Self-aggregation propensity of the Tat peptide revealed by UV-Vis, NMR and MD analyses.

By a combination of UV-Vis analyses, NMR-based diffusion measurements and MD simulations we have demonstrated for the first time that the HIV-1 Tat arginine-rich peptide (Tat) is able to self-aggregate in both its fluorescently labeled and unlabeled variants. We propose Tat dimerization as the dominant aggregation process and show that the associated equilibrium constant increases ten-fold by labeling with the standard TAMRA dye. Also, we extend similar conclusions to other cationic cell penetrating peptides (CPPs), such as Antennapedia (Ant) and nona-arginine (R9).

Spontaneous membrane-translocating peptides: influence of peptide self-aggregation and cargo polarity.

Peptides that translocate spontaneously across cell membranes could transform the field of drug delivery by enabling the transport of otherwise membrane-impermeant molecules into cells. In this regard, a 9-aminoacid-long motif (representative sequence: PLIYLRLLR, hereafter Translocating Motif 9, TM9) that spontaneously translocates across membranes while carrying a polar dye was recently identified by high-throughput screening. Here we investigate its transport properties by a combination of in cuvette physico-chemical assays, rational mutagenesis, live-cell confocal imaging and fluorescence correlation spectroscopy measurements.

Mechanistic insight into CM18-Tat11 peptide membrane-perturbing action by whole-cell patch-clamp recording.

The membrane-destabilization properties of the recently-introduced endosomolytic CM18-Tat11 hybrid peptide (KWKLFKKIGAVLKVLTTG-YGRKKRRQRRR, residues 1-7 of cecropin-A, 2-12 of melittin, and 47-57 of HIV-1 Tat protein) are investigated in CHO-K1 cells by using the whole-cell configuration of the patch-clamp technique. CM18-Tat11, CM18, and Tat11 peptides are administered to the cell membrane with a computer-controlled micro-perfusion system. CM18-Tat11 induces irreversible cell-membrane permeabilization at concentrations (≥4 µM) at which CM18 triggers transient pore formation, and Tat11 does not affect membrane integrity.

High-yield nontoxic gene transfer through conjugation of the CM₁₈-Tat₁₁ chimeric peptide with nanosecond electric pulses.

We report a novel nontoxic, high-yield, gene delivery system based on the synergistic use of nanosecond electric pulses (NPs) and nanomolar doses of the recently introduced CM18-Tat11 chimeric peptide (sequence of KWKLFKKIGAVLKVLTTGYGRKKRRQRRR, residues 1-7 of cecropin-A, 2-12 of melittin, and 47-57 of HIV-1 Tat protein). This combined use makes it possible to drastically reduce the required CM18-Tat11 concentration and confines stable nanopore formation to vesicle membranes followed by DNA release, while no detectable perturbation of the plasma membrane is observed. Two different experimental assays are exploited to quantitatively evaluate the details of NPs and CM18-Tat11 cooperation: (i) cytofluorimetric analysis of the integrity of synthetic 1,2-dioleoyl-sn-glycero-3-phosphocholine giant unilamellar vesicles exposed to CM18-Tat11 and NPs and (ii) the in vitro transfection efficiency of a green fluorescent protein-encoding plasmid conjugated to CM18-Tat11 in the presence of NPs.

Antimicrobial peptides design by evolutionary multiobjective optimization.

Antimicrobial peptides (AMPs) are an abundant and wide class of molecules produced by many tissues and cell types in a variety of mammals, plant and animal species. Linear alpha-helical antimicrobial peptides are among the most widespread membrane-disruptive AMPs in nature, representing a particularly successful structural arrangement in innate defense. Recently, AMPs have received increasing attention as potential therapeutic agents, owing to their broad activity spectrum and their reduced tendency to induce resistance.

In vitro efficient transfection by CM₁₈-Tat₁₁ hybrid peptide: a new tool for gene-delivery applications.

Cell penetrating peptides (CPPs) are actively researched as non-viral molecular carriers for the controlled delivery of nucleic acids into cells, but widespread application is severely hampered by their trapping into endosomes. Here we show that the recently introduced endosomolytic CM18-Tat11 hybrid peptide (KWKLFKKIGAVLKVLTTG-YGRKKRRQRRR, residues 1-7 of Cecropin-A, 2-12 of Melittin, and 47-57 of HIV-1 Tat protein) can be exploited to obtain a self-assembled peptide-DNA vector which maintains the CM18-Tat11 ability to enter cells and destabilize vesicular membranes, concomitantly yielding high DNA transfection efficiency with no detectable cytotoxic effects. Different peptide-DNA stoichiometric ratios were tested to optimize vector size, charge, and stability characteristics.

An automated plasma protein fractionation design: high-throughput perspectives for proteomic analysis.

Background: Human plasma, representing the most complete record of the individual phenotype, is an appealing sample for proteomics analysis in clinical applications. Up to today, the major obstacle in a proteomics study of plasma is the large dynamic range of protein concentration and the efforts of many researchers focused on the resolution of this important drawback.

Findings: In this study, proteins from pooled plasma samples were fractionated according to their chemical characteristics on a home-designed SPE automated platform.

A novel chimeric cell-penetrating peptide with membrane-disruptive properties for efficient endosomal escape.

Efficient endocytosis into a wide range of target cells and low toxicity make the arginine-rich Tat peptide (Tat(11): YGRKKRRQRRR, residues 47-57 of HIV-1 Tat protein) an excellent transporter for delivery purposes. Unfortunately, molecules taken up by endocytosis undergo endosomal entrapment and possible metabolic degradation. Escape from the endosome is therefore actively researched.

Ribozyme-mediated gene knock down strategy to dissect the consequences of PDGF stimulation in vascular smooth muscle cells.

Background: Vascular Smooth Muscle Cells (VSMCs), due to their plasticity and ability to shift from a physiological contractile-quiescent phenotype to a pathological proliferating-activated status, play a central role in the onset and progression of atherosclerosis and cardiovascular diseases. PDGF-BB, among a series of cytokines and growth factors, has been identified as the critical factor in this phenotypic switch. In order to obtain new insights on the molecular effects triggered by PDGF-BB, a hammerhead ribozyme targeting the membrane receptor PDGFR-β was applied to inhibit PDGF pathway in porcine VSMCs.

Vascular smooth-muscle-cell activation: proteomics point of view.

Vascular smooth-muscle cells (VSMCs) are the main component of the artery medial layer. Thanks to their great plasticity, when stimulated by external inputs, VSMCs react by changing morphology and functions and activating new signaling pathways while switching others off. In this way, they are able to increase the cell proliferation, migration, and synthetic capacity significantly in response to vascular injury assuming a more dedifferentiated state.

A proteomic study of microgravity cardiac effects: feature maps of label-free LC-MALDI data for differential expression analysis.

We present a computational analysis of Mass Spectrometry (MS) data based on a proteomic study of mice cardiac tissue samples whose measured changes in peptide and protein abundance were obtained under normal (control or CTRL) and simulated microgravity conditions (hind-limb unloading or HLU). A pipeline consisting of experimental and computational steps has been designed to achieve, respectively, pre-fractionation to simplify mouse heart protein extracts and data synthesis by feature consensus maps. Both acid and neutral protein fractions obtained from differential solubility have been digested, and peptide mixtures have been resolved by LC-MALDI.

A gel-free approach in vascular smooth muscle cell proteome: perspectives for a better insight into activation.

Background: The use of chromatography coupled with mass spectrometry (MS) analysis is a powerful approach to identify proteins, owing to its capacity to fractionate molecules according to different chemical features. The first protein expression map of vascular smooth muscle cells (VSMC) was published in 2001 and since then other papers have been produced. The most detailed two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) map was presented by Mayr et al who identified 235 proteins, corresponding to the 154 most abundant unique proteins in mouse aortic VSMC.

A proteomic approach to the investigation of early events involved in the activation of vascular smooth muscle cells.

Vascular smooth muscle cells (VSMC) are mature cells that maintain great plasticity. This distinctive quality is the basis of the migration and proliferation of VSMC in cardiovascular diseases. We have investigated, via a proteomic approach, the molecular changes that promote VSMC switching from a quiescent to an activated-proliferating phenotype.

A proteomic approach to the investigation of early events involved in vascular smooth muscle cell activation.

Vascular smooth muscle cells (VSMC) are mature cells that maintain great plasticity. This distinctive feature is the basis of the VSMC migration and proliferation involved in cardiovascular diseases. We have used a proteomic approach to the molecular changes that promote the switch of VSMC from having a quiescent to activated-proliferating phenotype.