Amyloid oligomer formation probed by water proton magnetic resonance spectroscopy.

Formation of amyloid oligomers, the most toxic species of amyloids in degenerative diseases, is critically coupled to the interplay with surrounding water. The hydrophobic force driving the oligomerization causes water removal from interfaces, changing the surface-hydration properties. Here, we show that such effects alter the magnetic relaxation response of local water in ways that may enable oligomer detection. By using water proton magnetic resonance spectroscopy, we measured significantly longer transverse magnetic relaxation (T(2)) times in mixtures of serum and amyloidogenic Aß(1-42) peptides versus similar concentration solutions of serum and nonamyloidogenic scrambled Aß(42-1) peptides. Immunochemistry with oligomer-specific antibodies, electron microscopy and computer simulations demonstrated that the hyperintense magnetic signal correlates with Aß(1-42) oligomerization. Finding early biophysical markers of the oligomerization process is crucial for guiding the development of new noninvasive imaging techniques, enabling timely diagnosis of amyloid-related diseases and pharmacological intervention.

Endoplasmic reticulum overcrowding as a mechanism of beta-cell dysfunction in diabetes.

This study suggests a molecular mechanism that explains the accumulation of denaturated proinsulin in the endoplasmic reticulum (ER) of beta-cells. Such states were frequently observed in beta-cells experiencing increased demand for insulin production and were shown to lead to secretory dysfunction and diabetes. Here, a self-consistent kinetic model is used to investigate changes in protein translation due to ER overloading. The model is based on a molecular theory that relates the molecular composition and level of molecular crowding in the ER to the kinetic rates of protein folding/misfolding and transit to the Golgi apparatus (GA). This study suggests that molecular crowding forces can increase protein misfolding and impair the transport to the GA, thus overwhelming the quality control mechanism in the ER. A continual accumulation of toxic residues in the ER enhances even further the molecular crowding, accelerating protein denaturation. This article shows that molecular crowding affects differently the transit of various proteins through the ER. Apparently, the molecular crowding level that can inhibit the transport of native proinsulin to the GA influences to a lesser extent the transit of proamylin, a much smaller peptide cosynthesized with proinsulin in the ER. Smaller-volume misfolded proinsulin species may also win the passage competition through the ER and move on the secretory track. However, misfolded proinsulin fails the conversion to active insulin. This study can help us to decipher circumstances leading to the alteration of the secretory function in susceptible beta-cells and the onset of diabetes.

Cholic acid micelles--controlling the size of the aqueous cavity by PEGylation.

Data show that cholic acid (CA) micelles are less densely packed and much smaller than micelles formed by typical surfactants, suggesting that CA derivatives can be used to synthesize drug nanocarriers. Presumably, the formation of internal cavities is favored by the facial characteristics of the CA molecule, i.e. the convex molecular structure that is hydrophobic on one side and hydrophilic on the other. Here, we present a thermodynamical approach to quantify the effect of facial characteristics on forces governing the self-assembling process of CA molecules. We show that facial characteristics favor the entrapment of water molecules at interfaces upon CA aggregation, which weakens the attraction between CA hydrophobic moieties. Our computer simulations suggest that these effects contribute significantly to the tendency of CA molecules to form small "hollow-core" micelles. The attachment of polyethylene glycol (PEG) molecular chains to CA increases the repulsive forces in the system, reducing even further the micelle size. We use the present molecular model and experimental critical micelle concentration (cmc) data for CA-PEG systems to predict the change of the micelle size and cavity volume with the increase of the PEG chain length (x). Our computations indicate that the CA-PEG micelles are good candidates for drug delivery. The structural stability of CA-PEG micelles was further assessed by molecular dynamics simulations. We also tested the drug loading efficiency of this system and found an average of 0.5 mg paclitaxel load per 20 mg of CA-PEG polymer. The present study helps to identify critical parameters that control structural properties of the CA based nanocarriers and suggests practical means to optimize the ratio between micelle size and volume of the internal cavity.

Dilation of the endoplasmic reticulum in beta cells due to molecular overcrowding? Kinetic simulations of extension limits and consequences on proinsulin synthesis.

Insulin regulates the energy homeostasis of the human body. This is synthesized in the endoplasmic reticulum (ER) of pancreatic beta cells from proinsulin. Chronic hyperglycemia increases considerably the proinsulin secretion, overcrowding the ER. Recent experimental evidence demonstrates that such states favor the proinsulin denaturation. The biophysical mechanism of this cellular dysfunction remains largely unknown. We use basic molecular principles and numerical simulations of time-dependent crowding conditions in the ER to show that crowding effects enhance the propensity of proinsulin molecules to (mis)fold in compressed, nonnative structures. Present results suggest: i) misfolding events and toxic accumulations increase dramatically if the proinsulin load exceeds 50% of the available space and ii) insufficient lag time for the relaxation of the ER between consecutive proinsulin uploads can cause irreversible alterations of folding capabilities. Present study may prove useful in generating new testable statements on circumstances leading to the development of diabetes.

Hydrophobe-water interactions: methane as a model.

Recent molecular-dynamics simulations have demonstrated that the use of an empirical hydrophobic potential displaying two minima, i.e., one for hydrophobes in close contact and one for hydrophobes separated by a hydration layer, leads to a marked improvement in protein structure prediction. This potential is supported by experimental data and simulations, but its physical origin and mathematical formulation have not been derived as yet. Here we show that water-mediated attraction (the "wetting regime") between two hydrophobic molecules originates in the interaction between the dipoles induced at the surface of the hydrophobes by the surrounding structured water. As an example, we derive the effective hydrophobic potential that describes the interaction between two methane molecules, a classical model of a double-well energy function. We found an excellent agreement with published results from all-atom, explicit solvent molecular-dynamics simulations of this interaction. The approach presented here provides the theoretical basis for implementing an adequate representation of the wetting regime of the hydrophobic interactions in force fields used for structure prediction. The results are useful for modeling both protein folding and binding.

The relative thermal stability of tissue macromolecules and cellular structure in burn injury.

When tissue is subjected to higher than physiological temperatures, protein and cell organelle structures can be altered resulting in cell death and subsequent tissue necrosis. A burn injury can be stratified into three main zones, coagulation, stasis and edema, which correlate with the extent of heat exposure and thermal properties of the tissue. While there has been considerable effort to characterize the time-temperature dependence of the injury, relatively little attention has been paid to the other important variable, the thermal susceptibility of the tissue. In the present study, we employ a standard physical chemistry approach to predict the level of denaturation at supraphysiological temperatures of 12 vital proteins as well as RNA, DNA and cell membrane components. Melting temperatures and unfolding enthalpies of the cellular components are used as input experimental parameters. This approach allows us to establish a relation between the level of denaturation of critical cellular components and clinical manifestations of the burn through the characteristic zones of the injury. Specifically, we evaluate the degree of molecular alteration for characteristic temperature profiles at two different depths (Mid-Dermis and Dermis-Fat interface) of 80 degrees C; 20s contact burn. The results of this investigation suggest that the thermal alteration of the plasma membrane is likely the most significant cause of the tissue necrosis. The lipid bilayer and membrane-bound ATPases show a high probability of thermal damage (almost 100% for the former and 85% for the latter) for short heat exposure times. These results suggest that strategies to minimize the damage in a burn injury might focus on the stabilization of the cellular membrane and membrane-bound ATPases. Further work will be required to validate these predictions in an in vivo model.

Stability of cellular proteins under supraphysiological temperatures.

We present quantitative analyses of the kinetics of cellular components confronted with the destabilizing effect of irreversible thermal denaturation. We examine the dependence of the thermal denaturation on the heating rate, relative stability, population and lifetime of the states involved in transition and crowding effects. We propose a mechanism for self-stabilization of proteins during unfolding in tightly packed fibers and membranes. Speaking in terms of vulnerability to thermal denaturation, our results suggest that the thermal alteration of the plasma membrane is likely to be the most significant cause of the tissue necrosis.

Magnetic resonance imaging of muscle electroporation injury.

Low frequency electrical currents traversing the body during electrical shock can produce tissue damage by effects of electrical forces on cellular organelles and proteins as well as by Joule heating beyond thermotolerance. Treatment for these different injuries are quite distinct. Therefore, it is important to accurately diagnose the form of injury. Here we discuss the use of MRI for this purpose.

On the doublet formation in the flocculation process of the yeast cells.

The combination of single cells to form doublets is regarded as the rate-limiting step of flocculation and requires the presence of surface proteins in active form. The process of activation of the flocculation proteins of yeast cells is described in the frame of the autocrine interaction regime (Science 224 (1984) 1312). The influence of several effectors (the cell efficiency to use sugars, the calcium content in the external medium and the probability that free cells collide each other under thermal motion conditions) on the initial rate of flocculation and on the fraction of remaining free cells in the steady state is briefly discussed in the paper. The present model offers a useful tool for further quantitative investigations in this topic. Also, it indicates qualitatively a way in which the regulation of flocculation might be controlled at the level of the expression of cell-surface activation abilities.

Diffusion model for growth factors--cell receptors interaction.

A theoretical model describing the interaction between growth factor molecules and their receptors from the cell surface within an 'off-centre' diffusion approximation is developed in this paper. It is assumed that a number of non interacting particles (growth-factor molecules) more diffusively in the presence of traps (specific receptors) which are located at the surfaces of a number of cells uniformly distributed in space. The diffusion equations system is solved by a perturbative method. The model predicts a nonlinear dependence of the receptor occupation and of the time needed to reach a threshold value of receptor occupation on the total number of cell receptors. An autocrine binding, when the ligand is secreted by the responsive cells accounts for the interaction between IL-2 and helper T-cells.