Chinese Journal of Biochemistry and Molecular Biology ERR . I2 (TNNI2) GST2TNNI2, 1. 1

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1 ISSN CN ΠQ Chinese Journal of Biochemistry and Molecular Biology 21 (5) : I2 ERR 1,,,,, (, ) 3 1 ( ERR 1) I2 (TNNI2) TNNI2 ERR 1, GST2TNNI2, 35 S ERR 1, GST,TNNI2 35 S ERR 1, TNNI2 ERR 1, I2 ERR 1 I 1 ( ERR 1) 2 Q51,Q78 Prokaryotic Expression of Troponin I2 and Its in vitro Interaction with ERR 1 LI Yu2Ping, CHEN Min, CHEN Bin, CHEN Jian, LI Qiang, ZHOU Du2Jin 3 ( Department of Biochemistry and Molecular Biology, Division of Basic Medicine, Third Military Medical University, Chongqing , China) Abstract Orphan receptor ERR 1 (estrogen receptor2related receptor 1) is discovered to be closely linked with breast cancer Distinct interaction between ERR 1 and TNNI2 (troponin I2) in breast tissue was found by yeast two2hybrid screening To further study the interaction between TNNI2 and ERR 1, fusion protein of GST2TNNI2 was expressed ERR 1 protein labeled with 35 S was translated in vitro and GST pull down assay was performed by incubating these two proteins together Result of autoradiography showed that TNNI2 could pull down ERR 1 labeled with 35 S The result demonstrates that TNNI2 can bind to ERR 1 directly, and suggests that TNNI2 may play a crucial role in the physiological process of ERR 1 Key words troponin I, estrogen receptor2related receptor 1 ( ERR 1), protein2protein interaction troponin I,TNNI2) [6 ], [1 ] TNNI2 1 ( estrogen receptor2related receptors 1, ERR 1) ER 1 [2,3 ] [4, ] ERR 1, 1 1 [2 ], : , : (No ,No ) [5 ], ERR 1 ERR 1 (ligand binding domain,lbd) (No 2458) 3 Tel : (023) ; Fax : (023) E2mail : com Received : October 21,2004 ;Accepted : July 13,2005 Supported by National Natural Science Foundation of China (No ), ERR 1ΠLBD I (fast skeletal muscle TNNI2 ERR 1 and Innovation Grant of Third Military Medical University (No 2458) 3 Corresponding author Tel : (023) , Fax : (023) E2mail : com

2 5 : I2 ERR B B2PER 300 l ( S2 (Amersham Pharmacia ) ; TNT g ) 200 gπml (Promega ) ;T4 DNA 1 min, 10 min 1 ml 1 10 ( ) ; B2PER 1 min rπmin Ampi Taq (Perkin2Elmer ) ;DNA 10 min 1 10 B2PER 1 (Qiagen ) ; IPTG(Sigma ) ;B2PER min l ( Pierce ) ; Protein Protein Dye Dye (Bio2Rad ) ; 10 %, p GEX2TK psg5 pact22 TNN I2 [6 ] ;psg522err 1 p GL3 ( SFRE) 3 2SV40 promoter2luciferase Dr1 Chen Shiuan ( Beckman Research Institute of City of 24 h Π 12 Hope, Duarte, CA, USA) ; DH5 HeLa Life Technologies, BL21 (DE3) Lipofectin DNA psg5 1 2 GST( )2TNNI2 3 5 h 15 % FBS( 2 ) Lipofectin pact22 TNN I2, DNA h PCR TNN I2 cdna, p GEX2TK, BamH EcoR, E coli ERR 1 DH5 35 S Promega 3 ml LBΠAmp (PEG ) 1 g psg52 ERR 1,BamH EcoR 40 l TNT g Π ( Promega),1 % TNT g Quick Master Mix 2 l 35 S2 PCR : 5 2GGC GGA TCC ATG GGA GAT GAG GAGAAG CGGAAC23 5 2GCC GAA TTC CTA, min - 70 GGA CTC GGA CTC AAA CAT 23, BamH EcoR 117 GST 1 3 GST GST2TNNI2 100 g GST GST2TNNI2 ( ) p GEX2TK2 TNN I2 30 l 50 % p GEX2TK Sepharose 4B (1 PBS ), 10 BL21 (DE3) ( CaCl 2 ) min, 1 PBS 3,, GST, ml 150 l 4 l 35 S2 25 gπml ( 25 mgπml ERR 1,4 2 h - 20 ) LB 37 BSA 3 50 l 3 4 h, A IPTG 1 SDS2PAGE, mmolπl ( 0125 molπl min 25 l, 10 % SDS2PAGE - 20 ) A S2ERR l B2PER, 1 min rπmin,5 min 60 2 h,, X 300 l B2PER, h 10 l SDS2PAGE HeLa 5 % FBS 011 gπ l Π D2MEM 5 % CO 2,37 (1 000 CiΠmmol) 50 l pgex2tk2 TNN I2

3 p GEX2TK2 TNN I2, (Fig13) ( Fig11), ERR 1 ERR 1 TNN I2 cdna ERR 1 GST2TNNI2 Fig 3 Transactivation functional analysis of ERR 1 in HeLa cells 1 :Reporter (p GL32(SFRE) 3 2SV402Promoter2Luciferase 100 ng) ; 2 :Reporter + psg52 ERR 1 10 ng ; 3 :Reporter + psg52 ERR 1 20 ng ; 4 :Reporter + psg52 ERR 1 40 ng Fig 1 Identification of p GEX2TK2 TNN I2 by restriction endonuclease digestion M: Marker ; 1 :Recombinant plasmid digested by BamH I and EcoR I The insert was about 564 bp 2 2 GST2 TNN I2 p GEX2TK2 TNN I2 38 kd (Fig12) GST 2 5 GST Fig 4 2GST2TNNI2 35 S2ERR 1 Fig 2 Expression and purification of GST2TNNI2 After IPTG induction at 37 for 4 hours, the purified fractions were analyzed by SDS2PAGE (12 %) M: Marker ; 1 : Whole cell lysate ; 2 : Soluble fraction ; 3 : Insoluble fraction 2 3 psg52err 1 ERR 1 cdna T7 2 4 psg52 ERR 1 psg52err 1 ERR 1 p GL32( SFRE) 3 2SV40 promoter2 Luciferase HeLa Fig 4 In vitro interaction of TNNI2 with ERR 1 Incubated proteins were separated by using 10 % SDS2polyacrylamide gel electrophoresis and then autoradiographed derived from 35 S2ERR 1 can be observed The autoradiographed bands M: Marker ; 1 : 35 S2ERR 1 by in vitro translation ; 2 : GST2TNNI2 incubated with 35 S2ERR 1 ; 3 : GST incubated with 35 S2ERR 1

4 5 : I2 ERR TNNI2 ERR 1 2GST2TNNI22 35 S2ERR 1 [11 10 %SDS2PAGE ] TNNI2, 35 S2ERR 1, LXXLL, 35 S2ERR 1 (LKALL,L,K,A,X GST 35 S2 ), ERR 1,TNNI2 ERR 1 (NR box) [5 ] 35 S2ERR 1 I, ERR 1 GST2TNNI2 ERR 1 LBD GST, 3, TNNI2 (coregulator) TNNI2 ERR 1, GST (coactivator) (corepressor) (glutathione S transferase, ) 2 TNNI2 ERR 1 GST,,, GST, TNNI2 GST2 [5 ] ERR 1 TNNI2, Π ( Promega) psg5 ( SV40, T7 ) ERR 1 LBD,, psg52err 1 ERR ERR 1ΠLBD [6 ] 3 ERR 1 ( 35 S2 ), [7 ],RBP4, 35 S2ERR 1, GST2 I2 TNNI2 SDS2PAGE (troponin,tn) GST2TNNI2 35 S2ERR 1 I (troponin I,TNNI) GST 35 S2, TNNI ERR 1, TNNI2 TNNI2 TNNI TNNI1 TNNI2 ERR 1 TNNI TNNI3 ctni [10 ] TNNI, TNNI2, I(TNNI3, ctni) [8 ] ; TNNI2 TNNI2,, TNNI2, [9 ] TNNI TNNI2 C ( Ca 2 + ) ( References) [11 ],TNNI TNNI2 NR, TNNI2 1 Margueron R, Duong V, Castet A, Cavailles V Histone deacetylase

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