Cell-penetrating peptides (CPPs) share the property of cellular internalization. The question of how these peptides reach the cytoplasm of cells is still widely debated. Herein, we have used a mass spectrometry-based method that enables quantification of internalized and membrane-bound peptides. Internalization of the most used CPP was studied at 37°C (endocytosis and translocation) and 4°C (translocation) in wild type and proteoglycandeficient Chinese hamster ovary cells. Both translocation and endocytosis are internalization pathways used by CPP. The choice of one pathway versus the other depends on the peptide sequence (not the number of positive changes), the extracellular peptide concentration, and the membrane components. There is no relationship between the high affinity of these peptides for the cell membrane and their internalization efficacy. Translocation occurs at low extracellular peptide concentration, whereas endocytosis, a saturable and cooperative phenomenon, is activated at higher concentrations. Translocation operates in a narrow time window, which implies a specific lipid/peptide co-import in cells.Cell-penetrating peptides (CPPs) 3 that share the activity of cellular entry are usually short peptides of less than 20 amino acids highly enriched in basic residues. Among them, Antp, Tat-(48 -60), and oligoarginine peptides are the most intensively studied. Despite the wide use of these CPPs as macromolecular delivery devices, the internalization mechanism of these peptides in cells still remains largely controversial. The energy dependence of the internalization mechanism is unique because all endocytotic pathways are inhibited at low temperature. Consequently, at low temperature, internalization likely reflects a direct translocation mechanism. Early studies proposed that Antp enters cells by an energy-independent membrane translocation mechanism (1). This first analysis was then contradicted by other studies that suggested, with the use of inhibitors, the involvement of endocytosis in the cellular internalization of cell-penetrating peptides (2-4).The hypothesis that endocytosis was the only internalization mechanism of CPP resulted from studies examining whether the temperature or the binding to cell-surface glycosaminoglycans (GAGs) were critical for peptide internalization. Most of these interpretations resulted from fluorescence microscopy data. For instance, it was reported that Antp, Tat, and oligoarginine peptides were not efficiently internalized in the Chinese hamster ovary (CHO) mutant pgsA-745 cell line, which does not produce cell-surface heparan sulfate or chondroitin sulfate (5, 6). However, recent data indicates that Tat-mediated transduction occurs in the absence of heparan sulfate and chondroitin sulfate (7). The discrepancies observed between studies may be explained in part by different incubation conditions (peptide/cells ratio and peptide concentration) (8), limits in fluorescence imaging, such as quenching (9), or fluorophore-dependent intracellular trafficking (10), as...

Induction of the C/EBP homologous protein (CHOP) is considered a key event for endoplasmic reticulum (ER) stress-mediated apoptosis. Type 1 diabetes (T1D) is characterized by an autoimmune destruction of the pancreatic b-cells. Pro-inflammatory cytokines are early mediators of b-cell death in T1D. Cytokines induce ER stress and CHOP overexpression in b-cells, but the role for CHOP overexpression in cytokine-induced b-cell apoptosis remains controversial. We presently observed that CHOP knockdown (KD) prevents cytokine-mediated degradation of the anti-apoptotic proteins B-cell lymphoma 2 (Bcl-2) and myeloid cell leukemia sequence 1 (Mcl-1), thereby decreasing the cleavage of executioner caspases 9 and 3, and apoptosis. Nuclear factor-jB (NF-jB) is a crucial transcription factor regulating b-cell apoptosis and inflammation. CHOP KD resulted in reduced cytokine-induced NF-jB activity and expression of key NF-jB target genes involved in apoptosis and inflammation, including iNOS, FAS, IRF-7, IL-15, CCL5 and CXCL10. This was due to decreased IjB degradation and p65 translocation to the nucleus. The present data suggest that CHOP has a dual role in promoting b-cell death: (1) CHOP directly contributes to cytokine-induced b-cell apoptosis by promoting cytokine-induced mitochondrial pathways of apoptosis; and (2) by supporting the NF-jB activation and subsequent cytokine/chemokine expression, CHOP may contribute to apoptosis and the chemo attraction of mononuclear cells to the islets during insulitis. Type 1 diabetes (T1D) is a severe chronic disease resulting from an autoimmune destruction of the pancreatic b-cells. The incidence of T1D has been rising steadily in developed countries from the 1950s to the present day, with the recent, alarming prediction that it will double in children under the age of 5 years by 2020. 1 b-cell loss in T1DM occurs slowly over years and 480% of the b-cell mass is usually lost at the time of diagnosis. Because of the excessive mortality associated with complications of T1D and the increasing incidence of childhood diabetes, 2 there is an ongoing effort to develop novel strategies for a better treatment and hopefully, prevention of T1D.In T1D, b-cells cooperate with the immune system to its own destruction by activating pro-apoptotic pathways and secreting chemokines/cytokines that contribute to islet inflammation. 3 These responses are mostly triggered via the secretion of the pro-inflammatory cytokines interleukin-1b (IL-1b), tumor necrosis factor-a (TNF-a) and interferon-g (IFN-g) by the infiltrated immune cells. The mechanisms regulating cytokine-mediated b-cell apoptosis and pro-inflammatory responses are intricate and include, but are not restricted to, the activation of the transcription factors nuclear factor-kB (NF-kB) and STAT-1, the c-Jun N-terminal kinases (JNK), endoplasmic reticulum (ER) stress pathways and the intrinsic mitochondrial apoptotic pathways. [3][4][5][6][7] NF-kB activation is due to cytokine-dependent activation of the inhibitor of k-light polypeptide gene enha...

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.