Efficient internalization of TAT peptide in zwitterionic DOPC phospholipid membrane revealed by neutron diffraction.

The aim of this study is to investigate the interactions between TAT peptides and a neutral DOPC bilayer by using neutron lamellar diffraction. The distribution of TAT peptides and the perturbation of water distribution across the DOPC bilayer were revealed. When compared to our previous study on an anionic DOPC/DOPS bilayer (X. Chen et al., Biochim Biophys Acta. 2013. 1828 (8), 1982-1988), a much deeper insertion of TAT peptides was found in the hydrophobic core of DOPC bilayer at a depth of 6.0Å from the center of the bilayer, a position close to the double bond of fatty acyl chain. We conclude that the electrostatic attractions between the positively charged TAT peptides and the negatively charged headgroups of phospholipid are not essential for the direct translocation. Furthermore, the interactions of TAT peptides with the DOPC bilayer were found to vary in a concentration-dependent manner. A limited number of peptides first associate with the phosphate moieties on the lipid headgroups by using the guanidinium ions pairing. Then the energetically favorable water defect structures are adopted to maintain the arginine residues hydrated by drawing water molecules and lipid headgroups into the bilayer core. Such bilayer deformations consequently lead to the deep intercalation of TAT peptides into the bilayer core. Once a threshold concentration of TAT peptide in the bilayer is reached, a significant rearrangement of bilayer will happen and steady-state water pores will form.

Insertion of TAT peptide and perturbation of negatively charged model phospholipid bilayer revealed by neutron diffraction.

TAT peptide is one of the best-characterized cell penetrating peptides derived from the transactivator of transcription protein from the human immunodeficiency virus 1. The aim of this study was to investigate the interaction between TAT peptide and partially negatively-charged phospholipid bilayer by using lamellar neutron diffraction. The main findings are the existence of a contiguous water channel across the bilayer in the presence of TAT peptide. Taken in combination with other observations, including thinning of the lipid bilayer, this unambiguously locates the peptide within the lipid bilayer. The interaction of TAT peptide with anionic lipid bilayer, composed of an 80:20 mixture of DOPC and DOPS, takes place at two locations. One is in the peripheral aqueous phase between adjacent bilayers and the second is below the glycerol backbone region of bilayer. A membrane thinning above a peptide concentration threshold (1mol%) was found, as was a contiguous transbilayer water channel at the highest peptide concentration (10mol%). This evidence leads to the suggestion that the toroidal pore model might be involved in the transmembrane of TAT peptide. We interpret the surface peptide distribution in the peripheral aqueous phase to be a massive exclusion of TAT peptide from its intrinsic location below the glycerol backbone region of the bilayer, due to the electrostatic attraction between the negatively-charged headgroups of phospholipids and the positively charged TAT peptides. Finally, we propose that the role that negatively-charged headgroups of DOPS lipids play in the transmembrane of TAT peptide is less important than previously thought.

Children's visual acuity tests without professional supervision: a prospective repeated measures study.

BACKGROUND: Home visual acuity tests could ease pressure on ophthalmic services by facilitating remote review of patients. Home tests may have further utility in giving service users frequent updates of vision outcomes during therapy, identifying vision problems in an asymptomatic population, and engaging stakeholders in therapy. METHODS: Children attending outpatient clinics had visual acuity measured 3 times at the same appointment: Once by a registered orthoptist per clinical protocols, once by an orthoptist using a tablet-based visual acuity test (iSight Test Pro, Kay Pictures), and once by an unsupervised parent/carer using the tablet-based test. RESULTS: In total, 42 children were recruited to the study. The mean age was 5.6 years (range 3.3 to 9.3 years). Median and interquartile ranges (IQR) for clinical standard, orthoptic-led and parent/carer-led iSight Test Pro visual acuity measurements were 0.155 (0.18 IQR), 0.180 (0.26 IQR), and 0.300 (0.33 IQR) logMAR respectively. The iSight Test Pro in the hands of parents/carers was significantly different from the standard of care measurements (P = 0.008). In the hands of orthoptists. There was no significant difference between orthoptists using the iSight Test Pro and standard of care (P = 0.289), nor between orthoptist iSight Test Pro and parents/carer iSight Test Pro measurements (P = 0.108). CONCLUSION: This technique of unsupervised visual acuity measures for children is not comparable to clinical measures and is unlikely to be valuable to clinical decision making. Future work should focus on improving the accuracy of the test through better training, equipment/software or supervision/support.

An Evidence for a Novel Antiviral Mechanism: Modulating Effects of Arg-Glc Maillard Reaction Products on the Phase Transition of Multilamellar Vesicles.

Maillard reaction products (MRPs) of protein, amino acids, and reducing sugars from many foods and aqueous extracts of herbs are found to have various bioactivities, including antiviral effects. A hypothesis was proposed that their antiviral activity is due to the interaction with the cellular membrane. Aiming to estimate the possible actions of MRPs on phospholipid bilayers, the Arg-Glc MRPs were prepared by boiling the pre-mixed solution of arginine and glucose for 60 min at 100°C and then examined at a series of concentrations for their effects on the phase transition of MeDOPE multilamellar vesicles (MLVs), for the first time, by using differential scanning calorimetry (DSC) and temperature-resolved small-angle X-ray scattering (SAXS). Arg-Glc MRPs inhibited the lamellar gel-liquid crystal (L ß-L α), lamellar liquid crystal-cubic (L α-Q II), and lamellar liquid crystal-inverted hexagonal (L α-H II) phase transitions at low concentration (molar ratio of lipid vs. MRPs was 100:1 or 100:2), but promoted all three transitions at medium concentration (100:5). At high concentration (10:1), the MRPs exhibited inhibitory effect again. The fusion peptide from simian immunodeficiency virus (SIV) induces membrane fusion by promoting the formation of a non-lamellar phase, e.g., cubic (Q II) phase, and inhibiting the transition to H II. Arg-Glc MRPs, at low concentration, stabilized the lamellar structure of SIV peptide containing lipid bilayers, but facilitated the formation of non-lamellar phases at medium concentration (100:5). The concentration-dependent activity of MRPs upon lipid phase transition indiciates a potential role in modulating some membrane-related biological events, e.g., viral membrane fusion.

Membrane-induced folding and structure of membrane-bound annexin A1 N-terminal peptides: implications for annexin-induced membrane aggregation.

Annexins constitute a family of calcium-dependent membrane-binding proteins and can be classified into two groups, depending on the length of the N-terminal domain unique for each individual annexin. The N-terminal domain of annexin A1 can adopt an alpha-helical conformation and has been implicated in mediating the membrane aggregation behavior of this protein. Although the calcium-independent interaction of the annexin A1 N-terminal domain has been known for some time, there was no structural information about the membrane interaction of this secondary membrane-binding site of annexin A1. This study used circular dichroism spectroscopy to show that a rat annexin A1 N-terminal peptide possesses random coil structure in aqueous buffer but an alpha-helical structure in the presence of small unilamellar vesicles. The binding of peptides to membranes was confirmed by surface pressure (Langmuir film balance) measurements using phosphatidylcholine/phosphatidylserine monolayers, which show a significant increase after injection of rat annexin A1 N-terminal peptides. Lamellar neutron diffraction with human and rat annexin A1 N-terminal peptides reveals an intercalation of the helical peptides with the phospholipid bilayer, with the helix axis lying parallel to the surface of membrane. Our findings confirm that phospholipid membranes assist the folding of the N-terminal peptides into alpha-helical structures and that this conformation enables favorable direct interactions with the membrane. The results are consistent with the hypothesis that the N-terminal domain of annexin A1 can serve as a secondary membrane binding site in the process of membrane aggregation by providing a peripheral membrane anchor.

Can the Cellular Internalization of Cargo Proteins Be Enhanced by Fusing a Tat Peptide in the Center of Proteins? A Fluorescence Study.

The aim of this study was to investigate whether the cellular uptake of cargo proteins can be enhanced by fusing a Tat peptide in the center of proteins; glutathione-S-transferase (GST)-Tat-green fluorescent protein (GFP) and GST-GFP-Tat proteins were first constructed and expressed. The cellular internalization of both proteins was then evaluated and compared in HeLa cells using fluorescent microscopy and flow cytometry, as well as the transdermal delivery in human skin using confocal microscopy. Results from in vitro cell experiments showed that GST-Tat-GFP protein efficiently internalized into HeLa cells when a Tat peptide was fused in the center of proteins, whereas its efficiency is lower than that of GST-GFP-Tat protein with a Tat peptide terminal fused. Ex vivo transdermal delivery data also demonstrated that the lower efficiency of GST-Tat-GFP penetrating through human stratum corneum layer when compared with GST-GFP-Tat. Furthermore, both GST-GFP-Tat and GST-Tat-GFP presented a various degree of a mixture of cytoplasmic diffuse staining and punctate surface staining, and the pattern of distribution varied considerably in HeLa cell experiments depending on the concentration of protein used. Therefore, an improved mechanism for Tat-conjugated proteins was proposed, in which Tat-conjugated proteins were first associated with cell membrane, then accumulated on the cell surface, and finally internalized into cells by pore formation mechanism.

A structural study of the myristoylated N-terminus of ARF1.

The effect of myristoylation on the 15-amino-acid peptide from the membrane-binding N-terminus of ADP ribosylation factor 1 (ARF1) was studied using neutron diffraction and circular dichroism. A previous study on the non-acylated form indicated that the peptide lies parallel to the membrane, at a shallow depth and in the vicinity of the phosphorylcholine headgroups. It was suggested that the helix does not extend past residue 12, an important consequence for the linking region of the ARF1 protein. In this paper, we show that the result of myristoylation is to increase the helical content reaching the peptide's C-terminus, resulting in the formation of a new hydrophobic face. This increased helicity may augment the entire protein's membrane-binding affinity, indicating that ARF1 effectively has two interdependent membrane-binding motifs.

Autoinsertion of soluble oligomers of Alzheimer's Abeta(1-42) peptide into cholesterol-containing membranes is accompanied by relocation of the sterol towards the bilayer surface.

BACKGROUND: Soluble Alzheimer's Abeta oligomers autoinsert into neuronal cell membranes, contributing to the pathology of Alzheimer's Disease (AD), and elevated serum cholesterol is a risk factor for AD, but the reason is unknown. We investigated potential connections between these two observations at the membrane level by testing the hypothesis that Abeta(1-42) relocates membrane cholesterol. RESULTS: Oligomers of Abeta(1-42), but not the monomeric peptide, inserted into cholesterol-containing phosphatidylcholine monolayers with an anomalously low molecular insertion area, suggesting concurrent lipid rearrangement. Membrane neutron diffraction, including isomorphous replacement of specific lipid hydrogens with highly-scattering deuterium, showed that Abeta(1-42) insertion was accompanied by outward displacement of membrane cholesterol, towards the polar surfaces of the bilayer. Changes in the generalised polarisation of laurdan confirmed that the structural changes were associated with a functional alteration in membrane lipid order. CONCLUSION: Cholesterol is known to regulate membrane lipid order, and this can affect a wide range of membrane mechanisms, including intercellular signalling. Previously unrecognised Abeta-dependent rearrangement of the membrane sterol could have an important role in AD.

Topical application of superoxide dismutase mediated by HIV-TAT peptide attenuates UVB-induced damages in human skin.

The purpose of this study was to evaluate whether topical application of superoxide dismutase with cell penetrating peptide (HIV-TAT) could protect against skin damage induced by UVB irradiation in humans. The permeability through stratum corneum of large proteins linked to TAT peptide was firstly confirmed by confocal microscopy and tape stripping. Ten healthy volunteers with either Fitzpatrick skin type II or III were recruited in this clinical study. TAT-SOD (300units/cm(2)) and vehicle cream were applied on two symmetric areas of both inner upper arms 1h prior to UVB irradiation. After one hour of pretreatment, subjects received 10 incremental doses of UVB on pretreated areas. 24h later, erythema, blood flow and apoptotic cells were measured. Pretreatment with TAT-SOD 1h prior to UVB radiation promoted a mean minimal erythema dose (MED) increase of 36.6±18.4% (p=0.013<0.05. n=10) compared to vehicle control. The median blood flow values of all subjects following 2 and 3-MED of UVB were 107.8±51.0units and 239.5±88.0units respectively, which account for 26% and 25% decrease with respect to vehicle groups. These data suggest that TAT-SOD significantly suppresses UVB induced erythema formation and blood flow rise. Furthermore, pretreatment with TAT-SOD 1h prior to 2-MED of UVB irradiation reduced the apoptotic sunburn cell formation by 47.6±8.6% (p<0.0001) in all subjects. Evaluating results generated from all measurements, we conclude that topical application of TAT-SOD significantly attenuates UVB-induced skin damage in man. These biological effects of TAT-SOD are probably mediated via its free radical scavenging properties, clearly differentiating it from other physical sunscreen agents.

The fusion peptide of simian immunodeficiency virus and the phase behaviour of N-methylated dioleoylphosphatidylethanolamine.

Temperature-scan X-ray scattering was used to study the effect of the fusion peptide of simian immunodeficiency virus (SIV) on the lipid polymorphism of N-methylated dioleoylphosphatidylethanolamine (DOPE-Me), in the presence and absence of one or both of the fusion inhibitors carbobenzoxy-D-phenylalanine-L-phenylalanine-glycine and 1-lauroyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC). Using X-ray diffraction at stations 2.1 and 8.2 of the Synchrotron Radiation Source at Daresbury Laboratory, UK, the structure of multilamellar vesicles (MLVs) was probed as the temperature was raised from 20 to 90 degrees C. The results are compared to those of similar studies, reported earlier, that used the fusion peptide of feline leukaemia virus (FeLV) which, at 28 amino acid residues in length, is considerably longer than the SIV peptide (12 amino acid residues). We interpret the results within the framework of current understanding of membrane fusion, and demonstrate how observed lipid polymorphism might describe the fusion process.