Short polyhistidine peptides penetrate effectively into Nicotiana tabacum-cultured cells and Saccharomyces cerevisiae cells

The polyhistidine peptides (PHPs) have been previously reported as novel cell-penetrating peptides and are efficiently internalized into mammal cells; however, penetration of PHPs into other cell types is unknown. In this study, the cellular uptake of PHPs in plant and yeast cells was found to be dependent on the number of histidines, and short PHPs (H6–H10 peptides) showed effective internalization. The H8 peptide showed the highest cell-penetrating capacity and localized to vacuoles in plant and yeast cells. Low-temperature conditions inhibited significantly the cellular uptake of short PHPs by both cells. However, net charge neutralization of PHPs also completely inhibited cellular uptake by plant cells, but not by yeast cells. These results indicate that short PHPs penetrate effectively into plant and yeast cells by similar mechanism with the exception of net charge dependency. The findings show the short PHPs are promising candidates for new delivery tools into plant and yeast cells.     

Simple Branched Arginine-Based Structures can Enhance the Cellular Uptake of Peptide Cargos

In an attempt to design delivery vehicles to enable epitope-based vaccine uptake, we investigated the properties of a variety of synthetic, branched cationic structures. We found that branched compounds based on arginine or lysine were able to facilitate internalization of peptide cargo into cells to different degrees. Branched constructs containing only two arginine residues (R₂) were not only able to bind to cells more efficiently than constructs with two lysine residues (K₂) but were also internalized within vesicle like compartments in the cell. The extent of binding and uptake was enhanced when additional arginine residues were incorporated to form a tetra arginine construct (R₄). An investigation into the kinetics and dose dependence of cellular uptake of these arginine-based constructs demonstrated that binding and internalization is a rapid and efficient event. We also found uptake of the peptide epitope TYQRTRALV was enhanced when it was coupled to R₄. This approach may prove useful for introducing peptide epitopes into antigen presenting cells as self-adjuvanting structures and also for delivery of other peptides into different specialized cells.     

Investigation of penetratin peptides. Part 2. In vitro uptake of penetratin and two of its derivatives

As endocytic uptake of the Antennapedia homeodomain‐derived penetratin peptide (RQIKIWFQNRRMKWKK) is finally being revealed, some of the early views about penetratin need to be reconsidered. Endocytic uptake seems to contradict the indispensability of tryptophans and also the minimum length of 16 amino acid residues for efficient internalization. To revise the membrane translocation of penetratin, two penetratin analogs were designed and synthesized: a peptide in which tryptophans were replaced by phenylalanines (Phe^{6, 14}‐penetratin, RQIKI F FQNRRMK F KK) and a shortened analog (dodeca‐penetratin, RQIKIWF‐R‐KWKK) made up of only 12 residues. The peptides were fluorescently labeled and applied to live, unfixed cells from various lines. Cellular uptake was analysed by confocal microscopy and flow cytometry. Low temperature or ATP‐depletion blocked the intracellular entry of all three penetratin peptides. A decrease in membrane fluidity or cholesterol depletion with methyl‐β‐cyclodextrin greatly inhibited peptide uptake, showing the involvement of cholesterol‐rich lipid rafts in internalization. Exogenous heparan sulfate also diminished the internalization of penetratin and its derivatives, reflecting the paramount importance of electrostatic interactions with polyanionic cell‐surface proteoglycans. The beneficial presence of tryptophans is supported by observations on the decreased cellular uptake of Phe^{6, 14}‐penetratin. The maintained translocational efficiency of dodeca‐penetratin demonstrates that a thorough understanding of penetratin internalization can yield new penetratin analogs with unaltered translocational abilities. This study provides evidence on the energy‐dependent and lipid raft‐mediated endocytic uptake of penetratin and highlights the necessity of revealing those pathways that cationic cell‐penetrating peptides employ to enter live cells. Copyright © 2005 European Peptide Society and John Wiley & Sons, Ltd.     

Cellular uptake and biological activity of peptide nucleic acids conjugated with peptides with and without cell‐penetrating ability

A 12‐mer peptide nucleic acid (PNA) directed against the nociceptin/orphanin FQ receptor mRNA was disulfide bridged with various peptides without and with cell‐penetrating features. The cellular uptake and the antisense activity of these conjugates were assessed in parallel. Quantitation of the internalized PNA was performed by using an approach based on capillary electrophoresis with laser‐induced fluorescence detection (CE‐LIF). This approach enabled a selective assessment of the PNA moiety liberated from the conjugate in the reducing intracellular environment, thus avoiding bias of the results by surface adsorption. The biological activity of the conjugates was studied by an assay based on the downregulation of the nociceptin/orphanin FQ receptor in neonatal rat cardiomyocytes (CM). Comparable cellular uptake was found for all conjugates and for the naked PNA, irrespective of the cell‐penetrating properties of the peptide components. All conjugates exhibited a comparable biological activity in the 100 nM range. The naked PNA also exhibited extensive antisense activity, which, however, proved about five times lower than that of the conjugates. The found results suggest cellular uptake and the bioactivity of PNA‐peptide conjugates to be not primarily related to the cell‐penetrating ability of their peptide components. Likewise from these results it can be inferred that the superior bioactivity of the PNA‐peptide conjugates in comparison with that of naked PNA rely on as yet unknown factors rather than on higher membrane permeability. Several hints point to the resistance against cellular export and the aggregation propensity combined with the endocytosis rate to be candidates for such factors. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Cellular uptake of antisense oligonucleotides after complexing or conjugation with cell-penetrating model peptides.

The uptake by mammalian cells of phosphorothioate oligonucleotides was compared with that of their respective complexes or conjugates with cationic, cell-penetrating model peptides of varying helix-forming propensity and amphipathicity. An HPLC-based protocol for the synthesis and purification of disulfide bridged conjugates in the 10-100 nmol range was developed. Confocal laser scanning microscopy (CLSM) in combination with gel-capillary electrophoresis and laser induced fluorescence detection (GCE-LIF) revealed cytoplasmic and nuclear accumulationin all cases. The uptake differences between naked oligonucleotides and their respective peptide complexes or conjugates were generally confined to one order of magnitude. No significant influence of the structural properties of the peptide components upon cellular uptake was found. Our results question the common belief that the increased biological activity of oligonucleotides after derivatization with membrane permeable peptides may be primarily due to improved membrane translocation.     

Assessment of the safety of the cationic arginine-rich peptides (CARPs) poly-arginine-18 (R18 and R18D) in ex vivo models of mast cell degranulation and red blood cell hemolysis

Our laboratory focuses on the development of novel neuroprotective cationic peptides, such poly-arginine-18 (R18: 18-mer of l-arginine; net charge +18) and its d-enantiomer R18D in stroke and other brain injuries. In the clinical development of R18/R18D, their cationic property raises potential safety concerns on their non-specific effects to induce mast cell degranulation and hemolysis. To address this, we first utilised primary human cultured mast cells (HCMCs) to examine anaphylactoid effects. We also included as controls, the well-characterised neuroprotective TAT-NR2B9c peptide and the widely used heparin reversal peptide, protamine. Degranulation assay based on β-hexosaminidase release demonstrated that R18 and R18D did not induce significant mast cell degranulation in both untreated (naïve) and IgE-sensitised HCMCs in a dose-response study to a maximum peptide concentration of 16 μM. Similarly, TAT-NR2B9c and protamine did not induce significant mast cell degranulation. To examine hemolytic effects, red blood cells (RBCs), were incubated with the peptides at a concentration range of 1–16 μM in the absence or presence of 2% plasma. Measurement of hemoglobin absorbance revealed that only R18 induced a modest, but significant degree of hemolysis at the 16 μM concentration, and only in the absence of plasma. This study addressed the potential safety concern of the application of the cationic neuroprotective peptides, especially, R18D, on anaphylactoid responses and hemolysis. The findings indicate that R18, R18D, TAT-NR2B9c and protamine are unlikely to induce histamine mediated anaphylactoid reactions or RBC hemolysis when administered intravenously to patients.     

Structural requirements for cellular uptake of α‐helical amphipathic peptides

The structure of the cell‐permeable α‐helical amphipathic model peptide FLUOS‐KLALKLALKALKAALKLA‐NH₂ ( I ) was modified stepwise with respect to its helix parameters hydrophobicity, hydrophobic moment and hydrophilic face as well as molecular size and charge. Cellular uptake and membrane destabilizing activity of the resulting peptides were studied using aortic endothelial cells and HPLC combined with CLSM. With the exceptions that a reduction of molecule size below 16 amino acid residues and the introduction of a negative net charge abolished uptake, none of the investigated structural parameters proved to be essential for the passage of these peptides across the plasma membrane. Membrane toxicity also showed no correlation to any of the parameters investigated and could be detected only at concentrations higher than 2 μm . These results implicate helical amphipathicity as the only essential structural requirement for the entry of such peptides into the cell interior, in accord with earlier studies. The pivotal role of helical amphipathicity was confirmed by uptake results obtained with two further pairs of amphipathic/non‐amphipathic 18‐mer peptides with different primary structure, net charge and helix parameters from I . The amphipathic counterparts were internalized into the cells to a comparable extent as I , whereas no cellular uptake could be detected for the non‐amphipathic analogues. The mode of uptake remains unclear and involves both temperature‐sensitive and ‐insensitive processes, indicating non‐endocytic contributions. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

综述: 细胞穿透肽及其结构改造在siRNA传递中的应用

小RNA药物应用于临床的主要技术瓶颈在于如何高效、低毒地将小RNA分子传递到它发挥功能的场所．基于细胞穿透肽在小RNA透皮给药的临床应用中所取得的进展,本文系统评述了近年来细胞穿透肽在小RNA的体内、体外传递方面的研究动态,分析了细胞穿透肽的结构改造对肽/小RNA复合物转染进入细胞发挥功能的影响,展望了细胞穿透肽作为小RNA的体内药物传递载体的发展方向．     

Cellular internalization of arginine‐rich peptides into tobacco suspension cells: a structure–activity relationship study

Translocation of several fluorescently labeled arginine‐rich peptides into intact plant cells was quantitatively examined in order to investigate the structural factors required for efficient cellular internalization, and thereby, to evaluate the potential of arginine‐rich peptides as intracellular delivery vectors in plants. Cell‐penetrating peptides (CPPs) such as arginine‐rich peptides permit the direct introduction of biologically active macromolecules into plant cytoplasm to manipulate various intracellular processes. While a significant level of adsorption of applied arginine‐rich peptides was observed in the cell walls rich in negative charges, removal of adsorbed peptides by trypsin treatment allowed determination of the amount of internalized peptides in a quantitative manner using spectrofluorometric analysis. The internalization of arginine‐rich peptides depended on the number of arginine residues, and the peptide containing eight arginine residues showed most effective internalization. Besides, the position of small cargoes attached to the arginine‐rich peptides markedly affected the internalization efficiency. The results obtained in this study provide useful information for the development of efficient intracellular delivery tools in plant science. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Enantiomer-specific bioactivities of peptidomimetic analogues of mastoparan and mitoparan: characterization of inverso mastoparan as a highly efficient cell penetrating peptide

Retro-inverso transformation has commonly been employed as a strategy both for the synthesis of proteolytically stable peptide analogues and for the detailed investigation of structure activity relationships. Herein, we adopted a similar strategy to probe the structure activity relationships of two biologically active tetradecapeptides. Analogues of the α-helical mastoparan, and the highly potent apoptogenic analogue mitoparan, were synthesized using d-amino acids assembled in both endogenous (inverso) and reverse (retro-inverso) orientations. For a more comprehensive comparison, our studies also included the retro l-enantiomer of both peptides. Contrary to expectation, comparative investigations of cytotoxicity, mast cell degranulation, and cellular penetration demonstrated that, while retro-inverso transformation abrogated the associated biological activities of these helical peptides, inverso homologues retained their bioactivities. Moreover, inverso mastoparan demonstrated the highest translocation efficacy of all analogues with much improved uptake kinetics compared to other cell penetrating peptides (CPPs) including the commonly employed inert vectors penetratin and tat. Data presented herein thus propound the utility of inverso mastoparan as a highly efficient peptide vector. Furthermore, correlation analysis of plasma membrane translocation and intracellular uptake efficacy further supports a two-compartment model of CPP import whereby the intracellular accumulation of polycationic peptides is dependent upon both the efficiency of transport into the cell and their subsequent accretion at distinct subcellular loci.     

Cellular uptake [correction of utake] of the Tat peptide: an endocytosis mechanism following ionic interactions

The cellular delivery of various biological compounds has recently been improved by conjugating them to short peptides known as protein transduction domains or cell penetrating peptides. These peptides include Tat, Antennapedia and arginine-rich peptides. The common feature of these peptides is their highly cationic nature. Up to now, the cellular uptake of about 50 different peptides and proteins coupled to Tat or Antennapedia peptides has been reported. The ability to deliver molecules into cells is not limited to peptide moieties, since oligonucleotides, peptide nucleic acids or other low molecular weight entities have been successfully internalized. Moreover, most of these examples have been accompanied by the expected biological response. More surprisingly, the uptake of large structures such as liposomes, phages, nanoparticles or adenoviruses has also been documented. Indeed the mechanism by which these very different entities could enter cells following a putative common pathway appeared more and more intriguing after each new reported example of cellular uptake mediated by these peptides. After a long period of uncertainty regarding the mechanism of entry, data from several groups now argue for an energy-dependent process of entry. The entry of most of these molecules is likely to be inhibited by low temperature incubation or in the presence of various drugs applied to inhibit the energy-dependent pathway of cell entry. Moreover, the binding of the highly cationic Tat peptide to various anionic membrane components probably initiates the first step of the cell internalization process.     

Designing Peptide Vaccines for Cellular Cross-Presentation

Synthetic peptides are safe and relatively cheap vaccine components. However, the efficiency of peptide vaccines is limited by peptide interaction with non-professional antigen-presenting cells, which may hamper induction of productive T-cell responses. This paper argues that peptide vaccines should be modified for exclusive uptake by cells with the capacity to prime T-cell responses. Moreover, design of peptide vaccines should take intracellular antigen processing into account and exploit cellular mechanisms of proteolysis, transport and HLA class I assembly of antigenic peptides to enhance efficiency of T-cell priming and stimulation.     

The presence of ANP in rat peritoneal mast cells

Atrial natriuretic peptide （ANP） is an important component of the natriuretic peptide system. A great role in many regulatory systems is played by mast cells. Meanwhile involvement of these cells in ANP activity is poorly studied. In this work, we have shown the presence of ANP in rat peritoneal mast cells. Pure fraction of mast cells was obtained by separation of rat peritoneal cells on a Percoll density gradient. By Westem blotting, two ANP-immunoreactive proteins of molecular masses of 2.5 kDa and 16.9 kDa were detected in lysates from these mast cells. Electron microscope immunogold labeling has revealed the presence of ANP-immunoreactive material in storage, secreting and released granules of mast cells. Our findings indicate the rat peritoneal mast cells to contain both ANP prohormone and ANP. These both peptides are located in mast cell secretory granules and released by mechanism of degranulation. It is discussed that many mast cell functions might be due to production of natriuretic peptides by these cells.     

The agony of choice: how to find a suitable CPP for cargo delivery

Successful and effective cellular delivery remains a main obstacles in the medical field. The use of cell‐penetrating peptides (CPPs) has become one of the most important tools for the internalisation of a wide range of molecules including pharmaceuticals. It is still difficult to choose one CPP for one biological application because there is no ubiquitous CPP meeting the diverse requirements. In our case, we are looking for a suitable CPP to deliver the pro‐apoptotic KLA peptide (KLAKLAKKLAKLAK) by a simple co‐incubation strategy. For that reason, we selected three different cell lines (fibroblastic, cancerous and macrophagic cells) and studied the uptake and subcellular localisation of six different CPPs alone as well as mixed with the KLA peptide. Furthermore, we used the CPPs with a carboxyamidated or a carboxylated C‐terminus and analysed the impact of the C‐termini on internalisation and cargo delivery. We could clearly showed that the cellular CPP uptake is not only dependent on the used CPP and cell line but also highly affected by its chemical nature of the C‐terminus (uptake: carboxyamidated CPPs > carboxylated CPPs) and can influence its cellular localisation. We successfully delivered the KLA peptide in the three cell lines and learned that here as well, the C‐terminus is crucial for an effective peptide delivery. Finally, we induced apoptosis in mouse leukaemic monocyte macrophage (RAW 264.7) and in human breast adenocarcinoma (MCF‐7) cells using the mixture of amidated MPG peptide : KLA and in african green monkey kidney fibroblast (Cos‐7) cells using carboxylated integrin peptide : KLA. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Translocation of analogues of the antimicrobial peptides magainin and buforin across human cell membranes

Cationic antimicrobial peptides play important roles in innate immunity. Compared with extensive studies on peptide-bacteria interactions, little is known about peptide-human cell interactions. Using human cervical carcinoma HeLa and fibroblastic TM12 cells, we investigated the cellular uptake of fluorescent analogues of the two representative antimicrobial peptides magainin 2 and buforin 2 in comparison with the representative Arg-rich cell-penetrating Tat-(47-57) peptide (YGRKKRRQRRR). The dose, time, temperature, and energy dependence of translocation suggested that the three peptides cross cell membranes through different mechanisms. The magainin peptide was internalized within a time scale of tens of minutes. The cooperative concentration dependence of uptake suggested that the peptide forms a pore as an intermediate similar to the observations in model membranes. Furthermore, the translocation was coupled with cytotoxicity, which was larger for tumor HeLa cells. In contrast, the buforin peptide translocated within 10 min by a temperature-independent, less concentration-dependent passive mechanism without showing any significant cytotoxicity at the highest concentration investigated (100 microm). The uptake of the Tat peptide was proportional to the peptide concentration, and the concentration dependence was lost upon ATP depletion. The peptide exhibited a moderate cytotoxicity at higher concentrations. The time course did not show saturation even after 120 min. The buforin peptide, covalently attached to the 28-kDa green fluorescent protein, also entered cells, suggesting a potency of the peptide as a vector for macromolecular delivery into cells. However, the mechanism appeared to be different from that of the parent peptide.     

Translocation of cell‐penetrating peptides into Candida fungal pathogens

Cell‐penetrating peptides (CPPs) are small peptides capable of crossing cellular membranes while carrying molecular cargo. Although they have been widely studied for their ability to translocate nucleic acids, small molecules, and proteins into mammalian cells, studies of their interaction with fungal cells are limited. In this work, we evaluated the translocation of eleven fluorescently labeled peptides into the important human fungal pathogens Candida albicans and C. glabrata and explored the mechanisms of translocation. Seven of these peptides (cecropin B, penetratin, pVEC, MAP, SynB, (KFF)₃K, and MPG) exhibited substantial translocation (>80% of cells) into both species in a concentration‐dependent manner, and an additional peptide (TP‐10) exhibiting strong translocation into only C. glabrata. Vacuoles were involved in translocation and intracellular trafficking of the peptides in the fungal cells and, for some peptides, escape from the vacuoles and localization in the cytosol were correlated to toxicity toward the fungal cells. Endocytosis was involved in the translocation of cecropin B, MAP, SynB, MPG, (KFF)₃K, and TP‐10, and cecropin B, penetratin, pVEC, and MAP caused membrane permeabilization during translocation. These results indicate the involvement of multiple translocation mechanisms for some CPPs. Although high levels of translocation were typically associated with toxicity of the peptides toward the fungal cells, SynB was translocated efficiently into Candida cells at concentrations that led to minimal toxicity. Our work highlights the potential of CPPs in delivering antifungal molecules and other bioactive cargo to Candida pathogens.     

Polyvalent human immunodeficiency virus synthetic immunogen comprised of envelope gp120 T helper cell sites and B cell neutralization epitopes

In previous studies, we have used antisera raised to envelope (env)-gene-encoded synthetic peptides to identify a region of (HIV) glycoprotein (gp) 120 env protein designated SP10 that contains a type-specific neutralizing determinant. To develop a polyvalent, synthetic peptide inoculum that can evoke both neutralizing antibodies and T cell proliferative responses to more than one HIV isolate, synthetic peptides containing type-specific neutralizing determinants of gp120 from HIV isolates HTLV-IIIB (IIIB), HTLV-IIIMN (MN) and HTLV-IIIRF (RF) were coupled to a 16 amino acid T cell epitope (T1) of HIV-IIIB gp120 and used to immunize goats. Goat antisera to each T1-SP10 peptide derived from the SP10 region of gp120 of IIIB, MN, and RF neutralized HIV isolates IIIB, MN and RF in a type-specific manner. Moreover, peripheral blood T cells from immunized goats also proliferated in a type-specific manner to peptides derived from gp120 of IIIB, MN, and RF. When combined in a trivalent inoculum, T1-SP10 peptides from HIV-1 isolates IIIB, MN, and RF evoked a high titered neutralizing antibody response to isolates IIIB, MN, and RF in goats and as well induced immune T cells to undergo blast transformation in the presence of peptides derived from gp120 of all three HIV isolates. The T1 portion of the T1-SP10 construct was shown to induce a B cell antibody response against determinants within the T1 peptide in addition to inducing T cell proliferative responses in immune goat T cells. Moreover, the SP10 portion of the T1-SP10 constructs not only induced B cell antibody production but also induced type-specific T cell proliferative responses localized to the C-terminal variable sequences of the SP10 peptides. Finally, the T1-SP10 peptide construct induced memory T cell proliferative responses to native gp120 env protein. Thus, combinations of homologous SP10 region synthetic peptides containing type-specific neutralizing determinants and T cell epitopes of HIV gp120 may be useful in man to elicit high titered neutralizing B cell responses and, as well, T cell responses to more than one HIV isolate.     

Probing the impact of valency on the routing of arginine-rich peptides into eukaryotic cells

Multivalency represents a critical parameter in cell biology responsible for the overall avidity of low-affinity interactions and the triggering of cellular events. Functions such as catalytic activity, cellular uptake, or localization are frequently linked to the oligomeric state of a protein. This study explores the impact of multivalency on the import and routing of peptides into cells. Specifically, cationic import sequences such as decaarginine, decalysine, and the HIV Tat peptide (GRKKRRQRRRAP, residues 48-59) as well as the nuclear localization sequence from SV40 large T-antigen were assembled into defined peptide oligomers by fusing them to the tetramerization domain of human p53 (residues 325-355, hp53(tet) domain). The resulting tetravalent peptides typically displayed between 10- and 100-fold enhancements in cellular import and intracellular routing properties in relation to their monomeric homologues. These peptides were not toxic to cells. Flow cytometry results and transfection assays indicated that tetravalent decaarginyl peptides (10R-p53(tet) and NLS-10R-p53(tet)) were the peptides most efficiently routed into cells. Their mechanism of import was subsequently examined on unfixed, viable cells using a combination of metabolic inhibitors, flow cytometry, and microscopy techniques. These studies revealed that tetravalent arginine-rich peptides bind to heparan sulfate on the cell surface, are internalized at 37 degrees C, but not at 4 degrees C, via a clathrin-mediated pathway, and accumulate into endosome-like acidic compartments. A fraction of these tetravalent peptides access the cytosol and accumulate in the nucleus of cells. This study concludes that the oligomerization of proteins harboring arginine-rich peptide chains may profoundly influence their ability to enter and be routed into cells.