LipL53, a temperature regulated protein from Leptospira interrogans that binds to extracellular matrix molecules

The regulation of gene expression by environmental signals, such as temperature and osmolarity, has been correlated with virulence. In this study, we characterize the protein LipL53 from Leptospira interrogans, previously shown to react with serum sample of individual diagnosed with leptospirosis and to be up-regulated by shift to physiological osmolarity. The recombinant protein was expressed in Escherichia coli system, in insoluble form, recovered by urea solubilization and further refolded by decreasing the denaturing agent concentration during the purification procedure. The secondary structure content of the recombinant LipL53, as assessed by circular dichroism, showed a mixture of beta-strands and alpha-helix. The presence of LipL53 transcript at 28 degrees C was only detected within the virulent strains. However, upon shifted of attenuated cultures of pathogenic strains from 28 degrees C to 37 degrees C and to 39 degrees C, this transcript could also be observed. LipL53 binds laminin, collagen IV, cellular and plasma fibronectin in dose-dependent and saturable manner. Animal challenge studies showed that LipL53, although immunogenic, elicited only partial protection in hamsters. LipL53 is probably surface exposed as seen through immunofluorescence confocal microscopy. Our results suggest that LipL53 is a novel temperature regulated adhesin of L. interrogans that may be relevant in the leptospiral pathogenesis. (C) 2009 Elsevier Masson SAS. All rights reserved.

Inhibitors of human immunodeficiency virus type 1 reverse transcriptase target distinct phases of early reverse transcription

Early HIV-1 reverse transcription can be separated into initiation and elongation phases. Here we show, using PCR analysis of negative-strand strong-stop DNA [(-)ssDNA] synthesis in intact virus, that different reverse transcriptase (RT) inhibitors affect distinct phases of early natural endogenous reverse transcription (NERT), The effects of nevirapine on NERT were consistent with a mechanism of action including both specific and nonspecific binding events. The nonspecific component of this inhibition targeted the elongation reaction, whereas the specific effect seemed principally to be directed at very early events (initiation or the initiation-elongation switch), In contrast, foscarnet and the nucleoside analog ddATP inhibited both early and late (-)ssDNA synthesis in a similar manner. We also examined compounds that targeted other viral proteins and found that Ro24-7429 (a Tat antagonist) and rosmarinic acid (an integrase inhibitor) also directly inhibited RT, Our results indicate that NERT can be used to identify and evaluate compounds that directly target the reverse transcription complex.

Recurrent gains and losses of large (84-109 bp) repeats in the rDNA internal transcribed spacer 2 (ITS2) of rhipicephaline ticks

We studied the internal transcribed spacer 2 (ITS2) in twenty-two spp. of ticks from the subfamily Rhipicephalinae. A 104-109 base pair (bp) region was Imperfectly repeated In most ticks studied. Mapping the number of repeat copies on to a phylogeny from the ITS2 showed that there have been many Independent gains and losses of repeats. Comparison of the sequences of the repeat copies Indicated that in most taxa concerted evolution had played little if any role in the evolution of these regions, as the copies clustered by sequence position rather than species, In our putative secondary structure, each repeat copy can fold into a distinct and almost identical stem-loop complex; a gain or loss of a repeat copy apparently does not impair the function of the ITS2 in these ticks.

Structural characterization of the N-terminal autoregulatory sequence of phenylalanine hydroxylase

Phenylalanine hydroxylase (PAH) is activated by its substrate phenylalanine, and through phosphorylation by cAMP-dependent protein kinase at Ser 16 in the N-terminal autoregulatory sequence of the enzyme. The crystal structures of phosphorylated and unphosphorylated forms of the enzyme showed that, in the absence of phenylalanine, in both cases the N-terminal 18 residues including the phosphorylation site contained no interpretable electron density. We used nuclear magnetic resonance (NMR) spectroscopy to characterize this N-terminal region of the molecule in different stages of the regulatory pathway. A number of sharp resonances are observed in PAH with an intact N-terminal region, but no sharp resonances are present in a truncation mutant lacking the N-terminal 29 residues. The N-terminal sequence therefore represents a mobile flexible region of the molecule. The resonances become weaker after the addition of phenylalanine, indicating a loss of mobility. The peptides corresponding to residues 2-20 of PAH have different structural characteristics in the phosphorylated and unphosphorylated forms, with the former showing increased secondary structure. Our results support the model whereby upon phenylalanine binding, the mobile N-terminal 18 residues of PAH associate with the folded core of the molecule; phosphorylation may facilitate this interaction.

Characterization of a chemsensory protein (ASP3c) from honeybee (Apis mellifera L.) as a brood pheromone carrier

Chemosensory proteins (CSPs) are ubiquitous soluble small proteins isolated from sensory organs of a wide range of insect species, which are believed to be involved in chemical communication. We report the cloning of a honeybee CSP gene called ASP3c, as well as the structural and functional characterization of the encoded protein. The protein was heterologously secreted by the yeast Pichia pastoris using the native signal peptide. ASP3c disulfide bonds were assigned after trypsinolysis followed by chromatography and mass spectrometry combined with microsequencing. The pairing (Cys(I)-Cys(II), Cys(III)-Cys(IV)) was found to be identical to that of Schistocerca gregaria CSPs, suggesting that this pattern occurs commonly throughout the insect CSPs. CD measurements revealed that ASP3c mainly consists of alpha-helices, like other insect CSPs. Gel filtration analysis showed that ASP3c is monomeric at neutral pH. Using ASA, a fluorescent fatty acid anthroyloxy analogue as a probe, ASP3c was shown to bind specifically to large fatty acids and ester derivatives, which are brood pheromone components, in the micromolar range. It was unable to bind tested general odorants and other tested pheromones (sexual and nonsexual). This is the first report on a natural pheromonal ligand bound by a recombinant CSP with a measured affinity constant.

Conformationally constrained macrocycles that mimic tripeptide beta-strands in water and aprotic solvents

The beta-strand conformation is unknown for short peptides in aqueous solution, yet it is a fundamental building block in proteins and the crucial recognition motif for proteolytic enzymes that enable formation and turnover of all proteins. To create a generalized scaffold as a peptidomimetic that is preorganized in a beta-strand, we individually synthesized a series of 15-22-membered macrocyclic analogues of tripeptides and analyzed their structures. Each cycle is highly constrained by two trans amide bonds and a planar aromatic ring with a short nonpeptidic linker between them. A measure of this ring strain is the restricted rotation of the component tyrosinyl aromatic ring (DeltaG(rot) 76.7 kJ mol(-1) (16-membered ring), 46.1 kJ mol(-1) (17-membered ring)) evidenced by variable temperature proton NMR spectra (DMF-d(7), 200-400 K). Unusually large amide coupling constants ((3)J(NH-CHalpha) 9-10 Hz) corresponding to large dihedral angles were detected in both protic and aprotic solvents for these macrocycles, consistent with a high degree of structure in solution. The temperature dependence of all amide NH chemical shifts (Deltadelta/T7-12 ppb/deg) precluded the presence of transannular hydrogen bonds that define alternative turn structures. Whereas similar sized conventional cyclic peptides usually exist in solution as an equilibrium mixture of multiple conformers, these macrocycles adopt a well-defined beta-strand structure even in water as revealed by 2-D NMR spectral data and by a structure calculation for the smallest (15-membered) and most constrained macrocycle. Macrocycles that are sufficiently constrained to exclusively adopt a beta-strand-mimicking structure in water may be useful pre-organized and generic templates for the design of compounds that interfere with beta-strand recognition in biology.

Effects of additional sequences directly downstream from the AUG on the expression of GFP gene

We have studied the expression of the green fluorescent protein (GFP) gene to gain more understanding of the effects of additional nucleotide triplets (codons) downstream from the initiation codon on the translation of the GFP mRNA in CHO and Cos1 cells. A leader sequence of six consecutive identical codons (GUG, CUC, AGU or UCA) was introduced into a humanized GFP (hm gfp) gene downstream from the AUG to produce four GFP gene variants. Northern blot and RT-PCR analysis indicated that mRNA transcription from the GFP gene was not significantly affected by any of the additional sequences. However, immunoblotting and FACS analysis revealed that AGU and UCA GFP variants produced GFP at a mean level per cell 3.5-fold higher than the other two GFP variants and the hm gfp gene. [35S]-Methionine labeling and immunoprecipitation demonstrate that GFP synthesis was very active in UCA variant transfected-cells, but not in GUG variant and hm gfp transfected-cells. Moreover, proteasome inhibitor MG-132 treatment indicated that the GFPs encoded by each of the GFP variants and the hm gfp were equally stable, and this together with the comparable mRNA levels observed for each construct suggested that the different steady-state GFP concentrations observed reflected different translation efficiencies of the various GFP genes. In addition, the CUC GFP variant, when transiently transfected into CHO or COS-1 cells, did not produce any GFP expressing cells (fully green cells), and the GUG variant produced GFP expressing cells less than 10%, while AGU and UCA GFP variants up to 30–35% in a time course study from 8 to 36 h posttransfection. Analysis of the potential secondary structure of the GFP variant mRNAs especially in the translation initiation region suggested that the secondary structure of the GFP mRNAs was unlikely to explain the different translation efficiencies of the GFP variants. The present findings indicate that a change of the initiation context of the GFP gene by addition of extra coding sequence can alter the translation efficiency of GFP mRNA, providing a means of more efficient expression of GFP in eukaryotic cells.

Studies of the Antiproliferative Activity of Ruthenium (II) Cyclopentadienyl-Derived Complexes with Nitrogen Coordinated Ligands

Four cationic ruthenium(II) complexes with the formula [Ru(eta(5)-C5H5)(PPh3)(2)](+), with L = 5-phenyl-1H-tetrazole (TzH) 1, imidazole (ImH) 2, benzo[1,2-b; 4,3-b'] dithio-phen-2-carbonitrile (Bzt) 3, and [5-(2-thiophen-2-yl)-vinyl]-thiophene-2-carbonitrile] (Tvt) 4 were prepared and characterized in view to evaluate their potentialities as antitumor agents. Studies by Circular Dichroism indicated changes in the secondary structure of ct-DNA. Changes in the tertiary structure of pBR322 plasmid DNA were also observed in gel electrophoresis experiment and the images obtained by atomic force microscopy (AFM) suggest strong interaction with pBR322 plasmid DNA; the observed decreasing of the viscosity with time indicates that the complexes do not intercalate between DNA base pairs. Compounds 1, 2, and 3 showed much higher cytotoxicity than the cisplatin against human leukaemia cancer cells (HL-60 cells).

Pseudomonas aeruginosa amidase: Aggregation in recombinant Escherichia coli

The effect of cultivation parameters such as temperature incubation, IPTG induction and ethanol shock on the production of Pseudomonasaeruginosa amidase (E.C.3.5.1.4) in a recombinant Escherichia coli strain in LB ampicillin culture medium was investigated. The highest yield of solubleamidase, relatively to other proteins, was obtained in the condition at 37 degrees C using 0.40 mM IPTG to induce growth, with ethanol. Our results demonstrate the formation of insoluble aggregates containing amidase, which was biologically active, in all tested growth conditions. Addition of ethanol at 25 degrees C in the culture medium improved amidase yield, which quantitatively aggregated in a biologically active form and exhibited in all conditions an increased specific activity relatively to the soluble form of the enzyme. Non-denaturing solubilization of the aggregated amidase was successfully achieved using L-arginine. The aggregates obtained from conditions at 37 degrees C by Furier transform infrared spectroscopy (FTIR) analysis demonstrated a lower content of intermolecular interactions, which facilitated the solubilization step applying non-denaturing conditions. The higher interactions exhibited in aggregates obtained at suboptimal conditions compromised the solubilization yield. This work provides an approach for the characterization and solubilization of novel reported biologically active aggregates of this amidase.

Structural characterization and immunogenicity in wildtype and immune tolerant mice of degraded recombinant human interferon alpha2b

Purpose - To study the influence of protein structure on the immunogenicity in wildtype and immune tolerant mice of well-characterized degradation products of recombinant human interferon alpha2b (rhIFNα2b). Methods - RhIFNα2b was degraded by metal catalyzed oxidation (M), crosslinking with glutaraldehyde (G), oxidation with hydrogen peroxide (H) and incubation in a boiling water bath (B). The products were characterized with UV absorption, circular dichroism and fluorescence spectroscopy, gel permeation chromatography, reversed-phase HPLC, SDS-PAGE, Western blotting and mass spectrometry. The immunogenicity of the products was evaluated in wildtype mice and in transgenic mice immune tolerant for hIFNα2. Serum antibodies were detected by ELISA or surface plasmon resonance. Results - M-rhIFNα2b contained covalently aggregated rhIFNα2b with three methionines partly oxidized to methionine sulfoxides. G-rhIFNα2b contained covalent aggregates and did not show changes in secondary structure. H-rhIFNα2b was only chemically changed with four partly oxidized methionines. B-rhIFNα2b was largely unfolded and heavily aggregated. Native (N) rhIFNα2b was immunogenic in the wildtype mice but not in the transgenic mice, showing that the latter were immune tolerant for rhIFNα2b. The antirhIFNα2b antibody levels in the wildtype mice depended on the degradation product: M-rhIFNα2b > H-rhIFNα2b ~ N-rhIFNα2b >> B-rhIFNα2b; G-rhIFNα2b did not induce anti-rhIFNα2b antibodies. In the transgenic mice, only M-rhIFNα2b could break the immune tolerance. Conclusions - RhIFNα2b immunogenicity is related to its structural integrity. Moreover, the immunogenicity of aggregated rhIFNα2b depends on the structure and orientation of the constituent protein molecules and/or on the aggregate size.

Structural characterization and immunogenicity in wild-type and immune tolerant mice of degraded recombinant human interferon Alpha2b

Purpose: This study was conducted to study the influence of protein structure on the immunogenicity in wild-type and immune tolerant mice of well-characterized degradation products of recombinant human interferon alpha2b (rhIFNα2b). Methods: RhIFNα2b was degraded by metal-catalyzed oxidation (M), cross-linking with glutaraldehyde (G), oxidation with hydrogen peroxide (H), and incubation in a boiling water bath (B). The products were characterized with UV absorption, circular dichroism and fluorescence spectroscopy, gel permeation chromatography, reverse-phase high-pressure liquid chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blotting, and mass spectrometry. The immunogenicity of the products was evaluated in wild-type mice and in transgenic mice immune tolerant for hIFNα2. Serum antibodies were detected by enzyme-linked immunosorbent assay or surface plasmon resonance. Results: M-rhIFNα2b contained covalently aggregated rhIFNα2b with three methionines partly oxidized to methionine sulfoxides. G-rhIFNα2b contained covalent aggregates and did not show changes in secondary structure. H-rhIFNα2b was only chemically changed with four partly oxidized methionines. B-rhIFNα2b was largely unfolded and heavily aggregated. Nontreated (N) rhIFNα2b was immunogenic in the wild-type mice but not in the transgenic mice, showing that the latter were immune tolerant for rhIFNα2b. The anti-rhIFNα2b antibody levels in the wild-type mice depended on the degradation product: M-rhIFNα2b > H-rhIFNα2b ∼ N-rhIFNα2b ≫ B-rhIFNα2b; G-rhIFNα2b did not induce anti-rhIFNα2b antibodies. In the transgenic mice, only M-rhIFNα2b could break the immune tolerance. Conclusions: RhIFNα2b immunogenicity is related to its structural integrity. Moreover, the immunogenicity of aggregated rhIFNα2b depends on the structure and orientation of the constituent protein molecules and/or on the aggregate size.

Metal ions modulate the folding and stability of the tumor suppressor protein S100A2

Febs Journal (2009)276:1776-1786

Role of a novel disulfide bridge within the all-beta fold of soluble Rieske proteins

Journal of Biological Inorganic Chemistry (2010)15: 271-281

Dimensionamento de Madres Enformadas a Frio Travadas por Painéis do Tipo Sandwich

O presente relatório de estágio foi elaborado no âmbito da Unidade Curricular de DIPRE, Dissertação/Projeto/Estágio, do 2.º ano de Mestrado em Engenharia Civil do Instituto Superior de Engenharia do Porto, do ramo de estruturas, tendo como principal foco, a análise e dimensionamento de madres de aço enformado a frio e sua utilização com painéis do tipo sandwich em coberturas. Seções enformadas a frio são cada vez mais utilizadas em construções modernas, especificamente como estrutura secundária em coberturas, onde geralmente são fixas a painéis através de ligações aparafusadas. A presença de painéis e fixações através de parafusos permitem a estabilização lateral e torsional de madres aumentando desta forma a capacidade resistente, mas por serem elementos estruturais de espessura reduzida, os enformados a frio são suscetíveis a fenómenos de instabilidade associados. Desta forma, a norma EN 1993-1-3 [4] permite a análise e dimensionamento deste tipo de elementos através das disposições regulamentares preconizadas nas partes 1-1 [3] e 1-5 [5] da mesma norma. Num primeiro estudo, o presente trabalho tem como objetivo o dimensionamento e verificação de segurança de elementos enformados a frio com base na seção efetiva determinada com o auxílio das normas EN 1993-1-1 (regras gerais) e EN 1993-1-5 (regras para elementos estruturais constituídos por placas). Numa segunda fase, este trabalho pretende apresentar um estudo do comportamento de interação entre os sistemas madres-painéis. Para tal, são quantificadas as rigidezes das conexões dos sistemas e dos painéis para se realizarem a análise relativamente à restrição lateral e restrição torsional de madres. Neste contexto, concluiu-se que os painéis, quando fixos de forma adequada às madres, contribuem para a estabilidade.