Amyloid formation by islet amyloid polypeptide (IAPP) contributes to β-cell dysfunction in type-2 diabetes. Perturbation of the β-cell membrane may contribute to IAPP induced toxicity. We examine the effects of lipid composition, salt and buffer on IAPP amyloid formation and on the ability of IAPP to induce leakage of model membranes. Even low levels of anionic lipids promote amyloid formation and membrane permeabilization. Increasing the percentage of the anionic lipids, POPS or DOPG, enhances the rate of amyloid formation and increases membrane permeabilization. The choice of zwitterionic lipid has no noticeable effect on membrane catalyzed amyloid formation, but in most cases affects leakage, which tends to decrease in the order DOPC>POPC>sphingomyelin. Uncharged lipids that increase membrane order reduce the ability of IAPP to induce leakage. Leakage is due predominately to pore formation rather than complete disruption of the vesicles under the conditions of these studies. Cholesterol at or below physiological levels significantly reduces the rate of vesicle catalyzed IAPP amyloid formation and decreases susceptibility to IAPP induced leakage. The effects of cholesterol on amyloid formation are masked by 25 mole percent POPS. Overall, there is a strong inverse correlation between the time to form amyloid and the extent of vesicle leakage. NaCl reduces the rate of membrane catalyzed amyloid formation by anionic vesicles, but accelerates amyloid formation in solution. The implications for IAPP membrane interactions are discussed, as is the possibility that loss of phosphatidylserine asymmetry enhances IAPP amyloid formation and membrane damage in vivo via a positive feedback loop.

Our understanding of membranes and membrane lipid function has lagged far behind that of nucleic acids and proteins, largely because it is difficult to manipulate cellular membrane lipid composition. To help solve this problem, we show that methyl-α-cyclodextrin (MαCD)-catalyzed lipid exchange can be used to maximally replace the sphingolipids and phospholipids in the outer leaflet of the plasma membrane of living mammalian cells with exogenous lipids, including unnatural lipids. In addition, lipid exchange experiments revealed that 70-80% of cell sphingomyelin resided in the plasma membrane outer leaflet; the asymmetry of metabolically active cells was similar to that previously defined for erythrocytes, as judged by outer leaflet lipid composition; and plasma membrane outer leaflet phosphatidylcholine had a significantly lower level of unsaturation than phosphatidylcholine in the remainder of the cell. The data also provided a rough estimate for the total cellular lipids residing in the plasma membrane (about half). In addition to such lipidomics applications, the exchange method should have wide potential for investigations of lipid function and modification of cellular behavior by modification of lipids.lipid exchange | plasma membrane outer leaflet | lipid asymmetry | methyl-alpha-cyclodextrin | mass spectrometry

The metabolism of host cholesterol by Mycobacterium tuberculosis (Mtb) is an important factor for both its virulence and pathogenesis, although how and why cholesterol metabolism is required is not fully understood. Mtb uses a unique set of catabolic enzymes that are homologous to those required for classical β-oxidation of fatty acids but are specific for steroid-derived substrates. Here, we identify and assign the substrate specificities of two of these enzymes, ChsE4-ChsE5 (Rv3504-Rv3505) and ChsE3 (Rv3573c), that carry out cholesterol side chain oxidation in Mtb. Steady-state assays demonstrate that ChsE4-ChsE5 preferentially catalyzes the oxidation of 3-oxo-cholest-4-en-26-oyl CoA in the first cycle of cholesterol side chain β-oxidation that ultimately yields propionyl-CoA, whereas ChsE3 specifically catalyzes the oxidation of 3-oxo-chol-4-en-24-oyl CoA in the second cycle of β-oxidation that generates acetyl-CoA. However, ChsE4-ChsE5 can catalyze the oxidation of 3-oxo-chol-4-en-24-oyl CoA as well as 3-oxo-4-pregnene-20-carboxyl-CoA. The functional redundancy of ChsE4-ChsE5 explains the in vivo phenotype of the igr knockout strain of Mycobacterium tuberculosis; the loss of ChsE1-ChsE2 can be compensated for by ChsE4-ChsE5 during the chronic phase of infection. The X-ray crystallographic structure of ChsE4-ChsE5 was determined to a resolution of 2.0 Å and represents the first high-resolution structure of a heterotetrameric acyl-CoA dehydrogenase (ACAD). Unlike typical homotetrameric ACADs that bind four flavin adenine dinucleotide (FAD) cofactors, ChsE4-ChsE5 binds one FAD at each dimer interface, resulting in only two substrate-binding sites rather than the classical four active sites. A comparison of the ChsE4-ChsE5 substrate-binding site to those of known mammalian ACADs reveals an enlarged binding cavity that accommodates steroid substrates and highlights novel prospects for designing inhibitors against the committed β-oxidation step in the first cycle of cholesterol side chain degradation by Mtb.

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cross-sectional area for the solution they're exposed to. Using VMD, both systems were split in half at the leaflet-leaflet interface and restacked such that there are two membranes, each with differing number of lipids in their two monolayers. Constant pressure and temperature (NPT) simulations for 0.8 ms with this system show 1) stable area/lipid, 2) no lipid flip flop, and 3) tens to hundreds of water translocations depending on temperature. With this innovative strategy for simulating osmotic-induced water movement, we hope to predict the effects of an osmotic gradient on alcohol partitioning in membranes. This will help us understand how alcohol affects membrane fusion as measured experimentally in our lab.

Molecular dynamics (MD) simulations have been extensively used to study lipid membranes in addition to experimental studies as they help better understand membranes in the atomic level. Computational models of bacterial (E. coli) membranes have been developed and applied to study the antimicrobial peptide proteins. Plant membranes are less frequently studied compared to the bacterial membrane. In this work, we will present the soybean plasma membranes models. The compositions of cell plasma membranes of soybean vary depending on the species, stage of development, and the part of the plant. The two parts of the plant that we study are the hypocotyl and the root. Each model consists of 100 lipids per leaflet, with the composition based on the weighted and averaged values from past experimental studies. Specifically, the hypocotyl membrane contains 7 types of unsaturated phospholipids and two types of sterols, while the root membrane contains 8 types of phospholipids and two types of sterols. All types of phospholipids in soybean contains the 18:2 (cis D9, 12) linoleoyl tail which was not well studied before, therefore, the simulations on the pure 18:0/18:2 and 18:2/18:2 phosphocholine (PC) lipid bilayers are also performed. The structural properties such as surface area per lipid, bilayer thicknesses, order parameters, and spin-lattice relaxation time are analyzed for all membranes. Moreover, the analyses of the sterols tilt angle distributions, hydrogen bonding, and clustering are also conducted for the soybean membranes. The structural properties of pure bilayers agree well with NMR experimental data validate the accuracy of 18:2 linoleoyl-containing lipids, based on which the soybean membrane models also result in reasonable structural properties. These results imply that the two soybean membrane models are realistic, and can facilitate the further study of soybean and other plant membranes.