Leich Franziska, Stöhr Nadine, Rietz Anne, Ulbrich-Hofmann Renate, Arnold Ulrich
Institute of Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, Halle, Germany.
J Biol Chem. 2007 Sep 21;282(38):27640-6. doi: 10.1074/jbc.M702240200. Epub 2007 Jul 17.
The cytotoxic action of ribonucleases (RNases) requires the interaction of the enzyme with the cellular membrane, its internalization, translocation to the cytosol, and the degradation of ribonucleic acid. The interplay of these processes as well as the role of the thermodynamic and proteolytic stability, the catalytic activity, and the evasion from the intracellular ribonuclease inhibitor (RI) has not yet been fully elucidated. As cytosolic internalization is indispensable for the cytotoxicity of extracellular ribonucleases, we investigated the extent of cytosolic internalization of a cytotoxic, RI-evasive RNase A variant (G88R-RNase A) and of various similarly cytotoxic but RI-sensitive RNase A tandem enzyme variants in comparison to the internalization of the non-cytotoxic and RI-sensitive RNase A. After incubation of K-562 cells with the RNase A variants for 36 h, the internalized amount of RNases was analyzed by rapid cell disruption followed by subcellular fractionation and semiquantitative immunoblotting. The data indicate that an enhanced cellular uptake and an increased entry of the RNases into the cytosol can outweigh the abolishment of catalytic activity by RI. As all RNase A variants proved to be resistant to the proteases present in the different subcellular fractions for more than 100 h, our results suggest that the cytotoxic potency of RNases is determined by an efficient internalization into the cytosol.
核糖核酸酶（RNases）的细胞毒性作用需要该酶与细胞膜相互作用、内化、转运至胞质溶胶以及核糖核酸的降解。这些过程之间的相互作用以及热力学和蛋白水解稳定性、催化活性以及逃避细胞内核糖核酸酶抑制剂（RI）的作用尚未完全阐明。由于胞质内化对于细胞外核糖核酸酶的细胞毒性不可或缺，我们研究了一种具有细胞毒性、可逃避RI的核糖核酸酶A变体（G88R-RNase A）以及各种类似的具有细胞毒性但对RI敏感的核糖核酸酶A串联酶变体与无细胞毒性且对RI敏感的核糖核酸酶A内化程度的比较。在用核糖核酸酶A变体孵育K-562细胞36小时后，通过快速细胞破碎、亚细胞分级分离和半定量免疫印迹分析核糖核酸酶的内化量。数据表明，核糖核酸酶增强的细胞摄取和增加的进入胞质溶胶的能力可以超过RI对催化活性的消除作用。由于所有核糖核酸酶A变体在超过100小时内对不同亚细胞组分中存在的蛋白酶均具有抗性，我们的结果表明核糖核酸酶的细胞毒性效力取决于有效内化至胞质溶胶中。
