Biomedical subjects

Antonio Romero

Publications and source records attributed to Antonio Romero.

15 recordsLinked to original sources

PubMed2006

Variable number of tandem repeats of TNF receptor type 2 promoter as genetic biomarker of susceptibility to develop invasive pulmonary aspergillosis.

Tumor necrosis factor alpha (TNF-alpha) and lymphotoxin alpha (LT-alpha) are pivotal mediators of inflammatory responses in fungal infection diseases. We hypothesized that polymorphisms in genes of these cytokines or their receptors might increase the susceptibility of hematologic patients to develop invasive pulmonary aspergillosis (IPA). One hundred two hematologic patients and 124 age-matched controls were enrolled in the study, and the following standard single nucleotide polymorphisms were investigated: TNF-alpha -308 and +489, LT-alpha +252 and Tumor Necrosis Factor Receptor 2 (TNFR2) +676. Variable number of tandem repeats (VNTRs) at position -322 of the TNFR2 gene were also studied. Genotypic and allelic frequencies were similar between patients and controls. IPA was diagnosed in 54 of the 102 patients according to consensus criteria published by the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group. TNF-alpha and LT-alpha polymorphisms were not associated with presence of IPA. Susceptibility to IPA was strongly associated with VNTR at position -322 in the promoter region of the TNR2 gene (p = 0.029) but was not associated with the presence of TNFR2 +676 polymorphism. A genetic difference in TNFR2 promoter VNTR may play a major role in susceptibility to IPA infection.

Aspergillosis↗

PubMed2006

Stereoselective synthesis of highly substituted gamma-lactams by the [3+2] annulation of alpha-siloxy allylic silanes with chlorosulfonyl isocyanate.

[reaction: see text] A stereoselective synthesis of gamma-lactams by the [3+2] annulation of alpha-siloxy allylic silanes with N-chlorosulfonyl isocyanate (ClSO(2)NCO) was developed. The use of these allylic silanes allowed for further diastereoselective substitution of the resultant N,O-acetal to give highly substituted gamma-lactams. Oxidation of the silyl group afforded access to complex beta-hydroxy-gamma-lactams.

Catalysis↗

PubMed2005

Ofloxacin-like antibiotics inhibit pneumococcal cell wall-degrading virulence factors.

The search for new drugs against Streptococcus pneumoniae (pneumococcus) is driven by the 1.5 million deaths it causes annually. Choline-binding proteins attach to the pneumococcal cell wall through domains that recognize choline moieties, and their involvement in pneumococcal virulence makes them potential targets for drug development. We have defined chemical criteria involved in the docking of small molecules from a three-dimensional structural library to the major pneumococcal autolysin (LytA) choline binding domain. These criteria were used to identify compounds that could interfere with the attachment of this protein to the cell wall, and several quinolones that fit this framework were found to inhibit the cell wall-degrading activity of LytA. Furthermore, these compounds produced similar effects on other enzymes with different catalytic activities but that contained a similar choline binding domain; that is, autolysin (LytC) and the phage lytic enzyme (Cpl-1). Finally, we resolved the crystal structure of the complex between the choline binding domain of LytA and ofloxacin at a resolution of 2.6 Angstroms. These data constitute an important launch pad from which effective drugs to combat pneumococcal infections can be developed.

Anti-Bacterial Agents↗

PubMed2005

Synthesis of the blood circulating C-terminal fragment of insulin-like growth factor (IGF)-binding protein-4 in its native conformation. Crystallization, heparin and IGF binding, and osteogenic activity.

Insulin-like growth factor-binding proteins play a critical role in a wide variety of important physiological processes. It has been demonstrated that both an N-terminal and a C-terminal fragment of insulin-like growth factor-binding protein-4 exist and accumulate in the circulatory system, these fragments accounting for virtually the whole amino acid sequence of the protein. The circulating C-terminal fragment establishes three disulfide bridges, and the binding pattern of these has recently been defined. Here we show that the monodimensional 1H NMR spectrum of the C-terminal fragment is typical of a protein with a relatively close packed tertiary structure. This fragment can be produced in its native conformation in Escherichia coli, without the requirement of further refolding procedures, when synthesis is coupled to its secretion from the cell. The recombinant protein crystallizes with the unit cell parameters of a hexagonal system. Furthermore, it binds strongly to heparin, acquiring a well defined oligomeric structure that interacts with insulin-like growth factors, and promotes bone formation in cultures of murine calvariae.

Amino Acid Sequence↗

PubMed2004

Growth-regulatory human galectin-1: crystallographic characterisation of the structural changes induced by single-site mutations and their impact on the thermodynamics of ligand binding.

Human galectin-1 is a potent multifunctional effector that participates in specific protein-carbohydrate and protein-protein (lipid) interactions. By determining its X-ray structure, we provide the basis to define the structure of its ligand-binding pocket and to perform rational drug design. We have also analysed whether single-site mutations introduced at some distance from the carbohydrate recognition domain can affect the lectin fold and influence sugar binding. Both the substitutions introduced in the C2S and R111H mutants altered the presentation of the loop, harbouring Asp123 in the common "jelly-roll" fold. The orientation of the side-chain was inverted 180 degrees and the positions of two key residues in the sugar-binding site of the R111H mutant were notably shifted, i.e. His52 and Trp68. Titration calorimetry was used to define the decrease in ligand affinity in both mutants and a significant increase in the entropic penalty was found to outweigh a slight enhancement of the enthalpic contribution. The position of the SH-groups in the galectin appeared to considerably restrict the potential to form intramolecular disulphide bridges and was assumed to be the reason for the unstable lectin activity in the absence of reducing agent. However, this offers no obvious explanation for the improved stability of the C2S mutant under oxidative conditions. The noted long-range effects in single-site mutants are relevant for the functional divergence of closely related galectins and in more general terms, the functionality definition of distinct amino acids.

Amino Acid Sequence↗

PubMed2004

Crystal structure of ovocleidin-17, a major protein of the calcified Gallus gallus eggshell: implications in the calcite mineral growth pattern.

Ovocleidin-17 (OC17) from Gallus gallus is one of the best candidates to control and regulate the deposition of calcium carbonate in the calcified eggshell layer. Here, the crystal structure of monomeric OC17, determined at a resolution of 1.5 A, was refined to a crystallographic R-factor of 20.1%. This is the first protein directly involved in a non-pathological biomineralization process resolved by x-ray diffraction to date. The protein has a mixed alpha/beta structure containing a single C-type lectin-like domain. However, although OC17 shares the conserved scaffold of the C-type lectins, it does not bind carbohydrates. Nevertheless, in vitro OC17 modifies the crystalline habit of calcium carbonate (CaCO3) and the pattern of crystal growth at intervals of 5-200 microg/ml. Determining the three-dimensional structure of OC17 contributes to a better understanding of the biological behavior of structurally related biomolecules and of the mechanisms involved in eggshell and other mineralization processes.

Amino Acid Sequence↗

PubMed2004

Gene design, expression, crystallization and preliminary diffraction analysis of two isolectins from the fungus Coprinus cinereus: a model for studying functional diversification of galectins in the same organism and their evolutionary pathways.

It is the aim of comparative structural biology to define the evolutionarily important traits of protein function and the points of diversification. Consequently, structural analysis, especially of distant members in a family which in this case are lectins involved in cell adhesion and growth regulation in animals (i.e. galectins), is required. For this purpose, recent work has been focused on the first galectins known from outside the animal kingdom. These are the two isolectins from the basidiomycete Coprinus cinereus (inky cap mushroom), termed Cgl-1 and Cgl-2. Additionally, the close similarity (83% deduced amino-acid identity) but the pronounced difference in the expression patterns of these two fungal lectins during fruiting-body formation affords a suitable object for study of the relation of structural difference to the observed functional disparity in the same organism. Both galectins were crystallized after recombinant production. Crystals belong to either the orthorhombic space group C222(1) (Cgl-1) or the monoclinic space group P2(1) (Cgl-2). The latter crystals diffracted to 1.6 A resolution using synchrotron radiation. To solve the phasing problem, a selenomethionine-containing variant of Cgl-1 was designed. Crystals isomorphous to those of the native counterpart were obtained. Their structural analysis will also be crucial to solving the structure of Cgl-2.

Cloning, Molecular↗

PubMed2004

The effect of increasing concentrations of precipitating salts used to crystallize proteins on the structure of the lipidic Q224 cubic phase.

A major obstacle to elucidating the structure of membrane proteins at high resolution is the difficulty of preparing these proteins as well as to grow well-ordered crystals. During the last few years several groups have considered the use of three-dimensional bicontinuous lipidic cubic phases as a possible crystallization matrix for such molecules. In a few cases these studies have been successfully approached by other laboratories, however, a number of questions remain, particularly in regard to the effects of solutes on the phase diagrams of lipid-water systems. In the present work we report the structural behavior of the lipidic Q224 (Pn3m), Q230 (Ia3d) and HII phases systematically studied in the presence of a range of concentrations of various salts and precipitating agents at various pH values. Some of the results reported here have been presented elsewhere Vargas et al. (2000) [Strategies in membrane protein crystallization. Chemical Prospectives in Crystallography of Molecular Biology. International School of Crystallography, NATO-ASI course, Erice (Italy)].

Crystallization↗

PubMed2003

How FMN binds to anabaena apoflavodoxin: a hydrophobic encounter at an open binding site.

Molecular recognition begins when two molecules approach and establish interactions of certain strength. The mechanisms of molecular recognition reactions between biological molecules are not well known, and few systems have been analyzed in detail. We investigate here the reaction between an apoprotein and its physiological cofactor (apoflavodoxin and flavin mononucleotide) that binds reversibly to form a non-covalent complex (flavodoxin) involved in electron transfer reactions. We have analyzed the fast binding reactions between the FMN cofactor (and shorter analogs) and wild type (and nine mutant apoflavodoxins where residues interacting with FMN in the final complex have been replaced). The x-ray structures of two such mutants are reported that show the mutations are well tolerated by the protein. From the calculated microscopic binding rate constants we have performed a Phi analysis of the transition state of complex formation that indicates that the binding starts by interaction of the isoalloxazine-fused rings in FMN with residues of its hydrophobic binding site. In contrast, the phosphate in FMN, known to contribute most to the affinity of the final holoflavodoxin complex, is not bound in the transition state complex. Both the effects of ionic strength and of phosphate concentration on the wild type complex rate constant agree with this scenario. As suggested previously by nmr data, it seems that the isoalloxazine-binding site may be substantially open in solution. Interestingly, although FMN is a charged molecule, electrostatic interactions seem not to play a role in directing the binding, unlike what has been reported for other biological complexes. The binding can thus be best described as a hydrophobic encounter at an open binding site.

Anabaena↗

PubMed2003

Leads for development of new naphthalenesulfonate derivatives with enhanced antiangiogenic activity: crystal structure of acidic fibroblast growth factor in complex with 5-amino-2-naphthalene sulfonate.

Inhibition of angiogenesis-promoting factors such as fibroblast growth factors is considered to be a potential procedure for inhibiting solid tumor growth. Although several peptide-based inhibitors are currently under study, the development of antiangiogenic compounds of small molecular size is a pharmacological goal of considerable interest. We have already shown that certain naphthalene sulfonates constitute minimal functional substitutes of the antiangiogenic compounds of the suramin and suradista family. Using those data as a lead, we have carried out a rational search for new angiogenesis inhibitors that could provide new pharmacological insights for the development of antiangiogenic treatments. The results of the study strongly underline the relevance of the stereochemistry for an efficient inhibition of acidic fibroblast growth factor mitogenic activity by the naphthalene sulfonate family and allow us to formulate rules to aid in searching for new inhibitors and pharmaceutical developments. To provide further leads for such developments and acquire a detailed insight into the basis of the inhibitory activity of the naphthalene sulfonate derivatives, we solved the three-dimensional structure of acidic fibroblast growth factor complexed to 5-amino-2-naphthalenesulfonate, the most pharmacologically promising of the identified inhibitors. The structure shows that binding of this compound would hamper the interaction of acidic fibroblast growth factor with the different components of the cell membrane mitogenesis-triggering complex.

Angiogenesis Inhibitors↗

PubMed2003

A conformational switch between transcriptional repression and replication initiation in the RepA dimerization domain.

Plasmids are natural vectors for gene transfer. In Gram-negative bacteria, plasmid DNA replication is triggered when monomers of an initiator protein (Rep) bind to direct repeats at the origin sequence. Rep dimers, which are inactive as initiators, bind to an inverse repeat operator, repressing transcription of the rep gene. Rep proteins are composed of N-terminal dimerization and C-terminal DNA-binding domains. Activation of Rep is coupled to dimer dissociation, converting the dimerization domain into a second origin-binding module. Although the structure of the monomeric F plasmid initiator (mRepE) has been determined, the molecular nature of Rep activation remains unknown. Here we report the crystal structure of the dimeric N-terminal domain of the pPS10 plasmid initiator (dRepA). dRepA has a winged-helix fold, as does its homologous domain in mRepE. However, dimerization transforms an interdomain loop and beta-strand (monomeric RepE) into an alpha-helix (dimeric RepA). dRepA resemble the C terminus of eukaryotic and archaeal Cdc6, giving clues to the phylogeny of DNA replication initiators.

Amino Acid Sequence↗

PubMed2002

Crystal structure of a prostate kallikrein isolated from stallion seminal plasma: a homologue of human PSA.

Prostate-specific kallikrein, a member of the gene family of serine proteases, was initially discovered in semen and is the most useful serum marker for prostate cancer diagnosis and prognosis. We report the crystal structure at 1.42A resolution of horse prostate kallikrein (HPK). This is the first structure of a serine protease purified from seminal plasma. HPK shares extensive sequence homology with human prostate-specific antigen (PSA), including a predicted chymotrypsin-like specificity, as suggested by the presence of a serine residue at position S1 of the specificity pocket. In contrast to other kallikreins, HPK shows a structurally distinct specificity pocket. Its entrance is blocked by the kallikrein loop, suggesting a possible protective or substrate-selective role for this loop. The HPK structure seems to be in an inactivated state and further processing might be required to allow the binding of substrate molecules. Crystal soaking experiments revealed a binding site for Zn(2+) and Hg(2+), two known PSA inhibitors.

Amino Acid Sequence↗

PubMed2002

Two new crystal forms of the choline-binding domain of the major pneumococcal autolysin: insights into the dynamics of the active homodimer.

Very little is known about the in vivo regulation of the catalytic activity of the major pneumococcal autolysin (LytA), a surface-exposed enzyme that rules the self-destruction of pneumococcal cells through degradation of their peptidoglycan backbone. Two new crystal forms of the cell wall anchoring domain of LytA were obtained, and their structures were solved and refined to 2.4A and 2.8A resolution. The domain is a homodimer with a boomerang-like shape in which the tertiary structure of each monomer is comprised by six independent beta hairpins arranged in a superhelical fashion. Choline molecules at the hydrophobic interface of consecutive hairpins maintain this unique structure. The C-terminal hairpin (last 13 residues of LytA) in the solenoid is responsible for the formation of the catalytically active homodimer. Although the general fold in the structures derived from both crystal forms is essentially the same, two different conformations of the basic homodimer are observed. Biochemical approaches have demonstrated the fundamental role of the 11 C-terminal residues in the catalytic activity of LytA. The studies reported here reveal the importance of some amino acid residues at the C terminus in the determination of the relative distance of the active dimeric form of the autolysin, which appears to be essential for the catalytic activity of this enzyme.

Amino Acid Sequence↗

PubMed2002

Expression, crystallization and preliminary X-ray diffraction studies on the complete choline-binding domain of the major pneumococcal autolysin.

The major pneumococcal autolysin (LytA), a virulence factor of this bacterium, is composed of an amino-terminal catalytic domain plus a carboxyl-terminal choline-binding domain (ChBD). This C-terminal domain, responsible for anchorage to the cell wall, is a tandem of six imperfect 20-residue repeats whose precise ends have been difficult to establish by sequence methods. The reported crystal structure of a shortened C-terminal fragment of the protein suggested that it might contain an additional repeat and thus an additional choline-binding site (ChBS). The complete recombinant choline-binding domain of LytA has now been overexpressed in soluble form using a secreting Escherichia coli strain which facilitates purification with a higher yield. It has been crystallized at room temperature using MPD as the main precipitant. The crystals belong to space group P2(1) and diffract to beyond 3.2 A resolution on a synchrotron-radiation source. The molecular-replacement solution indicates that a new ChBS which fits the topology of the solenoid structure is formed in the N-terminal region.

Binding Sites↗

PubMed2002

Sperm coating mechanism from the 1.8 A crystal structure of PDC-109-phosphorylcholine complex.

Bovine seminal plasma PDC-109 binds to sperm surface choline lipids and promotes sperm capacitation by stimulating the efflux of cholesterol and phospholipids. The structure of PDC-109 with bound phosphorylcholine was solved using MAD data of a single platinum site. Its two globular (40 x 50 x 20 A(3)) Fn2 domains are linked and clustered by a short polypeptide. The choline binding sites lie at the same face of the molecule. Phosphorylcholine binds to the Fn2 domains through a cation-pi interaction between the quaternary ammonium group and a core tryptophan, plus hydrogen bonding between hydroxyls of exposed tyrosines and the phosphate group. The structure of the PDC-109-oPC complex provides a structural ground for the sperm membrane-coating mechanism underlying PDC-109-induced capacitation.

Amino Acid Sequence↗