title: Identification and characterization of novel protein-derived arginine-rich cell-penetrating peptides.
abstract: 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.
authors.affiliation: Bioinformatics Centre, CSIR-Institute of Microbial Technology, Chandigarh, India.
authors.forename: Ankur
authors.initials: A
authors.lastname: Gautam
authors.affiliation: Bioinformatics Centre, CSIR-Institute of Microbial Technology, Chandigarh, India.
authors.forename: Minakshi
authors.initials: M
authors.lastname: Sharma
authors.affiliation: Bioinformatics Centre, CSIR-Institute of Microbial Technology, Chandigarh, India.
authors.forename: Pooja
authors.initials: P
authors.lastname: Vir
authors.affiliation: Bioinformatics Centre, CSIR-Institute of Microbial Technology, Chandigarh, India.
authors.forename: Kumardeep
authors.initials: K
authors.lastname: Chaudhary
authors.affiliation: Bioinformatics Centre, CSIR-Institute of Microbial Technology, Chandigarh, India.
authors.forename: Pallavi
authors.initials: P
authors.lastname: Kapoor
authors.affiliation: Bioinformatics Centre, CSIR-Institute of Microbial Technology, Chandigarh, India.
authors.forename: Rahul
authors.initials: R
authors.lastname: Kumar
authors.affiliation: Department of Protein Science and Engineering, CSIR-Institute of Microbial Technology, Chandigarh, India.
authors.forename: Samir K
authors.initials: SK
authors.lastname: Nath
authors.affiliation: Bioinformatics Centre, CSIR-Institute of Microbial Technology, Chandigarh, India. Electronic address: raghava@imtech.res.in.
authors.forename: Gajendra P S
authors.initials: GP
authors.lastname: Raghava
mesh_terms: D000818:Animals; D001120:Arginine / Q000737:chemistry* / Q000378:metabolism*; D016466:CHO Cells; D002460:Cell Line; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D057846:Cell-Penetrating Peptides / Q000737:chemistry* / Q000378:metabolism*; D003412:Cricetulus; D016503:Drug Delivery Systems / Q000379:methods; D004705:Endocytosis / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D018613:Microscopy, Confocal / Q000379:methods; D010873:Pinocytosis / Q000502:physiology; D021381:Protein Transport / Q000502:physiology; D011509:Proteoglycans / Q000737:chemistry / Q000378:metabolism
chemical_list: D057846:Cell-Penetrating Peptides; D011509:Proteoglycans; D001120:Arginine
keywords: Arginine-rich peptides; Cell-penetrating peptides; In silico screening; Intracellular delivery; Macropinocytosis; Uptake efficiency