Vyas D R, McCarthy J J, Tsika G L, Tsika R W
Department of Biochemistry, School of Medicine, School of Veterinary Medicine, and the Dalton Cardiovascular Research Center, University of Missouri, Columbia, Missouri 65211, USA.
J Biol Chem. 2001 Jan 12;276(2):1173-84. doi: 10.1074/jbc.M007750200.
To examine the role of the beta-myosin heavy chain (betaMyHC) distal muscle CAT (MCAT) element in muscle fiber type-specific expression and mechanical overload (MOV) responsiveness, we conducted transgenic and in vitro experiments. In adult transgenic mice, mutation of the distal MCAT element led to significant reductions in chloramphenicol acetyltransferase (CAT) specific activity measured in control soleus and plantaris muscles when compared with wild type transgene beta293WT but did not abolish MOV-induced CAT specific activity. Electrophoretic mobility shift assay revealed the formation of a specific low migrating nuclear protein complex (LMC) at the betaMyHC MCAT element that was highly enriched only when using either MOV plantaris or control soleus nuclear extract. Scanning mutagenesis of the betaMyHC distal MCAT element revealed that only the nucleotides comprising the core MCAT element were essential for LMC formation. The proteins within the LMC when using either MOV plantaris or control soleus nuclear extracts were antigenically related to nominal transcription enhancer factor 1 (NTEF-1), poly(ADP-ribose) polymerase (PARP), and Max. Only in vitro translated TEF-1 protein bound to the distal MCAT element, suggesting that this multiprotein complex is tethered to the DNA via TEF-1. Protein-protein interaction assays revealed interactions between nominal TEF-1, PARP, and Max. Our studies show that for transgene beta293 the distal MCAT element is not required for MOV responsiveness but suggest that a multiprotein complex likely comprised of nominal TEF-1, PARP, and Max forms at this element to contribute to basal slow fiber expression.
为了研究β-肌球蛋白重链（βMyHC）远端肌肉CAT（MCAT）元件在肌纤维类型特异性表达和机械过载（MOV）反应性中的作用，我们进行了转基因和体外实验。在成年转基因小鼠中，与野生型转基因β293WT相比，远端MCAT元件的突变导致在对照比目鱼肌和跖肌中测得的氯霉素乙酰转移酶（CAT）比活性显著降低，但并未消除MOV诱导的CAT比活性。电泳迁移率变动分析显示，在βMyHC MCAT元件处形成了一种特定的低迁移率核蛋白复合物（LMC），只有在使用MOV跖肌或对照比目鱼肌核提取物时，该复合物才高度富集。对βMyHC远端MCAT元件进行扫描诱变发现，只有构成核心MCAT元件的核苷酸对于LMC的形成至关重要。使用MOV跖肌或对照比目鱼肌核提取物时，LMC中的蛋白质与标称转录增强因子1（NTEF-1）、聚（ADP-核糖）聚合酶（PARP）和Max具有抗原相关性。只有体外翻译的TEF-1蛋白与远端MCAT元件结合，这表明这种多蛋白复合物是通过TEF-1与DNA相连的。蛋白质-蛋白质相互作用分析揭示了标称TEF-1、PARP和Max之间的相互作用。我们的研究表明，对于转基因β293，远端MCAT元件不是MOV反应性所必需的，但提示在该元件处可能形成了一种可能由标称TEF-1、PARP和Max组成的多蛋白复合物，以促进基础慢纤维表达。
