Evaluation of an LC8-binding peptide for the attachment of artificial cargo to dynein
Evaluation of an LC8-binding peptide for the attachment of artificial cargo to dynein
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DOI：
10.1021/mp060086o
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发表时间：
2007-01-01
影响因子：
4.9
通讯作者：
Pun, Suzie H.
中科院分区：
文献类型：
--
作者：
Bergen, Jamie M.;Pun, Suzie H.
非病毒基因载体有限的细胞质流动性可能是其低转染效率的原因。这一限制可以通过模仿病毒募集动力蛋白马达复合物以向宿主细胞核有效运输的策略来克服。将人工货物连接到动力蛋白的一种有前途的方法是通过衔接肽，该衔接肽结合动力蛋白复合物的货物结合区中发现的8 kDa轻链（LC 8）。几种结合LC 8的病毒蛋白共同具有由（K/R）XTQT定义的LC 8结合基序。含有该基序的短肽也已显示在体外结合重组LC 8。然而，由于大多数细胞内LC 8存在于动力蛋白复合物的外部，因此仍然不清楚显示该LC 8结合基序的肽是否可以接近并结合动力蛋白相关的LC 8。在这项研究中，我们采用生物化学分析，以调查的可行性，附加人工货物的动力蛋白马达复合物使用的肽显示良好的特点LC 8结合基序。我们报告说，游离细胞内LC 8结合特异性的LC 8结合（TQT）肽，而不是一个控制肽与突变的LC 8结合基序。然而，没有检测到TQT肽和细胞内动力蛋白之间的类似结合相互作用。为了确定TQT肽的动力蛋白结合是否由于与游离细胞内LC 8的竞争或由于肽不能接近其在动力蛋白复合物中的LC 8结合位点而被阻止，评价TQT肽结合纯化的LC 8或纯化的动力蛋白的能力。我们的结果表明，虽然TQT肽容易结合游离LC 8，但它不能结合动力蛋白相关LC 8。结果强调需要确定功能的动力蛋白结合肽，并强调设计结合完整的动力蛋白运动复合物的肽的重要性。
The limited cytoplasmic mobility of nonviral gene carriers is likely to contribute to their low transfection efficiency. This limitation could be overcome by mimicking the viral strategy of recruiting the dynein motor complex for efficient transport toward the host cell nucleus. A promising approach for attaching artificial cargo to dynein is through an adaptor peptide that binds the 8 kDa light chain (LC8) found in the cargo-binding region of the dynein complex. Several viral proteins that bind LC8 have in common an LC8-binding motif defined by (K/R)XTQT. Short peptides containing this motif have also been shown to bind recombinant LC8 in vitro. However, since the majority of intracellular LC8 exists outside of the dynein complex, it remains unclear whether peptides displaying this LC8-binding motif can access and bind to dynein-associated LC8. In this study, we employed biochemical analysis to investigate the feasibility of attaching artificial cargo to the dynein motor complex using a peptide displaying the well-characterized LC8-binding motif. We report that free intracellular LC8 bound specifically to an LC8-binding (TQT) peptide and not to a control peptide with a mutated LC8-binding motif. However, a similar binding interaction between the TQT peptide and intracellular dynein was not detected. To determine whether dynein binding of the TQT peptide was prevented by competition with free intracellular LC8 or due to the inability of the peptide to access its LC8 binding site in the dynein complex, the TQT peptide was evaluated for its ability to bind either purified LC8 or purified dynein. Our results demonstrate that, while the TQT peptide readily binds free LC8, it cannot bind to dynein-associated LC8. The results emphasize the need to identify functional dynein-binding peptides and highlight the importance of designing peptides that bind to the intact dynein motor complex.