Cholesterol inhibits the insertion of the Alzheimer’s peptide Aβ(25–35) in lipid bilayers

The physiological relationship between brain cholesterol content and the action of amyloid β (Aβ) peptide in Alzheimer’s disease (AD) is a highly controversially discussed topic. Evidences for modulations of the Aβ/membrane interaction induced by plasma membrane cholesterol have already been observed. We have recently reported that Aβ(25–35) is capable of inserting in lipid membranes and perturbing their structure. Applying neutron diffraction and selective deuteration, we now demonstrate that cholesterol alters, at the molecular level, the capability of Aβ(25–35) to penetrate into the lipid bilayers; in particular, a molar weight content of 20% of cholesterol hinders the intercalation of monomeric Aβ(25–35) completely. At very low cholesterol content (about 1% molar weight) the location of the C-terminal part of Aβ(25–35) has been unequivocally established in the hydrocarbon region of the membrane, in agreement with our previous results on pure phospholipids membrane. These results link a structural property to a physiological and functional behavior and point to a therapeutical approach to prevent the AD by modulation of membrane properties.     

Reflections on VDAC as a voltage-gated channel and a mitochondrial regulator

There is excellent agreement between the electrophysiological properties and the structure of the mitochondrial outer membrane protein, VDAC, ex vivo. However, the inference that the well-defined canonical “open” state of the VDAC pore is the normal physiological state of the channel in vivo is being challenged by several lines of evidence. Knowing the atomic structure of the detergent solubilized protein, a long sought after goal, will not be sufficient to understand the functioning of this channel protein. In addition, detailed information about VDAC’s topology in the outer membrane of intact mitochondria, and the structural changes that it undergoes in response to different stimuli in the cell will be needed to define its physiological functions and regulation.     

Coiled-coil binding of the leucine zipper domains of APOL1 is necessary for the open cation channel conformation

Apolipoprotein L-I (APOL1) is a channel-forming effector of innate immunity. The common human APOL1 variant G0 provides protection against infection with certain Trypanosoma and Leishmania parasite species, but it cannot protect against the trypanosomes responsible for human African trypanosomiasis. Human APOL1 variants G1 and G2 protect against human-infective trypanosomes but also confer a higher risk of developing chronic kidney disease. Trypanosome-killing activity is dependent on the ability of APOL1 to insert into membranes at acidic pH and form pH-gated cation channels. We previously mapped the channel’s pore-lining region to the C-terminal domain (residues 332–398) and identified a membrane-insertion domain (MID, residues 177–228) that facilitates acidic pH-dependent membrane insertion. In this article, we further investigate structural determinants of cation channel formation by APOL1. Using a combination of site-directed mutagenesis and targeted chemical modification, our data indicate that the C-terminal heptad-repeat sequence (residues 368–395) is a bona fide leucine zipper domain (ZIP) that is required for cation channel formation as well as lysis of trypanosomes and mammalian cells. Using protein-wide cysteine-scanning mutagenesis, coupled with the substituted cysteine accessibility method, we determined that, in the open channel state, both the N-terminal domain and the C-terminal ZIP domain are exposed on the intralumenal/extracellular side of the membrane and provide evidence that each APOL1 monomer contributes four transmembrane domains to the open cation channel conformation. Based on these data, we propose an oligomeric topology model in which the open APOL1 cation channel is assembled from the coiled-coil association of C-terminal ZIP domains.     

Asymmetric synthesis of β-amino alcohol by reductive cross-coupling of planar chiral ferrocenecarboxaldehyde with N-tosyl ferrocenylideneamine, and its application to asymmetric reaction

Samarium iodine-mediated cross-coupling of N-tosyl ferrocenylideneamine with planar chiral ferrocenecarboxaldehyde gave diastereoselectively anti-β-amino alcohol derivative in good yield. The obtained anti-β-amino alcohol with ferrocene ring at 1,2-positions was utilized as chiral auxiliary for asymmetric alkylation and acylation reactions.     

The number of subunits comprising the channel formed by the T domain of diphtheria toxin.

In the presence of a low pH environment, the channel-forming T domain of diphtheria toxin undergoes a conformational change that allows for both its own insertion into planar lipid bilayers and the translocation of the toxin's catalytic domain across them. Given that the T domain contributes only three transmembrane segments, and the channel is permeable to ions as large as glucosamine(+) and NAD(-), it would appear that the channel must be a multimer. Yet, there is substantial circumstantial evidence that the channel may be formed from a single subunit. To test the hypothesis that the channel formed by the T domain of diphtheria toxin is monomeric, we made mixtures of two T domain constructs whose voltage-gating characteristics differ, and then observed the gating behavior of the mixture's single channels in planar lipid bilayers. One of these constructs contained an NH(2)-terminal hexahistidine (H6) tag that blocks the channel at negative voltages; the other contained a COOH-terminal H6 tag that blocks the channel at positive voltages. If the channel is constructed from multiple T domain subunits, one expects to see a population of single channels from this mixture that are blocked at both positive and negative voltages. The observed single channels were blocked at either negative or positive voltages, but never both. Therefore, we conclude that the T domain channel is monomeric.     

Orientational constraints as three-dimensional structural constraints from chemical shift anisotropy: the polypeptide backbone of gramicidin A in a lipid bilayer.

Chemical shifts observed from samples that are uniformly aligned with respect to the magnetic field can be used as very high-resolution structural constraints. This constraint takes the form of an orientational constraint rather than the more familiar distance constraint. The accuracy of these constraints is dependent upon the quality of the tensor characterization. Both tensor element magnitudes and tensor orientations with respect to the molecular frame need to be considered. Here these constraints have been used to evaluate models for the channel conformation of gramicidin A. Of the three models used, the one experimentally derived model of gramicidin in sodium dodecyl sulfate micelles fits the data least well.     

Neutron Reflectometry and Molecular Simulations Demonstrate HIV-1 Nef Homodimer Formation on Model Lipid Bilayers

The HIV-1 Nef protein plays a critical role in viral infectivity, high-titer replication in vivo, and immune escape of HIV-infected cells. Nef lacks intrinsic biochemical activity, functioning instead through interactions with diverse host cell signaling proteins and intracellular trafficking pathways. Previous studies have established an essential role for Nef homodimer formation at the plasma membrane for most if not all its functions. Here we combined neutron reflectometry of full-length myristoylated Nef bound to model lipid bilayers with molecular simulations based on previous X-ray crystal structures of Nef homodimers. This integrated approach provides direct evidence that Nef associates with the membrane as a homodimer with its structured core region displaced from the membrane for partner protein engagement. Parallel studies of a dimerization-defective mutant, Nef-L112D, demonstrate that the helical dimerization interface present in previous crystal structures stabilizes the membrane-bound dimer. X-ray crystallography of the Nef-L112D mutant in complex with the SH3 domain of the Nef-associated host cell kinase Hck revealed a monomeric 1:1 complex instead of the 2:2 dimer complex formed with wild-type Nef. Importantly, the crystal structure of the Nef-L112D core and SH3 interface are virtually identical to the wild-type complex, indicating that this mutation does not affect the overall Nef fold. These findings support the intrinsic capacity of Nef to homodimerize at lipid bilayers using structural features present in X-ray crystal structures of dimeric complexes.     

Effect of carbohydrates and glycerol on the stability and surface properties of lyophilized liposomes

Summary The interaction of permeant molecules such as glycerol and urea and nonpermeants such as trehalose, sucrose, lactose and glucose with dipalmitoylphosphatidylcholine (DPPC) and egg yolk phosphatidylcholine (EPC) bilayers was studied by means of infrared spectroscopy in solid samples. The properties of the liposomes formed upon rehydration in different polyol solutions were determined by dynamic light scattering, fluorescence anisotropy, absorbance at 450 nm and merocyanine 540 spectra.Phospholipid samples dehydrated in the presence of urea and glycerol give v _1/2 values for the antisymmetric stretch (P=0 stretch) in the IR spectra; lower values are found for hydrated phospholipids.In contrast, the same procedure in the presence of carbohydrates, gives v _1/2 values close or higher to those found for hydrated phospholipids, following the sequence glucose>sucrose>trehalose. This order is similar to that found in hydrated bilayers for the 570/500 nm ratio determined in the MC 540 spectra as a function of the number of OH equatorial groups of the sugars.Liposomes lyophilized in the presence of those carbohydrates and rehydrated in buffer solution showed an increase in the 570/530 absorbance ratio in the MC spectra at temperatures below that corresponding to the gel-liquid crystalline transition. This is interpreted as an exposure of hydrophobic regions due to the carbohydrate-phospholipid interaction. In these conditions, the size at which liposomes spontaneously stabilize is a function of the type and concentration of the polyols in the aqueous solution. These changes in size are connected with packing and mechanical constraints of the bilayer for some of the sugars assayed.Similar results to those obtained with lyophilized liposomes were found after aging liposomes in high sugar concentration solutions. A clear distinction can be made between the effect of permeant and nonpermeant molecules in regard to size, packing and hydrophobic region exposure.     

Mechanisms of attack and defence at the cell surface: The use of phospholipid bilayers as models for cell membrane

Electrical conductivity across phospholipid bilayers induced by various cytotoxic proteins has been used to analyse the damaging action of such proteins on cells; the protective effect of divalent cations and protons against such attack has also been investigated. The predominant effect of divalent cations and protons is to promote the closed state of membrane pores, i.e. to gate protein-induced lesions.     

Effects of variation of ion and methylation of carrier on the rate constants of macrotetralide-mediated ion transport in lipid bilayers

Summary The effects of methylation on the rate constants of carrier-mediated ion transport have been studied on monooleindecane bilayers with K⁺, Rb⁺, NH ₄ ⁺ , and TI⁺ ions, using the series of homologue carriers, nonactin, monactin, dinactin, trinactin, and tetranactin, each member of the series differing from the previous one by only one methyl group. Measurements of the amplitude and time constant of the current relaxation after a voltage jump over a large domain of voltage and permeant ion concentration, together with a computer curve-fitting procedure, have allowed us, without the help of steady-state current-voltage data, to deduce and compare the values of the various rate constants for ion transport: formation (k _Ri) and dissociation (k _Di) of the ion-carrier complex at the interface, translocation across the membrane interior of the carrier (k _s) and the complex (k _is). With the additional information from steady-state low-voltage conductance measurements, we have obtained the value of the aqueous phase-membrane and torus-membrane partition coefficient of the carrier ({ie191-1} and {ie191-2}). From nonactin to tetranactin with the NH ₄ ⁺ ion,k _is, and {ie191-3} are found to increase by factors of 5 and 3, respectively,k _Di and {ie191-4} to decrease respectively by factors 8 and 2, whilek _Ri andk _s are practically invariant. Nearly identical results are found for K⁺, Rb⁺, and Tl⁺ ions.k _Ri,k _s andk _is are quite invariant from one ion to the other except for Tl⁺ wherek _Ri is about five times larger. On the other hand,k _Di depends strongly on the ion, indicating that dissociation is the determining step of the ionic selectivity of a given carrier. The systematic variations in the values of the rate constants with increasing methylation are interpreted in terms of modifications of energy barriers induced by the carrier increasing size. Within this framework, we have been able to establish and verify a fundamental relationship between the variations ofk _is andk _Di with methylation.