人Calpain1与心脏结构蛋白的相互作用

Interactions Between Human Calpain1 and Heart Structure Proteins

为探讨人calpain1与心脏结构蛋白之间的相互作用 ,采用酵母双杂交体系筛选与人calpain1大亚基相互作用的蛋白质 ,通过DNA序列测定及BLAST分析同源性 ,获得了阳性克隆 12 (pACT2 12 ) ,16 (pACT2 16 ) ,2 2 (pACT2 2 2 )和 37(pACT2 37)等 .12和 2 2分别与人心脏α肌动蛋白 (humancardiacmusclealpha actin ,ACTC)有 99%和 10 0 %同源性 .16和 37分别与人心脏的肌球蛋白结合蛋白C(humancardiacmyosin bindingproteinC ,MYBPC3)和人α2 辅肌动蛋白 (humancardiacalpha 2actinin ,ACTN2 )有 10 0 %同源性 .这些阳性克隆均属于心脏中心肌细胞的结构蛋白 ,参与心肌细胞的收缩运动 .为鉴定calpain1大亚基的哪些结构域可能参与这些相互作用 ,阳性克隆再分别与含有人calpain1大亚基结构域Ⅱ、Ⅲ、Ⅳ的诱饵质粒共同转化AH10 9进行酵母双杂交 ,发现pACT2 12(ACTC)和pACT2 16 (MYBPC3)均能与全长人calpain1大亚基的结构域Ⅱ和Ⅲ相互作用 ;pACT2 16(MYBPC3)还能与大亚基的结构域Ⅳ作用 ;而pACT2 37(ACTN)虽能与全长人calpain1大亚基作用 ,却不与其单独的结构域Ⅱ、Ⅲ或Ⅳ发生作用 .定量分析阳性克隆与人calpain1大亚基作用的强弱的结果显示 ,pACT2 12、16或 37分别与诱饵质粒pGBKT7 CANP共同转化AH10

To assess the relations of calpain 1 to heart structure proteins, positive clones No. 12(pACT2-12), No.16 (pACT2-16), No.22 (pACT2-22) and No. 37 (pACT2-37) were obtained following screening the proteins interacted with calpain1 large subunit using yeast two-hybrid system, sequencing and BLAST analysis of homology. The sequences of putative positive clones- No.12 and 22 have high homology with the 99% and 100% similarities to human cardiac muscle alpha-actin (ACTC). The sequences of the No. 16 and 37 have the 100% similarity to both human cardiac myosin-binding protein C (MYBPC3) and alpha 2 actinin (ACTN2). These clones all belong to the cytoskeletal proteins of cardiomyocytes. To examine which domains of calpain1 large subunit may involve in these interactions, positive clones were transformed into the AH109 with the baits of domain Ⅱ, Ⅲ, Ⅳ of human calpain1 large subunit, respectively. Both pACT2-12(ACTC) and pACT2-16(MYBPC3) could interact with domain Ⅱ or Ⅲ of the large subunit, respectively and pACT2-16(MYBPC3) could also interact with the domain IV of the large subunit; while pACT2-37(ACTN) could not interact with domain Ⅱ, Ⅲ and Ⅳ of the large subunit. Quantitative analysis of the interaction between the positive clones and human calpain1 large subunit was performed by detecting β-galactosidase (β-Gal) activity. When the positive clones- pACT2-12, 16 and 37 interacted with human calpain1 large subunit, β-Gal activities were 122.28, 142.23, 110.26 Units/mg, respectively. Our data indicated that human calpain1 large subunit could interact with heart structure proteins, including ACTC, MYBPC3 and ACTN2, respectively.