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- Development of novel alginate based hydrogel films for wound healing applicationsPublication . Pereira, Rúben; Carvalho, Anabela; Vaz, Daniela C.; Gil, M. H.; Mendes, Ausenda; Bártolo, PauloAlginate and Aloe vera are natural materials widely investigated and used in the biomedical field. In this research work, thin hydrogel films composed by alginate and Aloe vera gel in different proportions (95:5, 85:15 and 75:25, v/v) were prepared and characterized. The films were evaluated regarding the light transmission behavior, contact angle measurements, and chemical, thermal and mechanical properties. These thin hydrogel films, prepared by crosslinking reaction using 5% calcium chloride solution, were also investigated relatively to their water solubility and swelling behavior. Results showed that Aloe vera improved the transparency of the films, as well their thermal stability. The developed films present adequate mechanical properties for skin applications, while the solubility studies demonstrated the insolubility of the films after 24 h of immersion in distilled water. The water absorption and swelling behavior of these films were greatly improved by the increase in Aloe vera proportion.
- 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.
- BSA-PEG Hydrogel: A Novel Protein-Ligand Binding 3D MatrixPublication . Coelho, Carlos D. F.; Jesus, João A.; Vaz, Daniela C.; Lagoa, Ricardo; Moreno, Maria JoãoHydrogel materials have good biomimetic properties and high potential for biomedical and bioanalytical applications. In this work, a hydrogel of serum albumin crosslinked with poly-(ethylene glycol) was prepared and characterized for its water content, protein structure and stability. The ability of the hydrogel to bind small molecule ligands with different hydrophobicity was evaluated using a homologous series of amphiphiles (NBD-Cn, n = 4, 6 and 8) and the calculated binding affinities were similar to that of free protein in solution. Overall, the results indicate this type of hydrogel system as a convenient tool for studying the binding of xenobiotics to tissue proteins.
- Transthyretin mutagenesis: impact on amyloidogenesis and diseasePublication . Almeida, Zaida L.; Vaz, Daniela C.; Brito, Rui M. M.Transthyretin (TTR), a homotetrameric protein found in plasma, cerebrospinal fluid, and the eye, plays a pivotal role in the onset of several amyloid diseases with high morbidity and mortality. Protein aggregation and fibril formation by wild-type TTR and its natural more amyloidogenic variants are hallmarks of ATTRwt and ATTRv amyloidosis, respectively. The formation of soluble amyloid aggregates and the accumulation of insoluble amyloid fibrils and deposits in multiple tissues can lead to organ dysfunction and cell death. The most frequent manifestations of ATTR are polyneuropathies and cardiomyopathies. However, clinical manifestations such as carpal tunnel syndrome, leptomeningeal, and ocular amyloidosis, among several others may also occur. This review provides an up-to-date listing of all single amino-acid mutations in TTR known to date. Of approximately 220 single-point mutations, 93% are considered pathogenic. Aspartic acid is the residue mutated with the highest frequency, whereas tryptophan is highly conserved. “Hot spot” mutation regions are mainly assigned to β-strands B, C, and D. This manuscript also reviews the protein aggregation models that have been proposed for TTR amyloid fibril formation and the transient conformational states that convert native TTR into aggregation-prone molecular species. Finally, it compiles the various in vitro TTR aggregation protocols currently in use for research and drug development purposes. In short, this article reviews and discusses TTR mutagenesis and amyloidogenesis, and their implications in disease onset.
- Fotopolimerização de hidrogéis de ácido hialurónicoPublication . Duarte, Stacy; Pereira, Rúben; Campos, Carlos; Vaz, Daniela C.; Paulo Flores et al.O ácido hialurónico (HA) é um polissacarídeo amplamente presente no organismo humano e constitui um material com potencial para ser incorporado em hidrogéis direcionados para aplicações biomédicas. No entanto, as fracas propriedades mecânicas, rápida degradação e libertação in vivo do HA não reticulado solúvel limita muitas aplicações clínicas diretas. Deste modo, com vista melhorar as suas caraterísticas materiais procedemos à modificação química do HA com anidrido metacrílico (MA), com formação de ácido hialurónico metacrilado (MeHA), com propriedades fotopolimerizáveis, combinado ainda na presença e ausência de agente gelificante extra, tal como o polissacarídeo natural iota-carragenina. O HA utilizado foi caracterizado através de espectroscopia de ressonância magnética nuclear (RMN) e de dicroísmo circular (CD). Os hidrogéis produzidos foram analisados via testes de absorção de água e de degradação em meio aquoso, bem como através de análise mecânica dinâmica (DMA) à compressão. O estudo conduzido permitiu verificar que, enquanto os biofilmes de HA se mostraram quebradiços e com fracas propriedades mecânicas, o MeHA não só foi capaz de formar hidrogéis, por fotopolimerização, como a adição do iota-carragenina à formulação, possibilitou a formação de IPNs (Interpenetrating Polymer Networks) e de semi-IPNs com melhores propriedades físicas e mecânicas.
- Tetramer Dissociation and Monomer Partial Unfolding Precedes Protofibril Formation in Amyloidogenic Transthyretin VariantsPublication . Quintas, Alexandre; Vaz, Daniela C.; Cardoso, Isabel; Saraiva, Maria João M.; Brito, Rui M. M.Amyloid fibril formation and deposition is a common feature of a wide range of fatal diseases including spongiform encephalopathies, Alzheimer's disease, and familial amyloidotic polyneuropathies (FAP), among many others. In certain forms of FAP, the amyloid fibrils are mostly constituted by variants of transthyretin (TTR), a homotetrameric plasma protein. Recently, we showed that transthyretin in solution may undergo dissociation to a non-native monomer, even under close to physiological conditions of temperature, pH, ionic strength, and protein concentration. We also showed that this non-native monomer is a compact structure, does not behave as a molten globule, and may lead to the formation of partially unfolded monomeric species and high molecular mass soluble aggregates (Quintas, A., Saraiva, M. J. M., and Brito, R. M. M. (1999) J. Biol. Chem. 274, 32943–32949). Here, based on aging experiments of tetrameric TTR and chemically induced protein unfolding experiments of the non-native monomeric forms, we show that tetramer dissociation and partial unfolding of the monomer precedes amyloid fibril formation. We also show that TTR variants with the least thermodynamically stable non-native monomer produce the largest amount of partially unfolded monomeric species and soluble aggregates under conditions that are close to physiological. Additionally, the soluble aggregates formed by the amyloidogenic TTR variants showed morphological and thioflavin-T fluorescence properties characteristic of amyloid. These results allowed us to conclude that amyloid fibril formation by some TTR variants might be triggered by tetramer dissociation to a compact non-native monomer with low conformational stability, which originates partially unfolded monomeric species with a high tendency for ordered aggregation into amyloid fibrils. Thus, partial unfolding and conformational fluctuations of molecular species with marginal thermodynamic stability may play a crucial role on amyloid formationin vivo.
- Monitoring the modifications of Aloe vera by high performance liquid chromatographyPublication . Tojeira, Ana; Pereira, Rúben; Bártolo, Paulo; Mendes, Ausenda; Vaz, Daniela; Oliveira, Nelson SimõesAloe vera is a widely known and studied plant due to its therapeutic properties. The therapeutic properties exhibited by the Aloe vera exudates have been associated to the presence of certain compounds, such as, chromones, anthrones and anthraquinones. In this study, we have identified, and monitored by reversed phase high performance liquid chromatography (HPLC), six major compounds present in the Aloe vera exudates. The chromatographic profiles were followed in the course of time and at different wavelengths. This monitoring allowed us to verify the constitution and modifications of the samples, in order to identify the main changes responsible for the chemical degradation and loss of proprieties exhibited by the exudates over time.
- 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.
- Use of molecular interaction fields to understand drug resistance in HIV 1 protease caused by single point mutationsPublication . Vaz, Daniela C.; Brito, Rui M. M.; Mata, Ana I.; Alves, Nuno G.; Simões, Carlos J. V.; Vaz, João Pereira; Duque, VitorMolecular Interaction Fields (MIF) is an archetypal computational chemistry technique that can be applied to capture a singular fingerprint of an ensemble of atoms on a protein and encode its physicochemical environment. Thus, MIFs have particular relevance in the context of binding hot spots and binding site analysis. Taking HIV 1 Protease (HIVPR) as case study, the present work focuses on a MIF-based in silico approach to achieve a qualitative interpretation and quantitative determination of mutation effects on HIVPR’s binding site, to help to understand translated changes in the enzyme’s structure and physicochemical environment. Assuming that binding sites with similar chemical environments have similar affinity for inhibitors, our method calculates and compares MIF similarities, visually assessing structural differences and quantifying their overlap through a Tanimoto coefficient. To assess the method’s ability to capture mutation induced chemical perturbations within HIVPR’s binding site, we collected 48 X-ray structures from the Protein Data Bank (PDB), from HIV strains either resistant or susceptible to protease inhibitors and quantified their binding site MIF similarities against a high quality, susceptible, reference structure. We observed and defined a threshold that discriminated most susceptible and resistant structures, confirming the MIF's suitability for our approach. Subsequently, we built homology models containing different reported single point resistance-conferring mutations using a single high-quality PDB structure as template. Root-Mean-Square Deviation (RMSD) values between template and model structures were calculated on residue by residue basis, confirming that the mutation was the only structural change. Then, the MIF similarities were determined, showing that this technique effectively captured subtle changes on HIVPR’s binding sites induced by the studied mutations. Along with the perspective of following an equivalent ligand based approach, we believe our results can be a promising starting point for developing an algorithm with drug resistance predictive power.