Differential regulation of RasGAPs in cancer

Ever since their discovery as cellular counterparts of viral oncogenes more than 25 years ago, much progress has been made in understanding the complex networks of signal transduction pathways activated by oncogenic Ras mutations in human cancers. The activity of Ras is regulated by nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), and much emphasis has been put into the biochemical and structural analysis of the Ras/GAP complex. The mechanisms by which GAPs catalyze Ras-GTP hydrolysis have been clarified and revealed that oncogenic Ras mutations confer resistance to GAPs and remain constitutively active. However, it is yet unclear how cells coordinate the large and divergent GAP protein family to promote Ras inactivation and ensure a certain biological response. Different domain arrangements in GAPs to create differential protein-protein and protein-lipid interactions are probably key factors determining the inactivation of the 3 Ras isoforms H-, K-, and N-Ras and their effector pathways. In recent years, in vitro as well as cell- and animal-based studies examining GAP activity, localization, interaction partners, and expression profiles have provided further insights into Ras inactivation and revealed characteristics of several GAPs to exert specific and distinct functions. This review aims to summarize knowledge on the cell biology of RasGAP proteins that potentially contributes to differential regulation of spatiotemporal Ras signaling.

Proof of concept for microarray-based detection of DNA-binding oncogenes in cell extracts.

The function of DNA-binding proteins is controlled not just by their abundance, but mainly at the level of their activity in terms of their interactions with DNA and protein targets. Moreover, the affinity of such transcription factors to their target sequences is often controlled by co-factors and/or modifications that are not easily assessed from biological samples. Here, we describe a scalable method for monitoring protein-DNA interactions on a microarray surface. This approach was designed to determine the DNA-binding activity of proteins in crude cell extracts, complementing conventional expression profiling arrays. Enzymatic labeling of DNA enables direct normalization of the protein binding to the microarray, allowing the estimation of relative binding affinities. Using DNA sequences covering a range of affinities, we show that the new microarray-based method yields binding strength estimates similar to low-throughput gel mobility-shift assays. The microarray is also of high sensitivity, as it allows the detection of a rare DNA-binding protein from breast cancer cells, the human tumor suppressor AP-2. This approach thus mediates precise and robust assessment of the activity of DNA-binding proteins and takes present DNA-binding assays to a high throughput level.

Redes metalorgânicas e suas aplicações em catálise

Metal-organic frameworks (MOFs) form a new class of materials with well-defined yet tunable properties. These are crystalline, highly porous and exhibit strong metal-ligand interactions. Importantly, their physical and chemical properties, including pore size, pore structure, acidity, and magnetic and optical characteristics, can be tailored by choosing the appropriate ligands and metal precursors. Here we review the key aspects of synthesis and characterization of MOFs, focusing on lanthanide-based and vanadium-based materials. We also outline some of their applications in catalysis and materials science.

Nonconventional amide bond formation catalysis: programming enzyme specificity with substrate mimetics

This article reports on the design and characteristics of substrate mimetics in protease-catalyzed reactions. Firstly, the basis of protease-catalyzed peptide synthesis and the general advantages of substrate mimetics over common acyl donor components are described. The binding behavior of these artificial substrates and the mechanism of catalysis are further discussed on the basis of hydrolysis, acyl transfer, protein-ligand docking, and molecular dynamics studies on the trypsin model. The general validity of the substrate mimetic concept is illustrated by the expansion of this strategy to trypsin-like, glutamic acid-specific, and hydrophobic amino acid-specific proteases. Finally, opportunities for the combination of the substrate mimetic strategy with the chemical solid-phase peptide synthesis and the use of substrate mimetics for non-peptide organic amide synthesis are presented.

RNA and DNA aptamers as potential tools to prevent cell adhesion in disease

Recent research has shown that receptor-ligand interactions between surfaces of communicating cells are necessary prerequisites for cell proliferation, cell differentiation and immune defense. Cell-adhesion events have also been proposed for pathological conditions such as cancer growth, metastasis, and host-cell invasion by parasites such as Trypanosoma cruzi. RNA and DNA aptamers (aptus = Latin, fit) that have been selected from combinatorial nucleic acid libraries are capable of binding to cell-adhesion receptors leading to a halt in cellular processes induced by outside signals as a consequence of blockage of receptor-ligand interactions. We outline here a novel approach using RNA aptamers that bind to T. cruzi receptors and interrupt host-cell invasion in analogy to existing procedures of blocking selectin adhesion and function in vitro and in vivo.

Membrane proteins: functional and structural studies using reconstituted proteoliposomes and 2-D crystals

Reconstitution of membrane proteins into lipid bilayers is a powerful tool to analyze functional as well as structural areas of membrane protein research. First, the proper incorporation of a purified membrane protein into closed lipid vesicles, to produce proteoliposomes, allows the investigation of transport and/or catalytic properties of any membrane protein without interference by other membrane components. Second, the incorporation of a large amount of membrane proteins into lipid bilayers to grow crystals confined to two dimensions has recently opened a new way to solve their structure at high resolution using electron crystallography. However, reconstitution of membrane proteins into functional proteoliposomes or 2-D crystallization has been an empirical domain, which has been viewed for a long time more like "black magic" than science. Nevertheless, in the last ten years, important progress has been made in acquiring knowledge of lipid-protein-detergent interactions and has permitted to build upon a set of basic principles that has limited the empirical approach of reconstitution experiments. Reconstitution strategies have been improved and new strategies have been developed, facilitating the success rate of proteoliposome formation and 2-D crystallization. This review deals with the various strategies available to obtain proteoliposomes and 2-D crystals from detergent-solubilized proteins. It gives an overview of the methods that have been applied, which may be of help for reconstituting more proteins into lipid bilayers in a form suitable for functional studies at the molecular level and for high-resolution structural analysis.

Développements et applications de méthodes computationnelles pour l'étude de l'agrégation des protéines amyloïdes

Les protéines sont au coeur de la vie. Ce sont d'incroyables nanomachines moléculaires spécialisées et améliorées par des millions d'années d'évolution pour des fonctions bien définies dans la cellule. La structure des protéines, c'est-à-dire l'arrangement tridimensionnel de leurs atomes, est intimement liée à leurs fonctions. L'absence apparente de structure pour certaines protéines est aussi de plus en plus reconnue comme étant tout aussi cruciale. Les protéines amyloïdes en sont un exemple marquant : elles adoptent un ensemble de structures variées difficilement observables expérimentalement qui sont associées à des maladies neurodégénératives. Cette thèse, dans un premier temps, porte sur l'étude structurelle des protéines amyloïdes bêta-amyloïde (Alzheimer) et huntingtine (Huntington) lors de leur processus de repliement et d'auto-assemblage. Les résultats obtenus permettent de décrire avec une résolution atomique les interactions des ensembles structurels de ces deux protéines. Concernant la protéine bêta-amyloïde (AB), nos résultats identifient des différences structurelles significatives entre trois de ses formes physiologiques durant ses premières étapes d'auto-assemblage en environnement aqueux. Nous avons ensuite comparé ces résultats avec ceux obtenus au cours des dernières années par d'autres groupes de recherche avec des protocoles expérimentaux et de simulations variés. Des tendances claires émergent de notre comparaison quant à l'influence de la forme physiologique de AB sur son ensemble structurel durant ses premières étapes d'auto-assemblage. L'identification des propriétés structurelles différentes rationalise l'origine de leurs propriétés d'agrégation distinctes. Par ailleurs, l'identification des propriétés structurelles communes offrent des cibles potentielles pour des agents thérapeutiques empêchant la formation des oligomères responsables de la neurotoxicité. Concernant la protéine huntingtine, nous avons élucidé l'ensemble structurel de sa région fonctionnelle située à son N-terminal en environnement aqueux et membranaire. En accord avec les données expérimentales disponibles, nos résultats sur son repliement en environnement aqueux révèlent les interactions dominantes ainsi que l'influence sur celles-ci des régions adjacentes à la région fonctionnelle. Nous avons aussi caractérisé la stabilité et la croissance de structures nanotubulaires qui sont des candidats potentiels aux chemins d'auto-assemblage de la région amyloïde de huntingtine. Par ailleurs, nous avons également élaboré, avec un groupe d'expérimentateurs, un modèle détaillé illustrant les principales interactions responsables du rôle d'ancre membranaire de la région N-terminal, qui sert à contrôler la localisation de huntingtine dans la cellule. Dans un deuxième temps, cette thèse porte sur le raffinement d'un modèle gros-grain (sOPEP) et sur le développement d'un nouveau modèle tout-atome (aaOPEP) qui sont tous deux basés sur le champ de force gros-grain OPEP, couramment utilisé pour l'étude du repliement des protéines et de l'agrégation des protéines amyloïdes. L'optimisation de ces modèles a été effectuée dans le but d'améliorer les prédictions de novo de la structure de peptides par la méthode PEP-FOLD. Par ailleurs, les modèles OPEP, sOPEP et aaOPEP ont été inclus dans un nouveau code de dynamique moléculaire très flexible afin de grandement simplifier leurs développements futurs.

Targeted Stimuli-Responsive Dextran Conjugates for Doxorubicin Delivery to Hepatocytes

A targeted, stimuli-responsive, polymeric drug delivery vehicle is being developed in our lab to help alleviate severe side-effects caused by narrow therapeutic window drugs. Targeting specific cell types or organs via proteins, specifically, lectin-mediated targeting holds potential due to the high specificity and affinity of receptor-ligand interactions, rapid internalization, and relative ease of processing. Dextran, a commercially available, biodegradable polymer has been conjugated to doxorubicin and galactosamine to target hepatocytes in a three-step, one-pot synthesis. The loading of doxorubicin and galactose on the conjugates was determined by absorbance at 485 nm and elemental analysis, respectively. Conjugation efficiency based on the amount loaded of each reactant varies from 20% to 50% for doxorubicin and from 2% to 20% for galactosamine. Doxorubicin has also been attached to dextran through an acid-labile hydrazide bond. Doxorubicin acts by intercalating with DNA in the nuclei of cells. The fluorescence of doxorubicin is quenched when it binds to DNA. This allows a fluorescence-based cell-free assay to evaluate the efficacy of the polymer conjugates where we measure the fluorescence of doxorubicin and the conjugates in increasing concentrations of calf thymus DNA. Fluorescence quenching indicates that our conjugates can bind to DNA. The degree of binding increases with polymer molecular weight and substitution of doxorubicin. In cell culture experiments with hepatocytes, the relative uptake of polymer conjugates was evaluated using flow cytometry, and the killing efficiency was determined using the MTT cell proliferation assay. We have found that conjugate uptake is much lower than that of free doxorubicin. Lower uptake of conjugates may increase the maximum dose of drug tolerated by the body. Also, non-galactosylated conjugate uptake is lower than that of the galactosylated conjugate. Microscopy indicates that doxorubicin localizes almost exclusively at the nucleus, whereas the conjugates are present throughout the cell. Doxorubicin linked to dextran through a hydrazide bond was used to achieve improved killing efficiency. Following uptake, the doxorubicin dissociates from the polymer in an endosomal compartment and diffuses to the nucleus. The LC₅₀ of covalently linked doxorubicin is 7.4 μg/mL, whereas that of hydrazide linked doxorubicin is 4.4 μg/mL.

Molecular dynamics simulations of proteins: can the explicit water model be varied?

In molecular mechanics simulations of biological systems, the solvation water is typically represented by a default water model which is an integral part of the force field. Indeed, protein nonbonding parameters are chosen in order to obtain a balance between water-water and protein-water interactions and hence a reliable description of protein solvation. However, less attention has been paid to the question of whether the water model provides a reliable description of the water properties under the chosen simulation conditions, for which more accurate water models often exist. Here we consider the case of the CHARMM protein force field, which was parametrized for use with a modified TIP3P model. Using quantum mechanical and molecular mechanical calculations, we investigate whether the CHARMM force field can be used with other water models: TIP4P and TIP5P. Solvation properties of N-methylacetamide (NMA), other small solute molecules, and a small protein are examined. The results indicate differences in binding energies and minimum energy geometries, especially for TIP5P, but the overall description of solvation is found to be similar for all models tested. The results provide an indication that molecular mechanics simulations with the CHARMM force field can be performed with water models other than TIP3P, thus enabling an improved description of the solvent water properties.

Structural and thermodynamic analysis of thrombin:suramin interaction in solution and crystal phases

Suramin is a hexasulfonated naphthylurea which has been recently characterized as a non-competitive inhibitor of human alpha-thrombin activity over fibrinogen, although its binding site and mode of interaction with the enzyme remain elusive. Here, we determined two X-ray structure of the thrombin: suramin complex, refined at 2.4 angstrom resolution. While a single thrombin: suramin complex was found in the asymmetric unit cell of the crystal, some of the crystallographic contacts with symmetrically related molecules are mediated by both the enzyme and the ligand. Molecular dynamics simulations with the 1:1 complex demonstrate a large rearrangement of suramin in the complex, but with the protein scaffold and the more extensive protein-ligand regions keep unchanged. Small-angle X-ray scattering measurements at high micromolar concentration demonstrate a suramin-induced dimerization of the enzyme. These data indicating a dissimilar binding mode in the monomeric and oligomeric states, with a monomeric, 1:1 complex to be more likely to exist at the thrombin physiological, nanomolar concentration range. Collectively, close understanding on the structural basis for interaction is given which might establish a basis for design of suramin analogues targeting thrombin. Crown Copyright (C) 2009 Published by Elsevier B.V. All rights reserved.

Crystallographic Structure of Phosphofructokinase-2 from Escherichia coli in Complex with Two ATP Molecules. Implications for Substrate Inhibition

Phosphofructokinase-1 and -2 (Pfk-1 and Pfk-2, respectively) from Escherichia coli belong to different homologous superfamilies. However, in spite of the lack of a common ancestor, they share the ability to catalyze the same reaction and are inhibited by the substrate MgATP. Pfk-2, an ATP-dependent 6-phosphofructokinase member of the ribokinase-like superfamily, is a homodimer of 66 kDa subunits whose oligomerization state is necessary for catalysis and stability. The presence of MgATP favors the tetrameric form of the enzyme. In this work, we describe the structure of Pfk-2 in its inhibited tetrameric form, with each subunit bound to two ATP molecules and two Mg ions. The present structure indicates that substrate inhibition occurs due to the sequential binding of two MgATP molecules per subunit, the first at the usual site occupied by the nucleotide in homologous enzymes and the second at the allosteric site, making a number of direct and Mg-mediated interactions with the first. Two configurations are observed for the second MgATP, one of which involves interactions with Tyr23 from the adjacent subunit in the dimer and the other making an unusual non-Watson-Crick base pairing with the adenine in the substrate ATP. The oligomeric state observed in the crystal is tetrameric, and some of the structural elements involved in the binding of the Substrate and allosteric ATPs are also participating in the dimer-dimer interface. This structure also provides the grounds to compare analogous features of the nonhomologous phosphofructokinases from E. coli. (C) 2008 Elsevier Ltd. All rights reserved.

Double stranded DNA-binding studies of potential Rhodium(ll) antitumor complexes

In 1952, Dwyer and coworkers began testing a series of metal complexes for potential inhibition of cancer cell proliferation in animals.[l] The complexes tested were unsuitable for such studies due to their high toxicity. Therefore, no further work was done on the project. However, in 1965, Rosenberg and coworkers revisited the possibility of potential metal-based drugs. Serendipitously, they discovered that cis-diamminedichloroplatinum(lI) (cisplatin) inhibits cell division in E. coli.[2] Further studies of this and other platinum compounds revealed inhibition of tumor cell lines sarcoma 180 and leukemia LI2l0 in mice.[l] Cisplatin was approved by the Food and Drug Administration in 1970 as a chemical chemotherapeutic agent in the treatment of cancer. The drug has primarily been used in the treatment of testicular and ovarian cancers, although the powerful chemotherapeutic properties of the compound indicate use against a variety of other cancers.[3] The toxicity of this compound, however, warrants the development of other metal-based potential antitumor agents. The success of cisplatin, a transition-metal-based chemotherapeutic, opened the doors to a host of research on the antitumor effects of other transition-metal complexes. Beginning in the 1970s, researchers looked to rhodium for potential use in antitumor complexes. Dirhodium complexes with bridging equatorial ligands (Figure I) were the primary focus for this research. The overwhelming majority of these complexes were dirhodium(II) carboxylate complexes, containing two rhodium(II) centers, four equatorial ligands in a lantero formation around the metal center, and an axial ligand on either end. The family of complexes in Figure 1 will be referred to as dirhodium(II) carboxylate complexes. The dirhodium centers are each d? with a metal-metal bond between them. Although d? atoms are paramagnetic, the two unpaired electrons pair to make the complex diamagnetic. The basic formula of the dirhodium(lI) carboxylate complexes is Rh?(RCOO)?(L)? with R being methyl, ethyl, propyl, or butyl groups and L being water or the solvent in which the complex was crystalized. Of these dirbodium(II) carboxylate complexes, our research focuses on Rb la and two other similar complexes Rh2 and Rh3 (Figure 2). Rh2 is an activated form of Rhla, with four acetonitrile groups in place of two of the bidentate acetate ligands. Rh3 is similar to Rhla, with trifluoromethyl groups in place of the methyl groups on the acetate ligands.

Quantitative Analysis of Diepoxybutane Damage within the Chicken β-globin Domain

In industrial polymer and synthetic rubber production facilities, workers are exposed to 1,3-butadiene. This compound is converted in vivo to 1,2,3,4-diepoxybutane (DEB) and has been linked to increased incidences of cancer in these individuals. Carcinogenesis has been attributed to formation of DEB induced DNA interstrand cross-links. Previous studies have demonstrated that DEB cross-links deoxyguanosine residues within 5'-GNC sequences in synthetic DNA, in restriction fragments, and in defined sequence nucleosomes. The current study utilized the polymerase chain reaction (PCR) to examine DEB damage frequencies within nuclear genes, found within "open" regions of chromatin, as compared to regions of unexpressed sequence that reside in tightly packed, "closed" chromatin, to more closely model DEB reactivity in vivo. These initial studies have been performed in chicken liver homogenates. Preliminarily, we have found a dose-dependent DEB lesion-forming response within "open" chromatin. DEB appears to have little-to-no effect upon regions of "closed" chromatin.

Towards the prediction of essential genes by integration of network topology, cellular localization and biological process information

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Molecular models of NS3 protease variants of the Hepatitis C virus

Background: Hepatitis C virus (HCV) currently infects approximately three percent of the world population. In view of the lack of vaccines against HCV, there is an urgent need for an efficient treatment of the disease by an effective antiviral drug. Rational drug design has not been the primary way for discovering major therapeutics. Nevertheless, there are reports of success in the development of inhibitor using a structure-based approach. One of the possible targets for drug development against HCV is the NS3 protease variants. Based on the three-dimensional structure of these variants we expect to identify new NS3 protease inhibitors. In order to speed up the modeling process all NS3 protease variant models were generated in a Beowulf cluster. The potential of the structural bioinformatics for development of new antiviral drugs is discussed.Results: the atomic coordinates of crystallographic structure 1CU1 and 1DY9 were used as starting model for modeling of the NS3 protease variant structures. The NS3 protease variant structures are composed of six subdomains, which occur in sequence along the polypeptide chain. The protease domain exhibits the dual beta-barrel fold that is common among members of the chymotrypsin serine protease family. The helicase domain contains two structurally related beta-alpha-beta subdomains and a third subdomain of seven helices and three short beta strands. The latter domain is usually referred to as the helicase alpha-helical subdomain. The rmsd value of bond lengths and bond angles, the average G-factor and Verify 3D values are presented for NS3 protease variant structures.Conclusions: This project increases the certainty that homology modeling is an useful tool in structural biology and that it can be very valuable in annotating genome sequence information and contributing to structural and functional genomics from virus. The structural models will be used to guide future efforts in the structure-based drug design of a new generation of NS3 protease variants inhibitors. All models in the database are publicly accessible via our interactive website, providing us with large amount of structural models for use in protein-ligand docking analysis.