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首页> 外文期刊>Biochemistry >Translocation of beta-galactosidase mediated by the cell-penetrating peptide pep-1 into lipid vesicles and human HeLa cells is driven by membrane electrostatic potential
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Translocation of beta-galactosidase mediated by the cell-penetrating peptide pep-1 into lipid vesicles and human HeLa cells is driven by membrane electrostatic potential

机译：细胞渗透肽pep-1介导的β-半乳糖苷酶易位到脂质囊泡和人HeLa细胞中是由膜静电势驱动的

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摘要

The cell-penetrating peptide (CPP) pep-1 is capable of introducing large proteins into different cell lines, maintaining their biological activity. Two possible mechanisms have been proposed to explain the entrance of other CPPs in cells, endosomal-dependent and independent types. In this work, we evaluated the molecular mechanisms of pep-1-mediated cellular uptake of beta-galactosidase (beta-Gal) from Escherichia coli in large unilamellar vesicles (LUV) and HeLa cells. Fluorescence spectroscopy was used to evaluate the translocation process in model systems (LUV). Immunofluorescence microscopy was used to study the translocation in HeLa cells. Enzymatic activity detection enabled us to monitor the internalization of beta-Gal into LUV and the functionality of the protein in the interior of HeLa cells. beta-Gal translocated into LUV in a transmembrane potential-dependent manner. Likewise, the extent of beta-Gal incorporation was extensively decreased in depolarized cells. Furthermore, beta-Gal uptake efficiency and kinetics were temperature-independent, and beta-Gal did not colocalize with endosomes, lysosomes, or caveosomes. Therefore, beta-Gal translocation was not associated with the endosomal pathway. Although an excess of pep-1 was mandatory for beta-Gal translocation in vivo, transmembrane pores were not formed as concluded from the trypan blue exclusion method. These results altogether indicated that protein uptake both in vitro with LUV and in vivo with HeLa cells was mainly, if not solely, dependent on negative transmembrane potential across the bilayer, which suggests a physical mechanism governed by electrostatic interactions between pep-1 (positively charged) and membranes (negatively charged).
机译：细胞穿透肽（CPP）pep-1能够将大蛋白导入不同的细胞系，并保持其生物学活性。已经提出了两种可能的机制来解释细胞中其他CPP的进入：内体依赖性和非依赖性。在这项工作中，我们评估了大单层囊泡（LUV）和HeLa细胞中pep-1介导的从大肠埃希菌中摄取β-半乳糖苷酶（beta-Gal）的分子机制。荧光光谱法用于评估模型系统（LUV）中的转运过程。免疫荧光显微镜用于研究HeLa细胞中的易位。酶活性检测使我们能够监测β-Gal在LUV中的内在化以及HeLa细胞内部蛋白质的功能。 β-Gal以跨膜电位依赖性方式转移到LUV中。同样，去极化细胞中β-Gal掺入的程度大大降低。此外，β-Gal的吸收效率和动力学与温度无关，并且β-Gal不能与内体，溶酶体或小体共定位。因此，β-Gal易位与内体途径无关。尽管过量的pep-1是体内β-Gal转运所必需的，但并未如台盼蓝排除法所得出的那样形成跨膜孔。这些结果完全表明，LUV体外和HeLa细胞的体内蛋白质吸收主要（如果不是唯一的话）取决于跨双层的负跨膜电位，这表明由pep-1（带正电）之间的静电相互作用控制的物理机制。 ）和膜（带负电）。

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