Apolipoprotein Mimetic Peptide Inhibits Neutrophil-Driven Inflammatory Damage via Membrane Remodeling and Suppression of Cell Lysis.

Neutrophils are crucial for host defense but are notorious for causing sterile inflammatory damage. Activated neutrophils in inflamed tissue can liberate histone H4, which was recently shown to perpetuate inflammation by permeating membranes via the generation of negative Gaussian curvature (NGC), leading to lytic cell death. Here, we show that it is possible to build peptides or proteins that cancel NGC in membranes and thereby suppress pore formation, and demonstrate that they can inhibit H4 membrane remodeling and thereby reduce histone H4-driven lytic cell death and resultant inflammation. As a demonstration of principle, we use apolipoprotein A-I (apoA-I) mimetic peptide apoMP1. X-ray structural studies and theoretical calculations show that apoMP1 induces nanoscopic positive Gaussian curvature (PGC), which interacts with the NGC induced by the N-terminus of histone H4 (H4n) to inhibit membrane permeation. Interestingly, we show that induction of PGC can inhibit membrane-permeating activity in general and "turn off" diverse membrane-permeating molecules besides H4n. In vitro experiments show an apoMP1 dose-dependent rescue of H4 cytotoxicity. Using a mouse model, we show that tissue accumulation of neutrophils, release of neutrophil extracellular traps (NETs), and extracellular H4 all strongly correlate independently with local tissue cell death in multiple organs, but administration of apoMP1 inhibits histone H4-mediated cytotoxicity and strongly prevents organ tissue damage.

Evaluation of the energy resolution of a prompt gamma-ray imaging detector using LaBr3(Ce) scintillator and 8 × 8 array MPPC for an animal study of BNCT.

To develop boron neutron capture therapy (BNCT), it is desired to measure 10B concentration and obtain a two-dimensional 10B distribution in animal studies. In this research, we develop a prompt gamma-ray imaging detector to measure 10B distribution using a 50 mm × 50 mm x 10 mm LaBr3(Ce) scintillator and a multi-pixel photon counter (MPPC). To measure a two-dimensional 10B distribution, the 478 keV gamma-ray emitted from 10B(n,α)7Li reaction should be measured with the discrimination from 511 keV background gamma rays in each MPPC. Furthermore, as a characteristic of the detector, it is necessary to investigate whether the 478 keV events individually incident on the MPPC can be measured in two dimensions. In this study, we evaluated the energy resolution and performed a two-dimensional distribution measurement using a thermal neutron beam and prompt gamma rays from a boron sample. This system was able to obtain energy resolution as full width at half maximum at 511 keV of 5.0 ± 0.2% in all MPPC pixels, better than the 6.5% energy resolution required to discriminate between 478 and 511 keV gamma rays. When the region of interest (ROI) was set up from -3σ to - σ (first ROI) and -3σ to the median (second ROI) for the Gaussian distribution of a 478 keV gamma-ray peak using a 6.25 ppm sample, the detector count rate of the 478 keV gamma rays was 0.03 and 0.11 cps, respectively, without a collimator. Moreover, the effect due to the overlapping 511 keV gamma ray peak was approximately 2.0% in the first ROI and approximately 3.2% in the second ROI. In addition, the counts of 478 keV gamma rays were visualized in two-dimensional.

Determination of mosaicity in oriented stacks of lipid bilayers.

Two methods of measuring the misorientation of domains in oriented multilamellar stacks of lipid bilayers superficially appeared to give different values for the mosaic spread. It is first shown that the traditional rocking method and a newer ring method give the same value of the mosaic spread when the two types of data are similarly analyzed. Both indicate a long-tailed, nearly Lorentzian, mosaic distribution function. Our primary innovation is the analysis of ring data as a function of the rocking angle. For our best oriented DOPC sample, this analysis is consistent with a single Lorentzian mosaic distribution function with width 0.05°. In contrast, samples of DMPC indicate a more complex mosaic distribution and larger widths.

Structure of the DMPC lipid bilayer ripple phase.

High resolution structure is presented for the ripple (Pß') phase of the phospholipid dimyristoylphosphatidylcholine. Low angle X-ray scattering from oriented samples yielded 57 orders, more than twice as many as recorded previously. The determined electron density map has a sawtooth profile similar to the result from lower resolution data, but the features are sharper allowing better estimates for the modulated bilayer profile and the distribution of headgroups along the aqueous interface. Analysis of high resolution wide angle X-ray data shows that the hydrocarbon chains in the longer, major side of the asymmetric sawtooth are packed similarly to the LßF gel phase, with chains in both monolayers coupled and tilted by 18° in the same direction. The absence of Bragg rods that could be associated with the minor side is consistent with disordered chains, as often suggested in the literature. However, the new high resolution bilayer profile strongly suggests that the chains in the two monolayers in the minor side and the curved region are not in registry. This staggered monolayer modulated melting suggests a direction for improving theories of the ripple phase.

What are the true values of the bending modulus of simple lipid bilayers?

Values of the bending modulus KC are reviewed, and possible causes for the considerable differences are discussed. One possible cause is the use of glucose and sucrose in the classical micromechanical manipulation and shape analysis methods. New data, using the more recent low angle X-ray method, are presented that do not support an effect of glucose or sucrose on KC. Another possible cause is using an incomplete theory to interpret the data. Adding a tilt term to the theory clearly does not affect the value obtained from the shape analysis method. It is shown that a tilt term, using a value of the modulus Kθ indicated by simulations, theory, and estimated from order parameters obtained from NMR and from the wide angle X-ray method, should also not affect the value obtained using the micromechanical manipulation method, although it does require a small correction when determining the value of the area compressibility modulus KA. It is still being studied whether including a tilt term will significantly affect the values of KC obtained using low angle X-ray data. It remains unclear what causes the differences in the experimental values of KC for simple lipid bilayers.

HIV-1 Tat membrane interactions probed using X-ray and neutron scattering, CD spectroscopy and MD simulations.

We report the effect on lipid bilayers of the Tat peptide Y47GRKKRRQRRR57 from the HIV-1 virus transactivator of translation (Tat) protein. Synergistic use of low-angle X-ray scattering (LAXS) and atomistic molecular dynamic simulations (MD) indicate Tat peptide binding to neutral dioleoylphosphocholine (DOPC) lipid headgroups. This binding induced the local lipid phosphate groups to move 3Å closer to the center of the bilayer. Many of the positively charged guanidinium components of the arginines were as close to the center of the bilayer as the locally thinned lipid phosphate groups. LAXS data for DOPC, DOPC/dioleoylphosphoethanolamine (DOPE), DOPC/dioleoylphosphoserine (DOPS), and a mimic of the nuclear membrane gave similar results. Generally, the Tat peptide decreased the bilayer bending modulus KC and increased the area/lipid. Further indications that Tat softens a membrane, thereby facilitating translocation, were provided by wide-angle X-ray scattering (WAXS) and neutron scattering. CD spectroscopy was also applied to further characterize Tat/membrane interactions. Although a mechanism for translation remains obscure, this study suggests that the peptide/lipid interaction makes the Tat peptide poised to translocate from the headgroup region.

Comparing lipid membranes in different environments.

When engineering lipid membranes for applications, it is essential to characterize them to avoid artifacts introduced by manipulation and the experimental environment. Wide-angle X-ray scattering is a powerful structural characterization tool for well-ordered lipid systems. It reveals remarkable differences in rotational order parameters for samples prepared in different ways. New data and perspectives are presented here for multilamellar systems that support and extend the characterization work on unilamellar systems that is reported by Watkins et al. in this issue of ACS Nano.

Membrane structure correlates to function of LLP2 on the cytoplasmic tail of HIV-1 gp41 protein.

Mutation studies previously showed that the lentivirus lytic peptide (LLP2) sequence of the cytoplasmic C-terminal tail of the HIV-1 gp41 envelope protein inhibited viral-initiated T-cell death and T-cell syncytium formation, at which time in the HIV life cycle the gp41 protein is embedded in the T-cell membrane. In striking contrast, the mutants did not affect virion infectivity, during which time the gp41 protein is embedded in the HIV envelope membrane. To examine the role of LLP2/membrane interactions, we applied synchrotron x-radiation to determine structure of hydrated membranes. We focused on WT LLP2 peptide (+3 charge) and MX2 mutant (-1 charge) with membrane mimics for the T-cell and the HIV-1 membranes. To investigate the influence of electrostatics, cholesterol content, and peptide palmitoylation, we also studied three other LLP2 variants and HIV-1 mimics without negatively charged lipids or cholesterol as well as extracted HIV-1 lipids. All LLP2 peptides bound strongly to T-cell membrane mimics, as indicated by changes in membrane structure and bending. In contrast, none of the weakly bound LLP2 variants changed the HIV-1 membrane mimic structure or properties. This correlates well with, and provides a biophysical basis for, previously published results that reported lack of a mutant effect in HIV virion infectivity in contrast to an inhibitory effect in T-cell syncytium formation. It shows that interaction of LLP2 with the T-cell membrane modulates biological function.

Membrane morphology induced by anisotropic proteins.

There are a great many proteins that localize to and collectively generate curvature in biological fluid membranes. We study changes in the topology of fluid membranes due to the presence of highly anisotropic, curvature-inducing proteins. Generically, we find a surprisingly rich phase diagram with phases of both positive and negative Gaussian curvature. As a concrete example modeled on experiments, we find that a lamellar phase in a negative Gaussian curvature regime exhibits a propensity to form screw dislocations of definite Burgers scalar but of both chiralities.