Toledo M A S, Favaro M T P, Alves R F, Santos C A, Beloti L L, Crucello A, Santiago A S, Mendes J S, Horta M A C, Aparicio R, Souza A P, Azzoni A R
Appl Microbiol Biotechnol. 2014 Apr;98(8):3591-602. doi: 10.1007/s00253-013-5239-5.
Dynein light chains mediate the interaction between the cargo and the dynein motor complex during retrograde microtubule-mediated transport in eukaryotic cells. In this study, we expressed and characterized the recombinant human dynein light chain Rp3 and developed a modified variant harboring an N-terminal DNA-binding domain (Rp3-Db). Our approach aimed to explore the retrograde cell machinery based on dynein to enhance plasmid DNA (pDNA) traffic along the cytosol toward the nucleus. In the context of non-viral gene delivery, Rp3-Db is expected to simultaneously interact with DNA and dynein, thereby enabling a more rapid and efficient transport of the genetic material across the cytoplasm. We successfully purified recombinant Rp3 and obtained a low-resolution structural model using small-angle X-ray scattering. Additionally, we observed that Rp3 is a homodimer under reducing conditions and remains stable over a broad pH range. The ability of Rp3 to interact with the dynein intermediate chain in vitro was also observed, indicating that the recombinant Rp3 is correctly folded and functional. Finally, Rp3-Db was successfully expressed and purified and exhibited the ability to interact with pDNA and mediate the transfection of cultured HeLa cells. Rp3-Db was also capable of interacting in vitro with dynein intermediate chains, indicating that the addition of the N-terminal DNA-binding domain does not compromise its function. The transfection level observed for Rp3-Db is far superior than that reported for protamine and is comparable to that of the cationic lipid Lipofectamine™. This report presents an initial characterization of a non-viral delivery vector based on the dynein light chain Rp3 and demonstrates the potential use of modified human light chains as gene delivery vectors.
动力蛋白轻链在真核细胞中逆行微管介导的运输过程中，介导货物与动力蛋白运动复合体之间的相互作用。在本研究中，我们表达并鉴定了重组人动力蛋白轻链Rp3，并开发了一种带有N端DNA结合结构域的修饰变体（Rp3-Db）。我们的方法旨在探索基于动力蛋白的逆行细胞机制，以增强质粒DNA（pDNA）沿细胞质向细胞核的运输。在非病毒基因递送的背景下，Rp3-Db有望同时与DNA和动力蛋白相互作用，从而使遗传物质在细胞质中更快速、高效地运输。我们成功纯化了重组Rp3，并使用小角X射线散射获得了低分辨率结构模型。此外，我们观察到Rp3在还原条件下是同二聚体，并且在较宽的pH范围内保持稳定。还观察到Rp3在体外与动力蛋白中间链相互作用的能力，表明重组Rp3折叠正确且具有功能。最后，Rp3-Db成功表达并纯化，并表现出与pDNA相互作用并介导培养的HeLa细胞转染的能力。Rp3-Db在体外也能够与动力蛋白中间链相互作用，表明N端DNA结合结构域的添加不会损害其功能。观察到的Rp3-Db的转染水平远优于鱼精蛋白报道的水平，并且与阳离子脂质Lipofectamine™相当。本报告介绍了基于动力蛋白轻链Rp3的非病毒递送载体的初步表征，并证明了修饰的人轻链作为基因递送载体的潜在用途。
