Targeted removal of the FA2 site on human albumin prevents fatty acid-mediated inhibition of Zn2+ binding.

Zinc is required for virtually all biological processes. In plasma, Zn2+ is predominantly transported by human serum albumin (HSA), which possesses two Zn2+-binding sites of differing affinities (sites A and B). Fatty acids (FAs) are also transported by HSA, with seven structurally characterized FA-binding sites (named FA1-FA7) known. FA binding inhibits Zn2+-HSA interactions, in a manner that can impact upon hemostasis and cellular zinc uptake, but the degree to which binding at specific FA sites contributes to this inhibition is unclear. Wild-type HSA and H9A, H67A, H247A, and Y150F/R257A/S287A (FA2-KO) mutant albumins were expressed in Pichia pastoris. Isothermal titration calorimetry studies revealed that the Zn2+-binding capacity at the high-affinity Zn2+ site (site A) was reduced in H67A and H247A mutants, with site B less affected. The H9A mutation decreased Zn2+ binding at the lower-affinity site, establishing His9 as a site B ligand. Zn2+ binding to HSA and H9A was compromised by palmitate, consistent with FA binding affecting site A. 13C-NMR experiments confirmed that the FA2-KO mutations prohibited FA binding at site FA2. Zn2+ binding to the FA2-KO mutant was unaffected by myristate, suggesting binding at FA2 is solely responsible for inhibition. Molecular dynamics studies identified the steric obstruction exerted by bound FA in site FA2, which impedes the conformational change from open (FA-loaded) to closed (FA-free) states, required for Zn2+ to bind at site A. The successful targeting of the FA2 site will aid functional studies exploring the interplay between circulating FA levels and plasma Zn2+ speciation in health and disease.

Stacks of monogalactolipid bilayers can transform into a lattice of water channels.

The lipid matrix of thylakoid membranes is a lamellar bilayer, but under a certain condition it can convert locally into a nonlamellar structure. This is possible because one of the main membrane lipids, MGDG, promotes the formation of an inverse hexagonal phase. Here, the spontaneous transformation of aligned hydrated MGDG bilayers into nonlamellar structures is investigated using all-atom molecular dynamics simulation. Previous studies have demonstrated that MGDG polar head groups connect vertically across the interface. In this study, the evolution of the system's initial structure into a lattice of water channels and contacted surfaces created by numerous vertical MGDG connections depended on the width of the hydrating water layers. These widths controlled the bilayers' ability to bend, which was a prerequisite for channel formation. Locally, an intensive exchange of MGDG molecules between apposing bilayer leaflets occurred, although a stable semi-toroidal stalk did not develop.

Structural Impact of Selected Retinoids on Model Photoreceptor Membranes.

Photoreceptor membranes have a unique lipid composition. They contain a high level of polyunsaturated fatty acids including the most unsaturated fatty acid in nature, docosahexaenoic acid (22:6), and are enriched in phosphatidylethanolamines. The phospholipid composition and cholesterol content of the subcellular components of photoreceptor outer segments enables to divide photoreceptor membranes into three types: plasma membranes, young disc membranes, and old disc membranes. A high degree of lipid unsaturation, extended exposure to intensive irradiation, and high respiratory demands make these membranes sensitive to oxidative stress and lipid peroxidation. Moreover, all-trans retinal (AtRAL), which is a photoreactive product of visual pigment bleaching, accumulates transiently inside these membranes, where its concentration may reach a phototoxic level. An elevated concentration of AtRAL leads to accelerated formation and accumulation of bisretinoid condensation products such as A2E or AtRAL dimers. However, a possible structural impact of these retinoids on the photoreceptor-membrane properties has not yet been studied. In this work we focused just on this aspect. The changes induced by retinoids, although noticeable, seem not to be significant enough to be physiologically relevant. This is, however, an positive conclusion because it can be assumed that accumulation of AtRAL in photoreceptor membranes will not affect the transduction of visual signals and will not disturb the interaction of proteins engaged in this process.

Vemurafenib and Dabrafenib Downregulates RIPK4 Level.

Vemurafenib and dabrafenib are BRAF kinase inhibitors (BRAFi) used for the treatment of patients with melanoma carrying the V600E BRAF mutation. However, melanoma cells develop resistance to both drugs when used as monotherapy. Therefore, mechanisms of drug resistance are investigated, and new molecular targets are sought that could completely inhibit melanoma progression. Since receptor-interacting protein kinase (RIPK4) probably functions as an oncogene in melanoma and its structure is similar to the BRAF protein, we analyzed the impact of vemurafenib and dabrafenib on RIPK4 in melanomas. The in silico study confirmed the high similarity of BRAF kinase domains to the RIPK4 protein at both the sequence and structural levels and suggests that BRAFi could directly bind to RIPK4 even more strongly than to ATP. Furthermore, BRAFi inhibited ERK1/2 activity and lowered RIPK4 protein levels in BRAF-mutated melanoma cells (A375 and WM266.4), while in wild-type BRAF cells (BLM and LoVo), both inhibitors decreased the level of RIPK4 and enhanced ERK1/2 activity. The phosphorylation of phosphatidylethanolamine binding protein 1 (PEBP1)-a suppressor of the BRAF/MEK/ERK pathway-via RIPK4 observed in pancreatic cancer did not occur in melanoma. Neither downregulation nor upregulation of RIPK4 in BRAF- mutated cells affected PEBP1 levels or the BRAF/MEK/ERK pathway. The downregulation of RIPK4 inhibited cell proliferation and the FAK/AKT pathway, and increased BRAFi efficiency in WM266.4 cells. However, the silencing of RIPK4 did not induce apoptosis or necroptosis. Our study suggests that RIPK4 may be an off-target for BRAF inhibitors.

Lipid/water interface of galactolipid bilayers in different lyotropic liquid-crystalline phases.

In this study, carried out using computational methods, the organisation of the lipid/water interface of bilayers composed of galactolipids with both α-linolenoyl acyl chains is analysed and compared in three different lyotropic liquid-crystalline phases. These systems include the monogalactosyldiglyceride (MGDG) and digalactosyldiglyceride (DGDG) bilayers in the lamellar phase, the MGDG double bilayer during stalk phase formation and the inverse hexagonal MGDG phase. For each system, lipid-water and direct and water-mediated lipid-lipid interactions between the lipids of one bilayer leaflet and those of two apposing leaflets at the onset of new phase (stalk) formation, are identified. A network of interactions between DGDG molecules and its topological properties are derived and compared to those for the MGDG bilayer.

The importance of atomic partial charges in the reproduction of intermolecular interactions for the triacetin - a model of glycerol backbone.

Lipids play a central role within the cell. They not only encompass it but are also engaged in many processes such as cellular transport and energy production. Despite ongoing advances in experimental studies, computer simulations are a viable method to trace their behavior at the atomic level and on an elusive time scale. In molecular modeling studies, the quality of the obtained results is associated with the considered force field and its parameters. In the present work, the authors have investigated the procedure of partial charges fitting on the example of a triacetin molecule, containing chemical moieties present in the glycerol backbone. The goal of the study was to validate assigned partial charges based on the quality of the torsion profiles using optimally assigned torsional coefficients and reproduction of the condensed phase properties of triacetin. We applied various approaches and noticed a significant improvement in the parameterization of triacetin compared to the original one. The results showed that it is important to take into account the intermolecular interactions in the partial charges fitting procedure to obtain good quality validation results.

Extended Hierarchical Fuzzy Interpreted Petri Net.

Petri nets (PNs) have many advantages such as graphical representation, formal description, and the possibility of sequential and concurrent control. An important aspect of using PNs is hierarchical modeling, which may be provided in different ways. In this paper, a new concept and definition of the hierarchical structure for Fuzzy Interpreted Petri Net (FIPN) are proposed. The concept of macroplace with several input, output, and input-output places is introduced to the net. The functionality of the macroplace instances and the hierarchy graph are also proposed. They are implemented in a computer simulator called HFIPN-SML. In this study, FIPN is employed since it allows the use of analogue sensors directly for process control. Better visualization and more precise control are among advantages of the introduced approach.

Lutein and Zeaxanthin in the Lipid Bilayer-Similarities and Differences Revealed by Computational Studies.

Lutein and zeaxanthin are two similar carotenoids of the xanthophyll subgroup. Carotenoids are synthesized almost entirely by plants but are also present in significant amounts in animals. They are essential components of the lipid matrix of biomembranes, and one of their functions is to protect cells from light radiation, free radicals and oxidative stress. Carotenoids, depending on their chemical structure, can locate at various positions and in different orientations in the bilayer. Xanthophylls (XAN) are polar and in the bilayer are positionally restricted. In the case of lutein and zeaxanthin, whose both ionone rings are hydroxy-substituted and as such are anchored in the lipid bilayer interfaces, the position is generally transmembrane. However, both experimental and computer modelling studies indicate that lutein can also locate horizontally below the bilayer interface. This location has never been observed for zeaxanthin. To find a molecular-level explanation for the difference in the orientations of the XAN molecules in the bilayer, a number of phosphatidylcholine-XAN bilayers were constructed and molecular dynamics (MD) simulated for 1.1 µs each. The all-trans XAN molecules were initially placed either parallel or perpendicular to the bilayer surface. With the exception of one lutein, the horizontally placed molecules adopted the transmembrane orientation within 100-600 ns. On the basis of detailed analyses of the XAN orientations and the numbers and lifetimes of their interactions in the bilayer, a plausible explanation is offered as to why a lutein molecule may remain in the horizontal orientation while zeaxanthin does not. Contrary to common believe, lutein horizontal orientation is not related to the ε-ring rotation around the C6'-C7' bond.

Data for molecular dynamic simulations in the OPLSAA force field: Partial charges of cholesterol, C7-hydroxycholesterol and C7-hydroperoxycholesterol, torsional parameters for the hydroperoxy group of C7-hydroperoxycholesterol.

This data article contains partial charges generated for cholesterol, C7-hydroxycholesterol and C7-hydroperoxycholesterol and torsional parameters for hydroperoxy of C7-hydroperoxycholesterol for molecular dynamics simulations in the OPLSAA force field [1] using the package Gromacs [2]. The hydroperoxy group remained unparameterized in the OPLSAA force field and the parameters obtained have the potential for re-use in similar simulations. The atom-centred point charges on each sterol molecule were derived using the restrained electrostatic potential (RESP) approach [3]. The parameters for the C7-OET-OH-HO and C8-C7-OET-OH torsion angles were derived by fitting the parameters of the torsional term (Ryckaert-Bellemans function) of the OPLSAA potential energy function to the quantum mechanical rotational energy profile calculated at CCSD(T)/cc-pVQZ level of theory. This article presents data used in the research article "Chirality affects cholesterol-oxysterol association in water, a computational study" [4].

Chirality affects cholesterol-oxysterol association in water, a computational study.

Cholesterol (Chol) is the most prevalent sterol in the animal kingdom and an indispensable component of mammalian cell membranes. Chol content in the membrane is strictly controlled, although the oxidation of phospholipids may change the relative content of membrane Chol. An excess of it results in the formation of pure Chol microdomains in the membrane. It is likely that some Chol molecules detach from the domains and self-assemble in the aqueous environment. This may promote Chol microcrystallisation, which initiates the development of gallstones and atherosclerotic plaque. In this study, the molecular dynamics, free energy perturbation, umbrella sampling and Voronoi diagram methods are used to reveal the details of self-association of Chol and its oxidised forms (oxChol), namely 7α,ß-hydroxycholesterol and 7α,ß-hydroperoxycholesterol, in water. In the first part of the study the interactions between a sterol monomer and water over a short and longer timescale as well as the energy of hydration of each sterol are analysed. This helps one to understand Chol-Chol and Chol-OxChol with different chirality self-association in water better, which is analysed in the second part of the study. The Voronoi diagram approach is used to determine the relative arrangement of molecules in the dimer and, most importantly, to analyse the dehydration of the contacting surfaces of the assembling molecules. Free energy calculations indicate that Chol and 7ß-hydroxycholesterol associate into the most stable dimer and that Chol-Chol is the next most stable of the five dimers studied. Employing different computational methods enables us to obtain an adequate picture of Chol-sterol self-association in water, which includes dynamic, energetic and temporal aspects of the process.

Tauroursodeoxycholic Acid (TUDCA)-Lipid Interactions and Antioxidant Properties of TUDCA Studied in Model of Photoreceptor Membranes.

Tauroursodeoxycholic acid (TUDCA), a hydrophilic bile acid containing taurine conjugated with the ursodeoxycholic acid (UDCA), has been known and used from ancient times as a therapeutic compound in traditional Chinese medicine. TUDCA has recently been gaining significant interest as a neuroprotective agent, also exploited in the visual disorders. Among several mechanisms of TUDCA's protective action, its antioxidant activity and stabilizing effect on mitochondrial and plasma membranes are considered. In this work we investigated antioxidant activity of TUDCA and its impact on structural properties of model membranes of different composition using electron paramagnetic resonance spectroscopy and the spin labeling technique. Localization of TUDCA molecules in a pure POPC bilayer has been studied using a molecular dynamics simulation (MD). The obtained results indicate that TUDCA is not an efficient singlet oxygen (1O2 (1Δg)) quencher, and the determined rate constant of its interaction with 1O2 (1Δg) is only 1.9 × 105 M-1s-1. However, in lipid oxidation process induced by a Fenton reaction, TUDCA reveals substantial antioxidant activity significantly decreasing the rate of oxygen consumption in the system studied. In addition, TUDCA induces slight, but noticeable changes in the polarity and fluidity of the investigated model membranes. The results of performed MD simulation correspond very well with the experimental results.

Evidence of the milder phenotypic spectrum of c.1582G>A PIGT variant: Delineation based on seven novel Polish patients.

PIGT is one of over 29 glycosylphosphatidylinositol biosynthesis defect genes. Mutations cause genetically determined disorders characterized mainly by epilepsy with fever-sensitivity, central hypotonia, psychomotor delay and congenital malformations. The disease is known as multiple congenital anomalies-hypotonia-seizures syndrome 3 (MCAHS3) or glycosylphosphatidylinositol biosynthesis defect-7. Twenty-eight cases have been reported until today. We present seven novel Polish patients, all harboring 1582G>A variant in a homozygous or compound heterozygous state which seems to cause a milder phenotype of the disease.

The influence of PFK-II overexpression on neuroblastoma patients' survival may be dependent on the particular isoenzyme expressed, PFKFB3 or PFKFB4.

BACKGROUND/AIM: During cancer progression metabolic reprogramming is observed in parallel to the alternation in transcriptional profiles of malignant cells. Recent studies suggest that metabolic isoenzymes of phosphofructokinase II (PFK-II) - PFKFB3 and PFKFB4, often induced in hypoxic environment, significantly contribute to enhancement of glucose metabolism and in consequence cancer progression. MATERIALS AND METHODS: Using the publicly available data deposited in the R2 data base we performed a Kaplan-Meyer analysis for cancer patients divided into groups with high and low expression levels of PFKFB3/4, determined based on the median. RESULTS: Our data showed that high PFKFB3/4 expression significantly correlates with shorter overall survival in several cancers. Moreover, we found that neuroblastoma patients with poor overall survival and evidence free survival are characterized by high PFKFB3 and at the same time low PFKFB4 expression, whereas patients with high PFKFB4 expressions are characterized by significantly better overall survival/evidence free survival rates. CONCLUSION: Our analysis clearly indicates that expression of PFKFB3/4 isoenzymes may have a key prognostic value for several cancers. What's more, it seems that in neuroblastoma the prognostic value of PFK-II may be dependent on the relation between PFKFB3 and PFKFB4 isoenzyme expression, indicating that further studies analyzing the role of both cancer specific PFK-II isoenzymes are highly desired.

Asymmetric Spontaneous Intercalation of Lutein into a Phospholipid Bilayer, a Computational Study.

Lutein, a hydroxylated carotenoid, is a pigment synthesised by plants and bacteria. Animals are unable to synthesise lutein, nevertheless, it is present in animal tissues, where its only source is dietary intake. Both in plants and animals, carotenoids are associated mainly with membranes where they carry out important physiological functions. Their trafficking to and insertion into membranes are not well recognised due to experimental difficulties. In this paper, a computational approach is used to elucidate details of the dynamics and energetics of lutein intercalation from the water to the phospholipid bilayer phase. The dynamics is studied using molecular dynamics simulation, and the energetics using umbrella sampling. Lutein spontaneous insertion into the bilayer and translocation across it proceed via formation of hydrogen bonds between its hydroxyl groups and water and/or phospholipid oxygen atoms as well as desolvation of its polyene chain. As lutein molecule is asymmetric, its bilayer intercalation is also asymmetric. The course of events and timescale of the intercalation are different from those of helical peptides. The time of full lutein intercalation ranges from 20 to 100 ns and its final orientation is predominately vertical. Nevertheless, some lutein molecules are in the final horizontal position and some aggregate in the water phase and remain there for the whole simulation time. The highest energy barrier for the intercalation process is ~2.2 kcal/mol and the energy gain is ~18 kcal/mol. The results obtained for lutein can be applied to other xanthophylls and molecules of a similar structure.

Application of case study to introduce medical students to molecular biology techniques used in HIV diagnostics.

Diagnostic molecular biology is a fast developing discipline of laboratory medicine widely used in numerous medical branches such as oncology, hematology, immunology, internal medicine, or infectious diseases, which will certainly have a major impact on clinical medicine in the near future. Nowadays, educational process is forced to face the quickly growing overflow of easily accessible data and properly guide the students not to be lead astray in the information chaos. Hence, in view of the foregoing, it appears obvious that modern medical education should put particular stress on selective acquiring, interpreting, and applying integrated multidisciplinary knowledge rather than on just absorbing and memorizing huge amount of scattered information. The presented case study aims at familiarizing the students with basic molecular biology techniques such as enzyme-linked immunosorbent assay, Western blot, and quantitative reverse transcription-polymerase chain reaction. Importantly, it is not limited only to discussing and learning the principles of the assays mentioned earlier, but it also shows their practical application in a particular diagnostic process and give the guidelines on how to explain and interpret exemplary results. In parallel, the way the case study is constructed allows a tutor to lead students into discussion on clinical aspects related to HIV infection what should eventually create complete picture of a HIV diagnostic process, thereby integrating basic knowledge of molecular biology laboratory techniques, HIV biology, and immunological response. © 2019 International Union of Biochemistry and Molecular Biology, 47(3):355-360, 2019.

The Impact of Plaque Rupture on Mismatch Between Intravascular Ultrasound Minimal Lumen Area and Fractional Flow Reserve Measured in an Angiographically Borderline Left Main With Complex Left Anterior Descending Involvement.

Plaque rupture is known to affect the mismatch between anatomy and physiology. In this case, we relied on fractional flow reserve along with the tomographic perspective of intravascular ultrasound.

Is the tilt of the lipid head group correlated with the number of intermolecular interactions at the bilayer interface?

Lipid and water molecules comprising the bilayer form an integral entity owing to only weak physical interactions. At the bilayer interface, these interactions chiefly involve hydrogen bonding and charge pairing. Lipid head groups make hydrogen bonds (H-bonds) predominantly with water, whereas interlipid H-bonds and charge pairs are less numerous. Both interlipid H-bonding and charge pairing depend on the distance and relative orientation of the interacting head groups. In this computational paper, correlations are analysed between the orientation of the lipid head group and the number of interlipid interactions at the interface of a bilayer made of galactolipids, forming direct interlipid H-bonds, and of phosphatidylcholines forming interlipid charge pairs. The correlations are not strong, however, in both bilayers they show a similar trend.

Assessing gastric toxicity of xanthone derivatives of anti-inflammatory activity using simulation and experimental approaches.

Xanthones are tricyclic compounds of natural or synthetic origin exhibiting a broad spectrum of therapeutic activities. Three synthetic xanthone derivatives (KS1, KS2, and KS3) with properties typical for nonsteroidal anti-inflammatory drugs (NSAID) were objects of the presented model study. NSAIDs are in common use however; several of them exhibit gastric toxicity predominantly resulting from their direct interactions with the outermost lipid layer of the gastric mucosa that impair its hydrophobic barrier property. Among the studied xanthones, gastric toxicity of only KS2 has been determined in previous pharmacological studies, and it is low. In this study, carried out using X-ray diffraction and computer simulation, a palmitoyloleoylphosphatidylcholine-cholesterol bilayer (POPC-Chol) was used as a model of a hydrophobic layer of lipids protecting gastric mucosa as POPC and Chol are the main lipids in human mucus. X-ray diffraction data were used to validate the computer model. The aim of the study was to assess potential gastric toxicity of the xanthones by analysing their atomic level interactions with lipids, ions, and water in the lipid bilayer and their effect on the bilayer physicochemical properties. The results show that xanthones have small effect on the bilayer properties except for its rigidity whereas their interactions with water, ions, and lipids depend on their protonation state and for a given state, are similar for all the xanthones. As gastric toxicity of KS2 is low, based on MD simulations one can predict that toxicity of KS1 and KS3 is also low.