Structural and Biochemical Characterization of Peroxiredoxin Q beta from Xylella fastidiosa CATALYTIC MECHANISM and HIGH REACTIVITY

The phytopathogenic bacterium Xylella fastidiosa is the etiological agent of various plant diseases. To survive under oxidative stress imposed by the host, microorganisms express antioxidant proteins, including cysteine-based peroxidases named peroxiredoxins. This work is a comprehensive analysis of the catalysis performed by PrxQ from X. fastidiosa (XfPrxQ) that belongs to a peroxiredoxin class still poorly characterized and previously considered as moderately reactive toward hydroperoxides. Contrary to these assumptions, our competitive kinetics studies have shown that the second-order rate constants of the peroxidase reactions of XfPrxQ with hydrogen peroxide and peroxynitrite are in the order of 107 and 106 M(-1) s(-1), respectively, which are as fast as the most efficient peroxidases. The XfPrxQ disulfides were only slightly reducible by dithiothreitol; therefore, the identification of a thioredoxin system as the probable biological reductant of XfPrxQ was a relevant finding. We also showed by site-specific mutagenesis and mass spectrometry that an intramolecular disulfide bond between Cys-47 and Cys-83 is generated during the catalytic cycle. Furthermore, we elucidated the crystal structure of XfPrxQ C47S in which Ser-47 and Cys-83 lie similar to 12.3 angstrom apart. Therefore, significant conformational changes are required for disulfide bond formation. In fact, circular dichroism data indicated that there was a significant redox-dependent unfolding of alpha-helices, which is probably triggered by the peroxidatic cysteine oxidation. Finally, we proposed a model that takes data from this work as well data as from the literature into account.

Structural characterization and comparative analysis of human and piscine cartilage acidic protein (CRTAC1/CRTAC2)

Dissertação de mestrado, Biotecnologia, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2014

Análise da interação entre moléculas desenhadas a partir do ácido anacárdico e a proteína PPAR usando ferramentas in silico

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Biologia Molecular, 2016.

Understanding Co-translational Protein Targeting and Lithium Dendrite Formation through Free Energy Simulations and Coarse-Grained Models

We describe the application of alchemical free energy methods and coarse-grained models to study two key problems: (i) co-translational protein targeting and insertion to direct membrane proteins to the endoplasmic reticulum for proper localization and folding, (ii) lithium dendrite formation during recharging of lithium metal batteries. We show that conformational changes in the signal recognition particle, a central component of the protein targeting machinery, confer additional specificity during the the recognition of signal sequences. We then develop a three-dimensional coarse-grained model to study the long-timescale dynamics of membrane protein integration at the translocon and a framework for the calculation of binding free energies between the ribosome and translocon. Finally, we develop a coarse-grained model to capture the dynamics of lithium deposition and dissolution at the electrode interface with time-dependent voltages to show that pulse plating and reverse pulse plating methods can mitigate dendrite growth.

Liquid Mixtures Involving Hydrogenated and Fluorinated Alcohols: Thermodynamics, Spectroscopy, and Simulation

This article reports a combined thermodynamic, spectroscopic, and computational study on the interactions and structure of binary mixtures of hydrogenated and fluorinated substances that simultaneously interact through strong hydrogen bonding. Four binary mixtures of hydrogenated and fluorinated alcohols have been studied, namely, (ethanol + 2,2,2-trifluoroethanol (TFE)), (ethanol + 2,2,3,3,4,4,4-heptafluoro-1-butanol), (1-butanol (BuOH) + TFE), and (BuOH + 2,2,3,3,4,4,4-heptafluoro-1-butanol). Excess molar volumes and vibrational spectra of all four binary mixtures have been measured as a function of composition at 298 K, and molecular dynamics simulations have been performed. The systems display a complex behavior when compared with mixtures of hydrogenated alcohols and mixtures of alkanes and perfluoroalkanes. The combined analysis of the results from different approaches indicates that this results from a balance between preferential hydrogen bonding between the hydrogenated and fluorinated alcohols and the unfavorable dispersion forces between the hydrogenated and fluorinated chains. As the chain length increases, the contribution of dispersion increases and overcomes the contribution of H-bonds. In terms of the liquid structure, the simulations suggest the possibility of segregation between the hydrogenated and fluorinated segments, a hypothesis corroborated by the spectroscopic results. Furthermore, a quantitative analysis of the infrared spectra reveals that the presence of fluorinated groups induces conformational changes in the hydrogenated chains from the usually preferred all-trans to more globular arrangements involving gauche conformations. Conformational rearrangements at the CCOH dihedral angle upon mixing are also disclosed by the spectra.

Studies on the Hippo Pathway: new insights about a multifaceted signalling

The Hippo pathway is a well-known master regulator of cell growth and proliferation. Many studies have shed light on the centrality of Hippo functions, as this signalling is able to respond to different stimuli and translate them into distinct transcriptional outputs. Therefore, it is clearly implicated in a number of important processes, which alteration has consequences on the correct specification of the single cell, as well as the whole tissue. Even if the core of the signalling has been extensively characterized, it remains unclear which are the “co-workers” that permit the Hippo pathway to answer to so many different stimuli and act as a coordinator of the growth/differentiation balance. Taking advantage of the Drosophila model, which has witnessed most of the discoveries on this signalling pathway, this thesis aims to add some new knowledge about the Hippo pathway molecular mechanisms in different contexts, from development to disease. In the first part I studied the dynamics of the Hippo core kinase protein Warts in the development of the pupal eye. I have found out a critical time point in which the expression and the localization of Warts change suddenly, suggesting the intervention of upstream regulators modulating its activity in an extremely narrow time window. The second goal was investigating the role of the Hippo pathway in the neurodegenerative Gaucher disease. Indeed, I have produced some preliminary results which demonstrate a growth deficit associated with a massive reduction of some Yki targets, supporting a Hyper-Hippo condition underlying this neuropathic syndrome. Finally, I have evaluated the transcription factor Orthodenticle as a co-factor of Yorkie in driving tissue overgrowth, and my findings support a model of interaction of these two molecules based on Yki conformational changes. Altogether, my results lay the foundation for new important studies on the molecular mechanisms ruling Hippo pathway activity.

Ions and Small Molecules as Modulators of F1FO-ATPase, Mitochondrial Bioenergetics and Cell Metabolism

The properties of the mitochondrial F1FO-ATPase activated by the natural cofactor Mg2+ or by Ca2+, were studied, mainly on heart mitochondria from swine, widely used in translational medicine. The Ca2+ driven conformational changes in the F1FO-ATPase form the mitochondrial permeability transition pore (mPTP), which triggers regulated cell death and is involved in severe pathologies. The Ca2+-activated F1FO-ATPase hydrolyzes ATP with kinetics slightly different from those of the Mg2+-ATPase. Known F1-ATPase inhibitors inhibit both the Ca2+-activated F1FO-ATPase and the mPTP formation strengthening the molecular link between them. The different Gd3+ effects on the Ca2+- and Mg2+-activated F1FO-ATPases confirm their difference as also phenylglyoxal which preferentially inhibits the Ca2+-activated F1FO-ATPase. The effects of phenylarsine and dibromobimane, which interact with differently distant Cys thiols, show that mPTP opening is ruled by nearby or distant dithiols. Bergamot polyphenols and melatonin inhibit the mPTP and ROS formation. H2S, a known cardiovascular protector, unaffects the F1FO-ATPase, but inhibits Ca2+ absorption and indirectly the mPTP, both in swine heart and mussel midgut gland mitochondria. New generation triazoles inhibit the Ca2+-activated F1FO-ATPase and the mPTP, but unaffect the Mg2+-activated F1FOATPase. In parallel, the energy metabolism was investigated in mammalian cells. In boar sperm ATP is mainly produced by mitochondrial oxidative phosphorylation (OXPHOS), even if it decreases over time because of less active mitochondria. Insufficient ATP may induce sperm dysfunction. Also, canine mesenchymal stem cells rely on OXPHOS; those from umbilical cord which produce more ATP than those from adipose tissue, seem preferable for transplant studies. The intestinal porcine enterocyte cell line IPEC-J2, used for human gut research, responds to different fetal bovine serum concentrations by remodeling OXPHOS without altering the bioenergetic parameters. The IPEC-J2 bioenergetics is modulated by Vitamin K vitamers. These data shoulder cell bioenergetics as precious tool for medical research.

Incorporation of a S-glycosidic linkage into a glyconucleoside changes the conformational preference of both furanose sugars

A glyconucleoside containing a thioglycoside linkage, namely 1-(3-S-beta-D-ribofuranosyl-2,3-dideoxy-3-thio-beta-D-ribofuranosyl)-thy mine, has been prepared through condensation of a suitably protected derivative of 3'-thiothymidine with an activated ribose sugar. NMR has been used to study the conformation of the S-disaccharide and the unmodified O-disaccharide. A full pseudorotational analysis showed that for the S-disaccharide, the ribose and deoxy ribose sugars have a preference for the south and north pucker, respectively; which is the reverse of what is seen for the O-disaccharide. (c) 2006 Elsevier Ltd. All rights reserved.

Changes in optical properties caused by UV-irradiation of aquatic humic substances from the amazon river basin: Seasonal variability evaluation

Aquatic humic substances (AHS) isolated from two characteristic seasons of the Negro river, winter and summer corresponding to floody and dry periods, were structurally characterized by (13)C nuclear magnetic ressonance. Subsequently, AHS aqueous solutions were irradiated with a polychromatic lamp (290-475 nm) and monitored by its total organic carbon (TOC) content, ultraviolet-visible (UV-vis) absorbance, fluorescence and Fourier transformed infrared spectroscopy (FTIR). As a result, a photobleaching upto 80% after irradiation of 48 h was observed. Conformational rearrangements and formation of low molecular complexity structures were formed during the irradiation, as deduced from the pH decrement and the fluorescence shifting to lower wavelengths. Additionally a significant mineralization with the formation Of CO(2), CO, and inorganic carbon compounds was registered, as assumed by TOC losses of up to 70%. The differences in photodegradation between samples expressed by photobleaching efficiency were enhanced in the summer sample and related to its elevated aromatic content. Aromatic structures are assumed to have high autosensitization capacity effects mediated by the free radical generation from quinone and phenolic moieties.

Conformational selection of inhibitors and substrates by proteolytic enzymes: Implications for drug design and polypeptide processing

Inhibitors of proteolytic enzymes (proteases) are emerging as prospective treatments for diseases such as AIDS and viral infections, cancers, inflammatory disorders, and Alzheimer's disease. Generic approaches to the design of protease inhibitors are limited by the unpredictability of interactions between, and structural changes to, inhibitor and protease during binding. A computer analysis of superimposed crystal structures for 266 small molecule inhibitors bound to 48 proteases (16 aspartic, 17 serine, 8 cysteine, and 7 metallo) provides the first conclusive proof that inhibitors, including substrate analogues, commonly bind in an extended beta-strand conformation at the active sites of all these proteases. Representative superimposed structures are shown for (a) multiple inhibitors bound to a protease of each class, (b) single inhibitors each bound to multiple proteases, and (c) conformationally constrained inhibitors bound to proteases. Thus inhibitor/substrate conformation, rather than sequence/composition alone, influences protease recognition, and this has profound implications for inhibitor design. This conclusion is supported by NMR, CD, and binding studies for HIV-1 protease inhibitors/ substrates which, when preorganized in an extended conformation, have significantly higher protease affinity. Recognition is dependent upon conformational equilibria since helical and turn peptide conformations are not processed by proteases. Conformational selection explains the resistance of folded/structured regions of proteins to proteolytic degradation, the susceptibility of denatured proteins to processing, and the higher affinity of conformationally constrained 'extended' inhibitors/substrates for proteases. Other approaches to extended inhibitor conformations should similarly lead to high-affinity binding to a protease.

Transthyretin Amyloidosis: Chaperone Concentration Changes and Increased Proteolysis in the Pathway to Disease

Transthyretin amyloidosis is a conformational pathology characterized by the extracellular formation of amyloid deposits and the progressive impairment of the peripheral nervous system. Point mutations in this tetrameric plasma protein decrease its stability and are linked to disease onset and progression. Since non-mutated transthyretin also forms amyloid in systemic senile amyloidosis and some mutation bearers are asymptomatic throughout their lives, non-genetic factors must also be involved in transthyretin amyloidosis. We discovered, using a differential proteomics approach, that extracellular chaperones such as fibrinogen, clusterin, haptoglobin, alpha-1-anti-trypsin and 2-macroglobulin are overrepresented in transthyretin amyloidosis. Our data shows that a complex network of extracellular chaperones are over represented in human plasma and we speculate that they act synergistically to cope with amyloid prone proteins. Proteostasis may thus be as important as point mutations in transthyretin amyloidosis.

Switch-peptides: From conformational studies to Alzheimer's disease

Studies on designed peptides that exhibit high tendencies for medium-induced conformational transitions have recently attracted much attention because structural changes are considered as molecular key processes in degenerative diseases. The experimental access to these events has been limited so far mainly due to the intrinsic tendency of the involved polypeptides for self-association and aggregation, e.g. amyloid P plaque formation, thought to be at the origin of Alzheimer's disease. We have developed a new concept termed 'switch-peptides' which allows the controlled onset of polypeptide folding and misfolding in vitro and in vivo, starting from a soluble, non-toxic precursor molecule. As a major feature, the folding process is initiated by enzyme-triggered N,O-acyl migrations restoring the native peptide backbone in situ. As the folding is set off in the moment of creating the bioactive molecule ('in statu nascendi', ISN), our concept allows for the first time the investigation of the early steps of protein misfolding as relevant in degenerative diseases, opening new perspectives for the rational design of therapeutically relevant compounds.

Thiol and Sulfenic Acid Oxidation of AhpE, the One-Cysteine Peroxiredoxin from Mycobacterium tuberculosis: Kinetics, Acidity Constants, and Conformational Dynamics

Drug resistance and virulence of Mycobacterium tuberculosis are partially related to the pathogen`s antioxidant systems. Peroxide detoxification in this bacterium is achieved by the heme-containing catalase peroxidase and different two-cysteine peroxiredoxins. M. tuberculosis genome also codifies for a putative one-cysteine peroxiredoxin, alkyl hydroperoxide reductase E (MtAhpE). Its expression was previously demonstrated at a transcriptional level, and the crystallographic structure of the recombinant protein was resolved under reduced and oxidized states. Herein, we report that the conformation of MtAhpE changed depending on its single cysteine redox state, as reflected by different tryptophan fluorescence properties and changes in quaternary structure. Dynamics of fluorescence changes, complemented by competition kinetic assays, were used to perform protein functional studies. MtAhE reduced peroxynitrite 2 orders of magnitude faster than hydrogen peroxide (1.9 x 10(7) M(-1) s(-1) vs 8.2 x 10(4) M(-1) s(-1) at pH 7.4 and 25 degrees C, respectively). The latter also caused cysteine overoxidation to sulfinic acid, but at much slower rate constant (40 M(-1) s(-1)). The pK(a) of the thiol in the reduced enzyme was 5.2, more than one unit lower than that of the sulfenic acid in the oxidized enzyme. The pH profile of hydrogen peroxide-mediated thiol and sulfenic acid oxidations indicated thiolate and sulfenate as the reacting species. The formation of sulfenic acid as well as the catalytic peroxidase activity of MtAhpE was demonstrated using the artificial reducing substrate thionitrobenzoate. Taken together, our results indicate that MtAhpE is a relevant component in the antioxidant repertoire of M. tuberculosis probably involved in peroxide and specially peroxynitrite detoxification.

Low-Resolution Molecular Models Reveal the Oligomeric State of the PPAR and the Conformational Organization of Its Domains in Solution

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

Labaditin, a cyclic peptide with rich biotechnological potential: preliminary toxicological studies and structural changes in water and lipid membrane environment

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)