A genome-wide association scan on the levels of markers of inflammation in Sardinians reveals associations that underpin its complex regulation

Identifying the genes that influence levels of pro-inflammatory molecules can help to elucidate the mechanisms underlying this process. We first conducted a two-stage genome-wide association scan (GWAS) for the key inflammatory biomarkers Interleukin-6 (IL-6), the general measure of inflammation erythrocyte sedimentation rate (ESR), monocyte chemotactic protein-1 (MCP-1), and high-sensitivity C-reactive protein (hsCRP) in a large cohort of individuals from the founder population of Sardinia. By analysing 731,213 autosomal or X chromosome SNPs and an additional ∼1.9 million imputed variants in 4,694 individuals, we identified several SNPs associated with the selected quantitative trait loci (QTLs) and replicated all the top signals in an independent sample of 1,392 individuals from the same population. Next, to increase power to detect and resolve associations, we further genotyped the whole cohort (6,145 individuals) for 293,875 variants included on the ImmunoChip and MetaboChip custom arrays. Overall, our combined approach led to the identification of 9 genome-wide significant novel independent signals—5 of which were identified only with the custom arrays—and provided confirmatory evidence for an additional 7. Novel signals include: for IL-6, in the ABO gene (rs657152, p = 2.13×10⁻²⁹); for ESR, at the HBB (rs4910472, p = 2.31×10⁻¹¹) and UCN119B/SPPL3 (rs11829037, p = 8.91×10⁻¹⁰) loci; for MCP-1, near its receptor CCR2 (rs17141006, p = 7.53×10⁻¹³) and in CADM3 (rs3026968, p = 7.63×10⁻¹³); for hsCRP, within the CRP gene (rs3093077, p = 5.73×10⁻²¹), near DARC (rs3845624, p = 1.43×10⁻¹⁰), UNC119B/SPPL3 (rs11829037, p = 1.50×10⁻¹⁴), and ICOSLG/AIRE (rs113459440, p = 1.54×10⁻⁰⁸) loci. Confirmatory evidence was found for IL-6 in the IL-6R gene (rs4129267); for ESR at CR1 (rs12567990) and TMEM57 (rs10903129); for MCP-1 at DARC (rs12075); and for hsCRP at CRP (rs1205), HNF1A (rs225918), and APOC-I (rs4420638). Our results improve the current knowledge of genetic variants underlying inflammation and provide novel clues for the understanding of the molecular mechanisms regulating this complex process.     

Characterization of polar membrane lipids of the extremely halophilic bacterium Salinibacter ruber and possible role of cardiolipin

The lipid composition of the extremely halophilic bacterium Salinibacter ruber (Bacteroidetes) was investigated by thin layer chromatography, gas chromatography, high performance liquid chromatography and electrospray ionization-mass spectrometry. Polar lipids represent about 80% of the total lipid extract. The main polar lipids are a sulfonic acid analogue of ceramide (or capnine analogue), phosphatidylcholine, phosphatidylserine, dimethylphosphatidylethanolamine, phosphatidylglycerol, cardiolipin or bisphosphatidylglycerol, and a glycolipid. The major acyl chains in the phospholipids are C16:1 Δ9cis and C18:1 Δ11cis, while the sulfonolipid contains an amide-bound iso C15:0 fatty acid. On changing the salinity of the culture medium, no significant differences were found in the lipid profile or the unsaturation of the lipid fatty acyl chains. The structure of the cardiolipin, which represents 20% of polar lipids, has been elucidated by gas chromatography and electrospray ionization mass spectrometry analysis.     

Construction, purification and characterization of untagged human liver alanine-glyoxylate aminotransferase expressed in Escherichia coli

His-tagging is commonly used to aid and expedite the purification of recombinant proteins. It is commonly assumed, though less frequently tested, that the His-tag affects neither the structure nor the stability of the protein. Alanine:glyoxylate aminotransferase (AGT) is a peroxisomal pyridoxal 5'-phosphate (PLP) dependent enzyme which catalyzes the transamination of alanine and glyoxylate to pyruvate and glycine. AGT is a clinically relevant enzyme whose deficiency causes an inherited rare metabolic disorder named primary hyperoxaluria type I. Until now, the structure and function of this enzyme have been studied using recombinant wild-type AGT and variants purified using a hexa-histidine tag. However, the study of the functional roles of the N- and C-termini in the dimerization process and on the import into the peroxisome, respectively, requires the preparation of human liver AGT without histidine tags. We report for the first time the expression of untagged AGT together with a new rapid protocol for its purification. In addition, the kinetic parameters for the forward and reverse transamination catalyzed by untagged AGT as well as the spectroscopic features, the K(D(PLP)), the pH and thermal stability of the enzyme in the holo- and apo-form have been determined. This investigation will be the starting point for a detailed understanding of the contributions of the N- and C-termini on the dimerization and folding of AGT, and on its import into the peroxisome. This is prerequisite to understand how pathological mutations affect the proper native quaternary and tertiary structure, stability, and targeting of the enzyme.     

Calorimetric determination of thermodynamic parameters of reaction reveals different enthalpic compensations of the yeast hexokinase isozymes

The change in enthalpy and rate constants for the reactions of yeast hexokinase isozymes, PI (Hxk1) and PII (Hxk2), was determined at pH 7.6 and 25 degrees C by isothermal titration calorimetry. The reactions were done in five buffer systems with enthalpy of protonation varying from -1.22 kcal/mol (phosphate) to -11.51 kcal/mol (Tris), allowing the determination of the number of protons released during glucose phosphorylation. The reaction is exothermic for both isozymes with a small, but significant (p < 0.0001), difference in the enthalpy of reaction (Delta HR), with an Delta HR of -5.1 +/- 0.2 (mean +/- S.D.) kcal/mol for Hxk1, and an Delta HR of -3.3 +/- 0.3 (mean +/- S.D.) kcal/mol for Hxk2. The Km for ATP determined by ITC was very similar to those reported in the literature for both isozymes. The effect of NaCl and KCl, from 0 to 200 mM, showed that although the rate of reaction decreases with increasing ionic strength, no change in the Delta HR was observed suggesting an entropic nature for the ionic strength. The differences in Delta HR obtained here for both isozymes strongly suggest that, besides glucose phosphorylation, another side reaction such as ATP hydrolysis and/or enzyme phosphorylation is taking place.     

Behavior of fluorinated analogs of L-(3,4-dihydroxyphenyl)alanine and L-threo-(3,4-dihydroxyphenyl)serine as substrates for Dopa decarboxylase

We have determined the kinetic parameters for Dopa decarboxylase (DDC) of three ring-fluorinated analogs of 3,4-dihydroxyphenylalanine (Dopa). The rank order of catalytic efficiency of decarboxylation (k(cat)/K(m)) is Dopa>6-F-Dopa>2-F-Dopa>5-F-Dopa. This rank is consistent with previous in vivo and in vitro studies which indicate that, of the fluorinated analogs, 6-F-Dopa has pharmacokinetics that are most suited for positron emission tomographic (PET) evaluation of dopamine function. The effectiveness of PET as a diagnostic tool, the convenient half-life of (18)F (110 min) and the favorable pharmacokinetics of 6-[(18)F]FDOPA have combined to make this an extremely valuable reagent to study dopaminergic activity. The reactions of the related fluorinated DOPS analogs show that, while 6-F-threo-3,4-(dihydroxyphenyl)serine (DOPS) is decarboxylated at approximately the same rate as the non-fluorinated substrate, 2-F-threo-DOPS is not converted into the corresponding amine. In both cases a Pictet-Spengler condensation with the pyridoxal 5(')-phosphate (PLP) cofactor occurs to produce tetrahydroisoquinolines. Condensation of fluorinated catecholamines and catechol amino acids with endogenous aldehydes will be investigated as an approach to study possible mechanisms of L-Dopa-linked neurotoxicity.     

Membrane recognition by vesicular stomatitis virus involves enthalpy-driven protein-lipid interactions

Vesicular stomatitis virus (VSV) infection depends on the fusion of viral and cellular membranes, which is mediated by virus spike glycoprotein G at the acidic environment of the endosomal compartment. VSV G protein does not contain a hydrophobic amino acid sequence similar to the fusion peptides found among other viral glycoproteins, suggesting that membrane recognition occurs through an alternative mechanism. Here we studied the interaction between VSV G protein and liposomes of different phospholipid composition by force spectroscopy, isothermal titration calorimetry (ITC), and fluorescence spectroscopy. Force spectroscopy experiments revealed the requirement for negatively charged phospholipids for VSV binding to membranes, suggesting that this interaction is electrostatic in nature. In addition, ITC experiments showed that VSV binding to liposomes is an enthalpically driven process. Fluorescence data also showed the lack of VSV interaction with the vesicles as well as inhibition of VSV-induced membrane fusion at high ionic strength. Intrinsic fluorescence measurements showed that the extent of G protein conformational changes depends on the presence of phosphatidylserine (PS) on the target membrane. Although the increase in PS content did not change the binding profile, the rate of the fusion reaction was remarkably increased when the PS content was increased from 25 to 75%. On the basis of these data, we suggest that G protein binding to the target membrane essentially depends on electrostatic interactions, probably between positive charges on the protein surface and negatively charged phospholipids in the cellular membrane. In addition, the fusion is exothermic, indicating no entropic constraints to this process.     

Aluminum site structure in serum transferrin and lactoferrin revealed by synchrotron radiation X-ray spectroscopy

The Al site structure of serum transferrin and lactoferrin is investigated using X-ray absorption near edge structure (XANES) spectroscopy. Al K-edge spectra in the mono- and dialuminum forms of the proteins have been recorded for the first time. Our results show that the aluminium ion is hexa-coordinated in an octahedral-like symmetry and that the monoaluminum form, where only the C-terminal binding site is saturated, has an increased structural distortion around the metal site.     

Changes in Fe site structure from fetal to adult hemoglobin probed by XANES

Iron X-ray absorption near edge structure (XANES) spectra of human fetal (F) and adult (A) deoxyhemoglobin (deoxyHb) measured at the Frascati synchrotron radiation facility reveal the different geometrical structure of the Fe-porphyrin complexes in the two proteins. By this method, having determined for the first time the variation of atomic positions in fetal and adult hemoglobin in solution (close to the 'in vivo' situation), we give further insight into the structure-function relationship in hemoglobins.