Loss-of-function screening by randomized intracellular antibodies: Identification of hnRNP-K as a potential target for metastasis
Loss-of-function screening by randomized intracellular antibodies: Identification of hnRNP-K as a potential target for metastasis
复制标题
DOI：
10.1073/pnas.0607595104
复制
发表时间：
2007-05-22
影响因子：
11.1
通讯作者：
Wadhwa, Renu
中科院分区：
文献类型：
--
作者：
Inoue, Atsushi;Sawata, Shinya Y.;Wadhwa, Renu
我们开发了一种基于细胞内单域抗体表达的功能丧失筛选系统。我们证明了它在人类癌细胞转移的潜在靶标鉴定中的应用。随机的细胞内抗体在高转移细胞中表达，并且在趋化性实验中分离出迁移表型丧失的细胞衍生物池。从迁移表型缺失的细胞中分离抗体并鉴定其靶蛋白，揭示了异质性核核糖核蛋白K （hnRNP-K）这一多功能信号蛋白参与了转移。此外，我们发现hnRNP-K的细胞质积累对其在转移中的作用至关重要。结果表明：(1)我们的功能干扰筛选优于基因敲除和基因沉默，(2)hnRNP-K是转移的潜在靶点，(3)在体外细胞迁移试验中验证了潜在的抗转移肽。
We have developed a loss-of-function screening system on the basis of intracellular expression of single domain antibodies. We demonstrate its use in identification of potential targets of metastasis of human cancerous cells. Randomized intracellular antibodies were expressed in highly metastatic cells, and a derivative pool of cells with loss of migration phenotype in chemotaxis assay was isolated. isolation of antibodies from cells with loss of migration phenotype and identification of their target proteins revealed the involvement of the heterogeneous nuclear ribonucleoprotein K (hnRNP-K), a multifunctional signaling protein, in metastasis. Furthermore, we found that the cytoplasmic accumulation of hnRNP-K is crucial for its role in metastasis. The results demonstrate (t) the advantages of our functional interference screening over the gene-knockouts and gene-silencing, (it) hnRNP-K as a potential target of metastasis, and (iii) a potential anti-metastasis peptide validated in in vitro cell migration assays.