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Cell-penetrating peptides (CPPs) can cross the cell membrane and are widely used to deliver bioactive cargoes inside cells. The cargo and the CPP are often conjugated through a disulfide bridge with the common acceptation that this linker is stable in the extracellular biological medium and should not perturb the internalization process. However, with the use of thiol-specific reagents combined with mass spectrometry (as a quantitative method to measure intracellular concentrations of peptides) and confocal microscopy (as a qualitative method to visualize internalized peptides) analyses, we could show that, depending on the peptide sequence, thiol/disulfide exchange reactions could happen at the cell surface. These exchange reactions lead to the reduction of disulfide conjugates. In addition, it was observed that not only disulfide- but also thiol-containing peptides could cross-react with cell-surface thiols. The peptides cross-linked by thiol-containing membrane proteins were either trapped in the membrane or further internalized. Therefore, a new route of cellular uptake was unveiled that is not restricted to CPPs: a protein kinase C peptide inhibitor that is not cell permeant could cross cell membranes when an activated cysteine (with a 3-nitro-2-pyridinesulfenyl moiety) was introduced in its sequence.

a b s t r a c tIt is now becoming evident that hydrogen peroxide (H 2 O 2 ), which is constantly produced by nearly all cells, contributes to bona fide physiological processes. However, little is known regarding the distribution and functions of H 2 O 2 during embryonic development. To address this question, we used a dedicated genetic sensor and revealed a highly dynamic spatio-temporal pattern of H 2 O 2 levels during zebrafish morphogenesis. The highest H 2 O 2 levels are observed during somitogenesis and organogenesis, and these levels gradually decrease in the mature tissues. Biochemical and pharmacological approaches revealed that H 2 O 2 distribution is mainly controlled by its enzymatic degradation. Here we show that H 2 O 2 is enriched in different regions of the developing brain and demonstrate that it participates to axonal guidance. Retinal ganglion cell axonal projections are impaired upon H 2 O 2 depletion and this defect is rescued by H 2 O 2 or ectopic activation of the Hedgehog pathway. We further show that ex vivo, H 2 O 2 directly modifies Hedgehog secretion. We propose that physiological levels of H 2 O 2 regulate RGCs axonal growth through the modulation of Hedgehog pathway.

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...

No abstract

Cell-penetrating peptides are short, often hydrophilic peptides that get access to the intracellular milieu. They have aroused great interest both in academic and applied research. First, cellular internalization of CPPs often involves the crossing of a biological membrane (plasma or vesicular), thus challenging the view of the nonpermeability of these structures to large hydrophilic molecules. Secondly, CPPs can drive the internalization of hydrophilic cargoes into cells, a rate-limiting step in the development of many therapeutic substances. Interestingly, the two mostly used CPPs, TAT and Penetratin peptides, are derived from natural proteins, HIV Tat and Antennapedia homeoprotein, respectively. The identification of the Penetratin peptide, summarized in this review, is intimately linked to the study of its parental natural protein.

Cell-penetrating peptides are short, often hydrophilic peptides that get access to the intracellular milieu. They have aroused great interest both in academic and applied research. First, cellular internalization of CPPs often involves the crossing of a biological membrane (plasma or vesicular), thus challenging the view of the non-permeability of these structures to large hydrophilic molecules. Secondly, CPPs can drive the internalization of hydrophilic cargoes into cells, a rate-limiting step in the development of many therapeutic substances. Interestingly, the two most used CPPs, TAT and penetratin peptides, are derived from natural proteins, HIV Tat and Antennapedia homeoprotein, respectively. The identification of the penetratin peptide, summarized in this review, is intimately linked to the study of its parental natural protein.

Immortalized neural precursor cell lines carrying the polyoma large tumor (T) gene have been shown previously to retain a clear-cut contact inhibition of growth and to differentiate in vitro. In the present study, we have identified and isolated cDNA clones corresponding to RNA expressed preferentially when these cells reach confluence. One of them, NPDC-1, is expressed specifically in the nervous system. The transfection of dividing cells with a NPDC-1 expression vector results in the inhibition of cell proliferation. In addition, the stable introduction of NPDC-1 into transformed cells, even of nonneural origin, leads to the suppression of transformed characteristics.The identification of transcriptional factors has improved our knowledge of mechanisms underlying mammalian development and cell-lineage determination (for reviews, see refs.