Biophysical analysis of apolipoprotein E3 variants linked with development of type III hyperlipoproteinemia

Background

Apolipoprotein E (apoE) is a major protein of the lipoprotein transport system that plays important roles in lipid homeostasis and protection from atherosclerosis. ApoE is characterized by structural plasticity and thermodynamic instability and can undergo significant structural rearrangements as part of its biological function. Mutations in the 136–150 region of the N-terminal domain of apoE, reduce its low density lipoprotein (LDL) receptor binding capacity and have been linked with lipoprotein disorders, such as type III hyperlipoproteinemia (HLP) in humans. However, the LDL-receptor binding defects for these apoE variants do not correlate well with the severity of dyslipidemia, indicating that these variants may carry additional properties that contribute to their pathogenic potential.

Methodology/Principal Findings

In this study we examined whether three type III HLP predisposing apoE3 variants, namely R136S, R145C and K146E affect the biophysical properties of the protein. Circular dichroism (CD) spectroscopy revealed that these mutations do not significantly alter the secondary structure of the protein. Thermal and chemical unfolding analysis revealed small thermodynamic alterations in each variant compared to wild-type apoE3, as well as effects in the reversibility of the unfolding transition. All variants were able to remodel multillamelar 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) vesicles, but R136S and R145C had reduced kinetics. Dynamic light scattering analysis indicated that the variant R136S exists in a higher-order oligomerization state in solution. Finally, 1-anilinonaphthalene-8-sulfonic acid (ANS) binding suggested that the variant R145C exposes a larger amount of hydrophobic surface to the solvent.

Conclusions/Significance

Overall, our findings suggest that single amino acid changes in the functionally important region 136–150 of apoE3 can affect the molecule''s stability and conformation in solution and may underlie functional consequences. However, the magnitude and the non-concerted nature of these changes, make it unlikely that they constitute a distinct unifying mechanism leading to type III HLP pathogenesis.     

Afforestation for climate change mitigation: Potentials,risks and trade‐offs

Afforestation is considered a cost‐effective and readily available climate change mitigation option. In recent studies afforestation is presented as a major solution to limit climate change. However, estimates of afforestation potential vary widely. Moreover, the risks in global mitigation policy and the negative trade‐offs with food security are often not considered. Here we present a new approach to assess the economic potential of afforestation with the IMAGE 3.0 integrated assessment model framework. In addition, we discuss the role of afforestation in mitigation pathways and the effects of afforestation on the food system under increasingly ambitious climate targets. We show that afforestation has a mitigation potential of 4.9 GtCO₂/year at 200 US$/tCO₂ in 2050 leading to large‐scale application in an SSP2 scenario aiming for 2°C (410 GtCO₂ cumulative up to 2100). Afforestation reduces the overall costs of mitigation policy. However, it may lead to lower mitigation ambition and lock‐in situations in other sectors. Moreover, it bears risks to implementation and permanence as the negative emissions are increasingly located in regions with high investment risks and weak governance, for example in Sub‐Saharan Africa. Afforestation also requires large amounts of land (up to 1,100 Mha) leading to large reductions in agricultural land. The increased competition for land could lead to higher food prices and an increased population at risk of hunger. Our results confirm that afforestation has substantial potential for mitigation. At the same time, we highlight that major risks and trade‐offs are involved. Pathways aiming to limit climate change to 2°C or even 1.5°C need to minimize these risks and trade‐offs in order to achieve mitigation sustainably.     

Engineering cartilage-like tissue using human mesenchymal stem cells and silk protein scaffolds

Human mesenchymal stem cells (hMSC) derived from bone marrow aspirates can form the basis for the in vitro cultivation of autologous tissue grafts and help alleviate the problems of immunorejection and disease transmission associated with the use of allografts. We explored the utility of hMSC cultured on protein scaffolds for tissue engineering of cartilage. hMSC were isolated, expanded in culture, characterized with respect to the expression of surface markers and ability for chondrogenic and osteogenic differentiation, and seeded on scaffolds. Four different scaffolds were tested, formed as a highly porous sponge made of: 1) collagen, 2) cross-linked collagen, 3) silk, and 4) RGD-coupled silk. Cell-seeded scaffolds were cultured for up to 4 weeks in either control medium (DMEM supplemented with 10% fetal bovine serum) or chondrogenic medium (control medium supplemented with chondrogenic factors). hMSC attachment, proliferation, and metabolic activity were markedly better on slowly degrading silk than on fast-degrading collagen scaffolds. In chondrogenic medium, hMSC formed cartilaginous tissues on all scaffolds, but the extent of chondrogenesis was substantially higher for hMSC cultured on silk as compared to collagen scaffolds. The deposition of glycosaminoglycan (GAG) and type II collagen and the expression of type II collagen mRNA were all higher for hMSC cultured on silk than on collagen scaffolds. Taken together, these results suggest that silk scaffolds are particularly suitable for tissue engineering of cartilage starting from hMSC, presumably due to their high porosity, slow biodegradation, and structural integrity.     

MILVA: an interactive tool for the exploration of multidimensional microarray data

MOTIVATION: Clustering techniques such as k-means and hierarchical clustering are commonly used to analyze DNA microarray derived gene expression data. However, the interactions between processes underlying the cell activity suggest that the complexity of the microarray data structure may not be fully represented with discrete clustering methods. RESULTS: A newly developed software tool called MILVA (microarray latent visualization and analysis) is presented here to investigate microarray data without separating gene expression profiles into discrete classes. The underpinning of the MILVA software is the two-dimensional topographic representation of multidimensional microarray data. On this basis, the interactive MILVA functions allow a continuous exploration of microarray data driven by the direct supervision of the biologist in detecting activity patterns of co-regulated genes. AVAILABILITY: The MILVA software is freely available. The software and the related documentation can be downloaded from http://www.ncrg.aston.ac.uk/Projects/milva. User 'surrey' as username and '3245' as password to login. The software is currently available for Windows platform only.     

Deletions of helices 2 and 3 of human apoA-I are associated with severe dyslipidemia following adenovirus-mediated gene transfer in apoA-I-deficient mice

The objective of this study was to determine the effect of two amino-terminal apolipoprotein A-I (apoA-I) deletions on high-density lipoprotein (HDL) biosynthesis and lipid homeostasis. Adenovirus-mediated gene transfer showed that the apoA-I[Delta(89-99)] deletion mutant caused hypercholesterolemia, characterized by increased plasma cholesterol and phospholipids, that were distributed in the very low density/intermediate density/low-density lipoprotein (VLDL/IDL/LDL) region, and normal triglycerides. The capacity of the mutant protein to promote ATP-binding cassette transporter A1- (ABCA1-) mediated cholesterol efflux and to activate lecithin:cholesterol acyltranserase (LCAT) was approximately 70-80% of the wild-type (WT) control. The phospholipid transfer protein (PLTP) activity of plasma containing the apoA-I[Delta(89-99)] mutant was decreased to 32% of the WT control. Similar analysis showed that the apoA-I[Delta(62-78)] deletion mutant in apoA-I-deficient mice caused combined hyperlipidemia characterized by increased triglycerides, cholesterol, and phospholipids in the VLDL/IDL region. There was enrichment of the VLDL/IDL with mutant apoA-I that resulted in reduction of in vitro lipolysis. The capacity of this mutant to promote ABCA1-mediated cholesterol efflux was normal, and the capacity to activate LCAT in vitro was reduced by 53%. The WT apoA-I and the apoA-I[Delta(62-78)] mutant formed spherical HDL particles, whereas the apoA-I[Delta(89-99)] mutant formed discoidal HDL particles. We conclude that alterations in apoA-I not only may have adverse effects on HDL biosynthesis but also may promote dyslipidemia due to interference of the apoA-I mutants on the overall cholesterol and triglycerides homeostasis.     

ApoC-III deficiency prevents hyperlipidemia induced by apoE overexpression

Adenovirus-mediated overexpression of human apolipoprotein E (apoE) induces hyperlipidemia by stimulating the VLDL-triglyceride (TG) production rate and inhibiting the LPL-mediated VLDL-TG hydrolysis rate. Because apoC-III is a strong inhibitor of TG hydrolysis, we questioned whether Apoc3 deficiency might prevent the hyperlipidemia induced by apoE overexpression in vivo. Injection of 2 x 10(9) plaque-forming units of AdAPOE4 caused severe combined hyperlipidemia in Apoe-/- mice [TG from 0.7 +/- 0.2 to 57.2 +/- 6.7 mM; total cholesterol (TC) from 17.4 +/- 3.7 to 29.0 +/- 4.1 mM] that was confined to VLDL/intermediate density lipoprotein-sized lipoproteins. In contrast, Apoc3 deficiency resulted in a gene dose-dependent reduction of the apoE4-associated hyperlipidemia (TG from 57.2 +/- 6.7 mM to 21.2 +/- 18.5 and 1.5 +/- 1.4 mM; TC from 29.0 +/- 4.1 to 16.4 +/- 9.8 and 2.3 +/- 1.8 mM in Apoe-/-, Apoe-/-.Apoc3+/-, and Apoe-/-.Apoc3-/- mice, respectively). In both Apoe-/- mice and Apoe-/-.Apoc3-/- mice, injection of increasing doses of AdAPOE4 resulted in up to a 10-fold increased VLDL-TG production rate. However, Apoc3 deficiency resulted in a significant increase in the uptake of TG-derived fatty acids from VLDL-like emulsion particles by white adipose tissue, indicating enhanced LPL activity. In vitro experiments showed that apoC-III is a more specific inhibitor of LPL activity than is apoE. Thus, Apoc3 deficiency can prevent apoE-induced hyperlipidemia associated with a 10-fold increased hepatic VLDL-TG production rate, most likely by alleviating the apoE-induced inhibition of VLDL-TG hydrolysis.     

Evidence for increased local flexibility in psychrophilic alcohol dehydrogenase relative to its thermophilic homologue

The psychrophilic alcohol dehydrogenase (psADH) cloned from Antarctic Moraxella sp. TAE123 exhibits distinctive catalytic parameters in relation to the homologous thermophilic alcohol dehydrogenase (htADH) from Bacillus stearothermophilus LLD-R. Amide hydrogen-deuterium (H/D) exchange studies using Fourier-transformed infrared (FTIR) spectroscopy and mass spectrometry (MS) were conducted to investigate whether the differences are caused by variation in either global or regional protein flexibility. The FTIR H/D exchange study suggested that psADH does not share similar global flexibility with htADH at their physiologically relevant temperatures as has been predicted by the "corresponding state" hypothesis. However, the MS H/D exchange study revealed a more complicated picture concerning the flexibility of the two homologous enzymes. Analysis of the deuteration and exchange rates of protein-derived peptides suggested that only some functionally important regions in psADH that are involved in substrate and cofactor binding exhibit greater flexibility compared to htADH at low temperature (10 degrees C). These observations strongly suggest that variable conformational flexibility between the two protein forms is a local phenomenon, and that global H/D exchange measurement by FTIR can be misleading and should be used with discretion. These results are supportive of the idea that functionally important local flexibility can be uncoupled from global thermal stability. The structural factors underlying the differences in local protein flexibility and catalysis between htADH and psADH are discussed in conjunction with results from crystallographic and fluorescence spectroscopy studies.     

Bone osteonal tissues by Raman spectral mapping: orientation-composition

Bone is a composite material with a hierarchical structure. Its strength depends on its structural and material properties. In the present study, Raman microspectroscopic and Imaging analyses were employed to study 12 osteons in tissue sections from the femoral midshaft of a healthy human female, with a spatial resolution of approximately 1mum. Spatial changes in amount of mineral and organic matrix, as well as the variation in the mineral content were determined, imaged, and plotted as a function of the polarization of incident light. The results showed that the prominent bands, such as nu(1) PO(4) and amide I, commonly used for the determination of mineral and organic compositions, are quite sensitive to the orientation and the polarization direction of the incident light. On the other hand, bands such as amide III, nu(2) PO(4) and nu(4) PO(4) are less susceptible to the orientational effects. As a result, exclusive consideration of the nu(1) PO(4) and amide I bands for the calculation of material properties might lead to erroneous conclusions. Amide III, nu(2) PO(4) and nu(4) PO(4) Raman bands should also be taken into consideration for compositional analysis of bone structures, especially ones with unknown orientational features. Moreover, the results of the present study demonstrate the versatility of the analytical technique, and provide insights into the organization of bone tissue at the ultrastructural level.     

Zinc(II) complexes of the second-generation quinolone antibacterial drug enrofloxacin: Structure and DNA or albumin interaction

Zinc mononuclear complexes with the second-generation quinolone antibacterial drug enrofloxacin in the absence or presence of a nitrogen donor heterocyclic ligand 1,10-phenanthroline or 2,2′-bipyridine have been synthesized and characterized. Enrofloxacin is on deprotonated mode acting as a bidentate ligand coordinated to zinc ion through the ketone and a carboxylato oxygen atoms. The crystal structure of bis(enrofloxacinato)(1,10-phenanthroline)zinc(II), 2, has been determined by X-ray crystallography. The biological activity of the complexes has been evaluated by examining their ability to bind to calf-thymus DNA (CT DNA) with UV and fluorescence spectroscopies. UV studies of the interaction of the complexes with DNA have shown that they can bind to CT DNA and the DNA binding constants have been calculated. Competitive studies with ethidium bromide (EB) have shown that the complexes exhibit the ability to displace the DNA-bound EB indicating that they bind to DNA in strong competition with EB for the intercalative binding site. The complexes exhibit good binding propensity to human and bovine serum albumin proteins having relatively high binding constant values.     

Differential expression of Vegfr-2 and its soluble form in preeclampsia

Background

Several studies have suggested that the main features of preeclampsia (PE) are consequences of endothelial dysfunction related to excess circulating anti-angiogenic factors, most notably, soluble sVEGFR-1 (also known as sFlt-1) and soluble endoglin (sEng), as well as to decreased PlGF. Recently, soluble VEGF type 2 receptor (sVEGFR-2) has emerged as a crucial regulator of lymphangiogenesis. To date, however, there is a paucity of information on the changes of VEGFR-2 that occur during the clinical onset of PE. Therefore, the aim of our study was to characterize the plasma levels of VEGFR-2 in PE patients and to perform VEGFR-2 immunolocalization in placenta.

Methodology/Principal findings

By ELISA, we observed that the VEGFR-2 plasma levels were reduced during PE compared with normal gestational age matched pregnancies, whereas the VEGFR-1 and Eng plasma levels were increased. The dramatic drop in the VEGFR-1 levels shortly after delivery confirmed its placental origin. In contrast, the plasma levels of Eng and VEGFR-2 decreased only moderately during the early postpartum period. An RT-PCR analysis showed that the relative levels of VEGFR-1, sVEGFR-1 and Eng mRNA were increased in the placentas of women with severe PE. The relative levels of VEGFR-2 mRNA as well as expressing cells, were similar in both groups. We also made the novel finding that a recently described alternatively spliced VEGFR-2 mRNA variant was present at lower relative levels in the preeclamptic placentas.

Conclusions/Significance

Our results indicate that the plasma levels of anti-angiogenic factors, particularly VEGFR-1 and VEGFR-2, behave in different ways after delivery. The rapid decrease in plasma VEGFR-1 levels appears to be a consequence of the delivery of the placenta. The persistent circulating levels of VEGFR-2 suggest a maternal endothelial origin of this peptide. The decreased VEGFR-2 plasma levels in preeclamptic women may serve as a marker of endothelial dysfunction.