Protein transport in human cells mediated by covalently and noncovalently conjugated arginine-rich intracellular delivery peptides.
Generally, biomacromolecules, such as DNA, RNA, and proteins, cannot freely permeate into cells from outside the membrane. Protein transduction domains (PTDs) are peptides containing a large number of basic amino acids that can deliver macromolecules into living cells. Arginine-rich intracellular delivery (AID) peptides are more effective than other PTD peptides at carrying large molecules across cellular membranes. In the present study, we demonstrated that AID peptides are able to deliver cargo proteins into living cells in both covalent and noncovalent protein transductions (CNPT) synchronously. Human A549 cells were treated with a fluorescent protein (FP) that was noncovalently premixed with another AID-conjugated FP, which emitted a different color. After the delivery of carrier AID-FP and cargo FP into cells, the emission and merge of fluorescence were observed and recorded with a confocal microscope, while the internalization efficiency was quantitatively analyzed with a flow cytometer. The optimal molecular ratio between carrier AID-FP and cargo FP for CNPT is about 1:1/3. Fluorescence resonance energy transfer (FRET) assay further confirmed AID-conjugates can physically interact with its cargo FPs in CNPT in cells. Potential uptake mechanisms of CNPT may involve a combination of multiple internalization pathways. After delivery, intracellular distributions of AID-conjugates and FPs may possibly colocalize with lysosomes. These results will facilitate the understanding of multiple mechanisms of PTDs, and provide a powerful tool for simultaneously delivering several proteins or compounds in protein internalization.
Hu
Jia-Wei
JW
Department of Natural Resources and Environmental Studies, Institute of Biotechnology, National Dong Hwa University, No. 1 Sec. 2 Da-Hsueh Road, Hualien, Taiwan.
Liu
Betty Revon
BR
Wu
Chih-Yuan
CY
Lu
Shu-Wan
SW
Lee
Han-Jung
HJ
D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D001120:Arginine; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002704:Chlorates / Q000494:pharmacology; D015638:Cytochalasin D / Q000494:pharmacology; D004337:Drug Carriers / Q000378:metabolism* / Q000494:pharmacology; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D008247:Lysosomes / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D015739:Nocodazole / Q000494:pharmacology; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D010873:Pinocytosis / Q000187:drug effects; D010957:Plasmids / Q000235:genetics; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism / Q000494:pharmacology; D013696:Temperature; D047392:beta-Cyclodextrins / Q000494:pharmacology; D000097573:Red Fluorescent Protein
D000584:Amiloride; D001120:Arginine; D002704:Chlorates; D015638:Cytochalasin D; D004337:Drug Carriers; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D015739:Nocodazole; D009842:Oligopeptides; D010455:Peptides; D011993:Recombinant Fusion Proteins; D047392:beta-Cyclodextrins; D000097573:Red Fluorescent Protein; C108732:methyl-beta-cyclodextrin; C491427:nonaarginine; C032706:sodium chlorate; C039614:ethylisopropylamiloride