Martinelli Chiara, Colombo Emanuela, Piccini Daniele, Sironi Cristina, Pelicci Pier Giuseppe, de Marco Ario
IFOM-IEO Campus, Via Adamello 16, 20139 Milan, Italy.
Department of Experimental Oncology, IEO, Via Adamello 16, 20139 Milan, Italy.
Biotechnol Rep (Amst). 2014 May 27;3:27-33. doi: 10.1016/j.btre.2014.05.008. eCollection 2014 Sep.
The cytoplasmic accumulation of NPM1 (NPMc+) is found in acute myeloid leukemia (AML) with NPM1 mutation. NPM1 must shuttle between nucleus and cytoplasm to assure physiological protein synthesis and, therefore, the elimination of NPMc+ is not a suitable therapeutic option. We isolated, characterized, and produced a functional scFv intrabody fused to nuclear localization signal(s) (NLS) that does not recognize NPM1 but binds to the mutant-specific C-terminal NES (nuclear export signal) of NPMc+, responsible for its cytoplasmic accumulation. The scFv-NLS fusion accumulated in the nuclei of wild type cells and strongly bound to its antigen in the cytoplasm of NPMc+ expressing cells. However, it failed to relocate the majority of NPMc+ in the nucleus, even when fused to four NLS. Our results show the technical feasibility of producing recombinant intrabodies with defined sub-cellular targeting and nuclear accumulation but the lack of information concerning the features that confer variable strength to the signal peptides impairs the development of biomolecules able to counteract pathological sub-cellular distribution of shuttling proteins.
在伴有NPM1突变的急性髓系白血病（AML）中可发现NPM1在细胞质中积聚（NPMc +）。NPM1必须在细胞核和细胞质之间穿梭以确保生理蛋白质合成，因此，消除NPMc +并非合适的治疗选择。我们分离、鉴定并制备了一种与核定位信号（NLS）融合的功能性单链抗体片段（scFv）胞内抗体，该胞内抗体不识别NPM1，但能结合NPMc +突变特异性的C端核输出信号（NES），正是该信号导致其在细胞质中积聚。scFv-NLS融合蛋白在野生型细胞的细胞核中积聚，并在表达NPMc +的细胞的细胞质中与其抗原强烈结合。然而，即使与四个NLS融合，它也未能将大多数NPMc +重新定位到细胞核中。我们的结果表明，生产具有明确亚细胞靶向和核积聚功能的重组胞内抗体在技术上是可行的，但由于缺乏有关赋予信号肽不同强度特征的信息，阻碍了能够对抗穿梭蛋白病理性亚细胞分布的生物分子的开发。
