The plasma membrane represents an impermeable barrier for most macromolecules. Still some proteins and so‐called cell‐penetrating peptides enter cells efficiently. It has been shown that endocytosis contributes to the import of these molecules. However, conflicting results have been obtained concerning the nature of the endocytic process. In addition, there have been new findings for an endocytosis‐independent cellular entry. In this study, we provide evidence that the Antennapedia‐homeodomain‐derived antennapedia (Antp) peptide, nona‐arginine and the HIV‐1 Tat‐protein‐derived Tat peptide simultaneously use three endocytic pathways: macropinocytosis, clathrin‐mediated endocytosis and caveolae/lipid‐raft‐mediated endocytosis. Antennapedia differs from Tat and R9 by the extent by which the different import mechanisms contribute to uptake. Moreover, at higher concentrations, uptake occurs by a mechanism that originates from spatially restricted sites of the plasma membrane and leads to a rapid cytoplasmic distribution of the peptides. Endocytic vesicles could not be detected, suggesting an endocytosis‐independent mode of uptake. Heparinase treatment of cells negatively affects this import, as does the protein kinase C inhibitor rottlerin, expression of dominant‐negative dynamin and chlorpromazine. This mechanism of uptake was observed for a panel of different cell lines. For Antp, significantly higher peptide concentrations and inhibition of endocytosis were required to induce its uptake. The relevance of these findings for import of biologically active cargos is shown.

MHC-peptide complexes mediate key functions in adaptive immunity. In a classical view, MHC-I molecules present peptides from intracellular source proteins, whereas MHC-II molecules present antigenic peptides from exogenous and membrane proteins. Nevertheless, substantial crosstalk between these two pathways has been observed. We investigated the influence of autophagy on the MHC-II ligandome and demonstrated that peptide presentation is altered considerably upon induction of autophagy. The presentation of peptides from intracellular and lysosomal source proteins was strongly increased on MHC-II in contrast with peptides from membrane and secreted proteins. In addition, autophagy influenced the MHC-II antigen-processing machinery. Our study illustrates a profound influence of autophagy on the class II peptide repertoire and suggests that this finding has implications for the regulation of CD4 ؉ T cell-mediated processes.antigen processing ͉ lysosomal proteases ͉ T helper cells P eptides of foreign and self proteins are presented on MHC-I and MHC-II molecules at the cell surface and can be recognized by CD8ϩ and CD4 ϩ T lymphocytes, respectively (1, 2). From a classical point of view, MHC-I molecules present antigenic peptides derived from intracellular proteins, whereas MHC-II molecules do so for exogenous and membrane proteins (3). This phenomenon is reflected in the two major cellular breakdown pathways for proteins: proteasomal degradation, particularly relevant to the generation of MHC-I peptides (4), and degradation by the endosomal͞lysosomal system, which is responsible for the processing of MHC-II peptides (5). However, the separation of these distinct pools of source proteins is less stringent than originally believed. It is now well established that MHC-I molecules are able to present peptides derived from exogenous antigens (Ag) by a process known as cross presentation (6). On the other hand, intracellular proteins can be presented by MHC-II molecules (7,8), even though the underlying processes are less clear. It has been recently shown that peptides from cytosolic model proteins can be presented on MHC-II molecules through autophagy (9-11). Autophagy plays a role in the endosomal͞lysosomal degradation pathway and is responsible for feeding intracellular components into this pathway. It is thought to be required for normal turnover of cellular components, particularly in response to starvation (12). Against this background, we hypothesized that autophagy might mediate MHC-II presentation of intracellular Ag, meaning the contents of a cell contained within the plasma membrane, excluding large vacuoles and secretory or ingested material (Gene Ontology classifications), in general. Therefore, we performed a detailed characterization of the MHC-II ligand repertoire (ligandome) presented at the cell surface under normal conditions and after increased autophagy, leading to a comprehensive overall picture of changes in peptide processing and presentation. Materials and MethodsCell Culture and Autophagy Inducti...

The role of endosomal acidification and retrograde transport for the uptake of the highly basic cell-penetrating peptides penetratin, Tat, and oligoarginine was investigated. The effect of a panel of drugs that interfere with discrete steps of endocytosis or Golgi-mediated transport on uptake and cellular distribution of fluorescein-labeled peptide analogues was probed by confocal microscopy, flow cytometry, and fluorescence spectroscopy of whole cell lysates. The analyses were carried out in MC57 fibrosarcoma cells and in HeLa cells. While MC57 fibrosarcoma cells showed some vesicular fluorescence and a pronounced cytoplasmic fluorescence, in HeLa cells little cytoplasmic fluorescence was observed. In MC57 cells the inhibitors of endosomal acidification chloroquine and bafilomycin A1 abolished the release of the peptides into the cytoplasm. Release into the cytosol preserved endosomal integrity. In addition, cellular uptake of the peptides was inhibited by brefeldin A, a compound interfering with trafficking in the transGolgi network. In contrast, nordihydroguaiaretic acid, a drug that stimulates the rapid retrograde movement of both Golgi stacks and trans-Golgi network to the endoplasmic reticulum, promoted a cytoplasmic localization of Tat peptides in peptide-pulsed HeLa cells. The effects of these drugs on trafficking shared characteristics with those reported for the trafficking of plant and bacterial toxins, such as cholera toxin, which reach the cytoplasm by means of retrograde transport. A sequence comparison revealed a common stretch of 8 -10 amino acids with high sequence homology to the Tat peptide. The structural and functional data therefore strongly suggest a common mechanism of import for cationic cell-penetrating peptides and the toxins.

Cell-penetrating peptides (CPPs) have become widely used vectors for the cellular import of molecules in basic and applied biomedical research. Despite the broad acceptance of these molecules as molecular carriers, the details of the mode of cellular internalization and membrane permeation remain elusive. Within the last two years endocytosis has been demonstrated to be a route of uptake shared by several CPPs. These findings had a significant impact on CPP research. State-of-the-art cell biology is now required to advance the understanding of the intracellular fate of the CPP and cargo molecules. Owing to their presumed ability to cross lipid bilayers, CPPs also represent highly interesting objects of biophysical research. Numerous studies have investigated structure-activity relationships of CPPs with respect to their ability to bind to a lipid bilayer or to cross this barrier. Endocytosis route only relocates the membrane permeation from the cell surface to endocytic compartments. Therefore, biophysical experiments are key to a mechanistic molecular understanding of the cellular uptake of CPPs. However, biophysical investigations have to consider the molecular environment encountered by a peptide inside and outside a cell. In this contribution we will review biophysical and cell-biology data obtained for several prominent CPPs. Furthermore, we will summarize recent findings on the cell-penetrating characteristics of antimicrobial peptides and the antimicrobial properties of CPPs. Peptides of both groups have overlapping characteristics. Therefore, both fields may greatly benefit from each other. The review will conclude with a perspective of how biophysics and cell biology may synergize even more efficiently in the future.

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