Abstract
Targeting of peptides, proteins, and other functional cargo into living cells is contingent upon efficient transport across the plasma membrane barrier. We have harnessed the signal sequence hydrophobic region (SSHR) to deliver functional cargoes to cultured cells and to experimental animals. We now report evidence that two chirally distinct forms of SSHR composed of all l or all d amino acids showed similar membrane-translocating activity as assessed by confocal microscopy, flow cytometry, and direct fluorescence measurement. An attached nuclear localization sequence ferried by the SSHR enantiomers displayed similar intracellular function by inhibiting inducible nuclear import of transcription factor nuclear factor kappa B and suppressing nuclear factor kappa B-dependent gene expression of cytokines. A nuclear localization sequence comprised of a positively charged cluster of amino acids was rapidly translocated by SSHR enantiomers to the interior of unilamellar phospholipid vesicles. These findings indicate that the SSHR translocates functional peptides directly through the plasma membrane phospholipid bilayer without involving chirally specific receptor/transporter mechanisms. This mechanism of SSHR translocation is suitable for facile delivery of biologically active peptides for cell-based and animal-based functional proteomic studies.
Highlights
The plasma membrane imposes tight control on the access of extracellular peptides and proteins to the cell interior
If the signal sequence hydrophobic region (SSHR) composed of all L-amino acids is recognized by a receptor or transporter, a “mirror image” of the SSHR made of all D-amino acids is not
To characterize the plasma membrane-translocating mechanism of the SSHR, we designed two peptides, each with a chirally distinct membrane-translocating motif (MTM) based on SSHR but with an identical functional cargo
Summary
Distribution in multiple organs in mice, including the brain, which requires crossing the blood-brain barrier [15]. We employed a number of approaches using chirally distinct forms of SSHR They were analyzed by confocal laser scanning microscopy and flow cytometry of macrophages treated with SSHRcontaining peptides, and direct fluorescence monitoring of SSHR-based translocation of the nuclear localization sequence with its positively charged cluster of amino acids across unilamellar phospholipid vesicles. These approaches were coupled with the ultimate test of translocating efficiency: functional measurements of the intracellular effect of the cargo on nuclear import of proinflammatory transcription factor NFB and on expression of cytokine genes regulated by this transactivator in macrophages. These mechanistic findings will facilitate the rational design of a new generation of cell-permeant peptides for proteomic and drug delivery studies
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