On the cellular uptake and membrane effect of the multifunctional peptide, TatLK15.
Recently, the multifunctional peptide TatLK15 resulting from the fusion of the cell penetrating peptide Tat and the amphipathic peptide LK15 was shown to be efficient at mediating siRNA and shRNA delivery in leukemia cells to silence the bcr-abl oncoprotein. The present study focused on TatLK15 peptide cellular uptake and defining conditions for its use within a range of doses exhibiting minimal toxicity. The initial part of the study carried out in solution confirmed that the insertion of a glycine bridge allowed retention of the LK15 α-helicity, and fluorescence correlation spectroscopy did not reveal preferential conformations at the studied concentrations. In the second part, TatLK15 uptake mechanisms appeared peptide dose- and cell line- dependent as well as requiring membrane potential. Below a critical dose, TatLK15 toxicity appeared limited for approximately three hours as demonstrated by the combined use of lactate dehydrogenase release, MTT assays, and time-dependent observation of membrane-impermeant dye uptake using high content screening apparatus. Furthermore, toxicity was observed to occur rapidly at higher peptide doses. Finally, a comparison between TatLK15 and another Tat amphipathic peptide construct suggested that α-helix content should be viewed as a key element in the development of similar peptides.
School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester M13 9PT, UK.
Myasar
M
Alkotaji
Alain
A
Pluen
Egor
E
Zindy
Zahra
Z
Hamrang
Harmesh
H
Aojula
D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology; D015696:Gene Products, tat / Q000378:metabolism; D005998:Glycine / Q000378:metabolism; D019073:HT29 Cells; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008564:Membrane Potentials / Q000502:physiology; D010446:Peptide Fragments / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D017433:Protein Structure, Secondary
D015696:Gene Products, tat; D010446:Peptide Fragments; D010455:Peptides; D007770:L-Lactate Dehydrogenase; D005998:Glycine
Tat; cell; cell penetrating peptide; circular dichroism; diffusion; fluorescence spectroscopy; image analysis; membrane translocation; peptide delivery; toxicity