Cell-penetrating peptides induce apoptosis and necrosis through specific mechanism and cause impairment of Na+-K+-ATPase and mitochondria.

Cell-penetrating peptides (CPPs) are widely used in the development of various drug delivery systems because of their ability of penetrating plasma membrane. However, the safety of their application remains largely unknown. In this study, we found that the incubation of two main kinds of CPPs with human normal liver cells could cause the occurrence of apoptosis and necrosis, then the detailed apoptosis-related protein were detected out. To discover the specific way which leads to these results, several methods were used in this study. Several cytokines, such as Caspase3 and Bcl-2, were detected to prove that the damage happened after treated with different CPPs. Then shielding the positive charge of TAT and R8, depletion of Na+ in culturing medium and addition of several inhibitors of specific ATPase site were used to investigate whether the cytotoxicity were charge-dependent and ATPase-related. Furthermore, the membrane potential of mitochondria and the leakage of mitochondrial cytochrome c were detected after treated with CPPs to investigate the damage on mitochondria. In general, our results assess the cytotoxicity caused by two main kinds of CPPs and reveal the clear mechanism of how it occurs. This study reveals the essence of cytotoxicity caused by CPPs, and the methods we followed can be used to evaluate the biocompatibility of new-designed CPPs, which makes the application of CPPs better and safer.

Tackling amyloidogenesis in Alzheimer's disease with A2V variants of Amyloid-ß.

We developed a novel therapeutic strategy for Alzheimer's disease (AD) exploiting the properties of a natural variant of Amyloid-ß (Aß) carrying the A2V substitution, which protects heterozygous carriers from AD by its ability to interact with wild-type Aß, hindering conformational changes and assembly thereof. As prototypic compound we designed a six-mer mutated peptide (Aß1-6A2V), linked to the HIV-related TAT protein, which is widely used for brain delivery and cell membrane penetration of drugs. The resulting molecule [Aß1-6A2VTAT(D)] revealed strong anti-amyloidogenic effects in vitro and protected human neuroblastoma cells from Aß toxicity. Preclinical studies in AD mouse models showed that short-term treatment with Aß1-6A2VTAT(D) inhibits Aß aggregation and cerebral amyloid deposition, but a long treatment schedule unexpectedly increases amyloid burden, although preventing cognitive deterioration. Our data support the view that the AßA2V-based strategy can be successfully used for the development of treatments for AD, as suggested by the natural protection against the disease in human A2V heterozygous carriers. The undesirable outcome of the prolonged treatment with Aß1-6A2VTAT(D) was likely due to the TAT intrinsic attitude to increase Aß production, avidly bind amyloid and boost its seeding activity, warning against the use of the TAT carrier in the design of AD therapeutics.

Deferasirox-TAT(47-57) peptide conjugate as a water soluble, bifunctional iron chelator with potential use in neuromedicine.

Deferasirox (DFX), an orally active and clinically approved iron chelator, is being used extensively for the treatment of iron overload. However, its water insolubility makes it cumbersome for practical use. In addition to this, the low efficacy of DFX to remove brain iron prompted us to synthesize and evaluate a DFX-TAT(47-57) peptide conjugate for its iron chelation properties and permeability across RBE4 cell line, an in vitro model of the blood-brain barrier. The water-soluble conjugate was able to remove labile iron from buffered solution as well as from iron overloaded sera, and the permeability of DFX-TAT(47-57) conjugate into RBE4 cells was not affected compared to parent deferasirox. The iron bound conjugate was also able to translocate through the cell membrane.

Altering the Tat-derived peptide bioactivity landscape by changing the arginine side chain length.

Mutations of proteins with dual activities that lead to enhancement of one activity are frequently accompanied by attenuation of the other activity. However, this mutational negative trade-off phenomenon typically only involves the canonical 20 amino acids. To test the effect of non-canonical amino acids on the negative trade-off phenomenon, two bioactivities of HIV-1 Tat-derived peptides were monitored upon changing the Arg side chain length. In contrast to the expected mutational negative trade-off, shortening Arg by one methylene resulted in both higher TAR RNA binding specificity and higher cellular uptake. These results suggest that introducing previously unexploited building blocks, even if the difference is only one methylene, can alter the peptide bioactivity landscape leading to the enhancement of multiple bioactivities.

Enhancement of gene delivery using novel homodimeric tat peptide formed by disulfide bond.

Cationic liposomes have been actively used as gene delivery vehicle because of their minimal toxicity, but their relatively low efficiency of gene delivery is the major disadvantage of these vectors. Recently, cysteine residue incorporation to HIV-1 Tat peptide increased liposomemediated transfection compared with unmodified Tat peptide. Therefore, we designed a novel modified Tat peptide having a homodimeric (Tat-CTHD, Tat-NTHD) and closed structure (cyclic Tat) simply by using the disulfide bond between cysteines to develop a more efficient and safe nonviral gene delivery system. The mixing of Tat-CTHD and Tat-NTHD with DNA before mixing with lipofectamine increased the transfection efficiency compared with unmodified Tat peptide and lipofectamine only in MCF-7 breast cancer cells and rat vascular smooth muscle cells. However, cyclic Tat did not show any improvement in the transfection efficiency. In the gel retardation assay, Tat-CTHD and Tat-NTHD showed more strong binding with DNA than unmodified Tat and cyclic Tat peptide. This enhancement was only shown when Tat-CTHD and Tat-NTHD were mixed with DNA before mixing with lipofectamine. The effects of Tat- CTHD and Tat-NTHD were also valid in the experiment using DOTAP and DMRIE instead of lipofectamine. We could not find any significant cytotoxicity in the working concentration and more usage of these peptides. In conclusion, we have designed a novel transfection-enhancing peptide by easy homodimerization of Tat peptide, and the simple mix of these novel peptides with DNA increased the gene transfer of cationic lipids more efficiently with no additional cytotoxicity.

A modified Tat peptide for selective intracellular delivery of macromolecules.

OBJECTIVES: The Tat peptide has been widely used for the intracellular delivery of macromolecules. The aim of this study was to modify the peptide to enable regulation of cellular uptake through a dependency on activation by proteases present in the local environment. METHODS: The native Tat peptide sequence was altered to inhibit the initial interaction of the peptide with the cell membrane through the addition of the consensus sequence for urokinase plasminogen activator (uPA). uPA expression was characterised and semi-quantitatively rated in three cell lines (U251mg, MDA-MB-231 and HeLa). The modified peptide was incubated with both recombinant enzyme and with cells varying in uPA activity. Cellular uptake of the modified Tat peptide line was compared with that of the native peptide and rated according to uPA activity measured in each cell line. KEY FINDINGS: uPA activity was observed to be high in U251mg and MDA-MB-231 and low in HeLa. In MDA-MB-231 and HeLa, uptake of the modified peptide correlated with the level of uPA expression detected (93 and 52%, respectively). In U251mg, however, the uptake of the modified peptide was much less (19% observed reduction) than the native peptide despite a high level of uPA activity detected. CONCLUSIONS: Proteolytic activation represents an interesting strategy for the targeted delivery of macromolecules using peptide-based carriers and holds significant potential for further exploitation.

Synthesis and evaluation of a novel liposome containing BPA-peptide conjugate for BNCT.

We aimed at securing sufficient concentrations of (10)B in boron neutron capture therapy (BNCT) by developing a new drug delivery system. We have designed and developed a novel lipid analog and succeeded in using it to develop the new boron component liposome. It consisted of three different kinds of amino acid derivatives and two fatty acids, and could react directly with the peptide synthesized first on resin by Fmoc solid-phase synthesis. In this study, lipid analog conjugated with HIV-TAT peptide (domain of human immunodeficiency virus TAT protein) and boronophenylalanine (BPA) was synthesized and successfully incorporated into liposomes.

Side-chain assisted ligation in protein synthesis.

Chemical ligation methods for the assembly of functional proteins continue to advance our basic understanding of protein structure and function. In this work, we report on our progress towards the full synthesis of HIV-1 Tat utilizing our newly developed ligation method; side-chain assisted ligation. The HIV-1 Tat was assembled from three fragments wherein the two thioester peptides were synthesized efficiently using the side-chain anchoring strategy following Fmoc-SPPS. The side-chain assisted ligation step was efficient and provided the ligation product in good yield. Following this step, native chemical ligation was used to fully assemble the HIV-1 Tat protein. Although the removal of the auxiliary in small peptides was straightforward, in the case of HIV-1 Tat this step was inefficient thus hampering the completion of the synthesis.

Peptide-mediated cellular uptake of cryptophane.

Cryptophane-A has generated considerable interest based on its high affinity for xenon and potential for creating biosensors for (129)Xe nuclear magnetic resonance (NMR) spectroscopy. Here, we report the cellular delivery of three peptide-functionalized cryptophane biosensors. Cryptophanes were delivered using two cationic cell penetrating peptides into several human cancer and normal cell lines. An RGD peptide targeting alpha(v)beta(3) integrin receptor was shown to increase specificity of cryptophane cell uptake. Labeling the peptides with Cy3 made it possible to monitor cellular delivery using confocal laser scanning microscopy. The peptido-cryptophanes were determined to be relatively nontoxic by MTT assay at the micromolar cryptophane concentrations that are required for (129)Xe NMR biosensing experiments.

Modulation of microtubule assembly by the HIV-1 Tat protein is strongly dependent on zinc binding to Tat.

BACKGROUND: During HIV-1 infection, the Tat protein plays a key role by transactivating the transcription of the HIV-1 proviral DNA. In addition, Tat induces apoptosis of non-infected T lymphocytes, leading to a massive loss of immune competence. This apoptosis is notably mediated by the interaction of Tat with microtubules, which are dynamic components essential for cell structure and division. Tat binds two Zn2+ ions through its conserved cysteine-rich region in vitro, but the role of zinc in the structure and properties of Tat is still controversial. RESULTS: To investigate the role of zinc, we first characterized Tat apo- and holo-forms by fluorescence correlation spectroscopy and time-resolved fluorescence spectroscopy. Both of the Tat forms are monomeric and poorly folded but differ by local conformational changes in the vicinity of the cysteine-rich region. The interaction of the two Tat forms with tubulin dimers and microtubules was monitored by analytical ultracentrifugation, turbidity measurements and electron microscopy. At 20 degrees C, both of the Tat forms bind tubulin dimers, but only the holo-Tat was found to form discrete complexes. At 37 degrees C, both forms promoted the nucleation and increased the elongation rates of tubulin assembly. However, only the holo-Tat increased the amount of microtubules, decreased the tubulin critical concentration, and stabilized the microtubules. In contrast, apo-Tat induced a large amount of tubulin aggregates. CONCLUSION: Our data suggest that holo-Tat corresponds to the active form, responsible for the Tat-mediated apoptosis.

Effective antibacterial action of tat (47-58) by increased uptake into bacterial cells in the presence of trypsin.

In a previous study, we found an antifungal effect on human pathogenic fungi by the cell-penetrating peptide Tat (47-58) derived from HIV-1. Tat (47-58) immediately entered into the fungal nucleus and affected some physiological changes on the intracellular condition. In this study, Tat (47-58) showed a broad spectrum of antibacterial activity against pathogenic bacteria including bacterial clinical isolates. To improve resistance against proteases for use in vivo, we synthesized an analog of Tat (47-58) by substituting the L-amino acid for the D-amino acid. The D-enantiomer of Tat (47-58) also exhibited a broad spectrum of antibacterial activity at almost the same level of L-Tat (47-58) concentration. Unlike L-Tat (47-58), D-Tat (47-58) showed a significant proteolytic resistance against all proteases tested and antimicrobial activities in the presence of trypsin. Moreover, D-Tat (47-58) inhibited MRSA infection in human HeLa cells whereas L-Tat (47-58) partially allowed MRSA infection, and the results were due to the proteolytic resistance of D-Tat (47-58).

Effect of transcription peptide inhibitors on HIV-1 replication.

HIV-1 manipulates cellular machineries such as cyclin dependent kinases (cdks) and their cyclin elements, to stimulate virus production and maintain latent infection. Specifically, the HIV-1 viral protein Tat increases viral transcription by binding to the TAR promoter element. This binding event is mediated by the phosphorylation of Pol II by complexes such as cdk9/Cyclin T and cdk2/Cyclin E. Recent studies have shown that a Tat 41/44 peptide derivative prevents the loading of cdk2 onto the HIV-1 promoter, inhibiting gene expression and replication. Here we show that Tat peptide analogs computationally designed to dock at the cyclin binding site of cdk2 have the ability to bind to cdk2 and inhibit the association of cdk2 with the HIV promoter. Specifically, the peptide LAALS dissociated the complex and decreased kinase activity in vitro. We also describe our novel small animal model which utilizes humanized Rag2(-/-)gamma(c)(-/-) mice. This small peptide inhibitor induces a decrease in HIV-1 viral transcription in vitro and minimizes viral loads in vivo.