Cellular import mediated by nuclear localization signal sequences
Abstract
A series of NLS sequence peptides were synthesized with a fluorescein tag and their propensities to enter a number of different cancer cells lines was investigated by flow cytometry and confocal microscopy. All NLS peptides demonstrated rapid accumulation within cells when added to the cellular media. Differences were observed in the site of localization within the cells, with the more cationic peptides found within the cytoplasm, within acidic compartments. Covalent adducts of proteins and oligonucleotides with NLS peptides were also found to be effectively imported within cells. The NLS-NF-κB peptide was also evaluated for cellular uptake of the antitumor antibiotic adriamycin. When conjugated to the NLS-NF-κB peptide, adriamycin exhibited a two-fold increase in cell cytotoxicity relative to the free drug. The residues essential for cellular uptake of the NLS-NF-κB peptide were investigated via alanine scanning mutagenesis in MCF-7 cells (breast carcinoma). The alanine scanning study revealed that electrostatic interactions are not the primary driving force for membrane translocation. Hydrophobic interactions and hydrogen bonding interactions were determined to play a major role in cellular uptake of the NLS-NF-κB peptide. In an effort to elucidate the mechanism of cellular uptake with NLS peptides, temperature and energy dependent studies were carried out in MCF-7 cells. The data obtained indicates that cellular uptake of the NLS sequences is energy independent, and, therefore rule out a receptor-mediated transport pathway. NLS sequences show great promise as agents to promote the cellular uptake of attached cargo. An understanding of the structural and mechanistic properties of these sequences will provide great potential for the rational design of efficient and selective peptidic delivery systems.
Degree
Ph.D.
Advisors
Chmielewski, Purdue University.
Subject Area
Organic chemistry|Biochemistry|Cellular biology
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