Browsing by Author "Brito, Rui M. M."
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- Abieta-7,13-Diene in Nematode-Infected Pinewood Pinus pinaster Branch Extracts: Isolation and the Elucidation and Characterization of Its StructurePublication . Gaspar, Marisa C.; Cruz, Pedro F.; Brito, Rui M. M.; Sousa, Hermínio C. de; Braga, Mara E. M.The oleoresin and volatile fraction produced by conifers, such as Pinus pinaster, play a crucial role in plant defence, acting as precursors to resin acids and adapting in response to environmental stress or pathogen attacks. Abietadiene (abieta-7,13-diene), the biosynthetic precursor to abietic acid, has been identified as the most abundant compound in extracts from pinewood nematode (PWN)-infected P. pinaster trees. As abietadiene is not commercially available, this study aimed to achieve, for the first time, its isolation, structure elucidation, and detailed characterization from readily available forestry residues. Abietadiene was successfully isolated using thin-layer chromatography (TLC), and its purity and identity were evaluated using multiple analytical techniques: gas chromatography (GC), liquid chromatography (LC), nuclear magnetic resonance (NMR), and Fourier-transform infrared spectroscopy (FTIR). GC analysis indicated a purity of over 70% for the isolated compound, while LC provided the higher purity value of 98%. The identity of abietadiene was unequivocally confirmed through LC, FTIR, and NMR analysis. This work represents the first isolation and comprehensive characterization of abietadiene from a natural source, making detailed chemical data on this compound available to the scientific community. These findings may be used for future studies on the biological interactions and ecological roles of abietadiene, particularly in the context of plant defence and pathogen resistance.
- Corema album spp: Edible wild crowberries with a high content in minerals and organic acidsPublication . Brito, Catarina; Bertotti, Taciana; Primitivo, Maria João; Neves, Marta; Pires, Cristiana L.; Cruz, Pedro F.; Martins, Patrícia A. T.; Rodrigues, Ana Cristina; Moreno, Maria João; Brito, Rui M. M.; Campos, Maria; Vaz, Daniela C.; Pessoa, Maria Fernanda; Lidon, Fernando; Reboredo, Fernando; Ribeiro, Vânia S.Edible wild plants are part of the ethnobotanical and gastronomic heritage of different geographical areas. Corema album (L.) D. Don is an endemic species of the dune systems of the Atlantic coast of the Iberian Peninsula. The aerial parts of Corema album are a source of nutrients and antioxidants. The Corema album white berry (Portuguese crowberry) is rich in calcium, iron, and zinc. The plant also shows high phenolic content and antioxidant capacity associated with the leaves, fruit, and flowers. The presence of organic acids, namely phenolic acids, such as hydroxycinnamic acids, and long chain polyunsaturated fatty acids (PUFAs) omega-3 and omega-6 has also been confirmed. Toxicity studies evaluated by cell viability tests with human intestinal epithelium model cells (Caco-2) have shown that, at low concentrations, plant extracts may present beneficial effects.
- DIBMA nanodiscs keep α-synuclein foldedPublication . Adão, Regina; Cruz, Pedro F.; Vaz, Daniela C.; Fonseca, Fátima; Pedersen, Jannik Nedergaard; Ferreira-da-Silva, Frederico; Brito, Rui M. M.; Ramos, Carlos H. I.; Otzen, Daniel; Keller, Sandro; Bastos, Margaridaα-Synuclein (αsyn) is a cytosolic intrinsically disordered protein (IDP) known to fold into an α-helical structure when binding to membrane lipids, decreasing protein aggregation. Model membrane enable elucidation of factors critically affecting protein folding/aggregation, mostly using either small unilamellar vesicles (SUVs) or nanodiscs surrounded by membrane scaffold proteins (MSPs). Yet SUVs are mechanically strained, while MSP nanodiscs are expensive. To test the impact of lipid particle size on α-syn structuring, while overcoming the limitations associated with the lipid particles used so far, we compared the effects of large unilamellar vesicles (LUVs) and lipid-bilayer nanodiscs encapsulated by diisobutylene/maleic acid copolymer (DIBMA) on αsyn secondary-structure formation, using human-, elephant- and whale -αsyn. Our results confirm that negatively charged lipids induce αsyn folding in h-αsyn and e-αsyn but not in w-αsyn. When a mixture of zwitterionic and negatively charged lipids was used, no increase in the secondary structure was detected at 45 °C. Further, our results show that DIBMA/lipid particles (DIBMALPs) are highly suitable nanoscale membrane mimics for studying αsyn secondary-structure formation and aggregation, as folding was essentially independent of the lipid/protein ratio, in contrast with what we observed for LUVs having the same lipid compositions. This study reveals a new and promising application of polymer-encapsulated lipid-bilayer nanodiscs, due to their excellent efficiency in structuring disordered proteins such as αsyn into nontoxic α-helical structures. This will contribute to the unravelling and modelling aspects concerning protein-lipid interactions and α-helix formation by αsyn, paramount to the proposal of new methods to avoid protein aggregation and disease.
- Edible flowers of Helichrysum italicum: Composition, nutritive value, and bioactivitiesPublication . Primitivo, Maria João; Neves, Marta; Pires, Cristiana L.; Cruz, Pedro F.; Brito, Catarina; Rodrigues, Ana C.; Carvalho, Carla C. C. R. de; Mortimer, Megan M.; Moreno, Maria João; Brito, Rui M. M.; Taylor, Edward J.; Millson, Stefan H.; Reboredo, Fernando; Campos, Maria; Vaz, Daniela C.; Ribeiro, VâniaHelichrysum italicum (H. italicum) is a halophyte shrub with bright yellow flowers with a strong curry-like aroma. The essential oils of H. italicum have been used in the production of cosmetics and pharmaceuticals, due to their antiallergic and anti-inflammatory properties. In the agri-food sector, H. italicum flowers can be used for seasoning and flavoring food, and as natural food preservatives. Here, we report on the composition, bioactive compounds, and nutritive value of H. italicum flowers. Flowers were mainly composed of carbohydrates (>80 % dry weight), followed by minerals (6.31 ± 0.95 % dw), protein (5.44 ± 0.35 % dw), and lipids (3.59 % ± 0.53 % dw). High percentages of Fe, Zn, Ca, and K were found in the flower material, along with a high content in antioxidants, polyphenols, and carotenoids, as corroborated by the nuclear magnetic resonance (NMR) data. Flowers were mainly composed of saturated fatty acids (SFAs) (54.50 ± 0.95 % of total FA), followed by polyunsaturated fatty acids (PUFAs) (37.73 ± 1.25 % of total FA) and monounsaturated fatty acids (MUFAs) (7.77 ± 0.34 %), as detected by gas chromatography mass spectrometry (GC–MS). The omega-6 PUFA linoleic acid (22.55 ± 0.76 % of total FA) was the most abundant fatty acid found. Flower extracts showed antimicrobial activity against Saccharomyces cerevisiae and Komagataella phaffii, as well as against Gram-negative (Klebsiella pneumoniae) and Gram-positive (Staphylococcus aureus) bacteria. H. italicum flower material was nontoxic to human intestinal Caco-2 model cells at concentrations up to 1.0 % w/v.
- Enthalpic and entropic contributions mediate the role of disulfide bonds on the conformational stability of Interleukin-4Publication . Vaz, Daniela C.; Rodrigues, J. Rui; Sebald, Walter; Dobson, Christopher M.; Brito, Rui M. M.The role of disulfide bridges in the structure, stability, and folding pathways of proteins has been the subject of wide interest in the fields of protein design and engineering. However, the relative importance of entropic and enthalpic contributions for the stabilization of proteins provided by disulfides is not always clear. Here, we perform a detailed analysis of the role of disulfidesin the conformational stability of human Interleukin-4 (IL4), a four-helix bundle protein. In order to evaluate the contribution of two out of the three disulfides to the structure and stability of IL4, two IL4 mutants, C3T-IL4 and C24T-IL4, were used. NMR and ANS binding experiments were compatible with altered dynamics and an increase of the nonpolar solventaccessible surface area of the folded state of the mutant proteins. Chemical and thermal unfolding experiments followed by fluorescence and circular dichroism revealed that both mutant proteins have lower conformational stability than the wild-type protein. Transition temperatures of unfolding decreased 14C for C3T-IL4 and 10C for C24T-IL4, when compared toWT-IL4, and the conformational stability, at 25C, decreased 4.9 kcal/mol for C3T-IL4 and 3.2 kcal/mol for C24T-IL4. Interestingly, both the enthalpy and the entropy of unfolding, at the transition temperature, decreased in the mutant proteins. Moreover, a smaller change in heat capacity of unfolding was also observed for the mutants. Thus, disulfide bridges in IL4 play a critical role in maintaining the thermodynamic stability and core packing of the helix bundle.
- Lessons on protein structure from interleukin‐4: All disulfides are not created equalPublication . Vaz, Daniela C.; Rodrigues, J. Rui; Loureiro‐Ferreira, Nuno; Müller, Thomas D.; Sebald, Walter; Redfield, Christina; Brito, Rui M. M.Interleukin-4 (IL-4) is a hematopoietic cytokine composed by a four-helix bundle stabilized by an antiparallel beta-sheet and three disulfide bonds: Cys3-Cys127, Cys24-Cys65, and Cys46-Cys99. IL-4 is involved in several immune responses associated to infection, allergy, autoimmunity, and cancer. Besides its physiological relevance, IL-4 is often used as a “model” for protein design and engineering. Hence, to understand the role of each disulfide in the structure and dynamics of IL-4, we carried out several spectroscopic analyses (circular dichroism [CD], fluorescence, nuclear magnetic resonance [NMR]), and molecular dynamics (MD) simulations on wild-type IL-4 and four IL-4 disulfide mutants. All disulfide mutants showed loss of structure, altered interhelical angles, and looser core packings, showing that all disulfides are relevant for maintaining the overall fold and stability of the four-helix bundle motif, even at very low pH. In the absence of the disulfide connecting both protein termini Cys3-Cys127, C3T-IL4 showed a less packed protein core, loss of secondary structure ( 9%) and fast motions on the sub-nanosecond time scale (lower S2 order parameters and larger τc correlation time), especially at the two protein termini, loops, beginning of helix A and end of helix D. In the absence of Cys24-Cys65, C24T-IL4 presented shorter alpha-helices (14% loss in helical content), altered interhelical angles, less propensity to form the small anti-parallel beta-sheet and increased dynamics. Simultaneously deprived of two disulfides (Cys3-Cys127 and Cys24-Cys65), IL-4 formed a partially folded “molten globule” with high 8-anilino1-naphtalenesulphonic acid-binding affinity and considerable loss of secondary structure ( 50%decrease), as shown by the far UV-CD, NMR, and MD data.
- Morphological and Molecular Profiling of Amyloid-β Species in Alzheimer’s PathogenesisPublication . Almeida, Zaida L.; Vaz, Daniela C.; Brito, Rui M. M.Alzheimer’s disease (AD) is the most common form of dementia around the world (~ 65%). Here, we portray the neuropathology of AD, biomarkers, and classifcation of amyloid plaques (difuse, non-cored, dense core, compact). Tau pathology and its involvement with Aβ plaques and cell death are discussed. Amyloid cascade hypotheses, aggregation mechanisms, and molecular species formed in vitro and in vivo (on- and of-pathways) are described. Aβ42/Aβ40 monomers, dimers, trimers, Aβ‐derived difusible ligands, globulomers, dodecamers, amylospheroids, amorphous aggregates, protofbrils, fbrils, and plaques are characterized (structure, size, morphology, solubility, toxicity, mechanistic steps). An update on AD-approved drugs by regulatory agencies, along with new Aβ-based therapies, is presented. Beyond prescribing Aβ plaque disruptors, cholinergic agonists, or NMDA receptor antagonists, other therapeutic strategies (RNAi, glutaminyl cyclase inhibitors, monoclonal antibodies, secretase modulators, Aβ aggregation inhibitors, and anti-amyloid vaccines) are already under clinical trials. New drug discovery approaches based on “designed multiple ligands”, “hybrid molecules”, or “multitarget-directed ligands” are also being put forward and may contribute to tackling this highly debilitating and fatal form of human dementia.
- A New Folding Kinetic Mechanism for Human Transthyretin and the Influence of the Amyloidogenic V30M MutationPublication . Jesus, Catarina S. H.; Almeida, Zaida L.; Vaz, Daniela C.; Faria, Tiago Q.; Brito, Rui M. M.Protein aggregation into insoluble amyloid fibrils is the hallmark of several neurodegenerative diseases, chief among them Alzheimer’s and Parkinson’s. Although caused by different proteins, these pathologies share some basic molecular mechanisms with familial amyloidotic polyneuropathy (FAP), a rare hereditary neuropathy caused by amyloid formation and deposition by transthyretin (TTR) in the peripheral and autonomic nervous systems. Among the amyloidogenic TTR mutations known, V30M-TTR is the most common in FAP. TTR amyloidogenesis (ATTR) is triggered by tetramer dissociation, followed by partial unfolding and aggregation of the low conformational stability monomers formed. Thus, tetramer dissociation kinetics, monomer conformational stability and competition between refolding and aggregation pathways do play a critical role in ATTR. Here, we propose a new model to analyze the refolding kinetics of WT-TTR and V30M-TTR, showing that at pH and protein concentrations close to physiological, a two-step mechanism with a unimolecular first step followed by a second-order second step adjusts well to the experimental data. Interestingly, although sharing the same kinetic mechanism, V30M-TTR refolds at a much slower rate than WT-TTR, a feature that may favor the formation of transient species leading to kinetic partition into amyloidogenic pathways and, thus, significantly increasing the probability of amyloid formation in vivo.
- Nutritive Value and Bioactivities of a Halophyte Edible Plant: Crithmum maritimum L. (Sea Fennel)Publication . Correia, Iris; Antunes, Madalena; Tecelão, Carla; Neves, Marta; Pires, Cristiana L.; Cruz, Pedro F.; Peralta, Claúdia C.; Pereira, Cidália D.; Reboredo, Fernando; Moreno, Maria João; Brito, Rui M. M.; Ribeiro, Vânia S.; Vaz, Daniela C.; Campos, Maria; Rodrigues, Maria JorgeCrithmum maritimum L. (sea fennel), an edible xerophyte of coastal habitats, is considered an emerging cash crop for biosaline agriculture due to its salt-tolerance ability and potential applications in the agri-food sector. Here, the nutritional value and bioactive properties of sea fennel are described. Sea fennel leaves, flowers, and schizocarps are composed of carbohydrates (>65%) followed by ash, proteins, and lipids. Sea fennel’s salty, succulent leaves are a source of omega-6 and omega-3 polyunsaturated fatty acids, especially linoleic acid. Extracts obtained from flowers and fruits/schizocarps are rich in antioxidants and polyphenols and show antimicrobial activity against Staphylococcus aureus, Staphylococcus epidermis, Candida albicans, and Candida parapsilosis. Plant material is particularly rich in sodium (Na) but also in other nutritionally relevant minerals, such as calcium (Ca), chlorine (Cl), potassium (K), phosphorus (P), and sulfur (S), beyond presenting a potential prebiotic effect on Lactobacillus bulgaricus and being nontoxic to human intestinal epithelial Caco-2 model cells, up to 1.0% (w/v). Hence, the rational use of sea fennel can bring nutrients, aroma, and flavor to culinary dishes while balancing microbiomes and contributing to expanding the shelf life of food products.
- Oligomerization Profile of Human Transthyretin Variants with Distinct AmyloidogenicityPublication . Frangolho, Ana; Correia, Bruno E.; Vaz, Daniela C.; Almeida, Zaida L.; Brito, Rui M. M.One of the molecular hallmarks of amyloidoses is ordered protein aggregation involving the initial formation of soluble protein oligomers that eventually grow into insoluble fibrils. The identification and characterization of molecular species critical for amyloid fibril formation and disease development have been the focus of intense analysis in the literature. Here, using photo-induced cross-linking of unmodified proteins (PICUP), we studied the early stages of oligomerization of human transthyretin (TTR), a plasma protein involved in amyloid diseases (ATTR amyloidosis) with multiple clinical manifestations. Upon comparison, the oligomerization processes of wild-type TTR (TTRwt) and several TTR variants (TTRV30M, TTRL55P, and TTRT119M) clearly show distinct oligomerization kinetics for the amyloidogenic variants but a similar oligomerization mechanism. The oligomerization kinetics of the TTR amyloidogenic variants under analysis showed a good correlation with their amyloidogenic potential, with the most amyloidogenic variants aggregating faster (TTRL55P > TTRV30M > TTRwt). Moreover, the early stage oligomerization mechanism for these variants involves stepwise addition of monomeric units to the growing oligomer. A completely different behavior was observed for the nonamyloidogenic TTRT119M variant, which does not form oligomers in the same acidic conditions and even for longer incubation times. Thorough characterization of the initial steps of TTR oligomerization is critical for better understanding the origin of ATTR cytotoxicity and developing novel therapeutic strategies for the treatment of ATTR amyloidosis.