Hydrogen peroxide (H2O2) controls axon pathfinding during zebrafish development.

It is now becoming evident that hydrogen peroxide (H2O2), which is constantly produced by nearly all cells, contributes to bona fide physiological processes. However, little is known regarding the distribution and functions of H2O2 during embryonic development. To address this question, we used a dedicated genetic sensor and revealed a highly dynamic spatio-temporal pattern of H2O2 levels during zebrafish morphogenesis. The highest H2O2 levels are observed during somitogenesis and organogenesis, and these levels gradually decrease in the mature tissues. Biochemical and pharmacological approaches revealed that H2O2 distribution is mainly controlled by its enzymatic degradation. Here we show that H2O2 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 H2O2 depletion and this defect is rescued by H2O2 or ectopic activation of the Hedgehog pathway. We further show that ex vivo, H2O2 directly modifies Hedgehog secretion. We propose that physiological levels of H2O2 regulate RGCs axonal growth through the modulation of Hedgehog pathway.

Homeoproteins and homeoprotein-derived peptides: going in and out.

Since the initial evidence that antennapedia homeobox can cross cell membranes and internalize into cells, numerous peptides with similar translocation properties have been described. These peptides are referred to as cell-penetrating peptides (CPPs) or protein-transduction domains (PTDs). Reviews on reported CPP sequences have been recently published, together with reviews on their mechanisms of internalization. In this review, we will focus on natural homeoproteins and homeoprotein-derived peptides and describe results that have been obtained among different laboratories to unravel the different pathways by which these molecules reach the cell cytosol and nucleus or transfer from one cell to another. Using homeoproteins as a paradigm, we will also summarize recent evidences of the physiological functions of endogenous protein translocation.

Cell-surface thiols affect cell entry of disulfide-conjugated peptides.

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.

Identification of a signal peptide for unconventional secretion.

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.