Brigotti Maurizio, Alfieri Roberta, Sestili Piero, Bonelli Mara, Petronini Pier Giorgio, Guidarelli Andrea, Barbieri Luigi, Stirpe Fiorenzo, Sperti Simonetta
Dipartimento di Patologia Sperimentale, Università degli Studi di Bologna, Italy.
FASEB J. 2002 Mar;16(3):365-72. doi: 10.1096/fj.01-0521com.
Ribosome-inactivating proteins (RIPs) remove a specific adenine from 28S rRNA leading to inactivation of ribosomes and arrest of translation. Great interest as to a possible second physiological substrate for RIPs came from the observation that in vitro RIPs remove adenine from DNA. This paper addresses the problem of nuclear lesions induced by RIPs in human endothelial cells susceptible to the bacterial RIP Shiga toxin 1 and the plant RIP ricin. With both toxins, nuclear DNA damage as evaluated by two independent techniques (alkaline-halo assay and alkaline filter elution) appears early, concomitant with (ricin) or after (Shiga toxin 1) the inhibition of protein synthesis. At this time, the annexin V binding assay, caspase 3 activity, the formation of typical < or = 50 Kb DNA fragments, and changes in morphology associated with apoptosis were negative. Furthermore, a block of translation comparable to that induced by RIPs, but obtained with cycloheximide, did not induce nuclear damage. Such damage is consistent with the enzymatic activity (removal of adenine) of RIPs acting in vitro on RNA-free chromatin and DNA. The results unequivocally indicate that RIPs can damage nuclear DNA in whole cells by means that are not secondary to ribosome inactivation or apoptosis.
核糖体失活蛋白（RIPs）从28S核糖体RNA中去除特定腺嘌呤，导致核糖体失活并使翻译停滞。对RIPs可能的第二种生理底物产生了浓厚兴趣，这源于体外实验中观察到RIPs能从DNA中去除腺嘌呤。本文探讨了在对细菌RIP志贺毒素1和植物RIP蓖麻毒素敏感的人内皮细胞中，RIPs诱导的核损伤问题。使用这两种毒素时，通过两种独立技术（碱性晕圈试验和碱性滤膜洗脱法）评估的核DNA损伤出现得较早，与蛋白质合成抑制同时出现（蓖麻毒素）或在其之后出现（志贺毒素1）。此时，膜联蛋白V结合试验、半胱天冬酶3活性、典型的≤50 Kb DNA片段的形成以及与凋亡相关的形态变化均为阴性。此外，用放线菌酮获得的与RIPs诱导的翻译阻滞相当的情况，并未诱导核损伤。这种损伤与RIPs在体外对无RNA染色质和DNA起作用的酶活性（腺嘌呤去除）一致。结果明确表明，RIPs可通过并非继发于核糖体失活或凋亡的方式在全细胞中损伤核DNA。
