Internalization and Intracellular Trafficking of a PTD-Conjugated Anti-Fibrotic Peptide, AZX100, in Human Dermal Keloid Fibroblasts
Internalization and Intracellular Trafficking of a PTD-Conjugated Anti-Fibrotic Peptide, AZX100, in Human Dermal Keloid Fibroblasts
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DOI：
10.1002/jps.22087
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发表时间：
2010-07-01
影响因子：
3.8
通讯作者：
Brophy, Colleen M.
中科院分区：
文献类型：
--
作者：
Flynn, Charles R.;Cheung-Flynn, Joyce;Brophy, Colleen M.
先进药物递送的挑战是选择性地穿过质膜，这是一种阻止大多数基于肽和核酸的治疗剂的细胞内递送的屏障。20多年来，在病毒蛋白中首次发现的称为蛋白转导结构域（PTD）的各种短氨基酸序列已被用于将蛋白质非破坏性地递送到细胞中，然而，发生这种情况的机制是多种多样的并且是细胞特异性的。在这里，我们描述了AZX 100的活细胞成像实验的结果，AZX 100是一种具有“增强型”PTD（PTD 4）的细胞渗透性抗纤维化肽。我们监测荧光标记的AZX 100在细胞表面结合和随后的细胞内运输的细胞过程抑制剂和各种明确的荧光标记的货物的存在下。我们得出结论，AZX 100通过小窝迅速进入细胞，以一种独立于糖缀合物、肌动蛋白/微管聚合、发动蛋白、多种GTP酶和网格蛋白的方式，但与甲基-β-环糊精所揭示的脂筏相关。AZX 100处理增加了磷酸化小窝蛋白（Y14）的表达，这是粘着斑动力学的关键效应子，表明小窝蛋白-1磷酸化与肌动蛋白细胞骨架动力学之间存在机械联系。我们的结果揭示了PTD 4的新颖且有趣的特性，并为促进先进药物递送工具的细胞机制提供了新的见解。(C)2010 Wiley-Liss，Inc.和American Pharmacologist Association J Pharm Sci 99：3100-3121，2010
A challenge in advanced drug delivery is selectively traversing the plasma membrane, a barrier that prohibits the intracellular delivery of most peptide and nucleic acid-based therapeutics. A variety of short amino acid sequences termed protein transduction domains (PTDs) first identified in viral proteins have been utilized for over 20 years to deliver proteins nondestructively into cells, however, the mechanisms by which this occurs are varied and cell-specific. Here we describe the results of live cell imaging experiments with AZX100, a cell-permeable anti-fibrotic peptide bearing an "enhanced" PTD (PTD4). We monitored fluorescently labeled AZX100 upon cell surface binding and subsequent intracellular trafficking in the presence of cellular process inhibitors and various well-defined fluorescently labeled cargos. We conclude that AZX100 enters cells via caveolae rapidly, in a manner that is independent of glycoconjugates, actin/microtubule polymerization, dynamins, multiple GTPases, and clathrin, but is associated with lipid rafts as revealed by methyl-beta-cylodextrin. AZX100 treatment increases the expression of phospho-caveolin (Y14), a critical effector of focal adhesion dynamics, suggesting a mechanistic link between caveolin-1 phosphorylation and actin cytoskeleton dynamics. Our results reveal novel and interesting properties of PTD4 and offer new insight into the cellular mechanisms facilitating an advanced drug delivery tool. (C) 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:3100-3121, 2010