Release of extracellular vesicle miR-494-3p by ARPE-19 cells with impaired mitochondria.

BACKGROUND: Mitochondrial function in retinal pigmented epithelial (RPE) cells and extracellular vesicle (EV) formation/release are related through the lysosomal and exocytotic pathways that process and eliminate intracellular material, including mitochondrial fragments. We propose that RPE cells with impaired mitochondria will release EVs containing mitochondrial miRNAs that reflect the diminished capacity of mitochondria within these cells. METHODS: We screened ARPE-19 cells for miRNAs that localize to the mitochondria, exhibit biological activity, and are present in EVs released by both untreated cells and cells treated with rotenone to induce mitochondrial injury. EVs were characterized by vesicle size, size distribution, presence of EV biomarkers: CD81, CD63, and syntenin-1, miRNA cargo, and number concentration of EVs released per cell. RESULTS: We found that miR-494-3p was enriched in ARPE-19 mitochondria. Knockdown of miR-494-3p in ARPE-19 cells decreased ATP production and mitochondrial membrane potential in a dose-dependent manner, and decreased basal oxygen consumption rate and maximal respiratory capacity. Increased number of EVs released per cell and elevated levels of miR-494-3p in EVs released from ARPE-19 cells treated with rotenone were also measured. CONCLUSIONS: ARPE-19 mitochondrial function is regulated by miR-494-3p. Elevated levels of miR-494-3p in EVs released by ARPE-19 cells indicate diminished capacity of the mitochondria within these cells. GENERAL SIGNIFICANCE: EV miR-494-3p is a potential biomarker for RPE mitochondrial dysfunction, which plays a central role in non-neovascular age-related macular degeneration, and may be a diagnostic biomarker for monitoring the spread of degeneration to neighboring RPE cells in the retina.

Analysis of exosome release as a cellular response to MAPK pathway inhibition.

Exosome size distributions and numbers of exosomes released per cell are measured by asymmetric flow-field flow fractionation/multi-angle light scattering (A4F/MALS) for three thyroid cancer cell lines as a function of a treatment that inhibits MAPK signaling pathways in the cells. We show that these cell lines release exosomes with well-defined morphological features and size distributions that reflect a common biological process for their formation and release into the extracellular environment. We find that those cell lines with constitutive activation of the MAPK signaling pathway display MEK-dependent exosome release characterized by increased numbers of exosomes released per cell. Analysis of the measured exosome size distributions based on a generalized extreme value distribution model for exosome formation in intracellular multivesicular bodies highlights the importance of this experimental observable for delineating different mechanisms of vesicle formation and predicting how changes in exosome release can be modified by pathway inhibitors in a cell context-dependent manner.

Loop-Closure and Gaussian Models of Collective Structural Characteristics of Capped PEO Oligomers in Water.

Parallel-tempering MD results for a CH3(CH2-O-CH2)mCH3 chain in water are exploited as a database for analysis of collective structural characteristics of the PEO globule with a goal of defining models permitting statistical thermodynamic analysis of dispersants of Corexit type. The chain structure factor, relevant to neutron scattering from a deuterated chain in null water, is considered specifically. The traditional continuum-Gaussian structure factor is inconsistent with the simple k → ∞ behavior, but we consider a discrete-Gaussian model that does achieve that consistency. Shifting and scaling the discrete-Gaussian model helps to identify the low-k to high-k transition near k ≈ 2π/0.6 nm when an empirically matched number of Gaussian links is about one-third of the total number of effective atom sites. This short distance-scale boundary of 0.6 nm is directly verified with the r space distributions, and this distance is thus identified with a natural size for coarsened monomers. The probability distribution of Rg(2) is compared with the classic predictions for both the Gaussian model and freely jointed chains. ⟨Rg(2)(j)⟩, the contribution of the jth chain segment to ⟨Rg(2)⟩, depends on the contour index about as expected for Gaussian chains despite significant quantitative discrepancies that express the swelling of these chains in water. Monomers central to the chain contour occupy the center of the chain globule. The density profiles of chain segments relative to their center of mass can show distinctive density structuring for smaller chains due to the close proximity of central elements to the globule center. However, that density structuring washes out for longer chains where many chain elements additively contribute to the density profiles. Gaussian chain models thus become more satisfactory for the density profiles for longer chains.

Concentration dependence of the Flory-Huggins interaction parameter in aqueous solutions of capped PEO chains.

The dependence on volume fraction φ of the Flory-Huggins interaction parameter χwp(φ) describing the free energy of mixing of polymers in water is obtained by exploiting the connection of χwp(φ) to the chemical potential of the water, for which quasi-chemical theory is satisfactory. We test this theoretical approach with simulation data for aqueous solutions of capped PEO (polyethylene oxide) oligomers. For CH3(CH2-O-CH2)mCH3 (m = 11), χwp(φ) depends strongly on φ, consistent with experiment. These results identify coexisting water-rich and water-poor solutions at T = 300 K and p = 1 atm. Direct observation of the coexistence of these two solutions on simulation time scales supports that prediction for the system studied. This approach directly provides the osmotic pressures. The osmotic second virial coefficient for these chains is positive, reflecting repulsive interactions between the chains in the water, a good solvent for these chains.

Effect of PEG end-group hydrophobicity on lysozyme interactions in solution characterized by light scattering.

We compare protein-protein and protein-polymer osmotic virial coefficients measured by static light scattering for aqueous solutions of lysozyme with low-molecular-weight, hydroxy-terminated (hPEG) and methyl-terminated (mPEG) poly(ethylene glycol) at two solution conditions: pH 7.0 and 0.01 M ionic strength, and pH 6.2 and 0.8 M ionic strength. We find that adding PEG to aqueous lysozyme solutions makes a net repulsive contribution to lysozyme-lysozyme interactions, independent of ionic strength and PEG end-group hydrophobicity. PEG end-group hydrophobicity has a profound effect on the magnitude of this contribution, however, at low ionic strength where mPEG-lysozyme attractive interactions become significant. The enhanced attractions promote mPEG-lysozyme preferential interactions at the expense of lysozyme self-interactions, which leads to lysozyme-lysozyme interactions that are more repulsive in the presence of mPEG. These preferential interactions also lead to the preferential exclusion of diffusable ions locally around the protein, which results in a pronounced ionic strength dependence of mPEG-mediated lysozyme-lysozyme interactions.

Cosolvent preferential molecular interactions in aqueous solutions.

We extend the application of Kirkwood-Buff (KB) solution theory integrals to calculate cosolvent preferential interaction parameters from molecular simulations by deriving from a single simulation trajectory the excess chemical potential of the solute in addition to the solute-solvent molecular distribution functions. The solute excess chemical potential is derived from the potential distribution theorem (PDT) and used to define the local solvent domain around the solute, as distinguished from bulk solution. We show that this KB/PDT characterization of preferential molecular interactions resolves the problem of convergence of the preferential interaction parameter in the bulk solution limit, and as such, gives reliable estimates of preferential interaction parameters for methanol, ethanol, glycerol, and urea in aqueous cosolvent solutions with neopentane or tetramethyl ammonium ion as the solute. Preferential interaction parameters that are also calculated on the basis of cosolvent proximal distributions around the constituent methyl groups of the two solutes with the assumption of group additivity are in good agreement with those obtained by considering the molecular solute as a whole. The results suggest that this approach can be applied to estimate site-specific cosolvent preferential interaction parameters locally on the surface of complex, macromolecular solutes, such as proteins.

Novel cellular microarray assay for profiling T-cell peptide antigen specificities.

We present a novel cellular microarray assay using soluble peptide-loaded HLA A2-Ig dimer complexes that optimizes the avidity of peptide-HLA binding by preserving the molecular flexibility of the dimer complex while attaining much higher concentrations of the complex relative to cognate T-cell receptors. A seminal advance in assay development is made by separating the molecular T-cell receptor recognition event from the binding interactions that lead to antigen-specific cell capture on the microarray. This advance enables the quantitative determination of antigen-specific frequencies in heterogeneous T-cell populations without enumerating the number of cells captured on the microarray. The specificity of cell capture, sensitivity to low antigen-specific frequencies, and quantitation of antigenic T-cell specificities are established using CD8 T-cell populations with prepared antigen-specific CTL frequencies and heterogeneous T cells isolated from peripheral blood. The results demonstrate several advantages for high-throughput broad-based, quantitative assessments of low-frequency antigen specificities. The assay enables the use of cellular microarrays to determine the stability and flux of antigen-specific T-cell responses within and across populations.

Cooperative hydrophobic/hydrophilic interactions in the hydration of dimethyl ether.

Cooperative interactions in the hydration of dimethyl ether (DME) relative to its purely hydrophobic analog, propane, are analyzed by expressing the free energy of hydration in terms of an "inner-shell" contribution from water molecular packing and chemical association, and an "outer-shell" contribution described by the mean binding energy of the solute to the solution and fluctuations in this binding energy. We find that nonadditive, cooperative interactions associated with strong correlations in the binding energy fluctuations of the methyl groups and ether oxygen play a dominant role in the hydration of DME relative to propane. The electrostatic nature of these interactions is revealed in a multi-Gaussian analysis of hydration substates, which shows that the formation of favorable ether oxygen-water hydrogen bonds is correlated with less favorable methyl group-water interactions, and vice versa. We conclude that the group additive distinction between the hydrophobic hydration of the DME methyl groups and hydrophilic hydration of the ether oxygen is lost in the context of these cooperative interactions. Our results also suggest that the binding energy fluctuations of constituent hydrophobic/hydrophilic groups are more sensitive than local water density fluctuations for characterizing the hydration of heterogeneous interfaces.

Ion selectivity from local configurations of ligands in solutions and ion channels.

Probabilities of numbers of ligands proximal to an ion lead to simple, general formulae for the free energy of ion selectivity between different media. That free energy does not depend on the definition of an inner shell for ligand-counting, but other quantities of mechanistic interest do. If analysis is restricted to a specific coordination number, then two distinct probabilities are required to obtain the free energy in addition. The normalizations of those distributions produce partition function formulae for the free energy. Quasi-chemical theory introduces concepts of chemical equilibrium, then seeks the probability that is simplest to estimate, that of the most probable coordination number. Quasi-chemical theory establishes the utility of distributions of ligand-number, and sharpens our understanding of quasi-chemical calculations based on electronic structure methods. This development identifies contributions with clear physical interpretations, and shows that evaluation of those contributions can establish a mechanistic understanding of the selectivity in ion channels.

Distinguishing thermodynamic and kinetic views of the preferential hydration of protein surfaces.

Motivated by a quasi-chemical view of protein hydration, we define specific hydration sites on the surface of globular proteins in terms of the local water density at each site relative to bulk water density. The corresponding kinetic definition invokes the average residence time for a water molecule at each site and the average time that site remains unoccupied. Bound waters are identified by high site occupancies using either definition. In agreement with previous molecular dynamics simulation studies, we find only a weak correlation between local water densities and water residence times for hydration sites on the surface of two globular proteins, lysozyme and staphylococcal nuclease. However, a strong correlation is obtained when both the average residence and vacancy times are appropriately taken into account. In addition, two distinct kinetic regimes are observed for hydration sites with high occupancies: long residence times relative to vacancy times for a single water molecule, and short residence times with high turnover involving multiple water molecules. We also correlate water dynamics, characterized by average occupancy and vacancy times, with local heterogeneities in surface charge and surface roughness, and show that both features are necessary to obtain sites corresponding to kinetically bound waters.

Balancing local order and long-ranged interactions in the molecular theory of liquid water.

A molecular theory of liquid water is identified and studied on the basis of computer simulation of the TIP3P model of liquid water. This theory would be exact for models of liquid water in which the intermolecular interactions vanish outside a finite spatial range, and therefore provides a precise analysis tool for investigating the effects of longer-ranged intermolecular interactions. We show how local order can be introduced through quasichemical theory. Long-ranged interactions are characterized generally by a conditional distribution of binding energies, and this formulation is interpreted as a regularization of the primitive statistical thermodynamic problem. These binding-energy distributions for liquid water are observed to be unimodal. The Gaussian approximation proposed is remarkably successful in predicting the Gibbs free energy and the molar entropy of liquid water, as judged by comparison with numerically exact results. The remaining discrepancies are subtle quantitative problems that do have significant consequences for the thermodynamic properties that distinguish water from many other liquids. The basic subtlety of liquid water is found then in the competition of several effects which must be quantitatively balanced for realistic results.

Non-van der Waals treatment of the hydrophobic solubilities of CF4.

A quasi-chemical theory implemented on the basis of molecular simulation is derived and tested for the hydrophobic hydration of CF4(aq). The theory formulated here subsumes a van der Waals treatment of solvation and identifies contributions to the hydration free energy of CF4(aq) that naturally arise from chemical contributions defined by quasi-chemical theory and fluctuation contributions analogous to Debye-Hückel or random phase approximations. The resulting Gaussian statistical thermodynamic model avoids consideration of hypothetical drying-then-rewetting problems and is physically reliable in these applications as judged by the size of the fluctuation contribution. The specific results here confirm that unfavorable tails of binding energy distributions reflect few-body close solute-solvent encounters. The solvent near-neighbors are pushed by the medium into unfavorable interactions with the solute, in contrast to the alternative view that a preformed interface is pulled by the solute-solvent attractive interactions into contact with the solute. The polyatomic model of CF4(aq) studied gives a satisfactory description of the experimental solubilities including the temperature dependence. The proximal distributions evaluated here for polyatomic solutes accurately reconstruct the observed distributions of water near these molecules which are nonspherical. These results suggest that drying is not an essential consideration for the hydrophobic solubilities of CF4, or of C(CH3)4 which is more soluble.

An analysis of molecular packing and chemical association in liquid water using quasichemical theory.

We calculate the hydration free energy of liquid TIP3P water at 298 K and 1 bar using a quasi-chemical theory framework in which interactions between a distinguished water molecule and the surrounding water molecules are partitioned into chemical associations with proximal (inner-shell) waters and classical electrostatic-dispersion interactions with the remaining (outer-shell) waters. The calculated free energy is found to be independent of this partitioning, as expected, and in excellent agreement with values derived from the literature. An analysis of the spatial distribution of inner-shell water molecules as a function of the inner-shell volume reveals that water molecules are preferentially excluded from the interior of large volumes as the occupancy number decreases. The driving force for water exclusion is formulated in terms of a free energy for rearranging inner-shell water molecules under the influence of the field exerted by outer-shell waters in order to accommodate one water molecule at the center. The results indicate a balance between chemical association and molecular packing in liquid water that becomes increasingly important as the inner-shell volume grows in size.

Light-scattering studies of protein solutions: role of hydration in weak protein-protein interactions.

We model the hydration contribution to short-range electrostatic/dispersion protein interactions embodied in the osmotic second virial coefficient, B(2), by adopting a quasi-chemical description in which water molecules associated with the protein are identified through explicit molecular dynamics simulations. These water molecules reduce the surface complementarity of highly favorable short-range interactions, and therefore can play an important role in mediating protein-protein interactions. Here we examine this quasi-chemical view of hydration by predicting the interaction part of B(2) and comparing our results with those derived from light-scattering measurements of B(2) for staphylococcal nuclease, lysozyme, and chymotrypsinogen at 25 degrees C as a function of solution pH and ionic strength. We find that short-range protein interactions are influenced by water molecules strongly associated with a relatively small fraction of the protein surface. However, the effect of these strongly associated water molecules on the surface complementarity of short-range protein interactions is significant, and must be taken into account for an accurate description of B(2). We also observe remarkably similar hydration behavior for these proteins despite substantial differences in their three-dimensional structures and spatial charge distributions, suggesting a general characterization of protein hydration.

A consistent experimental and modeling approach to light-scattering studies of protein-protein interactions in solution.

The osmotic second virial coefficient, B(2), obtained by light scattering from protein solutions has two principal components: the Donnan contribution and a contribution due to protein-protein interactions in the limit of infinite dilution. The Donnan contribution accounts for electroneutrality in a multicomponent solution of (poly)electrolytes. The importance of distinguishing this ideal contribution to B(2) is emphasized, thereby allowing us to model the interaction part of B(2) by molecular computations. The model for protein-protein interactions that we use here extends earlier work (Neal et al., 1998) by accounting for long-range electrostatic interactions and the specific hydration of the protein by strongly associated water molecules. Our model predictions are compared with measurements of B(2) for lysozyme at 25 degrees C over pH from 5.0 to 9.0, and 7-60 mM ionic strength. We find that B(2) is positive at all solution conditions and decreases with increasing ionic strength, as expected, whereas the interaction part of B(2) is negative at all conditions and becomes progressively less negative with increasing ionic strength. Although long-range electrostatic interactions dominate this contribution, particularly at low ionic strength, short-range electrostatic/dispersion interactions with specific hydration are essential for an accurate description of B(2) derived from experiment.

Effect of solute size and solute-water attractive interactions on hydration water structure around hydrophobic solutes.

Using Monte Carlo simulations, we investigated the influence of solute size and solute-water attractive interactions on hydration water structure around spherical clusters of 1, 13, 57, 135, and 305 hexagonally close-packed methanes and the single hard-sphere (HS) solute analogues of these clusters. We obtain quantitative results on the density of water molecules in contact with the HS solutes as a function of solute size for HS radii between 3.25 and 16.45 A. Analysis of these results based on scaled-particle theory yields a hydration free energy/surface area coefficient equal to 139 cal/(mol A2), independent of solute size, when this coefficient is defined with respect to the van der Waals surface of the solute. The same coefficient defined with respect to the solvent-accessible surface decreases with decreasing solute size for HS radii less than approximately 10 A. We also find that solute-water attractive interactions play an important role in the hydration of the methane clusters. Water densities in the first hydration shell of the three largest clusters are greater than bulk water density and are insensitive to the cluster size. In contrast, contact water densities for the HS analogues of these clusters decrease with solute size, falling below the bulk density of water for the two largest solutes. Thus, the large HS solutes dewet, while methane clusters of the same size do not.

Conformational equilibria of alkanes in aqueous solution: relationship to water structure near hydrophobic solutes.

Conformational free energies of butane, pentane, and hexane in water are calculated from molecular simulations with explicit waters and from a simple molecular theory in which the local hydration structure is estimated based on a proximity approximation. This proximity approximation uses only the two nearest carbon atoms on the alkane to predict the local water density at a given point in space. Conformational free energies of hydration are subsequently calculated using a free energy perturbation method. Quantitative agreement is found between the free energies obtained from simulations and theory. Moreover, free energy calculations using this proximity approximation are approximately four orders of magnitude faster than those based on explicit water simulations. Our results demonstrate the accuracy and utility of the proximity approximation for predicting water structure as the basis for a quantitative description of n-alkane conformational equilibria in water. In addition, the proximity approximation provides a molecular foundation for extending predictions of water structure and hydration thermodynamic properties of simple hydrophobic solutes to larger clusters or assemblies of hydrophobic solutes.

Hydration and conformational equilibria of simple hydrophobic and amphiphilic solutes.

We consider whether the continuum model of hydration optimized to reproduce vacuum-to-water transfer free energies simultaneously describes the hydration free energy contributions to conformational equilibria of the same solutes in water. To this end, transfer and conformational free energies of idealized hydrophobic and amphiphilic solutes in water are calculated from explicit water simulations and compared to continuum model predictions. As benchmark hydrophobic solutes, we examine the hydration of linear alkanes from methane through hexane. Amphiphilic solutes were created by adding a charge of +/-1e to a terminal methyl group of butane. We find that phenomenological continuum parameters fit to transfer free energies are significantly different from those fit to conformational free energies of our model solutes. This difference is attributed to continuum model parameters that depend on solute conformation in water, and leads to effective values for the free energy/surface area coefficient and Born radii that best describe conformational equilibrium. In light of these results, we believe that continuum models of hydration optimized to fit transfer free energies do not accurately capture the balance between hydrophobic and electrostatic contributions that determines the solute conformational state in aqueous solution.

The pressure dependence of hydrophobic interactions is consistent with the observed pressure denaturation of proteins.

Proteins can be denatured by pressures of a few hundred MPa. This finding apparently contradicts the most widely used model of protein stability, where the formation of a hydrophobic core drives protein folding. The pressure denaturation puzzle is resolved by focusing on the pressure-dependent transfer of water into the protein interior, in contrast to the transfer of nonpolar residues into water, the approach commonly taken in models of protein unfolding. Pressure denaturation of proteins can then be explained by the pressure destabilization of hydrophobic aggregates by using an information theory model of hydrophobic interactions. Pressure-denatured proteins, unlike heat-denatured proteins, retain a compact structure with water molecules penetrating their core. Activation volumes for hydrophobic contributions to protein folding and unfolding kinetics are positive. Clathrate hydrates are predicted to form by virtually the same mechanism that drives pressure denaturation of proteins.

The hydration of proteins in nearly anhydrous organic solvent suspensions.

Water sorption isotherms at 27 degrees C have been measured for lysozyme and chymotrypsin in suspensions of toluene, di(n-butyl) ether, n-propanol, and a solution of 1M n-propanol in benzene. Sorption isotherms for the different suspensions are compared by converting solvent water content to the thermodynamic activity of water in each solvent. The sorption behavior is also compared to that for the two proteins hydrated from the vapor phase. At low water activities, all sorption isotherms are similar when compared on the basis of water activity. However, at higher activities, water sorption by the proteins in the organic suspensions is suppressed relative to the sorption of water vapor. The greatest suppression is observed for n-propanol, which suggests that the suppression may be due to a competition for water-binding sites on the protein by the organic solvent. Sorption isotherms at low water activities have also been predicted using a thermodynamic model in which it is assumed that water binds selectively to the ionizable residues on the surface of the protein. A comparison of predicted and measured sorption isotherms shows that the model can provide reasonable estimates of water sorption in nonpolar or moderately polar organic solvent suspensions at low levels of hydration.