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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Magnetic nanoparticle-based isolation of endocytic vesicles reveals a role of the heat shock protein GRP75 in macromolecular delivery
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Magnetic nanoparticle-based isolation of endocytic vesicles reveals a role of the heat shock protein GRP75 in macromolecular delivery

机译:基于磁性纳米粒子的内吞囊泡分离揭示了热激蛋白GRP75在大分子传递中的作用

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An increased understanding of cellular uptake mechanisms of macromolecules remains an important challenge in cell biology with implications for viral infection and macromolecular drug delivery. Here, we report a strategy based on antibody-conjugated magnetic nanoparticles for the isolation of endocytic vesicles induced by heparan sulfate proteoglycans (HSPGs), key cell-surface receptors of macromolecular delivery. We provide evidence for a role of the glucose-regulated protein (GRP)75/PBP74/mtHSP70/mortalin (hereafter termed "GRP75") in HSPG-mediated endocytosis of macromolecules. GRP75 was found to be a functional constituent of intra-cellular vesicles of a nonclathrin-, noncaveolin- dependent pathway that was sensitive to membrane cholesterol depletion and that showed colocalization with the membrane raft marker cholera toxin subunit B. We further demonstrate a functional role of the RhoA GTPase family member CDC42 in this transport pathway; however, the small GTPase dynamin appeared not to be involved. Interestingly, we provide evidence of a functional role of GRP75 using RNAi-mediated down-regulation of GRP75 and GRP75-blocking antibodies, both of which inhibited macromolecular endocytosis. We conclude that GRP75, a chaperone protein classically found in the endoplasmic reticulum and mitochondria, is a functional constituent of noncaveolar, membrane raft-associated endocytic vesicles. Our data provide proof of principle of a strategy that should be generally applicable in the molecular characterization of selected endocytic pathways involved in macromolecular uptake by mammalian cells.
机译:对大分子细胞摄取机制的加深了解仍然是细胞生物学中的重要挑战,对病毒感染和大分子药物传递具有影响。在这里,我们报告了一种基于抗体结合的磁性纳米颗粒的策略,该策略用于分离硫酸乙酰肝素蛋白聚糖(HSPG)(大分子传递的关键细胞表面受体)诱导的内吞囊泡。我们提供了葡萄糖调节蛋白(GRP)75 / PBP74 / mtHSP70 / mortalin(以下称为“ GRP75”)在HSPG介导的大分子内吞作用中的作用的证据。发现GRP75是非clathrin-,non-caveolin依赖性途径的细胞内囊泡的功能成分,该途径对膜胆固醇消耗敏感,并且与膜筏标记物霍乱毒素亚基B共定位。我们进一步证明了GRP75的功能作用。该转运途径中的RhoA GTPase家族成员CDC42;但是,小的GTPase动力蛋白似乎没有参与。有趣的是,我们提供了RNAi介导的GRP75和GRP75阻断抗体的下调,GRP75的功能性作用的证据,两者均抑制了大分子内吞作用。我们得出的结论是,GRP75是一种典型地在内质网和线粒体中发现的分子伴侣蛋白,是非牙槽膜,膜筏相关的内吞囊泡的功能成分。我们的数据提供了一种策略原理的证明,该策略应普遍适用于与哺乳动物细胞吸收大分子有关的所选内吞途径的分子特征。

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