No entry for TAT(44–57) into liposomes and intact MDCK cells: novel approach to study membrane permeation of cell-penetrating peptides

Homeoproteins are a class of transcription factors defined by the structure of their DNA-binding domain, the homeodomain. In addition to their nuclear cell-autonomous activities, homeoproteins transfer between cells, thanks to two separate steps of secretion and internalization, which both rely on unconventional mechanisms. Internalization is driven by the third helix of the homeodomain (Penetratin) through a non-vesicular and endocytosis-independent mechanism. In contrast, homeoprotein secretion involves vesicular compartments and requires the presence of a sequence of 11 amino acids (Sec sequence) spanning between the second and third helix of the homeodomain. In this study, we report that the SecPen polypeptide, which combines the two identified domains, Penetratin and Sec, bears all of the necessary information to go in and out of cells. We have analyzed key mechanisms and demonstrated that this peptide can efficiently cross a tight junction epithelium.Homeoproteins belong to a class of transcription factors with key developmental and adult functions. In addition to their nuclear cell-autonomous activities, they also have the ability to transfer between cells (1). The latter property likely serves specific paracrine functions. For example, the extracellular application of a gradient of Engrailed homeoprotein attracts or repulses the nasal or temporal growth cones of retinal ganglion cells, respectively, suggesting a non-cell autonomous function of Engrailed in axonal guidance (2). Homeoprotein intercellular transfer involves two separate steps of secretion and internalization. The homeodomain (the DNA-binding domain that defines homeoproteins) is necessary and sufficient for transfer (3), even though other parts of the protein are endowed with transfer regulatory functions (4). The two sequences required for secretion and internalization are highly conserved, and indeed, intercellular transfer is a shared property of several homeoproteins. Mutational analysis indicates that internalization and secretion rely on two distinct mechanisms (5). Internalization is driven by the third helix of the homeodomain (6). This 16-amino-acid-long peptide (thereafter Penetratin or Pen) 2 is internalized by live cells through a mechanism independent of endocytosis (7) and has been used extensively to address exogenous compounds into the cell cytoplasm and nucleus (see Ref. 8 for review). Homeoproteins are also secreted despite the absence of classical secretion signals. Using Engrailed-1 (En1) and Engrailed-2 (En2) as prototypic homeoproteins, it was shown that they are present in vivo in vesicles enriched in cholesterol and glycosphingolipids (9). The addressing of Engrailed proteins to this compartment and their secretion are abolished upon deletion of 11 amino acids spanning between the second and third helix of the homeodomain (5, 10), which was called the Sec sequence (see Fig. 3A). It thus suggests that this sequence participates in unconventional homeoprotein secretion (in the absence of secretion signal sequence).In t...

Section: Discussionsupporting

confidence: 90%

Identification of a Signal Peptide for Unconventional Secretion

Dupont¹,

Prochiantz

Joliot³

2007

“…For about a decade, it was commonly accepted that, despite their highly polar nature, PTDs are able to rapidly and efficiently translocate directly through the lipid bilayer, thus delivering their cargo into the cytoplasm and nucleus in a receptor-and energy-independent way (14,15). This model was supported by a large number of indirect evidence including temperature, ATP, and receptor independence of cellular uptake (11)(12)(13)(14), but attempts to directly validate this model on protein-free lipid bilayer systems gave contradictory results (31,32). Moreover, it was recently shown that apparent ATP and temperature independence and fast kinetics of PTD uptake result from artifacts of cell fixation and incomplete removal of surface-bound peptide (18,19).…”

Section: Discussionmentioning

confidence: 99%

Cellular Uptake of Unconjugated TAT Peptide Involves Clathrin-dependent Endocytosis and Heparan Sulfate Receptors

Richard

Melikov

Brooks

et al. 2005

532

462

Delivery of macromolecules mediated by protein transduction domains (PTDs) attracts a lot of interest due to its therapeutic and biotechnological potential. A major reevaluation of the mechanism of PTD-mediated internalization and the role of endocytosis in this mechanism has been recently initiated. Here, we demonstrate that the entry of TAT peptide (one of the most widely used PTDs) into different primary cells is ATP-and temperature-dependent, indicating the involvement of endocytosis. Specific inhibitors of clathrin-dependent endocytosis partially inhibit TAT peptide uptake, implicating this pathway in TAT peptide entry. In contrast, the caveolindependent pathway is not essential for the uptake of unconjugated TAT peptide as evidenced by the efficient internalization of TAT in the presence of the known inhibitors of raft/caveolin-dependent pathway and for cells lacking or deficient in caveolin-1 expression. Whereas a significant part of TAT peptide uptake involves heparan sulfate receptors, efficient internalization of peptide is observed even in their absence, indicating the involvement of other receptors. Our results suggest that unconjugated peptide might follow endocytic pathways different from those utilized by TAT peptide conjugated to different proteins.Recent advances in the identification of new molecular therapy targets and disease-relevant proteins, accelerated by the completion of the human genome project, emphasized an importance of high molecular weight information-rich biomolecules, such as peptides, proteins, antisense DNA, and small interfering RNA, for molecular therapy. However, the delivery of proteins and nucleic acids into cells is greatly hampered by the low permeability of the cell plasma membrane to polar molecules. Not surprisingly, the discovery that a number of cationic peptides known as protein transduction domains (PTDs) 1 can facilitate cytoplasmic and nuclear delivery of a conjugated cargo has attracted a lot of interest (1-3). Up to date, a wide range of cargo molecules, including low molecular weight drugs (4), oligonucleotides (5), peptides (6) and even full-length proteins (7-10), have been successfully delivered into cells using PTDs and, most importantly, the functional activity of the delivered cargo has been observed (7-10).Despite significant progress in the cytoplasmic and nuclear delivery of various cargo molecules using PTDs, the underlying mechanisms remain under active debate. Until recently, it was widely assumed that the internalization of cationic PTDs is an energy-and receptor-independent process based on direct transport through the lipid bilayer (11-15). On the other hand, there have been indications that uptake of full-length TAT protein, from which one of the most commonly used PTDs referred to as TAT peptide is derived, occurs via endocytosis and depends on cell surface heparan sulfate receptors (16). Moreover, the validity of some of the important data, supporting a direct transport model for synthetic TAT peptide, has been questioned in several recent ...

“…This energy-independent internalization property of ZEBRA-MD-EGFP is highly favorable because endosomal trapping can be circumvented, which seems to be the major bottleneck for most CPP, as, for example, TAT, which enters cell by macropinocytosis (15) and fails to enter into liposomes (43,49). Further study to determine the amino acids from the MD responsible for the translocation properties will permit a better understanding of the molecular mechanism of ZEBRA cellular uptake.…”

Section: Discussionmentioning

confidence: 99%

Characterization of the Cell-penetrating Properties of the Epstein-Barr Virus ZEBRA trans-Activator

Rothe

Liguori

Villegas-Mendéz

et al. 2010

The Epstein-Barr virus basic leucine zipper transcriptional activator ZEBRA was shown recently to cross the outer membrane of live cells and to accumulate in the nucleus of lymphocytes. We investigated the potential application of the EpsteinBarr virus trans-activator ZEBRA as a transporter protein to facilitate transduction of cargo proteins. Analysis of different truncated forms of ZEBRA revealed that the minimal domain (MD) required for internalization spans residues 170 -220. MD efficiently transported reporter proteins such as enhanced green fluorescent protein (EGFP) and ␤-galactosidase in several normal and tumor cell lines. Functionality of internalized cargo proteins was confirmed by ␤-galactosidase activity in transduced cells, and no MD-associated cell toxicity was detected. Translocation of MD through the cell membrane required binding to cell surface-associated heparan sulfate proteoglycans as shown by strong inhibition of protein uptake in the presence of heparin. We found that internalization was blocked at 4°C, whereas no ATP was required as shown by an only 25% decreased uptake efficiency in energy-depleted cells. Common endocytotic inhibitors such as nystatin, chlorpromazine, and wortmannin had no significant impact on MD-EGFP uptake. Only methyl-␤-cyclodextrin inhibited MD-EGFP uptake by 40%, implicating the lipid raft-mediated endocytotic pathway. These data suggest that MD-reporter protein transduction occurs mostly via direct translocation through the lipid bilayer and not by endocytosis. This mechanism of MD-mediated internalization is suitable for the efficient delivery of biologically active proteins and renders ZEBRA-MD a promising candidate for therapeutic protein delivery applications.Recently, cell-penetrating peptides (CPPs) 2 were discovered that have the ability to cross lipid bilayers of mammalian cells, which naturally constitute a tight biological barrier allowing only the uptake of non-polar molecules Ͻ500 Da into the cell (1). Coupling of CPPs to molecules such as small interfering RNA, peptide nucleic acids, and bioactive proteins facilitates delivery of therapeutic agents into live cells (2-5). The most intensively studied CPPs include the Tat peptide, which is derived from human immunodeficiency virus protein-1 (6, 7); the third helix of the Antennapedia homeodomain (also referred to as Penetratin) from Drosophila (8); and the VP22 peptide of the herpes simplex virus (9, 10).The activity of therapeutic molecules depends on their availability and functionality after their delivery into cells, both of which are determined by the transduction mechanism of the particular CPP (11). Consequently, extensive research was directed to elucidate pathways underlying protein transduction. Because of its apparent temperature and ATP dependence, it is widely assumed that cell entry occurs via endocytosis after binding of the CPP to negatively charged heparan sulfate proteoglycans (HSPGs) located on the cell surface (12-14). Tat fusion proteins were shown to be internalized via macropinoc...