Gautam Ankur, Sharma Minakshi, Vir Pooja, Chaudhary Kumardeep, Kapoor Pallavi, Kumar Rahul, Nath Samir K, Raghava Gajendra P S
Bioinformatics Centre, CSIR-Institute of Microbial Technology, Chandigarh, India.
Department of Protein Science and Engineering, CSIR-Institute of Microbial Technology, Chandigarh, India.
Eur J Pharm Biopharm. 2015 Jan;89:93-106. doi: 10.1016/j.ejpb.2014.11.020. Epub 2014 Nov 29.
Cell-penetrating peptides (CPPs) have proven their potential as an efficient delivery system due to their intrinsic ability to traverse biological membranes and transport various cargoes into the cells. In the present study, we have identified novel natural protein-derived CPPs using an integrated (in silico and experimental) approach. First, using bioinformatics approach, arginine-rich peptide segments were extracted from SwissProt proteins and their cell-penetrating properties were predicted. Finally, eight peptides were selected and their internalization was validated using experimental techniques. Laser scanning confocal microscopy and flow cytometry confirmed that seven out of eight peptides were internalized into live cells with varying efficiencies without significant cytotoxicity. Three peptides have shown higher internalization efficacy than TAT peptide, the most widely used CPP. Among these three peptides, one peptide (P8), derived from voltage-dependent L-type calcium channel subunit alpha-1D, was able to accumulate inside in a variety of cell types very efficiently through a rapid dose-dependent process. Further, experiments involving inhibition with various endocytic inhibitors along with co-localization studies indicate that the uptake mechanism of P8 is macropinocytosis, a fluid-phase endocytosis process. In addition, competitive inhibition with heparin revealed the involvement of cell-surface proteoglycans in P8 uptake. In summary, the present study provides evidence that an integrated in silico and experimental approach is an effective strategy for the identification of novel CPPs and CPPs identified in the present study have promising perspectives for future drug delivery applications.
细胞穿透肽（CPPs）已证明其作为一种高效递送系统的潜力，这归因于其穿越生物膜并将各种货物转运到细胞内的内在能力。在本研究中，我们使用一种综合（计算机模拟和实验）方法鉴定了新型天然蛋白质衍生的CPPs。首先，利用生物信息学方法，从SwissProt蛋白质中提取富含精氨酸的肽段，并预测其细胞穿透特性。最后，选择了8种肽，并使用实验技术验证了它们的内化情况。激光扫描共聚焦显微镜和流式细胞术证实，8种肽中有7种以不同效率内化到活细胞中，且无明显细胞毒性。3种肽的内化效率高于最常用的CPP——TAT肽。在这3种肽中，一种源自电压依赖性L型钙通道亚基α-1D的肽（P8）能够通过快速的剂量依赖性过程非常有效地在多种细胞类型内积累。此外，涉及用各种内吞抑制剂进行抑制以及共定位研究的实验表明，P8的摄取机制是巨胞饮作用，一种液相内吞过程。此外，用肝素进行的竞争性抑制揭示了细胞表面蛋白聚糖参与P8的摄取。总之，本研究提供了证据，表明综合计算机模拟和实验方法是鉴定新型CPPs的有效策略，并且本研究中鉴定的CPPs在未来药物递送应用中有广阔前景。
