGGAACTTTTCA23 ; C 378 S (Cys378 Ser378) C 373 378 S IgG ECL Western HSF1 PCR HSF1 DNA 116 ( EMSA) [5 ] pjc20 HSF1 DNA HSF1 cpm 25 000 C 36 T C 103 Y PflM Nco 30 000 32 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 373 378 S Nco Bgl HSF1 DNA 42 60 min ; pjc20 HS F1 ( Nco ΠBgl ) C 378 S Bpu10 Bgl pjc20 C 373 378 S C 378 S pjc20 HS F1 Bpu10 ΠBgl ( Eppendorf 2510 ) DNA DH5 DNA (QIAfilter Midi QIAGEN) DNA DNA (1 2 500) 85 90 kd ngaan2 HSE 500 g poly (diπdc) 25 30 20 mmolπl 5 min 25 ; DTT 5 mmolπl 5 min 25 2 211 HSF1 6 C 36 T C 103 Y C 153 S C 373 S C 378 S ; C 373 378 S DNA ABI310 ( Pirken2Elmer) (TCA) 114 Promerga TNT g [ 35 S ] T7 20 000 25 000 ( 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 373 378 S DNA ( 5 mmolπl DDT 3) HSF1 15 min 1 mmolπl DDT SD2Dry 213 HSF1 HSF1
344 21 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) ( 2 4 6 ) C 153 S C 373 378 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
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 Ref21 153 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 373 378 S HSF1 ox2hsf1 HSF1 C 36 T C 103 Y HSF1 C 373 S C 378 S 153 2 HSF1 373 378 ox2hsf1 ox2hsf1 HSF1 C 153
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