Polyhedral 3D structure of human plasma very low density lipoproteins by individual particle cryo-electron tomography1

Human VLDLs assembled in the liver and secreted into the circulation supply energy to peripheral tissues. VLDL lipolysis yields atherogenic LDLs and VLDL remnants that strongly correlate with CVD. Although the composition of VLDL particles has been well-characterized, their 3D structure is elusive because of their variations in size, heterogeneity in composition, structural flexibility, and mobility in solution. Here, we employed cryo-electron microscopy and individual-particle electron tomography to study the 3D structure of individual VLDL particles (without averaging) at both below and above their lipid phase transition temperatures. The 3D reconstructions of VLDL and VLDL bound to antibodies revealed an unexpected polyhedral shape, in contrast to the generally accepted model of a spherical emulsion-like particle. The smaller curvature of surface lipids compared with HDL may also reduce surface hydrophobicity, resulting in lower binding affinity to the hydrophobic distal end of the N-terminal β-barrel domain of cholesteryl ester transfer protein (CETP) compared with HDL. The directional binding of CETP to HDL and VLDL may explain the function of CETP in transferring TGs and cholesteryl esters between these particles. This first visualization of the 3D structure of VLDL could improve our understanding of the role of VLDL in atherogenesis.     

Different duplex/quadruplex junctions determine the properties of anti-thrombin aptamers with mixed folding

Mixed duplex/quadruplex oligonucleotides have attracted great interest as therapeutic targets as well as effective biomedical aptamers. In the case of thrombin-binding aptamer (TBA), the addition of a duplex motif to the G-quadruplex module improves the aptamer resistance to biodegradation and the affinity for thrombin. In particular, the mixed oligonucleotide RE31 is significantly more effective than TBA in anticoagulation experiments and shows a slower disappearance rate in human plasma and blood. In the crystal structure of the complex with thrombin, RE31 adopts an elongated structure in which the duplex and quadruplex regions are perfectly stacked on top of each other, firmly connected by a well-structured junction. The lock-and-key shape complementarity between the TT loops of the G-quadruplex and the protein exosite I gives rise to the basic interaction that stabilizes the complex. However, our data suggest that the duplex motif may have an active role in determining the greater anti-thrombin activity in biological fluids with respect to TBA. This work gives new information on mixed oligonucleotides and highlights the importance of structural data on duplex/quadruplex junctions, which appear to be varied, unpredictable, and fundamental in determining the aptamer functional properties.     

Epigenetic perturbations in aging stem cells

Stem cells maintain homeostasis in all regenerating tissues during the lifespan of an organism. Thus, age-related functional decline of such tissues is likely to be at least partially explained by molecular events occurring in the stem cell compartment. Some of these events involve epigenetic changes, which may dictate how an aging genome can lead to differential gene expression programs. Recent technological advances have made it now possible to assess the genome-wide distribution of an ever-increasing number of epigenetic marks. As a result, the hypothesis that there may be a causal role for an altered epigenome contributing to the functional decline of cells, tissues, and organs in aging organisms can now be explored. In this paper, we review recent developments in the field of epigenetic regulation of stem cells, and how this may contribute to aging.     

How do acetylcholine receptor ligands reach their binding sites?

The access pathway to the binding sites for large competitive antagonists of the nicotinic acetylcholine receptor from Torpedo californica electric tissue was analyzed by binding and photolabeling experiments with alpha-neurotoxins. Binding assays with [125I]alpha-bungarotoxin showed an increase in the number of accessible binding sites upon stepwise solubilization of the receptor-rich membranes. Similarily, ligand binding is facilitated upon fluidization of the membrane by increasing the temperature. The access to the binding sites seems to be sterically 'hindered' in the densely packed membrane state. Using a novel series of large biotinylated photoactivatable derivatives of neurotoxin II, we observed that the accessibility to the alpha/gamma- but not to the alpha/delta-binding site was considerably decreased for some derivatives under native conditions. This effect was less apparent at higher temperatures and could be abolished by complete solubilization. These observations support the nonequivalence of the receptor's binding sites. Together, our data suggest (a) that alpha-neurotoxins approach their binding sites from the membrane-facing periphery of the receptor's extramembrane domain rather than through the channel mouth and (b) that different entrance pathways to each binding site exist which vary in their sensitivity to the physical state of the plasma membrane.     

Dissociable and nondissociable forms of platelet-activating factor acetylhydrolase in human plasma LDL: implications for LDL oxidative susceptibility

Platelet-activating factor acetylhydrolase (PAF-AH) is transported by lipoproteins in plasma and is thought to possess both anti-inflammatory and anti-oxidative activity. It has been reported that PAF-AH is recovered primarily in small, dense LDL and HDL following ultracentrifugal separation of lipoproteins. In the present studies, we aimed to further define the distribution of PAF-AH among lipoprotein fractions and subfractions, and to determine whether these distributions are affected by the lipoprotein isolation strategy (FPLC versus sequential ultracentrifugation) and LDL particle distribution profile. When lipoproteins were isolated by FPLC, the bulk (～85%) of plasma PAF-AH activity was recovered within LDL-containing fractions, whereas with ultracentrifugation, there was a redistribution to HDL (which contained ～18% of the activity) and the d>1.21 g/ml fraction (which contained ～32%). Notably, re-ultracentrifugation of isolated LDL did not result in any further movement of PAF-AH to higher densities, suggesting the presence of dissociable and nondissociable forms of the enzyme on LDL. Differences were noted in the distribution of PAF-AH activity among LDL subfractions from subjects exhibiting the pattern A (primarily large, buoyant LDL) versus pattern B (primarily small, dense LDL) phenotype. In the latter group, there was a relative depletion of PAF-AH activity in subfractions in the intermediate to dense range (d=1.039–1.047 g/ml) with a corresponding increase in enzyme activity recovered within the d>1.21 g/ml ultracentrifugal fraction. Thus, there appears to be a greater proportion of the dissociable form of PAF-AH in pattern B subjects. In both populations, most of the nondissociable activity was recovered in a minor small, dense LDL subfraction. Based on conjugated dienes as a measure of lipid peroxidation, variations in PAF-AH activity appeared to contribute to variations in oxidative behavior among ultracentrifugally isolated LDL subfractions. The physiologic relevance of PAF-AH dissociability and the minor PAF-AH-enriched oxidation-resistant LDL subpopulation remains to be determined.     

A validated antibody panel for the characterization of tau post-translational modifications

Background

Tau is a microtubule-binding protein, which is subject to various post-translational modifications (PTMs) including phosphorylation, methylation, acetylation, glycosylation, nitration, sumoylation and truncation. Aberrant PTMs such as hyperphosphorylation result in tau aggregation and the formation of neurofibrillary tangles, which are a hallmark of Alzheimer’s disease (AD). In order to study the importance of PTMs on tau function, antibodies raised against specific modification sites are widely used. However, quality control of these antibodies is lacking and their specificity for particular modifications is often unclear.

Methods

In this study, we first designed an online tool called ‘TauPTM’, which enables the visualization of PTMs and their interactions on human tau. Using TauPTM, we next searched for commercially available antibodies against tau PTMs and characterized their specificity by peptide array, immunoblotting, electrochemiluminescence ELISA and immunofluorescence technologies.

Results

We demonstrate that commercially available antibodies can show a significant lack of specificity, and PTM-specific antibodies in particular often recognize non-modified versions of the protein. In addition, detection may be hindered by other PTMs in close vicinity, complicating the interpretation of results. Finally, we compiled a panel of specific antibodies and show that they are useful to detect PTM-modified endogenous tau in hiPSC-derived neurons and mouse brains.

Conclusion

This study has created a platform to reliably and robustly detect changes in localization and abundance of post-translationally modified tau in health and disease. A web-based version of TauPTM is fully available at http://www.tauptm.org.

     

Pharmacometabolomic signature links simvastatin therapy and insulin resistance

Introduction

Statins, widely prescribed drugs for treatment of cardiovascular disease, inhibit the biosynthesis of low density lipoprotein cholesterol (LDL-C). Despite providing major benefits, sub populations of patients experience adverse effects, including muscle myopathy and development of type II diabetes mellitus (T2DM) that may result in premature discontinuation of treatment. There are no reliable biomarkers for predicting clinical side effects in vulnerable individuals. Pharmacometabolomics provides powerful tools for identifying global biochemical changes induced by statin treatment, providing insights about drug mechanism of action, development of side effects and basis of variation of response.

Objective

To determine whether statin-induced changes in intermediary metabolism correlated with statin-induced hyperglycemia and insulin resistance; to identify pre-drug treatment metabolites predictive of post-drug treatment increased diabetic risk.

Methods

Drug-naïve patients were treated with 40 mg/day simvastatin for 6 weeks in the Cholesterol and Pharmacogenetics (CAP) study; metabolomics by gas chromatography-time-of-flight mass-spectrometry (GC–TOF–MS) was performed on plasma pre and post treatment on 148 of the 944 participants.

Results

Six weeks of simvastatin treatment resulted in 6.9% of patients developing hyperglycemia and 25% developing changes consistent with development of pre-diabetes. Altered beta cell function was observed in 53% of patients following simvastatin therapy and insulin resistance was observed in 54% of patients. We identified initial signature of simvastatin-induced insulin resistance, including ethanolamine, hydroxylamine, hydroxycarbamate and isoleucine which, upon further replication and expansion, could be predictive biomarkers of individual susceptibility to simvastatin-induced new onset pre-type II diabetes mellitus. No patients were clinically diagnosed with T2DM.

Conclusion

Within this short 6 weeks study, some patients became hyperglycemic and/or insulin resistant. Diabetic markers were associated with decarboxylated small aminated metabolites as well as a branched chain amino acid directly linked to glucose metabolism and fatty acid biosynthesis. Pharmacometabolomics provides powerful tools for precision medicine by predicting development of drug adverse effects in sub populations of patients. Metabolic profiling prior to start of drug therapy may empower physicians with critical information when prescribing medication and determining prognosis.

     

Quantification of the Influence of Extracellular Laccase and Intracellular Reactions on the Isomer-Specific Biotransformation of the Xenoestrogen Technical Nonylphenol by the Aquatic Hyphomycete Clavariopsis aquatica

The aquatic hyphomycete Clavariopsis aquatica was used to quantify the effects of extracellular laccase and intracellular reactions on the isomer-specific biotransformation of technical nonylphenol (t-NP). In laccase-producing cultures, maximal removal rates of t-NP and the isomer 4-(1-ethyl-1,4-dimethylpentyl)phenol (NP112) were about 1.6- and 2.4-fold higher, respectively, than in laccase-lacking cultures. The selective suppression of either laccase or intracellular reactions resulted in essentially comparable maximal removal rates for both compounds. Evidence for an unspecific oxidation of t-NP isomers was consistently obtained from laccase-expressing fungal cultures when intracellular biotransformation was suppressed and from reaction mixtures containing isolated laccase. This observation contrasts with the selective degradation of t-NP isomers by bacteria and should prevent the enrichment of highly estrogenic isomers in remaining t-NP. In contrast with laccase reactions, intracellular fungal biotransformation caused a significant shift in the isomeric composition of remaining t-NP. As a result, certain t-NP constituents related to more estrogenic isomers were less efficiently degraded than others. In contrast to bacterial degradation via ipso-hydroxylation, the substitution pattern of the quaternary α-carbon of t-NP isomers does not seem to be very important for intracellular transformation in C. aquatica. As-yet-unknown intracellular enzymes are obviously induced by nonylphenols. Mass spectral data of the metabolites resulting from the intracellular oxidation of t-NP, NP112, and 4-(1-ethyl-1,3-dimethylpentyl)phenol indicate nonyl chain hydroxylation, further oxidation into keto or aldehyde compounds, and the subsequent formation of carboxylic acid derivatives. Further metabolites suggest nonyl chain desaturation and methylation of carboxylic acids. The phenolic moieties of the nonylphenols remained unchanged.Nonylphenol ethoxylates (NPEOs) represent a major group of industrial nonionic surfactants. Technical nonylphenol (t-NP), used for the production of NPEOs, is synthesized by Friedel-Crafts alkylation of phenol with a mixture of differently branched nonenes. It therefore comprises a great variety of mainly para-substituted isomers, with variously branched nonyl chains. About 50 to 80 t-NP isomers were estimated to occur in environmentally relevant matrices (19). The incomplete bioconversion of NPEOs in wastewater treatment plants results in the formation of the less biodegradable t-NP and is considered a major source of this contaminant in the aquatic environment (57). The recalcitrance of t-NP to biodegradation is partly due to the presence in more than 85% of the t-NP isomers of a quaternary α-carbon in the branched nonyl chain. Such structural characteristics are considered to limit biological nonyl chain oxidation (11, 53, 55). Nonylphenols are known to disrupt normal endocrine functions in vertebrates (57). Certain isomers contained in t-NP have been reported to possess a considerably higher estrogenic activity than the t-NP mixture (15). Due to increasing concerns with respect to their largely unknown environmental fate and potentially adverse environmental and human health effects, nonylphenols have been listed as priority hazardous substances in the EU water framework directive.In light of the concerns above, microbial reactions with the potential to reduce nonylphenol concentrations in the environment but also offering new possibilities for applications such as effluent treatment have received increasing attention (11). Among environmental microorganisms, both aquatic and terrestrial fungi, as well as bacteria, have been shown to degrade t-NP (11). Fungal attack on nonylphenols differs from bacterial nonylphenol degradation. In the case of intracellular nonylphenol biotransformation reactions catalyzed by fungi, only metabolites modified in the alkyl chain have been described (23, 52). Metabolites indicative of oxidation of the phenolic ring have not been described to date. Bacterial degradation pathways have only been documented in the genera Sphingomonas and Sphingobium. Bacterial mineralization of the aromatic moiety of t-NP isomers to CO₂ and H₂O is initiated via ring hydroxylation at the ipso (C-4) position of the phenolic ring, and nonanols are produced from the nonyl chains (10, 11, 15, 16). Bacteria have been shown to utilize branched-chain nonylphenols as growth substrates (11, 12, 17, 43). In contrast, only one report describes the growth of a fungus, the yeast Candida aquaetextoris, on nonylphenol (the isomer 4-n-NP containing a linear nonyl chain) (52). With respect to fungal attack on t-NP, cometabolism seems to be the dominating process (11). Recent literature data indicate that certain t-NP isomers with an estrogenic potency higher than those of the original t-NP mixture can be enriched in remaining t-NP. This results from the selective removal of individual isomers upon bacterial ipso-substitution degradation mechanisms (15). However, the effects of fungal biotransformation reactions on the isomeric profile of t-NP have not yet been quantified.Laccases are extracellular multicopper oxidases. These have most frequently been described in white-rot basidiomycetes, which unspecifically oxidize via one-electron abstraction certain lignin constituents, as well many xenobiotic compounds. Thereby, organic radicals are generated as the primary oxidation products (3). Among the several groups of fungi found in aquatic environments, aquatic hyphomycetes (AQH) are a phylogenetically diverse group of mitosporic fungi specifically adapted to their exclusively aquatic lifestyle. AQH have been shown to metabolize several organic environmental pollutants, including t-NP (23), polycyclic musk fragrances (31), pesticide metabolites (2), and synthetic dyes (22). Therefore, with respect to the fungal attack on organic pollutants found in aquatic ecosystems, AQH are of special importance. Laccase production by strictly aquatic fungi such as AQH has already been demonstrated and discussed in the context of lignocellulose decay in aquatic ecosystems (1). A role of this enzyme in the AQH-catalyzed breakdown of aquatic environmental pollutants has been recently suggested. Here, laccase isolated from the AQH Clavariopsis aquatica was shown to act on nonylphenol (23) and polycyclic musk fragrances (31). Laccase has also been implicated in nonylphenol degradation by white-rot fungi (44, 45). Isolated extracellular laccases from several aquatic and terrestrial fungi were shown to catalyze the formation of oligomeric coupling products from nonylphenols via organic radical intermediates (6, 11). However, the effects of laccase reactions on the isomeric patterns of t-NP have not been assessed to date.The aim of the present study was to quantify the influence of extracellular laccase catalysis and intracellular biotransformation on nonylphenol removal rates and on the isomeric composition of t-NP. For this, C. aquatica was used as a model organism. The derived data were compared to effects of bacteria on nonylphenol isomers reported by other authors (15), and environmental and biotechnological implications of fungal t-NP biotransformation were deduced. At the same time we addressed metabolite formation from t-NP and the two major t-NP isomers 4-(1-ethyl-1,3-dimethylpentyl)phenol (NP111) and 4-(1-ethyl-1,4-dimethylpentyl)phenol (NP112) (Fig. ​(Fig.1).1). This was done to substantiate the apparent differences between fungi and bacteria in the intracellular oxidation of t-NP (11, 15).Open in a separate windowFIG. 1.Chemical structures of the nonylphenol isomers NP111 and NP112.     

Is there evidence of adaptation to tidal flooding in saplings of baldcypress subjected to different salinity regimes?

Plant populations may adapt to environmental conditions over time by developing genetically based morphological or physiological characteristics. For tidal freshwater forested wetlands, we hypothesized that the conditions under which trees developed led to ecotypic difference in response of progeny to hydroperiod. Specifically, we looked for evidence of ecotypic adaptation for tidal flooding at different salinity regimes using growth and ecophysiological characteristics of two tidal and two non-tidal source collections of baldcypress (Taxodium distichum (L.) L.C. Rich) from the southeastern United States. Saplings were subjected to treatments of hydrology (permanent versus tidal flooding) and salinity (0 versus ～2 g l^?1) for two and a half growing seasons in a greenhouse environment. Saplings from tidal sources maintained 21–41% lower overall growth and biomass accumulation than saplings from non-tidal sources, while saplings from non-tidal sources maintained 14–19% lower overall rates of net photosynthetic assimilation, leaf transpiration, and stomatal conductance than saplings from tidal sources. However, we found no evidence for growth or physiological enhancement of saplings from tidal sources to tide, or of saplings from non-tidal sources to no tide. All saplings growing under permanent flooding exhibited reduced growth and leaf gas exchange regardless of source, with little evidence for consistent salinity effects across hydroperiods. While we reject our original hypothesis, we suggest that adaptations of coastal baldcypress to broad (rather than narrow) environmental conditions may promote ecophysiological and growth enhancements under a range of global-change-induced stressors, perhaps reflecting a natural resilience to environmental change while precluding adaptations for specific flood regimes.     

Inhibition of canonical Wnt signaling promotes gliogenesis in P0-NSCs

Wnt signaling plays an essential role in the development of mammalian central nervous system. We investigated the impact of activation/inhibition of the Wnt signaling pathway on neuronal/glial differentiation in neurospheres derived from neonatal mouse forebrains. For short term alterations, neurospheres were stimulated with recombinant Wnt-3a, Wnt-5a and the Wnt inhibitor Dickkopf-1 (Dkk1). Furthermore, neurospheres were transduced with retroviral vectors encoding Wnt-3a, Wnt-7a and their inhibitors Dkk1 and soluble Frizzled related protein-5 (sFRP5). Long-term activation of Wnt pathway by Wnt-7a or by treatment with GSK3 inhibitors promoted a moderate increase of the neuronal differentiation and blocked gliogenesis. In contrast, Wnt pathway inhibition in neurospheres, induced by retroviral overexpression of either Dkk1 or sFRP5, robustly increased the gliogenesis at the expense of neurogenesis. In summary, our data demonstrate that activation or inhibition of Wnt/β-catenin signaling in neurospheres regulates neuronal and glial differentiation, respectively. Thus, our results suggest that Wnt signaling may also contribute to regulate these processes in the neonatal brain.