Department of Biotechnology, The Catholic University of Korea, 43 Jibong-ro, Wonmi-gu, Bucheon-si, Gyeonggi do, 420-743, Republic of Korea; Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan.
Colloids Surf B Biointerfaces. 2017 Dec 1;160:682-687. doi: 10.1016/j.colsurfb.2017.10.023. Epub 2017 Oct 7.
One of the major concerns in target identification has been the need for new methods to detect target molecules in the native cellular environment. In conventional target identification, affinity-based pull down has been conducted using cell lysates. However, interactions in cell lysate do not reflect real endogenous interactions in living cells, and can produce false-positive or false-negative results. This study aimed to develop a new method of target protein identification in living cells. Targeting probes were conjugated onto magnetic nanoparticles (MNPs). After nanoparticle cellular uptake, identification and recruitment of target proteins were conducted in living cells, and the target protein was finally recovered under the magnetic field. As a proof-of-concept study, we developed a functionalized MNP (PTS1-MNP) to mimic a peroxisomal protein containing a synthetic peroxisomal targeting signal 1 (PTS1). The PTS1-MNPs were imported into human hepatoma HepG2 cells to recruit PTS1-receptor protein Pex5p. Successful peroxisomal translocation of PTS-MNPs was achieved via transient interaction with Pex5p. Pull-down of Pex5p in lysed or living HepG2 cells confirmed the selective recruiting functionality of synthetic PTS1. The specific detection of Pex5p before complete PTS1-MNPs translocation in living HepG2 cells further demonstrated the transient interaction between Pex5p and PTS1-MNPs. This is the first report showing the peroxisomal translocation of nanostructured materials in living cells. This approach can be applied as a new concept to study transient interactions and target identification or recruiting in living cells.
在目标识别中，主要关注的问题之一是需要新的方法来检测天然细胞环境中的靶分子。在传统的靶标识别中，使用细胞裂解物进行基于亲和力的下拉。然而，细胞裂解物中的相互作用不能反映活细胞中真实的内源性相互作用，并且可能产生假阳性或假阴性结果。本研究旨在开发一种在活细胞中鉴定靶蛋白的新方法。将靶向探针连接到磁性纳米颗粒（MNPs）上。在纳米颗粒被细胞摄取后，在活细胞中进行靶蛋白的鉴定和募集，然后在磁场下回收靶蛋白。作为概念验证研究，我们开发了一种功能化的 MNP（PTS1-MNP）来模拟含有合成过氧化物酶体靶向信号 1（PTS1）的过氧化物酶体蛋白。将 PTS1-MNPs 导入人肝癌 HepG2 细胞中，募集 PTS1 受体蛋白 Pex5p。通过与 Pex5p 的瞬时相互作用，成功实现了 PTS-MNPs 的过氧化物酶体易位。在裂解或活 HepG2 细胞中 Pex5p 的下拉证实了合成 PTS1 的选择性募集功能。在活 HepG2 细胞中完全 PTS1-MNPs 易位之前对 Pex5p 的特异性检测进一步证明了 Pex5p 和 PTS1-MNPs 之间的瞬时相互作用。这是首次报道在活细胞中过氧化物酶体转运纳米结构材料。这种方法可以作为一种新概念应用于研究活细胞中瞬时相互作用和靶标鉴定或募集。
