Cicchillitti Lucia, Penci Roberta, Di Michele Michela, Filippetti Flavia, Rotilio Domenico, Donati Maria Benedetta, Scambia Giovanni, Ferlini Cristiano
Department of Oncology, Catholic University of the Sacred Heart, Largo A. Gemelli, 1-86100, Campobasso, Italy.
Mol Cancer Ther. 2008 Jul;7(7):2070-9. doi: 10.1158/1535-7163.MCT-07-2370.
Class III beta-tubulin (TUBB3) has been discovered as a marker of drug resistance in human cancer. To get insights into the mechanisms by which this protein is involved in drug resistance, we analyzed TUBB3 in a panel of drug-sensitive and drug-resistant cell lines. We identified two main different isoforms of TUBB3 having a specific electrophoretic profile. We showed that the apparently higher molecular weight isoform is glycosylated and phosphorylated and it is localized in the cytoskeleton. The apparently lower molecular weight isoform is instead found exclusively in mitochondria. We observed that levels of phosphorylation and glycosylation of TUBB3 are associated with the resistant phenotype and compartmentalization into cytoskeleton. By two-dimensional nonreduced/reduced SDS-PAGE analysis, we also found that TUBB3 protein in vivo forms protein complexes through intermolecular disulfide bridges. Through TUBB3 immunoprecipitation, we isolated protein species able to interact with TUBB3. Following trypsin digestion, these proteins were characterized by mass spectrometry analysis. Functional analysis revealed that these proteins are involved in adaptation to oxidative stress and glucose deprivation, thereby suggesting that TUBB3 is a survival factor able to directly contribute to drug resistance. Moreover, glycosylation of TUBB3 could represent an attractive pathway whose inhibition could hamper cytoskeletal compartmentalization and TUBB3 function.
III类β-微管蛋白（TUBB3）已被发现是人类癌症中耐药性的标志物。为了深入了解该蛋白参与耐药性的机制，我们在一组药物敏感和耐药细胞系中分析了TUBB3。我们鉴定出TUBB3有两种主要的不同同工型，具有特定的电泳图谱。我们发现，分子量明显较高的同工型是糖基化和磷酸化的，并且定位于细胞骨架中。而分子量明显较低的同工型则仅存在于线粒体中。我们观察到，TUBB3的磷酸化和糖基化水平与耐药表型以及在细胞骨架中的区室化有关。通过二维非还原/还原SDS-PAGE分析，我们还发现TUBB3蛋白在体内通过分子间二硫键形成蛋白复合物。通过TUBB3免疫沉淀，我们分离出了能够与TUBB3相互作用的蛋白质种类。经胰蛋白酶消化后，这些蛋白质通过质谱分析进行了表征。功能分析表明，这些蛋白质参与了对氧化应激和葡萄糖剥夺的适应，从而表明TUBB3是一种能够直接导致耐药性的生存因子。此外，TUBB3的糖基化可能代表了一条有吸引力的途径，对其抑制可能会阻碍细胞骨架区室化和TUBB3的功能。
