Translocation of a beta-peptide across cell membranes.

Short cationic peptides derived from DNA-binding proteins, of which HIV Tat is a prototype, can cross the membranes of living cells, and they can bring covalently attached moieties (proteins, drugs) along with them. We show that a beta-amino acid analogue of Tat 47-57 enters HeLa cells with comparable efficiency to Tat 47-57 itself (YGRKKRRQRRR). The beta-peptide is comprised of residues that bear the appropriate side chain at the beta-carbon. Both the alpha- and the beta-peptide were conjugated to fluorescein at the N terminus, and cell penetration was monitored by confocal fluorescence microscopy. Deletion of the three C-terminal arginine residues from the alpha-peptide abolished translocation activity, consistent with prior reports, and deletion of the three C-terminal beta3-homoarginine residues from the beta-peptide had a similarly adverse effect. Thus, alpha- and beta-peptide translocation processes show similar length/charge dependence. The beta-peptide appeared to be largely unfolded in water, which is consistent with the behavior of short Tat-derived alpha-peptides, but in methanol the beta-peptide adopted a helical conformation, in contrast to short Tat-derived alpha-peptides. Our results show that neither altering the oligomeric backbone (amide group spacing) nor increasing the intrinsic propensity to adopt a specific secondary structure affects translocation activity.     

Cell–Membrane Penetration of Tat‐Conjugated Polymeric Micelles: Effect of Tat Coating Density

Considerable efforts have been devoted to enhancing the cell penetration of nanoparticles by coating cell‐penetrating peptides (CPPs) on the surface. However, the internalization mechanism for a CPP at different concentrations varies a lot. It is acknowledged that the mechanism is restricted to endocytic pathway at relatively low concentrations; however, direct translocation becomes dominant at high concentrations. This raises an interesting question on how the surface Tat coating density of the nanoparticles would influence their cell–membrane interaction and the consequent internalization behavior. This study systematically investigates the effect of Tat peptides on the internalization behavior of polymeric micelles by tuning surface Tat coating density, incubation concentrations, incubation time, and other factors using poly(ethylene glycol)–poly(ε‐caprolactone) copolymer (PEG‐PCL) micelles. It is found that both energy‐dependent and energy‐independent pathways are involved in the cellular uptake process, and the Tat‐conjugated polymeric micelles strongly accumulated on the cell surface at initial stage. The membrane‐anchoring and internalization rate increase with the increasing Tat coating density. Furthermore, the increasing of Tat coating density accelerates the energy‐independent pathway. It is envisioned that this finding will further shed light on the surface modification of nanoparticles for enhanced cell penetration and direct translocation into cell cytoplasm.     

Development of an efficient transdermal delivery system of small interfering RNA using functional peptides, Tat and AT-1002

Topical use of small interfering RNA (siRNA) as a therapeutic nucleic acid is increasingly studied for the treatment of skin diseases and for the improvement of skin properties. However, naked siRNA transdermal delivery is limited by its low stability in the body and low permeability into target cells. This is due to various skin barriers such as the stratum corneum that has multiple lipid bilayers and epidermal layers that have tight junctions. In this study, we investigate non-invasive transdermal siRNA delivery using two functional peptides: AT1002, which is a tight junction modulator and 6-mer synthetic peptide belonging to a novel class of compounds that reversibly increases paracellular transport of molecules across the epithelial barrier; and Tat, which is a cell-penetrating peptide applicable as a transdermal siRNA delivery enhancer. We examined whether expression of the tight junction protein zonula occludens protein 1 (ZO-1) was detected in mouse skin applied with AT1002. Additionally, siRNA stabilities for RNaseA using Tat and AT1002 were assessed. We also determined the intradermal delivery efficiency of siRNA using functional peptides by confocal laser microscopy of fluorescently labeled siRNA in mouse skin. We found that the Tat analog and AT1002 strongly increased siRNA stability against RNaseA. In addition, ZO-1 disappeared from the skin after treatment with AT1002, yet recovered with time after washing. Finally, we also found that Tat and AT1002 peptides accelerate transdermal siRNA delivery both widely and effectively. Thus, combination of Tat and AT1002 is expected to be a transdermal delivery enhancer of siRNA.     

TAT peptide internalization: seeking the mechanism of entry

During the last decade several peptides have been extensively studied for their ability to translocate across the plasma membrane. These peptides have been called "cell penetrating peptides" (CPP) or "protein transduction domains" (PTD). These peptides also promote the cellular uptake of various cargo molecules. Their mechanism of cellular entry appeared very intriguing since most publications in the field highlighted an energy-independent process. Indeed, cellular uptake of these peptides was still observed by fluorescence microscopy at low temperature or in the presence of several drugs known to inhibit active transport. In addition, internalization was reported to be much faster than known endocytic processes. However the involvement of a specific cellular component responsible for this uptake process appeared unlikely following intensive structure activity relationship studies using a wide panel of Tat analogues. Several reports about a possible artefactual redistribution of CPPs, and their associated cargos, during the cell fixation step commonly used for fluorescence microscopy have recently emerged in the literature. Moreover strong ionic interactions of CPPs with the cell surface also led to an overestimation of the recorded cell-associated fluorescent signal. It now seems well established that arginine-rich peptides are internalized by an energy dependent process involving endocytosis. Whatever the case, however, an increasing number of data indicate that the conjugation of non-permeant molecules to these CPPs allows their cellular uptake and leads to the expected biological responses, thus pointing to the interest of this delivery strategy. However, initial structure activity relationship studies of these CPPs will have to be reconsidered and the relative potency of each peptide (and their analogues) to vectorize the cargos to their most appropriate subcellular compartment will require careful re-evaluation.     

Cellular uptake of aminoglycosides, guanidinoglycosides, and poly-arginine

Aminoglycosides (including neomycin B and tobramycin) exhibit poor uptake by eukaryotic cell lines. When the amines of these natural products are converted into guanidine groups, their cellular uptake is dramatically enhanced. We have synthesized BODIPY-containing aminoglycosides and guanidinoglycosides to evaluate their cellular uptake properties. Fluorescence activated cell sorting (FACS) and fluorescence microscopy are used to compare the membrane translocation and the cellular localization of these compounds. Upon guanidinylation, the cellular uptake efficiencies of tobramycin and neomycin B are enhanced by 10-fold and 20-fold, respectively. Guanidino-neomycin B exhibits a highly efficient uptake, superior to a fluorescent poly-arginine peptide. Interestingly, the cellular uptake of this common transduction peptide is inhibited by guanidine-neomycin B, suggesting a similar uptake mechanism for both the arginine-rich peptides and the guanidinoglycosides.     

Acetylation of the HIV-1 Tat protein: an in vitro study

In the last few years, the understanding of lysine acetylation as a regulatory post-translational modification of proteins in cell signalling cascades has increased. It is now known that not only histones but also non-histone factors can serve as substrates of different acetyltransferase enzymes. Acetylated lysine residues in non-histone factors are often identified using radioactive labelling experiments and immunochemical analysis of synthetic peptides. In this study of the human immunodeficiency virus 1 (HIV-1) Tat protein, we demonstrate the benefits of matrix-assisted laser desorption/ionisation mass spectrometry, proteolytic digestion and Edman sequencing for the mapping of acetylation sites. We confirmed that the HIV-1 Tat protein is acetylated in vitro by the acetyltransferase p300 at a specific lysine residue at position 50 in its RNA binding region. Furthermore, we showed that the Tat cysteine-rich region is acetylated at multiple cysteine residues in the absence of enzyme. Since this non-enzymatic cysteine acetylation occurs independently from the surrounding peptide sequence, we consider the presence of cysteine residues in acetylated peptides an important factor for the interpretation of in vitro acetylation assays in general.Abbreviations aa Amino acid - AcCoA Acetyl coenzyme A - acm Acetamidomethyl - ARM Arginine-rich motif - CRR Cysteine-rich region - HAT Histone acetyltransferaseThis article is dedicated to Harald zur Hausen on the occasion of his retirement as head of the German Cancer Research Center (Deutsches Krebsforschungszentrum) with gratitude and appreciation for 20 years of leadership     

Cellular uptake of an α-AApeptide

Some short and cationic peptides such as the Tat peptide can cross the cell membrane and function as vectors for intracellular delivery. Here we show that an α-AApeptide is able to penetrate the membranes of living cells from an extracellular environment and enter the endosome and cytoplasm of cells. The efficiency of the cellular uptake is comparable to a Tat peptide (48-57) of the same length and is unexpectedly superior to an α-peptide with identical functional groups. The mechanism of uptake is similar to that of the Tat peptide and is through endocytosis by an energy-dependent pathway. Due to the easy synthesis of the α-AApeptides, their resistance to proteolytic hydrolysis, and their low cytotoxicity, α-AApeptides represent a new class of transporters for the delivery of drugs.     

Derivatisation of arginine residues with malondialdehyde for the analysis of peptides and protein digests by LC-ESI-MS/MS

In this study a selective tagging strategy for the derivatisation of arginine residues in peptides is presented. It is based on the reaction of the guanidine group of the arginine side-chain with malondialdehyde (MDA) under strongly acidic conditions, in which a stable pyrimidine ring is formed. The reaction conditions have been optimised so that quantitative modification can be achieved for a variety of peptides. The label has a strong influence on the polarity and basicity of the arginine side-chain and thus on the chromatographic and mass spectrometric properties of arginine-containing peptides. For example, retention, particularly of small and polar peptides as well as arginine-rich peptides, is significantly increased by derivatisation, and therefore sensitivity is also enhanced in liquid chromatography-mass spectrometry (LC-MS). The arginine side-chain also has a strong impact on the fragmentation behaviour of peptides in tandem mass spectrometry. This has been investigated for standard peptides for which, in some cases, significantly more fragment ions were formed after derivatisation. Finally, the method was tested for tryptic digests of standard proteins to demonstrate how the tagging strategy can give improved or complementary information for protein identification.     

Highly efficient (R-X-R)-type carbamates as molecular transporters for cellular delivery

The (R-X-R) motif-containing arginine-rich peptides are among the most effective cell-penetrating peptides. The replacement of amide linkages in the (R-X-R) motif by carbamate linkages as in (r-ahx-r)(4) or (r-ahx-r-r-apr-r)(2) increases the efficacy of such oligomers several-fold. Internalization of these oligomers in mammalian cell lines occurs by an energy-independent process. These oligomers show efficient delivery of biologically active plasmid DNA into CHO-K1 cells.     

Calcium ions effectively enhance the effect of antisense peptide nucleic acids conjugated to cationic tat and oligoarginine peptides

Cell-penetrating peptides have been widely used to improve cellular delivery of a variety of proteins and antisense agents. However, recent studies indicate that such cationic peptides are predominantly entering cells via an endosomal pathway. We now show that the nuclear antisense effect in HeLa cells of a variety of peptide nucleic acid (PNA) peptide conjugates is significantly enhanced by addition of 6 mM Ca(2+) (as well as by the lysosomotrophic agent chloroquine). In particular, the antisense activities of Tat(48-60) and heptaarginine-conjugated PNAs were increased 44-fold and 8.5-fold, respectively. Evidence is presented that the mechanism involves endosomal release. The present results show that Ca(2+) can be used as an effective enhancer for in vitro cellular delivery of cationic peptide-conjugated PNA oligomers, and also emphasize the significance of the endosomal escape route for such peptides.     

RNA architecture dictates the conformations of a bound peptide.

BACKGROUND: The biological function of several viral and bacteriophage proteins, and their arginine-rich subdomains, involves RNA-mediated interactions. It has been shown recently that bound peptides adopt either beta-hairpin or alpha-helical conformations in viral and phage peptide-RNA complexes. We have compared the structures of the arginine-rich peptide domain of HIV-1 Rev bound to two RNA aptamers to determine whether RNA architecture can dictate the conformations of a bound peptide. RESULTS: The core-binding segment of the HIV-1 Rev peptide class II RNA aptamer complex spans the two-base bulge and hairpin loop of the bound RNA and the carboxy-terminal segment of the bound peptide. The bound peptide is anchored in place by backbone and sidechain intermolecular hydrogen bonding and van der Waals stacking interactions. One of the bulge bases participates in U*(A*U) base triple formation, whereas the other is looped out and flaps over the bound peptide in the complex. The seven-residue hairpin loop is closed by a sheared G*A mismatch pair with several pyrimidines looped out of the hairpin fold. CONCLUSIONS: Our structural studies establish that RNA architecture dictates whether the same HIV-1 Rev peptide folds into an extended or alpha-helical conformation on complex formation. Arginine-rich peptides can therefore adapt distinct secondary folds to complement the tertiary folds of their RNA targets. This contrasts with protein-RNA complexes in which elements of RNA secondary structure adapt to fit within the tertiary folds of their protein targets.     

Dendrimer Functionalization with a Membrane‐Interacting Domain of Herpes Simplex Virus Type 1: Towards Intracellular Delivery

A poly(amide)‐based dendrimer was synthesized and functionalized with the membrane‐interacting peptide gH(625–644) (gH625) derived from the herpes simplex virus type 1 (HSV‐1) envelope glycoprotein H, which has previously been shown to assist in delivering large cargoes across the cellular membrane. We demonstrate that the attachment of the gH625 peptide sequence to the termini of a dendrimer allows the conjugate to penetrate into the cellular matrix, whereas the unfunctionalized dendrimer is excluded from translocation. The peptide‐functionalized dendrimer is rapidly taken into the cells mainly through a non‐active translocation mechanism. Our results suggest that the presented peptidodendrimeric scaffold may be a promising material for efficient drug delivery.     

Highly efficient, nonpeptidic oligoguanidinium vectors that selectively internalize into mitochondria

Oligoguanidinium-based cell delivery systems have gained broad interest in the drug delivery field since one decade ago. Thus, arginine-containing peptides as Tat or Antp, oligoarginine peptides, and derived peptoids have been described as shuttles for delivering nonpermeant drugs inside cancer cells. Herein we report a new family of tetraguanidinium cell penetrating vectors efficiently internalized in human tumor cells. Their high internalization, studied by confocal microscopy and flow cytometry, as well as their specific accumulation in mitochondria makes these new vectors likely vehicles for the targeted delivery of anticancer drugs to mitochondria.     

Peptide Inhibitors of HIV-1 Virus Infection Based on Cullin-5

Virion infectivity factor（Vif） is one of the six accessory proteins of HIV-1 and is necessary for viral infectivity. Human Apolipoprotein B editing complex protein 3G（h-APOBEC3G） is a cytidine deaminase only expressed in ＂nonpermissive＂ cells and exhibits virus suppressive activity. With the aid of a Cullin-5 E3 ligase, Vif induces h-APOBEC3G degradation and with the destruction of this ligase, Vif is functionally inactive. Therefore, it is expected that blocking this E3 pathway would be a new therapeutic strategy against HIV-1 infection. In this article, the authors＇ took sequence alignment of the N-termini of Cullin-5 and three other members of the Cullin protein family, respectively. A set of small peptides has been synthesized based on the sequence comparison results and possible Vif-Cullin-5 interaction domains. Moreover, it has been demonstrated that several peptides can reduce virus infectivity in ＂nonpermissive＂ cells with a dose-responsive manner, but not in ＂permissive＂ cells. The results also indicate that the loss of viral infectivity may be because of the increase of APOBEC3G amount in the peptide-treated cells. It is concluded that peptides derived from Cullin-5 can block the APOBEC3G degradation induced by Vif and suppress HIV-1 infectivity. Therefore this study starts a novel strategy for the development of a new HIV-1 inhibitor.     

Investigation of RNA-protein and RNA-metal ion interactions by electron paramagnetic resonance spectroscopy. The HIV TAR-Tat motif

Electron paramagnetic resonance (EPR) spectroscopy was used to investigate changes in dynamics of spin-labeled nucleotides in the TAR RNA (U23, U25, U38, and U40) upon binding to cations, argininamide, and two peptides derived from the Tat protein. Nearly identical changes in dynamics were obtained for either calcium or sodium ions, indicating the absence of a calcium-specific structural change for the TAR RNA in solution that had previously been suggested by crystallographic data. Similar dynamic signatures were obtained for two Tat-derived peptides that have the same important binding determinant (R52) and similar binding affinities to the TAR RNA. However, U23 and U38 were substantially less mobile for the wild-type peptide (YGRKKRRQRRR) than for the mutant (YKKKKRKKKKA), demonstrating that, flanking R52, amino acids in the wild-type sequence make specific contacts to the RNA.     

Negatively charged phospholipids trigger the interaction of a bacterial Tat substrate precursor protein with lipid monolayers

Folded proteins can be translocated across biological membranes via the Tat machinery. It has been shown in vitro that these Tat substrates can interact with membranes prior to translocation. Here we report a monolayer and infrared reflection-absorption spectroscopic (IRRAS) study of the initial states of this membrane interaction, the binding to a lipid monolayer at the air/water interface serving as a model for half of a biological membrane. Using the model Tat substrate HiPIP (high potential iron-sulfur protein) from Allochromatium vinosum, we found that the precursor preferentially interacts with monolayers of negatively charged phospholipids. The signal peptide is essential for the interaction of the precursor protein with the monolayer because the mature HiPIP protein showed no interaction with the lipid monolayer. However, the individual signal peptide interacted differently with the monolayer compared to the complete precursor protein. IRRA spectroscopy indicated that the individual signal peptide forms mainly aggregated β-sheet structures. This β-sheet formation did not occur for the signal peptide when being part of the full length precursor. In this case it adopted an α-helical structure upon membrane insertion. The importance of the signal peptide and the mature domain for the membrane interaction is discussed in terms of current ideas of Tat substrate-membrane interactions.