Browsing by Author "Melo, Eduardo"
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- Cartilage acidic protein 1, a new member of the beta-propeller protein family with amyloid propensityPublication . Anjos, Lliana; Morgado, Isabel; Guerreiro, Marta; Cardoso, João CR; Melo, Eduardo; Power, DeborahCartilage acidic protein1 (CRTAC1) is an extracellular matrix protein of chondrogenic tissue in humans and its presence in bacteria indicate it is of ancient origin. Structural modeling of piscine CRTAC1 reveals it belongs to the large family of beta-propeller proteins that in mammals have been associated with diseases, including amyloid diseases such as Alzheimer's. In order to characterize the structure/function evolution of this new member of the beta-propeller family we exploited the unique characteristics of piscine duplicate genes Crtac1a and Crtac1b and compared their structural and biochemical modifications with human recombinant CRTAC1. We demonstrate that CRTAC1 has a beta-propeller structure that has been conserved during evolution and easily forms high molecular weight thermo-stable aggregates. We reveal for the first time the propensity of CRTAC1 to form amyloid-like structures, and hypothesize that the aggregating property of CRTAC1 may be related to its disease-association. We further contribute to the general understating of CRTAC1's and beta-propeller family evolution and function. Proteins 2017; 85:242-255. (c) 2016 Wiley Periodicals, Inc.
- Cartilage Acidic Protein 2 a hyperthermostable, high affinity calcium-binding proteinPublication . Anjos, Liliana; Melo, Eduardo; Canario, Adelino V. M.; Power, DeborahCartilage Acidic Protein 2 (CRTAC2) is a novel protein present fromprokaryotes to vertebrateswith abundant expression in the teleost fish pituitary gland and an isoformof CRTAC1, a chondrocyte marker in humans. The two proteins are non-integrins containingN-terminal integrin-like Ca2+-bindingmotifs and their structure and function remain to be assigned. Structural studies of recombinant sea bream (sb)CRTAC2 revealed it is composed of 8.8% α-helix, 33.4% β-sheet and 57.8% unordered protein. sbCRTAC2 bound Ca2+ with high affinity (Kd= 1.46 nM) and favourable Gibbs free energy (ΔG=−12.4 kcal/mol). The stoichiometry for Ca2+ bound to sbCRTAC2 at saturation indicated six Ca2+ ligand-binding sites exist per protein molecule. No conformational change in sbCRTAC2 occurred in the presence of Ca2+. Fluorescence emission revealed that the tertiary structure of the protein is hyperthermostable between 25 °C and 95 °C and the fully unfolded state is only induced by chemical denaturing (4 MGndCl). sbCRTAC has awidespread tissue distribution and is present as highmolecular weight aggregates, although strong reducing conditions promote formation of the monomer. sbCRTAC2 promotes epithelial cell outgrowth in vitro suggesting it may share functional homology with mammalian CRTAC1, recently implicated in cell–cell and cell–matrix interactions.
- Improving kinetic or thermodynamic stability of an azoreductase by directed evolutionPublication . Brissos, Vânia; Gonçalves, Nádia; Melo, Eduardo; Martins, Lígia O.Protein stability arises from a combination of factors which are often difficult to rationalise. Therefore its improvement is better addressed through directed evolution than by rational design approaches. In this study, five rounds of mutagenesis/recombination followed by high-throughput screening (approximate to 10,000 clones) yielded the hit 1B6 showing a 300-fold higher half life at 50 degrees C than that exhibited by the homodimeric wild type PpAzoR azoreductase from Pseudomonas putida MET94. The characterization using fluorescence, calorimetry and light scattering shows that 1B6 has a folded state slightly less stable than the wild type (with lower melting and optimal temperatures) but in contrast is more resistant to irreversible denaturation. The superior kinetic stability of 1B6 variant was therefore related to an increased resistance of the unfolded monomers to aggregation through the introduction of mutations that disturbed hydrophobic patches and increased the surface net charge of the protein. Variants 2A1 and 2A1-Y179H with increased thermodynamic stability (10 to 20 degrees C higher melting temperature than wild type) were also examined showing the distinctive nature of mutations that lead to improved structural robustness: these occur in residues that are mostly involved in strengthening the solvent-exposed loops or the inter-dimer interactions of the folded state.
- Intact protein folding in the glutathione-depleted endoplasmic reticulum implicates alternative protein thiol reductantsPublication . Tsunoda, Satoshi; Avezov, Edward; Zyryanova, Alisa; Konno, Tasuku; Leonardo Mendes-Silva; Melo, Eduardo; Harding, Heather P.; Ron, DavidProtein folding homeostasis in the endoplasmic reticulum (ER) requires efficient protein thiol oxidation, but also relies on a parallel reductive process to edit disulfides during the maturation or degradation of secreted proteins. To critically examine the widely held assumption that reduced ER glutathione fuels disulfide reduction, we expressed a modified form of a cytosolic glutathione-degrading enzyme, ChaC1, in the ER lumen. ChaC1(CtoS) purged the ER of glutathione eliciting the expected kinetic defect in oxidation of an ER-localized glutathione-coupled Grx1-roGFP2 optical probe, but had no effect on the disulfide editing-dependent maturation of the LDL receptor or the reduction-dependent degradation of misfolded alpha-1 antitrypsin. Furthermore, glutathione depletion had no measurable effect on induction of the unfolded protein response (UPR); a sensitive measure of ER protein folding homeostasis. These findings challenge the importance of reduced ER glutathione and suggest the existence of alternative electron donor(s) that maintain the reductive capacity of the ER.
- Intracellular sources of ROS/H2O2 in health and neurodegeneration: spotlight on endoplasmic reticulumPublication . Konno, Tasuku; Melo, Eduardo; Chambers, Joseph E.; Avezov, EdwardReactive oxygen species (ROS) are produced continuously throughout the cell as products of various redox reactions. Yet these products function as important signal messengers, acting through oxidation of specific target factors. Whilst excess ROS production has the potential to induce oxidative stress, physiological roles of ROS are supported by a spatiotemporal equilibrium between ROS producers and scavengers such as antioxidative enzymes. In the endoplasmic reticulum (ER), hydrogen peroxide (H2O2), a non-radical ROS, is produced through the process of oxidative folding. Utilisation and dysregulation of H2O2, in particular that generated in the ER, affects not only cellular homeostasis but also the longevity of organisms. ROS dysregulation has been implicated in various pathologies including dementia and other neurodegenerative diseases, sanctioning a field of research that strives to better understand cell-intrinsic ROS production. Here we review the organelle-specific ROS-generating and consuming pathways, providing evidence that the ER is a major contributing source of potentially pathologic ROS.
- Live-cell FRET imaging reveals clustering of the prion protein at the cell surface induced by infectious prionsPublication . Tavares, Evandro; Macedo, J.A.; Paulo, Pedro M. R.; Sousa, Catarina; Lopes, Carlos; Melo, EduardoPrion diseases are associated to the conversion of the prion protein into a misfolded pathological isoform. The mechanism of propagation of protein misfolding by protein templating remains largely unknown. Neuroblastoma cells were transfected with constructs of the prion protein fused to both CFP-GPI-anchored and to YFP-GPI-anchored and directed to its cell membrane location. Live-cell FRET imaging between the prion protein fused to CFP or YFP was measured giving consistent values of 10 +/- 2%. This result was confirmed by fluorescence lifetime imaging microscopy and indicates intermolecular interactions between neighbor prion proteins. In particular, considering that a maximum FRET efficiency of 17 +/- 2% was determined from a positive control consisting of a fusion CFP-YFP-GPI-anchored. A stable cell clone expressing the two fusions containing the prion protein was also selected to minimize cell-to-cell variability. In both, stable and transiently transfected cells, the FRET efficiency consistently increased in the presence of infectious prions - from 4 +/- 1% to 7 +/- 1% in the stable clone and from 10 +/- 2% to 16 +/- 1% in transiently transfected cells. These results clearly reflect an increased clustering of the prion protein on the membrane in the presence of infectious prions, which was not observed in negative control using constructs without the prion protein and upon addition of non-infected brain. Our data corroborates the recent view that the primary site for prion conversion is the cell membrane. Since our fluorescent cell clone is not susceptible to propagate infectivity, we hypothesize that the initial event of prion infectivity might be the clustering of the GPI-anchored prion protein. (C) 2014 Elsevier B.V. All rights reserved.
- Oxidative protein folding by an endoplasmic reticulum-localized peroxiredoxinPublication . Zito, Ester; Melo, Eduardo; Yang, Yun; Wahlander, Asa; Neubert, Thomas A.; Ron, DavidEndoplasmic reticulum (ER) oxidation 1 (ERO1) transfers disulfides to protein disulfide isomerase (PDI) and is essential for oxidative protein folding in simple eukaryotes such as yeast and worms. Surprisingly, ERO1-deficient mammalian cells exhibit only a modest delay in disulfide bond formation. To identify ERO1-independent pathways to disulfide bond formation, we purified PDI oxidants with a trapping mutant of PDI. Peroxiredoxin IV (PRDX4) stood out in this list, as the related cytosolic peroxiredoxins are known to form disulfides in the presence of hydroperoxides. Mouse embryo fibroblasts lacking ERO1 were intolerant of PRDX4 knockdown. Introduction of wild-type mammalian PRDX4 into the ER rescued the temperature-sensitive phenotype of an ero1 yeast mutation. In the presence of an H2O2-generating system, purified PRDX4 oxidized PDI and reconstituted oxidative folding of RNase A. These observations implicate ER-localized PRDX4 in a previously unanticipated, parallel, ERO1-independent pathway that couples hydroperoxide production to oxidative protein folding in mammalian cells.
- Photophysics and photochemistry of horseradish peroxidase A2 upon ultraviolet illuminationPublication . Neves-Petersen, Maria Teresa; Klitgaard, Soren; Leitao Carvalho, Ana Sofia; Petersen, Steffen B.; de Barros, Maria Raquel Aires; Melo, EduardoDetailed analysis of the effects of ultraviolet (UV) and blue light illumination of horseradish peroxidase A2, a heme-containing enzyme that reduces H2O2 to oxidize organic and inorganic compounds, is presented. The effects of increasing illumination time on the protein's enzymatic activity, Reinheitzahl value,. fluorescence emission,. fluorescence lifetime distribution,. fluorescence mean lifetime, and heme absorption are reported. UV illumination leads to an exponential decay of the enzyme activity followed by changes in heme group absorption. Longer UV illumination time leads to lower T-m values as well as helical content loss. Prolonged UV illumination and heme irradiation at 403 nm has a pronounced effect on the. fluorescence quantum yield correlated with changes in the prosthetic group pocket, leading to a pronounced decrease in the heme's Soret absorbance band. Analysis of the picosecond-resolved. fluorescence emission of horseradish peroxidase A2 with streak camera shows that UV illumination induces an exponential change in the preexponential factors distribution associated to the protein's. fluorescence lifetimes, leading to an exponential increase of the mean. fluorescence lifetime. Illumination of aromatic residues and of the heme group leads to changes indicative of heme leaving the molecule and/or that photoinduced chemical changes occur in the heme moiety. Our studies bring new insight into light-induced reactions in proteins. We show how streak camera technology can be of outstanding value to follow such ultrafast processes and how streak camera data can be correlated with protein structural changes.
- Rationally guided improvement of NOV1 Dioxygenase for the conversion of lignin-derived isoeugenol to vanillinPublication . De Simone, Mario; Alvigini, Laura; Alonso-Cotchico, Lur; Brissos, Vânia; Caroli, Jonatan; Lucas, Maria Fátima; Monza, Emanuele; Melo, Eduardo; Mattevi, Andrea; Martins, Lígia O.Biocatalysis is a key tool in both green chemistry and biorefinery fields. NOV1 is a dioxygenase that catalyzes the one-step, coenzyme-free oxidation of isoeugenol into vanillin and holds enormous biotechnological potential for the complete valorization of lignin as a sustainable starting material for biobased chemicals, polymers, and materials. This study integrates computational, kinetic, structural, and biophysical approaches to characterize a new NOV1 variant featuring improved activity and stability compared to those of the wild type. The S283F replacement results in a 2-fold increased turnover rate (kcat) for isoeugenol and a 4-fold higher catalytic efficiency (kcat/Km) for molecular oxygen compared to those of the wild type. Furthermore, the variant exhibits a half-life that is 20-fold higher than that of the wild type, which most likely relates to the enhanced stabilization of the iron cofactor in the active site. Molecular dynamics supports this view, revealing that the S283F replacement decreases the optimal pKa and favors conformations of the iron-coordinating histidines compatible with an increased level of binding to iron. Importantly, whole cells containing the S283F variant catalyze the conversion of <= 100 mM isoeugenol to vanillin, yielding >99% molar conversion yields within 24 h. This integrative strategy provided a new enzyme for biotechnological applications and mechanistic insights that will facilitate the future design of robust and efficient biocatalysts.
- Retarded PDI diffusion and a reductive shift in poise of the calcium depleted endoplasmic reticulumPublication . Avezov, Edward; Konno, Tasuku; Zyryanova, Alisa; Chen, Weiyue; Laine, Romain; Crespillo-Casado, Ana; Melo, Eduardo; Ushioda, Ryo; Nagata, Kazuhiro; Kaminski, Clemens F.; Harding, Heather P.; Ron, DavidBackground: Endoplasmic reticulum (ER) lumenal protein thiol redox balance resists dramatic variation in unfolded protein load imposed by diverse physiological challenges including compromise in the key upstream oxidases. Lumenal calcium depletion, incurred during normal cell signaling, stands out as a notable exception to this resilience, promoting a rapid and reversible shift towards a more reducing poise. Calcium depletion induced ER redox alterations are relevant to physiological conditions associated with calcium signaling, such as the response of pancreatic cells to secretagogues and neuronal activity. The core components of the ER redox machinery are well characterized; however, the molecular basis for the calcium-depletion induced shift in redox balance is presently obscure. Results: In vitro, the core machinery for generating disulfides, consisting of ERO1 and the oxidizing protein disulfide isomerase, PDI1A, was indifferent to variation in calcium concentration within the physiological range. However, ER calcium depletion in vivo led to a selective 2.5-fold decline in PDI1A mobility, whereas the mobility of the reducing PDI family member, ERdj5 was unaffected. In vivo, fluorescence resonance energy transfer measurements revealed that declining PDI1A mobility correlated with formation of a complex with the abundant ER chaperone calreticulin, whose mobility was also inhibited by calcium depletion and the calcium depletion-mediated reductive shift was attenuated in cells lacking calreticulin. Measurements with purified proteins confirmed that the PDI1A-calreticulin complex dissociated as Ca2+ concentrations approached those normally found in the ER lumen ([Ca2+] K-0.5max = 190 mu M). Conclusions: Our findings suggest that selective sequestration of PDI1A in a calcium depletion-mediated complex with the abundant chaperone calreticulin attenuates the effective concentration of this major lumenal thiol oxidant, providing a plausible and simple mechanism for the observed shift in ER lumenal redox poise upon physiological calcium depletion.