Department of Molecular Spectroscopy, Max Planck Institute for Polymer Research , D-55128 Mainz, Germany.
Anal Chem. 2017 Nov 7;89(21):11310-11317. doi: 10.1021/acs.analchem.7b01895. Epub 2017 Oct 27.
Cell-penetrating peptides (CPPs) are short peptide sequences that can translocate across cellular plasma membranes and are thus potential delivery vectors for diagnostic and therapeutic applications. Many CPPs exhibit some sort of structural polymorphism, where the secondary structure of the peptide is altered strongly by its local environment, which is believed to facilitate membrane translocation and uptake. However, much less is known about the fate and structure of CPPs within cells largely due to measurement difficulty. Here we employ isotopic labeling combined with hyperspectral, quantitative coherent Raman microscopy to localize a model CPP-penetratin-and determine its secondary structure in different cellular compartments. Our results show that penetratin is mostly α-helical in the cytosol and acquires a more β-sheet and random coil character in the nucleus. The increased helicity in the cytosol is similar to that seen in previous studies with model lipid membranes, suggesting that the peptide is associated with membranes in, e.g., endosomes (or lysosomes) in the cytosol. The ability to both localize and determine the secondary structure of a CPP within cells is critical for clarifying the mechanism of peptide-mediated translocation and delivery of cargo molecules to specific cellular destinations.
细胞穿透肽（CPPs）是能够穿过细胞膜的短肽序列，因此是诊断和治疗应用的潜在递药载体。许多 CPP 表现出某种结构多态性，其中肽的二级结构被其局部环境强烈改变，这被认为有助于膜转运和摄取。然而，由于测量困难，对于 CPP 在细胞内的命运和结构知之甚少。在这里，我们采用同位素标记与超光谱、定量相干拉曼显微镜相结合的方法，定位模型 CPP-穿透肽，并确定其在不同细胞区室中的二级结构。我们的结果表明，穿透肽在细胞质中主要呈α-螺旋构象，在核中呈现更多的β-折叠和无规卷曲特征。细胞质中增加的螺旋度与以前在模型脂质膜中观察到的相似，这表明肽与膜相关，例如在细胞质中的内体（或溶酶体）中。能够在细胞内定位和确定 CPP 的二级结构对于阐明肽介导的转位和货物分子递送到特定细胞靶位的机制至关重要。
