Inhibition of Nuclear Translocation of Transcription Factor NF-B by a Synthetic Peptide Containing a Cell Membrane-permeable Motif and Nuclear Localization Sequence

doi:10.1074/jbc.270.24.14255

Inhibition of Nuclear Translocation of Transcription Factor NF-B by a Synthetic Peptide Containing a Cell Membrane-permeable Motif and Nuclear Localization Sequence (*)

From the (1)Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2363

^§ To whom correspondence and reprint requests should be addressed. Tel.: 615-343-8277 or 615-343-8280; Fax: 615-343-7392.

Abstract

To control agonist-induced nuclear translocation of transcription factor κB (NF-κB) in intact cells, cell-permeable synthetic peptides were devised. Their import into intact cells was dependent on a hydrophobic region selected from the signal peptide sequences and was verified by their inaccessibility to extracellular proteases and by confocal laser scanning microscopy. When a cell-permeable peptide carried a functional cargo representing the nuclear localization sequence of NF-κB p50, it inhibited in a concentration-dependent manner nuclear translocation of NF-κB in cultured endothelial and monocytic cells stimulated with lipopolysaccharide or tumor necrosis factor-α. Synthetic peptide analogues with deleted hydrophobic cell membrane-permeable motif or with a mutated nuclear localization sequence were inactive. Cell membrane-permeable peptides were not cytotoxic within the concentration range used in these experiments. These results suggest that cell-permeable synthetic peptides carrying a functional cargo can be applied to control signal transduction-dependent subcellular traffic of transcription factors mediating the cellular responses to different agonists. Moreover, this approach can be used to study other intracellular processes involving proteins with functionally distinct domains.

Footnotes

↵* This research was supported by National Institutes of Health Grants HL45994 and HL30647 and a Mellon Foundation Award for Faculty Development. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

NLS

nuclear localization sequence

CPPI

cell-permeable peptide import

EMSA

electrophoretic mobility shift assay

FBS

fetal bovine serum

PBS

phosphate-buffered saline

IκBα

inhibitor κBα

K-FGF

Kaposi fibroblast growth factor

LPS

lipopolysaccharide

NF-κB

nuclear factor κB

TNFα

tumor necrosis factor-α

HPLC

high performance liquid chromatography

DMEM

Dulbecco's modified Eagle's medium.
↵²A. Burzynski and J. Hawiger, unpublished results.
↵³X.-Y. Liu, S. Timmons, Y.-Z. Lin, and J. Hawiger, unpublished results.