Publications by authors named "João V Rodrigues"

29 Publications

  • Page 1 of 1

Development of antibacterial compounds that constrain evolutionary pathways to resistance.

Elife 2021 07 19;10. Epub 2021 Jul 19.

Department of Chemistry and Chemical Biology, Harvard University, Cambridge, United States.

Antibiotic resistance is a worldwide challenge. A potential approach to block resistance is to simultaneously inhibit WT and known escape variants of the target bacterial protein. Here, we applied an integrated computational and experimental approach to discover compounds that inhibit both WT and trimethoprim (TMP) resistant mutants of dihydrofolate reductase (DHFR). We identified a novel compound (CD15-3) that inhibits WT DHFR and its TMP resistant variants L28R, P21L and A26T with IC 50-75 µM against WT and TMP-resistant strains. Resistance to CD15-3 was dramatically delayed compared to TMP in in vitro evolution. Whole genome sequencing of CD15-3-resistant strains showed no mutations in the target folA locus. Rather, gene duplication of several efflux pumps gave rise to weak (about twofold increase in IC) resistance against CD15-3. Altogether, our results demonstrate the promise of strategy to develop evolution drugs - compounds which constrain evolutionary escape routes in pathogens.
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http://dx.doi.org/10.7554/eLife.64518DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8331180PMC
July 2021

Adaptation to mutational inactivation of an essential gene converges to an accessible suboptimal fitness peak.

Elife 2019 10 1;8. Epub 2019 Oct 1.

Department of Chemistry and Chemical Biology, Harvard University, Cambridge, United States.

The mechanisms of adaptation to inactivation of essential genes remain unknown. Here we inactivate dihydrofolate reductase (DHFR) by introducing D27G,N,F chromosomal mutations in a key catalytic residue with subsequent adaptation by an automated serial transfer protocol. The partial reversal G27- > C occurred in three evolutionary trajectories. Conversely, in one trajectory for D27G and in all trajectories for D27F,N strains adapted to grow at very low metabolic supplement (folAmix) concentrations but did not escape entirely from supplement auxotrophy. Major global shifts in metabolome and proteome occurred upon DHFR inactivation, which were partially reversed in adapted strains. Loss-of-function mutations in two genes, and , ensured adaptation to low folAmix by rerouting the 2-Deoxy-D-ribose-phosphate metabolism from glycolysis towards synthesis of dTMP. Multiple evolutionary pathways of adaptation converged to a suboptimal solution due to the high accessibility to loss-of-function mutations that block the path to the highest, yet least accessible, fitness peak.
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http://dx.doi.org/10.7554/eLife.50509DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6828540PMC
October 2019

Chimeric dihydrofolate reductases display properties of modularity and biophysical diversity.

Protein Sci 2019 07 30;28(7):1359-1367. Epub 2019 May 30.

Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts.

While reverse genetics and functional genomics have long affirmed the role of individual mutations in determining protein function, there have been fewer studies addressing how large-scale changes in protein sequences, such as in entire modular segments, influence protein function and evolution. Given how recombination can reassort protein sequences, these types of changes may play an underappreciated role in how novel protein functions evolve in nature. Such studies could aid our understanding of whether certain organismal phenotypes related to protein function-such as growth in the presence or absence of an antibiotic-are robust with respect to the identity of certain modular segments. In this study, we combine molecular genetics with biochemical and biophysical methods to gain a better understanding of protein modularity in dihydrofolate reductase (DHFR), an enzyme target of antibiotics also widely used as a model for protein evolution. We replace an integral α-helical segment of Escherichia coli DHFR with segments from a number of different organisms (many nonmicrobial) and examine how these chimeric enzymes affect organismal phenotypes (e.g., resistance to an antibiotic) as well as biophysical properties of the enzyme (e.g., thermostability). We find that organismal phenotypes and enzyme properties are highly sensitive to the identity of DHFR modules, and that this chimeric approach can create enzymes with diverse biophysical characteristics.
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http://dx.doi.org/10.1002/pro.3646DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7663999PMC
July 2019

Proteostasis Environment Shapes Higher-Order Epistasis Operating on Antibiotic Resistance.

Genetics 2019 06 23;212(2):565-575. Epub 2019 Apr 23.

Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138.

Recent studies have affirmed that higher-order epistasis is ubiquitous and can have large effects on complex traits. Yet, we lack frameworks for understanding how epistatic interactions are influenced by central features of cell physiology. In this study, we assess how protein quality control machinery-a critical component of cell physiology-affects epistasis for different traits related to bacterial resistance to antibiotics. Specifically, we disentangle the interactions between different protein quality control genetic backgrounds and two sets of mutations: (i) SNPs associated with resistance to antibiotics in an essential bacterial enzyme (dihydrofolate reductase, or DHFR) and (ii) differing DHFR bacterial species-specific amino acid background sequences (, , and ). In doing so, we improve on generic observations that epistasis is widespread by discussing how patterns of epistasis can be partly explained by specific interactions between mutations in an essential enzyme and genes associated with the proteostasis environment. These findings speak to the role of environmental and genotypic context in modulating higher-order epistasis, with direct implications for evolutionary theory, genetic modification technology, and efforts to manage antimicrobial resistance.
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http://dx.doi.org/10.1534/genetics.119.302138DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6553834PMC
June 2019

Evolution on the Biophysical Fitness Landscape of an RNA Virus.

Mol Biol Evol 2018 10;35(10):2390-2400

Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA.

Viral evolutionary pathways are determined by the fitness landscape, which maps viral genotype to fitness. However, a quantitative description of the landscape and the evolutionary forces on it remain elusive. Here, we apply a biophysical fitness model based on capsid folding stability and antibody binding affinity to predict the evolutionary pathway of norovirus escaping a neutralizing antibody. The model is validated by experimental evolution in bulk culture and in a drop-based microfluidics that propagates millions of independent small viral subpopulations. We demonstrate that along the axis of binding affinity, selection for escape variants and drift due to random mutations have the same direction, an atypical case in evolution. However, along folding stability, selection and drift are opposing forces whose balance is tuned by viral population size. Our results demonstrate that predictable epistatic tradeoffs between molecular traits of viral proteins shape viral evolution.
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http://dx.doi.org/10.1093/molbev/msy131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6188569PMC
October 2018

Differential Enzyme Flexibility Probed Using Solid-State Nanopores.

ACS Nano 2018 05 17;12(5):4494-4502. Epub 2018 Apr 17.

Department of Physics , Northeastern University , Boston , Massachusetts 02115 , United States.

Enzymes and motor proteins are dynamic macromolecules that coexist in a number of conformations of similar energies. Protein function is usually accompanied by a change in structure and flexibility, often induced upon binding to ligands. However, while measuring protein flexibility changes between active and resting states is of therapeutic significance, it remains a challenge. Recently, our group has demonstrated that breadth of signal amplitudes in measured electrical signatures as an ensemble of individual protein molecules is driven through solid-state nanopores and correlates with protein conformational dynamics. Here, we extend our study to resolve subtle flexibility variation in dihydrofolate reductase mutants from unlabeled single molecules in solution. We first demonstrate using a canonical protein system, adenylate kinase, that both size and flexibility changes can be observed upon binding to a substrate that locks the protein in a closed conformation. Next, we investigate the influence of voltage bias and pore geometry on the measured electrical pulse statistics during protein transport. Finally, using the optimal experimental conditions, we systematically study a series of wild-type and mutant dihydrofolate reductase proteins, finding a good correlation between nanopore-measured protein conformational dynamics and equilibrium bulk fluorescence probe measurements. Our results unequivocally demonstrate that nanopore-based measurements reliably probe conformational diversity in native protein ensembles.
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http://dx.doi.org/10.1021/acsnano.8b00734DOI Listing
May 2018

Stability of the Influenza Virus Hemagglutinin Protein Correlates with Evolutionary Dynamics.

mSphere 2018 Jan-Feb;3(1). Epub 2018 Jan 3.

W. Harry Feinstone Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, USA.

Protein thermodynamics are an integral determinant of viral fitness and one of the major drivers of protein evolution. Mutations in the influenza A virus (IAV) hemagglutinin (HA) protein can eliminate neutralizing antibody binding to mediate escape from preexisting antiviral immunity. Prior research on the IAV nucleoprotein suggests that protein stability may constrain seasonal IAV evolution; however, the role of stability in shaping the evolutionary dynamics of the HA protein has not been explored. We used the full coding sequence of 9,797 H1N1pdm09 HA sequences and 16,716 human seasonal H3N2 HA sequences to computationally estimate relative changes in the thermal stability of the HA protein between 2009 and 2016. Phylogenetic methods were used to characterize how stability differences impacted the evolutionary dynamics of the virus. We found that pandemic H1N1 IAV strains split into two lineages that had different relative HA protein stabilities and that later variants were descended from the higher-stability lineage. Analysis of the mutations associated with the selective sweep of the higher-stability lineage found that they were characterized by the early appearance of highly stabilizing mutations, the earliest of which was not located in a known antigenic site. Experimental evidence further suggested that H1N1 HA stability may be correlated with virus production and infection. A similar analysis of H3N2 strains found that surviving lineages were also largely descended from viruses predicted to encode more-stable HA proteins. Our results suggest that HA protein stability likely plays a significant role in the persistence of different IAV lineages. One of the constraints on fast-evolving viruses, such as influenza virus, is protein stability, or how strongly the folded protein holds together. Despite the importance of this protein property, there has been limited investigation of the impact of the stability of the influenza virus hemagglutinin protein-the primary antibody target of the immune system-on its evolution. Using a combination of computational estimates of stability and experiments, our analysis found that viruses with more-stable hemagglutinin proteins were associated with long-term persistence in the population. There are two potential reasons for the observed persistence. One is that more-stable proteins tolerate destabilizing mutations that less-stable proteins could not, thus increasing opportunities for immune escape. The second is that greater stability increases the fitness of the virus through increased production of infectious particles. Further research on the relative importance of these mechanisms could help inform the annual influenza vaccine composition decision process.
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http://dx.doi.org/10.1128/mSphereDirect.00554-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5750392PMC
January 2018

Rational Design of Novel Allosteric Dihydrofolate Reductase Inhibitors Showing Antibacterial Effects on Drug-Resistant Escherichia coli Escape Variants.

ACS Chem Biol 2017 07 31;12(7):1848-1857. Epub 2017 May 31.

Center for the Study of Systems Biology, School of Biology, Georgia Institute of Technology , 950 Atlantic Drive, Atlanta, Georgia 30332, United States.

In drug discovery, systematic variations of substituents on a common scaffold and bioisosteric replacements are often used to generate diversity and obtain molecules with better biological effects. However, this could saturate the small-molecule diversity pool resulting in drug resistance. On the other hand, conventional drug discovery relies on targeting known pockets on protein surfaces leading to drug resistance by mutations of critical pocket residues. Here, we present a two-pronged strategy of designing novel drugs that target unique pockets on a protein's surface to overcome the above problems. Dihydrofolate reductase, DHFR, is a critical enzyme involved in thymidine and purine nucleotide biosynthesis. Several classes of compounds that are structural analogues of the substrate dihydrofolate have been explored for their antifolate activity. Here, we describe 10 novel small-molecule inhibitors of Escherichia coli DHFR, EcDHFR, belonging to the stilbenoid, deoxybenzoin, and chalcone family of compounds discovered by a combination of pocket-based virtual ligand screening and systematic scaffold hopping. These inhibitors show a unique uncompetitive or noncompetitive inhibition mechanism, distinct from those reported for all known inhibitors of DHFR, indicative of binding to a unique pocket distinct from either substrate or cofactor-binding pockets. Furthermore, we demonstrate that rescue mutants of EcDHFR, with reduced affinity to all known classes of DHFR inhibitors, are inhibited at the same concentration as the wild-type. These compounds also exhibit antibacterial activity against E. coli harboring the drug-resistant variant of DHFR. This discovery is the first report on a novel class of inhibitors targeting a unique pocket on EcDHFR.
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http://dx.doi.org/10.1021/acschembio.7b00175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5819740PMC
July 2017

Biophysical principles predict fitness landscapes of drug resistance.

Proc Natl Acad Sci U S A 2016 Mar 29;113(11):E1470-8. Epub 2016 Feb 29.

Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138;

Fitness landscapes of drug resistance constitute powerful tools to elucidate mutational pathways of antibiotic escape. Here, we developed a predictive biophysics-based fitness landscape of trimethoprim (TMP) resistance for Escherichia coli dihydrofolate reductase (DHFR). We investigated the activity, binding, folding stability, and intracellular abundance for a complete set of combinatorial DHFR mutants made out of three key resistance mutations and extended this analysis to DHFR originated from Chlamydia muridarum and Listeria grayi We found that the acquisition of TMP resistance via decreased drug affinity is limited by a trade-off in catalytic efficiency. Protein stability is concurrently affected by the resistant mutants, which precludes a precise description of fitness from a single molecular trait. Application of the kinetic flux theory provided an accurate model to predict resistance phenotypes (IC50) quantitatively from a unique combination of the in vitro protein molecular properties. Further, we found that a controlled modulation of the GroEL/ES chaperonins and Lon protease levels affects the intracellular steady-state concentration of DHFR in a mutation-specific manner, whereas IC50 is changed proportionally, as indeed predicted by the model. This unveils a molecular rationale for the pleiotropic role of the protein quality control machinery on the evolution of antibiotic resistance, which, as we illustrate here, may drastically confound the evolutionary outcome. These results provide a comprehensive quantitative genotype-phenotype map for the essential enzyme that serves as an important target of antibiotic and anticancer therapies.
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http://dx.doi.org/10.1073/pnas.1601441113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4801265PMC
March 2016

Resistance to dual-gene Bt maize in Spodoptera frugiperda: selection, inheritance, and cross-resistance to other transgenic events.

Sci Rep 2015 Dec 17;5:18243. Epub 2015 Dec 17.

Departamento de Entomologia, Universidade Federal de Viçosa, Viçosa, MG, 36570-900, Brazil.

Transgenic crop "pyramids" producing two or more Bacillus thuringiensis (Bt) toxins active against the same pest are used to delay evolution of resistance in insect pest populations. Laboratory and greenhouse experiments were performed with fall armyworm, Spodoptera frugiperda, to characterize resistance to Bt maize producing Cry1A.105 and Cry2Ab and test some assumptions of the "pyramid" resistance management strategy. Selection of a field-derived strain of S. frugiperda already resistant to Cry1F maize with Cry1A.105 + Cry2Ab maize for ten generations produced resistance that allowed the larvae to colonize and complete the life cycle on these Bt maize plants. Greenhouse experiments revealed that the resistance was completely recessive (Dx = 0), incomplete, autosomal, and without maternal effects or cross-resistance to the Vip3Aa20 toxin produced in other Bt maize events. This profile of resistance supports some of the assumptions of the pyramid strategy for resistance management. However, laboratory experiments with purified Bt toxin and plant leaf tissue showed that resistance to Cry1A.105 + Cry2Ab2 maize further increased resistance to Cry1Fa, which indicates that populations of fall armyworm have high potential for developing resistance to some currently available pyramided maize used against this pest, especially where resistance to Cry1Fa was reported in the field.
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http://dx.doi.org/10.1038/srep18243DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4682147PMC
December 2015

Free Superoxide is an Intermediate in the Production of H2O2 by Copper(I)-Aβ Peptide and O2.

Angew Chem Int Ed Engl 2016 Jan 2;55(3):1085-9. Epub 2015 Dec 2.

CNRS, LCC (Laboratoire de Chimie de Coordination), 205 route de Narbonne, BP 44099, 31077, Toulouse Cedex 4, France.

Oxidative stress is considered as an important factor and an early event in the etiology of Alzheimer's disease (AD). Cu bound to the peptide amyloid-β (Aβ) is found in AD brains, and Cu-Aβ could contribute to this oxidative stress, as it is able to produce in vitro H2O2 and HO˙ in the presence of oxygen and biological reducing agents such as ascorbate. The mechanism of Cu-Aβ-catalyzed H2O2 production is however not known, although it was proposed that H2O2 is directly formed from O2 via a 2-electron process. Here, we implement an electrochemical setup and use the specificity of superoxide dismutase-1 (SOD1) to show, for the first time, that H2O2 production by Cu-Aβ in the presence of ascorbate occurs mainly via a free O2˙(-) intermediate. This finding radically changes the view on the catalytic mechanism of H2O2 production by Cu-Aβ, and opens the possibility that Cu-Aβ-catalyzed O2˙(-) contributes to oxidative stress in AD, and hence may be of interest.
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http://dx.doi.org/10.1002/anie.201508597DOI Listing
January 2016

Ethylmalonic encephalopathy ETHE1 R163W/R163Q mutations alter protein stability and redox properties of the iron centre.

PLoS One 2014 8;9(9):e107157. Epub 2014 Sep 8.

Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal.

ETHE1 is an iron-containing protein from the metallo β-lactamase family involved in the mitochondrial sulfide oxidation pathway. Mutations in ETHE1 causing loss of function result in sulfide toxicity and in the rare fatal disease Ethylmalonic Encephalopathy (EE). Frequently mutations resulting in depletion of ETHE1 in patient cells are due to severe structural and folding defects. However, some ETHE1 mutations yield nearly normal protein levels and in these cases disease mechanism was suspected to lie in compromised catalytic activity. To address this issue and to elicit how ETHE1 dysfunction results in EE, we have investigated two such pathological mutations, ETHE1-p.Arg163Gln and p.Arg163Trp. In addition, we report a number of benchmark properties of wild type human ETHE1, including for the first time the redox properties of the mononuclear iron centre. We show that loss of function in these variants results from a combination of decreased protein stability and activity. Although structural assessment revealed that the protein fold is not perturbed by mutations, both variants have decreased thermal stabilities and higher proteolytic susceptibilities. ETHE1 wild type and variants bind 1 ± 0.2 mol iron/protein and no zinc; however, the variants exhibited only ≈ 10% of wild-type catalytically activity. Analysis of the redox properties of ETHE1 mononuclear iron centre revealed that the variants have lowered reduction potentials with respect to that of the wild type. This illustrates how point mutation-induced loss of function may arise via very discrete subtle conformational effects on the protein fold and active site chemistry, without extensive disruption of the protein structure or protein-cofactor association.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0107157PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4157841PMC
January 2016

Simplified 2,4-dinitrophenylhydrazine spectrophotometric assay for quantification of carbonyls in oxidized proteins.

Anal Biochem 2014 Aug 6;458:69-71. Epub 2014 May 6.

Center of Chemistry, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal. Electronic address:

This work proposes a modification of the 2,4-dinitrophenylhydrazine (DNPH) spectrophotometric assay commonly used to evaluate the concentration of carbonyl groups in oxidized proteins. In this approach NaOH is added to the protein solution after the addition of DNPH, shifting the maximum absorbance wavelength of the derivatized protein from 370 to 450nm. This reduces the interference of DNPH and allows the direct quantification in the sample solution without the need for the precipitation, washing, and resuspension steps that are carried out in the traditional DNPH method. The two methods were compared under various conditions and are statistically equivalent.
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http://dx.doi.org/10.1016/j.ab.2014.04.034DOI Listing
August 2014

Cofactors and metabolites as protein folding helpers in metabolic diseases.

Curr Top Med Chem 2012 ;12(22):2546-59

Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.

In the past few decades, improved early diagnosis methods, technological developments and an increasing crosstalk between clinicians and researchers has led to the identification of an increasing number of inborn metabolic diseases. In these disorders, missense mutations are the most frequent type of genetic defects, frequently resulting in defective protein folding. A better understanding at the molecular level of protein misfolding and its role in disease has prompted the emergence of therapies based in the use of small molecules that have the ability to correct protein folding defects. Well-known cases are reported for phenylketonuria and Gaucher's disease. Most of these compounds have a specific mechanism of action interacting directly with a particular protein, the so called pharmacological chaperones. Among such small molecules are protein ligands, either natural substrates or synthetic derivatives, cofactors, competitive inhibitors, and agonist/antagonists. In this review we will start by briefly overviewing the mechanisms through which such ligands exert a stabilizing action, and then move on to an extended discussion on therapeutic approaches and use of vitamins and substrates to correct protein misfolding in metabolic disorders. Examples of vitamins that have been successfully prescribed to rescue some cases of inborn errors of metabolism will be presented. In particular, the role of riboflavin supplementation in the treatment of fatty acid β-oxidation disorders will be thoroughly analyzed, focusing on recent reports that shed light on the molecular basis of vitamin responsiveness. Moreover, we will highlight the latest studies that point to a synergistic effect of cofactors and metabolites in the rescue of defective fatty acid β-oxidation enzymes. The synergism of multiple small molecules may underlie a promising general pharmacological strategy for the treatment of metabolic diseases in general.
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http://dx.doi.org/10.2174/1568026611212220009DOI Listing
September 2013

Mechanism of superoxide and hydrogen peroxide generation by human electron-transfer flavoprotein and pathological variants.

Free Radic Biol Med 2012 Jul 26;53(1):12-9. Epub 2012 Apr 26.

Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, 2780-157 Oeiras, Portugal.

Reactive oxygen species production by mitochondrial enzymes plays a fundamental role both in cellular signaling and in the progression of dysfunctional states. However, sources of reactive oxygen species and the mechanisms by which enzymes produce these reactive species still remain elusive. We characterized the generation of reactive oxygen species by purified human electron-transfer flavoprotein (ETF), a mitochondrial enzyme that has a central role in the metabolism of lipids, amino acids, and choline. The results showed that ETF produces significant amounts of both superoxide and hydrogen peroxide in the presence of its partner enzyme medium-chain acyl-CoA dehydrogenase (MCAD). ETF-mediated production of reactive oxygen species is partially inhibited at high MCAD/ETF ratios, whereas it is enhanced at high ionic strength. Determination of the reduction potentials of ETF showed that thermodynamic properties of the FAD cofactor are changed upon formation of a complex between ETF and MCAD, supporting the notion that protein:protein interactions modulate the reactivity of the protein with dioxygen. Two pathogenic ETF variants were also studied to determine which factors modulate the reactivity toward molecular oxygen and promote reactive oxygen species production. The results obtained show that destabilized conformations and defective protein:protein interactions increase the ability of ETF to generate reactive oxygen species. A possible role for these processes in mitochondrial dysfunction in metabolic disorders of fatty acid β-oxidation is discussed.
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http://dx.doi.org/10.1016/j.freeradbiomed.2012.04.016DOI Listing
July 2012

Mutations at the flavin binding site of ETF:QO yield a MADD-like severe phenotype in Drosophila.

Biochim Biophys Acta 2012 Aug 9;1822(8):1284-92. Epub 2012 May 9.

Instituto Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.

Following a screening on EMS-induced Drosophila mutants defective for formation and morphogenesis of epithelial cells, we have identified three lethal mutants defective for the production of embryonic cuticle. The mutants are allelic to the CG12140 gene, the fly homologue of electron transfer flavoprotein:ubiquinone oxidoreductase (ETF:QO). In humans, inherited defects in this inner membrane protein account for multiple acyl-CoA dehydrogenase deficiency (MADD), a metabolic disease of β-oxidation, with a broad range of clinical phenotypes, varying from embryonic lethal to mild forms. The three mutant alleles carried distinct missense mutations in ETF:QO (G65E, A68V and S104F) and maternal mutant embryos for ETF:QO showed lethal morphogenetic defects and a significant induction of apoptosis following germ-band elongation. This phenotype is accompanied by an embryonic accumulation of short- and medium-chain acylcarnitines (C4, C8 and C12) as well as long-chain acylcarnitines (C14 and C16:1), whose elevation is also found in severe MADD forms in humans under intense metabolic decompensation. In agreement the ETF:QO activity in the mutant embryos is markedly decreased in relation to wild type activity. Amino acid sequence analysis and structural mapping into a molecular model of ETF:QO show that all mutations map at FAD interacting residues, two of which at the nucleotide-binding Rossmann fold. This structural domain is composed by a β-strand connected by a short loop to an α-helix, and its perturbation results in impaired cofactor association via structural destabilisation and consequently enzymatic inactivation. This work thus pinpoints the molecular origins of a severe MADD-like phenotype in the fruit fly and establishes the proof of concept concerning the suitability of this organism as a potential model organism for MADD.
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http://dx.doi.org/10.1016/j.bbadis.2012.05.003DOI Listing
August 2012

Cofactors and metabolites as potential stabilizers of mitochondrial acyl-CoA dehydrogenases.

Biochim Biophys Acta 2011 Dec 24;1812(12):1658-63. Epub 2011 Sep 24.

Instituto Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.

Protein misfolding is a hallmark of a number of metabolic diseases, in which fatty acid oxidation defects are included. The latter result from genetic deficiencies in transport proteins and enzymes of the mitochondrial β-oxidation, and milder disease conditions frequently result from conformational destabilization and decreased enzymatic function of the affected proteins. Small molecules which have the ability to raise the functional levels of the affected protein above a certain disease threshold are thus valuable tools for effective drug design. In this work we have investigated the effect of mitochondrial cofactors and metabolites as potential stabilizers in two β-oxidation acyl-CoA dehydrogenases: short chain acyl-CoA dehydrogenase and the medium chain acyl-CoA dehydrogenase as well as glutaryl-CoA dehydrogenase, which is involved in lysine and tryptophan metabolism. We found that near physiological concentrations (low micromolar) of FAD resulted in a spectacular enhancement of the thermal stabilities of these enzymes and prevented enzymatic activity loss during a 1h incubation at 40°C. A clear effect of the respective substrate, which was additive to that of the FAD effect, was also observed for short- and medium-chain acyl-CoA dehydrogenase but not for glutaryl-CoA dehydrogenase. In conclusion, riboflavin may be beneficial during feverish crises in patients with short- and medium-chain acyl-CoA dehydrogenase as well as in glutaryl-CoA dehydrogenase deficiencies, and treatment with substrate analogs to butyryl- and octanoyl-CoAs could theoretically enhance enzyme activity for some enzyme proteins with inherited folding difficulties.
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http://dx.doi.org/10.1016/j.bbadis.2011.09.009DOI Listing
December 2011

Protein stability in an ionic liquid milieu: on the use of differential scanning fluorimetry.

Phys Chem Chem Phys 2011 Aug 27;13(30):13614-6. Epub 2011 Jun 27.

Instituto Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. República 127, 2780-756 Oeiras, Portugal.

Protein stability is a major bottleneck in the biotechnological application of ionic liquid-containing solvents, either in the frame of biocatalysis or protein storage. Herein, differential scanning fluorimetry was successfully implemented as a high throughput method to fast scan the impact of a number of cholinium-based ionic liquids on the stability of proteins.
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http://dx.doi.org/10.1039/c1cp21187kDOI Listing
August 2011

Enhanced superoxide and hydrogen peroxide detection in biological assays.

Free Radic Biol Med 2010 Jul 21;49(1):61-6. Epub 2010 Mar 21.

Instituto Tecnologia Química e Biológica, Universidade Nova de Lisboa, Avenida República, EAN, 2785-572 Oeiras, Portugal.

Superoxide reductase (SOR) is an enzyme that converts superoxide into hydrogen peroxide at a twofold higher yield than canonical superoxide dismutases (SOD). Superoxide radical detection was investigated using the Amplex red (AR)/peroxidase system to measure the difference in hydrogen peroxide production yield in the presence of SOR or SOD. We found that reduced SOR reacts with the AR oxidation intermediate, a one-electron reduced AR(*) radical, by reducing this intermediate back to the initial AR leuco compound. Ascorbate also quenched this radical in a concentration-dependent manner and could be used to compete efficiently with SOR; at concentrations of ascorbate higher than 5 microM, SOR no longer interfered with the detection of H(2)O(2). By using xanthine/xanthine oxidase as a superoxide-generating system, it was possible to successfully quantify superoxide and hydrogen peroxide in vitro using the AR/peroxidase/SOR system, either by visible absorption or by fluorescence emission, with a considerable low detection limit of 10nM/min. The use of enzymes with diffusion-limited reactivity toward superoxide substantially increases specificity and detection threshold for superoxide and turns this approach into a powerful system to detect ROS in biological systems.
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http://dx.doi.org/10.1016/j.freeradbiomed.2010.03.014DOI Listing
July 2010

Purification, crystallization and X-ray crystallographic analysis of Archaeoglobus fulgidus neelaredoxin.

Acta Crystallogr Sect F Struct Biol Cryst Commun 2010 Mar 24;66(Pt 3):316-9. Epub 2010 Feb 24.

Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Apartado 127, 2781-901 Oeiras, Portugal.

Neelaredoxins are a type of superoxide reductase (SOR), which are blue 14 kDa metalloproteins with a catalytic nonhaem iron centre coordinated by four histidines and one cysteine in the ferrous form. Anaerobic organisms such as Archaeoglobus fulgidus, a hyperthermophilic sulfate-reducing archaeon, have developed defence mechanisms against toxic oxygen species in which superoxide reductases play a key role. SOR is responsible for scavenging toxic superoxide anion radicals (O(2)(*-)), catalysing the one-electron reduction of superoxide to hydrogen peroxide. Crystals of recombinant A. fulgidus neelaredoxin in the oxidized form (13.7 kDa, 125 residues) were obtained using polyethylene glycol and ammonium sulfate. These crystals diffracted to 1.9 A resolution and belonged to the tetragonal space group P4(1)2(1)2, with unit-cell parameters a = b = 75.72, c = 185.44 A. Cell-content analysis indicated the presence of a tetramer in the asymmetric unit, with a Matthews coefficient (V(M)) of 2.36 A(3) Da(-1) and an estimated solvent content of 48%. The three-dimensional structure was determined by the MAD method and is currently under refinement.
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http://dx.doi.org/10.1107/S1744309110000916DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2833046PMC
March 2010

Reductive elimination of superoxide: Structure and mechanism of superoxide reductases.

Biochim Biophys Acta 2010 Feb 24;1804(2):285-97. Epub 2009 Oct 24.

Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Avenida da República (EAN), 2780-157 Oeiras, Portugal.

Superoxide anion is among the deleterious reactive oxygen species, towards which all organisms have specialized detoxifying enzymes. For quite a long time, superoxide elimination was thought to occur through its dismutation, catalyzed by Fe, Cu, and Mn or, as more recently discovered, by Ni-containing enzymes. However, during the last decade, a novel type of enzyme was established that eliminates superoxide through its reduction: the superoxide reductases, which are spread among anaerobic and facultative microorganisms, from the three life kingdoms. These enzymes share the same unique catalytic site, an iron ion bound to four histidines and a cysteine that, in its reduced form, reacts with superoxide anion with a diffusion-limited second order rate constant of approximately 10(9) M(-1) s(-1). In this review, the properties of these enzymes will be thoroughly discussed.
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http://dx.doi.org/10.1016/j.bbapap.2009.10.011DOI Listing
February 2010

Resonance Raman study of the superoxide reductase from Archaeoglobus fulgidus, E12 mutants and a 'natural variant'.

Phys Chem Chem Phys 2009 Mar 28;11(11):1809-15. Epub 2009 Jan 28.

Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Avenida da Republica 127, 2780-157 Oeiras, Portugal.

The resonance Raman (RR) spectra of the oxidized wild-type Archaeoglobus fuglidus 1Fe-Superoxide reductase (SOR), E12V and E12Q mutants were studied at different pH conditions upon excitation in resonance with the pH-dependent charge transfer transition to the ferric iron. The wild-type SOR from Nanoarchaeum equitans that lacks the highly conserved glutamate residue was investigated as a 'natural variant'. No substantial differences were observed in the RR spectra of the active sites of the A. fulgidus proteins. Based on the component analysis in the metal-ligand stretching region the modes involving the Fe-S(Cys) stretching coordinates have been identified. The frequencies of these modes reflect the electronic properties of the Fe-S bond which are related to the catalytic activity of SORs, including reduction of superoxide and product dissociation. Moreover, hydroxide binding to the E12 mutant proteins was demonstrated at high pH. It was further observed that the ferric active site of all three SORs from A. fulgidus senses the presence of phosphate, which possibly replaces the hydroxide at high pH.
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http://dx.doi.org/10.1039/b815489aDOI Listing
March 2009

Role of flavinylation in a mild variant of multiple acyl-CoA dehydrogenation deficiency: a molecular rationale for the effects of riboflavin supplementation.

J Biol Chem 2009 Feb 16;284(7):4222-9. Epub 2008 Dec 16.

Instituto Tecnologia Química e Biológica, Universidade Nova de Lisboa, 2780-756 Oeiras, Portugal.

Mutations in the genes encoding the alpha-subunit and beta-subunit of the mitochondrial electron transfer flavoprotein (ETF) and the electron transfer flavoprotein:ubiquinone oxidoreductase (ETF:QO) cause multiple acyl-CoA dehydrogenation deficiency (MADD), a disorder of fatty acid and amino acid metabolism. Point mutations in ETF, which may compromise folding, and/or activity, are associated with both mild and severe forms of MADD. Here we report the investigation on the conformational and stability properties of the disease-causing variant ETFbeta-D128N, and our findings on the effect of flavinylation in modulating protein conformational stability and activity. A combination of biochemical and biophysical methods including circular dichroism, visible absorption, flavin, and tryptophan fluorescence emission allowed the analysis of structural changes and of the FAD moiety. The ETFbeta-D128N variant retains the overall fold of the wild type, but under stress conditions its flavin becomes less tightly bound. Flavinylation is shown to improve the conformational stability and biological activity of a destabilized D128N variant protein. Moreover, the presence of flavin prevented proteolytic digestion by avoiding protein destabilization. A patient homozygous for the ETFbeta-D128N mutation developed severe disease symptoms in association with a viral infection and fever. In agreement, our results suggest that heat inactivation of the mutant may be more relevant at temperatures above 37 degrees C. To mimic a situation of fever in vitro, the flavinylation status was tested at 39 degrees C. FAD exerts the effect of a pharmacological chaperone, improving ETF conformation, and yielding a more stable and active enzyme. Our results provide a structural and functional framework that could help to elucidate the role that an increased cellular FAD content obtained from riboflavin supplementation may play in the molecular pathogenesis of not only MADD, but genetic disorders of flavoproteins in general.
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http://dx.doi.org/10.1074/jbc.M805719200DOI Listing
February 2009

Superoxide reduction by Nanoarchaeum equitans neelaredoxin, an enzyme lacking the highly conserved glutamate iron ligand.

J Biol Inorg Chem 2008 Feb 30;13(2):219-28. Epub 2007 Oct 30.

Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República (EAN), 2781-901, Oeiras, Portugal.

Superoxide reductases (SORs) are antioxidant enzymes present in many prokaryotes, either anaerobes or microaerophiles, which detoxify superoxide by reducing it to hydrogen peroxide. The reaction mechanism involves the diffusion-limited encounter of superoxide with the reduced iron site and concomitant formation of an Fe(3+)-(hydro)peroxo adduct that, upon protonation, leads to the formation of hydrogen peroxide. By the end of this process, a glutamate residue coordinates the ferric ion, acting as a sixth ligand. Although this residue is able to shuttle protons to the intermediate at low pH, it seems to have a minor relevance to the overall reduction mechanism. Nevertheless, this ligand is conserved in most SORs known thus far, with the notable exception of neelaredoxin from Nanoarchaeum equitans. The protein of this organism was cloned and overexpressed, and its spectroscopic characterization revealed distinct pH-equilibrium properties in comparison with those of glutamate-containing SORs. A three-dimensional model of this protein was generated in an effort to identify structural properties that could explain these distinct features. Pulse radiolysis measurements showed that the efficiency of this enzyme in reducing superoxide is comparable to that of glutamate-containing SORs, thus definitely ruling out the requirement for such a ligand in the reduction mechanism.
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http://dx.doi.org/10.1007/s00775-007-0313-zDOI Listing
February 2008

Kinetics of electron transfer from NADH to the Escherichia coli nitric oxide reductase flavorubredoxin.

FEBS J 2007 Feb 20;274(3):677-86. Epub 2006 Dec 20.

Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.

Escherichia coli flavorubredoxin (FlRd) belongs to the family of flavodiiron proteins (FDPs), microbial enzymes that are expressed to scavenge nitric oxide (NO) under anaerobic conditions. To degrade NO, FlRd has to be reduced by NADH via the FAD-binding protein flavorubredoxin reductase, thus the kinetics of electron transfer along this pathway was investigated by stopped-flow absorption spectroscopy. We found that NADH, but not NADPH, quickly reduces the FlRd-reductase (k = 5.5 +/- 2.2 x 10(6) M(-1).s(-1) at 5 degrees C), with a limiting rate of 255 +/- 17 s(-1). The reductase in turn quickly reduces the rubredoxin (Rd) center of FlRd, as assessed at 5 degrees C working with the native FlRd enzyme (k = 2.4 +/- 0.1 x 10(6) m(-1).s(-1)) and with its isolated Rd-domain (k approximately 1 x 10(7) M(-1).s(-1)); in both cases the reaction was found to be dependent on pH and ionic strength. In FlRd the fast reduction of the Rd center occurs synchronously with the formation of flavin mononucleotide semiquinone. Our data provide evidence that (a) FlRd-reductase rapidly shuttles electrons between NADH and FlRd, a prerequisite for NO reduction in this detoxification pathway, and (b) the electron accepting site in FlRd, the Rd center, is in very fast redox equilibrium with the flavin mononucleotide.
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http://dx.doi.org/10.1111/j.1742-4658.2006.05612.xDOI Listing
February 2007

Superoxide reduction by Archaeoglobus fulgidus desulfoferrodoxin: comparison with neelaredoxin.

J Biol Inorg Chem 2007 Feb 26;12(2):248-56. Epub 2006 Oct 26.

Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República (EAN), 2784-505, Oeiras, Portugal.

Superoxide reductases (SORs) are non-heme iron-containing enzymes that remove superoxide by reducing it to hydrogen peroxide. The active center of SORs consists of a ferrous ion coordinated by four histidines and one cysteine in a square-pyramidal geometry. In the 2Fe-SOR, a distinct family of SORs, there is an additional desulforedoxin-like site that does not appear to be involved in SOR activity. Our previous studies on recombinant Archaeoglobus fulgidus neelaredoxin (1Fe-SOR) have shown that the reaction with superoxide involves the formation of a transient ferric form that, upon protonation, decays to yield an Fe(3+)-OH species, followed by binding of glutamate to the ferric ion via replacement of hydroxide (Rodrigues et al. in Biochemistry 45:9266-9278, 2006). Here, we report the characterization of recombinant desulfoferrodoxin from the same organism, which is a member of the 2Fe-SOR family, and show that the steps involved in the superoxide reduction are similar in both families of SOR. The electron donation to the SOR from its redox partner, rubredoxin, is also presented here.
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http://dx.doi.org/10.1007/s00775-006-0182-xDOI Listing
February 2007

Superoxide reduction mechanism of Archaeoglobus fulgidus one-iron superoxide reductase.

Biochemistry 2006 Aug;45(30):9266-78

Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República (EAN), 2784-505 Oeiras, Portugal.

Superoxide reductases (SORs), iron-centered enzymes responsible for reducing superoxide (O2(-)) to hydrogen peroxide, are found in many anaerobic and microaerophilic prokaryotes. The rapid reaction with an exogenous electron donor renders the reductase activity catalytic. Here, we demonstrate using pulse radiolysis that the initial reaction between O2(-) and Archaeoglobus fulgidus neelaredoxin, a one-iron SOR, leads to a short-lived transient that immediately disappears to yield a solvent-bound ferric species in acid-base equilibrium. Through comparison of wild-type neelaredoxin with mutants lacking the ferric ion coordinating glutamate, we demonstrate that the remaining step is related to the final coordination of this ligand to the oxidized metal center and kinetically characterize it for the first time, by pulse radiolysis and stopped-flow kinetics. The way exogenous phosphate perturbs the kinetics of superoxide reduction by neelaredoxin and mutant proteins was also investigated.
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http://dx.doi.org/10.1021/bi052489kDOI Listing
August 2006

Rubredoxin acts as an electron donor for neelaredoxin in Archaeoglobus fulgidus.

Biochem Biophys Res Commun 2005 Apr;329(4):1300-5

Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República (EAN) Apt 127, 2781-901 Oeiras, Portugal.

Archaeoglobus fulgidus neelaredoxin (Nlr) is an electron donor:superoxide oxidoreductase. The reaction of superoxide with reduced Nlr is almost diffusion-limited, but the overall efficiency for detoxifying superoxide in vivo depends on the rate of reduction of Nlr by electron donors. Here, we report the purification and characterization of the two type I rubredoxins from A. fulgidus (AF0880 and AF1349) and show that they act as efficient electron donors for neelaredoxin, in vitro, with a second-order rate constant of 10(7)M(-1)s(-1) at 10 degrees C and pH 7.2.
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http://dx.doi.org/10.1016/j.bbrc.2005.02.114DOI Listing
April 2005
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