TAT protein transduction into isolated perfused hearts -: TAT-apoptosis repressor with caspase recruitment domain is cardioprotective
TAT protein transduction into isolated perfused hearts -: TAT-apoptosis repressor with caspase recruitment domain is cardioprotective
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DOI：
10.1161/01.cir.0000023943.50821.f7
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发表时间：
2002-08-06
期刊：
影响因子：
37.8
通讯作者：
Gottlieb, RA
中科院分区：
文献类型：
--
作者：
Gustafsson, ÅB;Sayen, MR;Gottlieb, RA
背景-HIV达特的11个氨基酸转导结构域与异源蛋白质的连接允许蛋白质容易地转导到细胞中。方法和结果-在这项研究中，我们将凋亡阻遏物与半胱天冬酶募集结构域（ARC）或β-半乳糖苷酶一起插入将（β-gal）cDNA插入pTAT-血凝素细菌表达载体中以产生遗传框内TAT-ARC或TAT-β-gal。gal融合蛋白用于成年大鼠心脏的细胞培养和Langendorff灌注。TAT-beta-gal和TAT-ARC与德克萨斯红缀合，并且可以在>95%的细胞中检测到。TAT-ARC能够保护H9 c2细胞免受过氧化氢介导的细胞死亡，如通过保护线粒体膜电位的丧失和核形态的保存所测量的。用重组TAT-β-gal或TAT-ARC（20 nmol/L）灌注离体成人心脏15分钟，然后进行30分钟的全脑无血流缺血，随后再灌注2小时。通过细胞裂解物和胞质和线粒体组分的蛋白质印迹以及德克萨斯红缀合的达特蛋白的荧光显微镜来评估蛋白质转导。TAT-beta-gal和TAT-ARC很容易转导到灌注的心脏中，并且均匀分布。通过氯化2，3，5-三苯基四唑染色确定细胞大小，并测量肌酸激酶释放。TAT-ARC的转导是cardioprotective管理时，全球缺血和reperfusion. Conclusions，我们的研究结果表明，TAT连接的融合蛋白转导到心肌是可行的，TAT-ARC的转导是保护细胞培养和灌注心脏。
Background-Linkage of the 11-amino-acid transduction domain of HIV TAT to a heterologous protein allows the protein to be transduced readily into cells.Methods and Results-In this study, we inserted the apoptosis repressor with caspase recruitment domain (ARC) or beta-galactosidase (beta-gal) cDNA into the pTAT-hemagglutinin bacterial expression vector to produce genetic in-frame TAT-ARC or TAT-beta-gal fusion proteins for use in cell culture and in Langendorff perfusion of adult rat hearts. TAT-beta-gal and TAT-ARC were conjugated with Texas Red and could be detected in >95% of cells. TAT-ARC was able to protect H9c2 cells against cell death mediated by hydrogen peroxide, as measured by protection against the loss of mitochondrial membrane potential and preservation of nuclear morphology. Isolated adult hearts were perfused with recombinant TAT-beta-gal or TAT-ARC (20 nmol/L) for 15 minutes and then subjected to 30 minutes of global no-flow ischemia, followed by 2 hours of reperfusion. Protein transduction was assessed by Western blotting of cell lysates and cytosolic and mitochondrial fractions and by fluorescence microscopy of Texas Red-conjugated TAT proteins. TAT-beta-gal and TAT-ARC readily transduced into perfused hearts and were homogeneously distributed. Infarct size was determined by 2,3,5-triphenyltetrazolium chloride staining, and creatine kinase release was measured. Transduction of TAT-ARC was cardioprotective when administered before global ischemia and reperfusion.Conclusions-Our results demonstrate that TAT-linked fusion protein transduction into the myocardium is feasible and that transduction of TAT-ARC is protective in cell culture and in the perfused heart.