Alvarez-García Elisa, Martínez-del-Pozo Alvaro, Gavilanes José G
Departamento de Bioquímica y Biología Molecular I, Facultad de Ciencias Químicas, Universidad Complutense, Avenida Complutense s/n, Ciudad Universitaria, Madrid, Spain.
Arch Biochem Biophys. 2009 Jan 1;481(1):37-44. doi: 10.1016/j.abb.2008.10.012. Epub 2008 Oct 12.
Ribotoxins are a family of toxic extracellular fungal RNases that first enter into the cells and then exert a highly specific ribonucleolytic activity on the larger rRNA molecule, leading to protein synthesis inhibition and cell death by apoptosis. alpha-Sarcin is the best characterized ribotoxin. Previous characterization of a deletion variant of this protein showed that its long NH(2)-terminal beta-hairpin is essential for its cytotoxicity. Docking, enzymatic, and lipid-protein interaction studies suggested that this beta-hairpin establishes specific interactions with ribosomal proteins and that it is a region involved in the interaction with cell membranes. Consequently, in order to assess the influence of the basic character of this NH(2)-terminal beta-hairpin (there are 1 arginine and 4 lysines along its 16 residues) on the ribotoxins cytotoxic ability, five individual mutants substituting these five basic residues by glutamic acid were produced, purified to homogeneity, and characterized. Regarding ribosomal recognition, all mutants showed a diminished activity in a cell-free reticulocyte lysate, whereas the activity against an oligoribonucleotide mimicking the sarcin/ricin loop rRNA (SRL) or the homopolymer poly(A) remained unaffected, confirming that the mutated basic residues participate in electrostatic interactions with other ribosomal elements apart from this SRL. The study of the interaction with phospholipid vesicles showed that Lys 17, Arg 22, and, most importantly, Lys 14 and Lys 21, are crucial residues in the first stages of the aggregation phenomenon, where protein-vesicle and protein-protein interactions are required. The data obtained reveal that electrostatic interactions involving basic residues of the beta-hairpin are required not only for establishing specific interactions with ribosomal regions other than the SRL but also to explain the ability of the protein to interact with acid phospholipid bilayers.
核糖体毒素是一类有毒的细胞外真菌核糖核酸酶，它们首先进入细胞，然后对较大的核糖体RNA分子发挥高度特异性的核糖核酸水解活性，导致蛋白质合成抑制并通过凋亡使细胞死亡。α-肌动蛋白是特征最明确的核糖体毒素。此前对该蛋白一个缺失变体的表征表明，其长的NH₂-末端β-发夹结构对其细胞毒性至关重要。对接、酶学和脂蛋白相互作用研究表明，这个β-发夹结构与核糖体蛋白建立了特定的相互作用，并且它是参与与细胞膜相互作用的区域。因此，为了评估这个NH₂-末端β-发夹结构（其16个残基中有1个精氨酸和4个赖氨酸）的碱性特征对核糖体毒素细胞毒性能力的影响，制备了五个将这五个碱性残基替换为谷氨酸的单个突变体，纯化至同质，并进行了表征。关于核糖体识别，所有突变体在无细胞网织红细胞裂解物中的活性均降低，而针对模拟肌动蛋白/蓖麻毒素环rRNA（SRL）的寡核糖核苷酸或均聚物聚（A）的活性不受影响，这证实突变的碱性残基除了与这个SRL之外，还参与与其他核糖体元件的静电相互作用。与磷脂囊泡相互作用的研究表明，赖氨酸17、精氨酸22，以及最重要的赖氨酸14和赖氨酸21，是聚集现象第一阶段的关键残基，在这个阶段需要蛋白质-囊泡和蛋白质-蛋白质相互作用。所获得的数据表明，涉及β-发夹结构碱性残基的静电相互作用不仅是与SRL以外的核糖体区域建立特定相互作用所必需的，也是解释该蛋白与酸性磷脂双层相互作用能力所必需的。
