Solution structure of the apo and copper(I)-loaded human metallochaperone HAH1

The human metallochaperone HAH1 has been produced in Escherichia coli with four additional amino acids at the C-terminus and characterized in solution by NMR spectroscopy, both with and without copper(I). The solution structure of the apo-HAH1 monomer has a root-mean-square-deviation (RMSD) of 0.50 A for the coordinates of the backbone atoms and 0.96 A for all heavy atoms. These values compare, respectively, with 0.45 and 0.95 A for copper(I)-HAH1. There are only minor structural rearrangements upon copper(I) binding. In particular, the variation of interatomic interactions around the metal-binding region is limited to a movement of Lys60 toward the metal site. The protein structures are similar to those obtained by X-ray crystallography in a variety of derivatives, with backbone RMSD values below 1 A. In the holoprotein, copper(I) is confirmed to be two coordinated. If these data are compared with those of orthologue proteins, we learn that HAH1 has a lower tendency to change coordination number from two to three. Such a switch in coordination is a key step in copper transfer.     

The unusually stable quaternary structure of human Cu,Zn-superoxide dismutase 1 is controlled by both metal occupancy and disulfide status

The eukaryotic copper,zinc superoxide dismutases are remarkably stable dimeric proteins that maintain an intrasubunit disulfide bond in the reducing environment of the cytosol and are active under a variety of stringent denaturing conditions. The structural interplay of conserved disulfide bond and metal-site occupancy in human copper,zinc superoxide dismutase (hSOD1) is of increasing interest as these post-translational modifications are known to dramatically alter the catalytic chemistry, the subcellular localization, and the susceptibility of the protein to aggregation. Using biophysical methods, we find no significant change in the gross secondary or tertiary structure of the demetallated form upon reduction of the disulfide. Interestingly, reduction does lead to a dramatic change in the quaternary structure, decreasing the monomer-to-dimer equilibrium constant by at least four orders of magnitude. This reduced form of hSOD1 is monomeric, even at concentrations well above the physiological range. Either the addition of Zn(II) or the formation of the disulfide leads to a shift in equilibrium that favors the dimeric species, even at low protein concentrations (i.e. micromolar range). We conclude that only the most immature form of hSOD1, i.e. one without any post-translational modifications, favors the monomeric state under physiological conditions. This finding provides a basis for understanding the selectivity of mitochondrial SOD1 import and may be relevant to the toxic properties of mutant forms of hSOD1 that can cause the familial form of amyotrophic lateral sclerosis.     

Bioinformatic comparison of structures and homology-models of matrix metalloproteinases

The entire family of human matrix metalloproteinases (MMPs) was investigated using phylogenetic trees and homology modeling. The phylogenetic analysis indicates that individual domains of each MMP have evolved in a correlated manner. Despite their high sequence similarity, the phylogenetic tree of the catalytic domains already allows functional (e.g., linked to regulation and substrate recognition) homologies between different MMPs to be identified. The same pattern of functional homologies is confirmed by the phylogenetic analysis of the mature proteins. Structural models were built for the catalytic domains of the entire MMP family, for twelve hemopexin domains and for twelve mature proteins. The surface properties around the active site cleft of the modeled and experimental structures are quite conserved, whereas the hemopexin domains are more differentiated, possibly indicating a role in determining substrate specificity. The analysis of mature MMPs showed that the area of the interface between the catalytic and hemopexin domains is essentially conserved, with both hydrophobic and hydrophilic amino acids at the interface. The absence of specific conserved interdomain contacts suggests that the interface is tolerant to amino acid replacements, and that there may be a certain degree of plasticity with respect to the reciprocal orientation of the two domains.     

Biodiversity of brachyuran crabs (Crustacea: Decapoda) from non-consolidated sublittoral bottom on the northern coast of São Paulo State, Brazil

The brachyuran community of the coast of São Paulo State is represented by about 188 species of crabs inhabiting different kinds of coastal marine environments. The biodiversity of brachyurans found on non-consolidated sublittoral bottom was investigated. The Ubatuba region (Ubatumirim, Ubatuba and Mar Virado bays, Couves and Mar Virado Islands, offshore region) was sampled for 3 years (1998–2000), at depths of 2–40 m. All sampling was performed using a fishing boat equipped with two double-rig nets. We collected 79 brachyuran species representing 9 superfamilies (4 Dromioidea, 1 Homoloidea, 2 Calappoidea, 5 Leucosioidea, 20 Majoidea, 7 Parthenopoidea, 17 Portunoidea, 18 Xanthoidea, and 5 Pinnotheroidea) and 41 genera. Ubatuba bay showed the greatest species richness with 50 species, followed by Ubatumirim with 45 and Mar Virado with 29. The number of species collected represents about 57% of the known species of crabs already reported for the shore of São Paulo State. It is worth noticing that this percentage is restricted only to non-consolidated sublittoral bottom. This fact indicates a great biodiversity of the habitat in this studied region, probably one to the diversity of habitat types present in the bays.     

Neutral mixed-ligand complexes of 2,2′-bipyridinedichloroplatinum(II) with dianionic aromatic chelating ligands as potential photosensitizers

The electronic spectra of NCS^? and I^? adducts of cobalt(II) human carbonic anhydrase I are pH dependent at pH values below 7. The pK_a of such equilibrium is dependent on the anion concentration and varies between 4.6 and 6.6. The ¹H NMR spectra show that the three histidine residues are bound to the metal ion over the entire pH range investigated. It is supposed that a Glu residue triggers the change in stereochemistry around the metal ion. It is possible that such a Glu residue is Glu 106 present in the active cavity.     

Backbone dynamics of plastocyanin in both oxidation states. Solution structure of the reduced form and comparison with the oxidized state

A model-free analysis based on (15)N R(1), (15)N R(2), and (15)N-(1)H nuclear Overhauser effects was performed on reduced (diamagnetic) and oxidized (paramagnetic) forms of plastocyanin from Synechocystis sp. PCC6803. The protein backbone is rigid, displaying a small degree of mobility in the sub-nanosecond time scale. The loops surrounding the copper ion, involved in physiological electron transfer, feature a higher extent of flexibility in the longer time scale in both redox states, as measured from D(2)O exchange of amide protons and from NH-H(2)O saturation transfer experiments. In contrast to the situation for other electron transfer proteins, no significant difference in the dynamic properties is found between the two redox forms. A solution structure was also determined for the reduced plastocyanin and compared with the solution structure of the oxidized form in order to assess possible structural changes related to the copper ion redox state. Within the attained resolution, the structure of the reduced plastocyanin is indistinguishable from that of the oxidized form, even though small chemical shift differences are observed. The present characterization provides information on both the structural and dynamic behavior of blue copper proteins in solution that is useful to understand further the role(s) of protein dynamics in electron transfer processes.     

BACE1 inhibitory activities of enantiomerically pure, variously substituted N-(3-(4-benzhydrylpiperazin-1-yl)-2-hydroxypropyl) arylsulfonamides

β-Secretase (BACE1) has been widely recognized as one of the possible therapeutic targets for the treatment of Alzheimer's disease. In this paper, we report the synthesis and the BACE1 inhibitory activity of new, variously substituted N-(3-(4-benzhydrylpiperazin-1-yl)-2-hydroxypropyl) arylsulfonamides. Each enantiomeric form was separately evaluated in BACE1 inhibition assays and IC(50) values were obtained in the low micromolar range. According to our biological results and docking studies, it can be asserted that the stereochemistry around the OH group in the central hydroxyethylamino linker does not significantly influence the BACE1 inhibitory activity of this type of molecules.     

Evolution of mitochondrial-type cytochrome c domains and of the protein machinery for their assembly

Proteins containing mitochondrial-type cytochrome c domains, defined here as protein domains having the mitochondrial cytochrome c fold, are found in organisms from all domains of life, and constitute essential components in several different metabolic pathways. The number of cytochrome c domains present in a given organism as well as their functional roles can vary widely even for quite closely related organisms. In this work, we have analysed in detail the distribution of mitochondrial-type cytochrome c domains along the tree of life and attempted to define the evolutionary relationships among them. In parallel, we have similarly analysed also the occurrence and distribution of the different machineries for cytochrome c assembly. It is found that the first appearance of mitochondrial-type cytochrome c domains has likely happened in the bacterial world, together with the first apparatus for their assembly. Evolution of cytochrome c domains has been extensive, involving several gene duplication and gene transfer events. Of particular relevance are gene transfer events from Bacteria to Eukarya and Archaea. The transfer of genes encoding cytochrome c domains has generally co-occurred with transfer of the assembly machinery. This has occurred also in Eukarya, where however the latter machinery has been subsequently replaced by a new one. It is possible that of the three known enzymatic systems for cytochrome c assembly, system II (found, among others, in cyanobacteria and Gram-positive bacteria) is the most ancient. Archaea have inherited from Bacteria system I or, possibly, an evolutionary intermediate between system II and system I.     

Bone mass and quality in patients with juvenile idiopathic arthritis: longitudinal evaluation of bone-mass determinants by using dual-energy x-ray absorptiometry,peripheral quantitative computed tomography,and quantitative ultrasonography

Introduction

Our objective was to evaluate longitudinally the main bone-mass and quality predictors in young juvenile idiopathic arthritis (JIA) patients by using lumbar spine dual-energy X-ray absorptiometry (DXA) scan, radius peripheral quantitative computed tomography (pQCT), and phalangeal quantitative ultrasonography (QUS) at the same time.

Methods

In total, 245 patients (172 females, 73 males; median age, 15.6 years: 148 oligoarticular, 55 polyarticular, 20 systemic, and 22 enthesitis-related-arthritis (ERA) onset) entered the study. Of these, 166 patients were evaluated longitudinally. Data were compared with two age- and sex-matched control groups.

Results

In comparison with controls, JIA patients, but not with ERA, had a reduced spine bone-mineral apparent density (BMAD) standard deviation score (P < 0.001) and musculoskeletal deficits, with significantly lower levels of trabecular bone mineral density (TrabBMD) (P < 0.0001), muscle cross-sectional area (CSA) (P < 0.005), and density-weighted polar section modulus (SSIp) (P < 0.05). In contrast, JIA showed fat CSA significantly higher than controls (P < 0.0001). Finally, JIA patients had a significant reduced amplitude-dependent speed of sound (AD-SoS) (P < 0.001), and QUS z score (P < 0.005).Longitudinally, we did not find any difference in all JIA patients in comparison with baseline, except for the SSIp value that normalized. Analyzing the treatments, a significant negative correlation among spine BMAD values, TrabBMD, AD-SoS, and systemic and/or intraarticular corticosteroids, and a positive correlation among TNF-α-blocking agents and spine BMAD, TrabBMD, and AD-SoS were observed.

Conclusions

JIA patients have a low bone mass that, after a first increase due to the therapy, does not reach the normal condition over time. The pronounced bone deficits in JIA are greater than would be expected because of reduction in muscle cross-sectional area. Thus, bone alterations in JIA likely represent a mixed defect of bone accrual and lower muscle forces.     

Global metabolomics characterization of bacteria: pre-analytical treatments and profiling

Pre-analytical treatments of bacteria are crucial steps in bacterial metabolomics studies. In order to achieve reliable samples that can best represent the global metabolic profile in vivo both qualitatively and quantitatively, many sample treatment procedures have been developed. The use of different methods makes it difficult to compare the results among different groups. In this work, E. coli samples were tested by using NMR spectroscopy. Both liquid N₂ and cold methanol quenching procedures reduce the cell membrane integrity and cause metabolites leakage. However, liquid N₂ quenching affected the cell viability and the NMR metabolites’ profile less than cold methanol procedure. Samples obtained by metabolite extraction were significantly superior over cell suspensions and cell lysates, with a higher number of detectable metabolites. Methanol/chloroform extraction proved most efficient at extraction of intracellular metabolites from both qualitative and quantitative points of view. Finally, standard operating procedures of bacterial sample treatments for NMR metabolomics study are presented.