Percorrer por autor "Macedo, Anjos L."
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- Characterization of recombinant desulfovibrio gigas ferredoxinPublication . Rodrigues, Pedro; Graça, Fernando; Macedo, Anjos L.; Moura, Isabel; Moura, José J. G.Dg ferredoxin gene was cloned using the polymerase chain reaction (PCR), inserted into vector pT7-7, and overexpressed in Escherichia coli (E. coli) grown in aerobic media. The recombinant protein is a dimer and contains a [3Fe–4S] cluster per monomer. EPR and 1H NMR data of recombinant and wild-type protein are compared.
- Desulfovibrio gigas ferredoxin II: redox structural modulation of the [3Fe-4S] clusterPublication . Rodrigues, Pedro; Macedo, Anjos L.; Goodfellow, B. J.; Moura, Isabel; Moura, José J. G.Abstract Desulfovibrio gigas ferredoxin II (DgFdII) is a small protein with a polypeptide chain composed of 58 amino acids, containing one Fe3S4 cluster per monomer. Upon studying the redox cycle of this protein, we detected a stable intermediate (FdIIint) with four 1H resonances at 24.1, 20.5, 20.8 and 13.7 ppm. The differences between FdIIox and FdIIint were attributed to conformational changes resulting from the breaking/formation of an internal disulfide bridge. The same 1H NMR methodology used to fully assign the three cysteinyl ligands of the [3Fe–4S] core in the oxidized state (DgFdIIox) was used here for the assignment of the same three ligands in the intermediate state (DgFdIIint). The spin-coupling model used for the oxidized form of DgFdII where magnetic exchange coupling constants of around 300 cm 1 and hyperfine coupling constants equal to 1 MHz for all the three iron centres were found, does not explain the isotropic shift temperature dependence for the three cysteinyl cluster ligands in DgFdIIint. This study, together with the spin delocalization mechanism proposed here for DgFdIIint, allows the detection of structural modifications at the [3Fe-4S] cluster in DgFdIIox and DgFdIIint.
- Gla rich protein (GRP) mediates vascular smooth muscle cell (VSMC) osteogenic differentiation, extracellular vesicle (EV) calcification propensity, and immunomodulatory propertiesPublication . Maia, Teresa M.; Macedo, Anjos L.; Matos, António P.; Neves, José; Viegas, Carla; Camilo Carreira, Joana Sofia; Simes, DinaVascular calcification (VC) is a complex process involving vascular smooth muscle cell (VSMC) osteogenic differentiation, inflammation, and extracellular vesicle (EV) calcification and communication networks. Gla rich protein (GRP) is a calcification inhibitor involved in most of these processes. However, the molecular mechanism of GRP in VC and the specific characteristics, cargo, and functionality of calcifying EVs require further elucidation. Here, we use a combination of human ex vivo aortic fragments and primary vascular smooth muscle cell (VSMC) models to obtain new information on GRP function in VC and EVs released by VSMCs. We demonstrate that GRP inhibits VSMC osteogenic differentiation through downregulation of bone-related proteins and upregulation of mineralization inhibitors, with decreased mineral crystallinity in EVs deposited into the tissue extracellular matrix (ECM). EVs isolated by ultracentrifugation at 30K and 100K from the cell media (CM) and deposited in the ECM from control (CTR) and mineralizing (MM) VSMCs were biochemically, physically, and proteomically characterized. Four different EV populations were identified with shared markers commonly present in all EVs but with unique protein cargo and specific molecular profiles. Comparative proteomics identified several regulated proteins specifically loaded into MM EV populations associated with multiple processes involved in VC. Functional analysis demonstrated that 30K and 100K ECM-MM EVs with higher calcium and lower GRP levels induced macrophage inflammation. Our findings reinforce the functional relevance of GRP in multiple VC processes and suggest that ECM EVs released under calcification stress function as a new signaling axis on the calcification-inflammation cycle.
- Gla-rich protein function as an anti-inflammatory agent in monocytes/macrophages: implications for calcification-related chronic inflammatory diseasesPublication . Viegas, Carla; Costa, Ruben M.; Santos, Lúcia; Videira, Paula A.; Silva, Zelia; Araujo, Nuna C. P.; Macedo, Anjos L.; Matos, Antonio P.; Vermeer, Cees; Simes, DinaCalcification-related chronic inflammatory diseases are multifactorial pathological processes, involving a complex interplay between inflammation and calcification events in a positive feed-back loop driving disease progression. Gla-rich protein (GRP) is a vitamin K dependent protein (VKDP) shown to function as a calcification inhibitor in cardiovascular and articular tissues, and proposed as an anti-inflammatory agent in chondrocytes and synoviocytes, acting as a new crosstalk factor between these two interconnected events in osteoarthritis. However, a possible function of GRP in the immune system has never been studied. Here we focused our investigation in the involvement of GRP in the cell inflammatory response mechanisms, using a combination of freshly isolated human leucocytes and undifferentiated/differentiated THP-1 cell line. Our results demonstrate that VKDPs such as GRP and matrix gla protein (MGP) are synthesized and gamma-carboxylated in the majority of human immune system cells either involved in innate or adaptive immune responses. Stimulation of THP-1 monocytes/macrophages with LPS or hydroxyapatite (HA) up-regulated GRP expression, and treatments with GRP or GRP-coated basic calcium phosphate crystals resulted in the down-regulation of mediators of inflammation and inflammatory cytokines, independently of the protein gamma-carboxylation status. Moreover, overexpression of GRP in THP-1 cells rescued the inflammation induced by LPS and HA, by down-regulation of the proinflammatory cytokines TNF alpha, IL-1 beta and NFkB. Interestingly, GRP was detected at protein and mRNA levels in extracellular vesicles released by macrophages, which may act as vehicles for extracellular trafficking and release. Our data indicate GRP as an endogenous mediator of inflammatory responses acting as an anti-inflammatory agent in monocytes/macrophages. We propose that in a context of chronic inflammation and calcification-related pathologies, GRP might act as a novel molecular mediator linking inflammation and calcification events, with potential therapeutic application.
- Nanoencapsulation as a novel delivery approach for therapeutic applications of gla-rich protein (GRP)Publication . Araújo, Nuna; Viegas, Carla; Pontes, Jorge Filipe; Marreiros, Catarina; Raimundo, Pedro; Macedo, Anjos L.; Alves de Matos, António; Grenha, Ana; Vermeer, Cees; Simes, DinaGla rich protein (GRP) is a vitamin K dependent protein, shown to function as an inhibitor of pathological calcification and as an anti-inflammatory agent, with potential therapeutic use for age-related diseases such as osteoarthritis (OA) [1,2]. OA is a leading cause of disability and morbidity in the older population and constitutes a major world wide challenge for our health system. Presently, there are no drugs approved that can prevent, stop, or even restrain progression of OA. GRP has been shown to be able to lower inflammation and mineralisation processes in the articular tissue. Chitosan/tripolyphosphate (TPP) nanoparticles were selected for this study due to their biocompatibility, biodegradability and capacity to overcome the problem of low solubility of GRP in physiological conditions. This study aims to produce and characterise chitosan/TPP nanoparticles as GRP-delivery vehicles and test its anti-inflammatory potential in human macrophages.
- Nanoencapsulation of Gla-Rich Protein (GRP) as a novel approach to target inflammationPublication . Viegas, Carla; Araújo, Nuna; Carreira, Joana; Pontes, Jorge Filipe; Macedo, Anjos L.; Vinhas, Maurícia; Moreira, Ana S.; Faria, Tiago Q.; Grenha, Ana; de Matos, António A.; Schurgers, Leon; Vermeer, Cees; Simes, DinaChronic inflammation is a major driver of chronic inflammatory diseases (CIDs), with a tremendous impact worldwide. Besides its function as a pathological calcification inhibitor, vitamin K-dependent protein Gla-rich protein (GRP) was shown to act as an anti-inflammatory agent independently of its gamma-carboxylation status. Although GRP’s therapeutic potential has been highlighted, its low solubility at physiological pH still constitutes a major challenge for its biomedical application. In this work, we produced fluorescein-labeled chitosan-tripolyphosphate nanoparticles containing non-carboxylated GRP (ucGRP) (FCNG) via ionotropic gelation, increasing its bioavailability, stability, and anti-inflammatory potential. The results indicate the nanosized nature of FCNG with PDI and a zeta potential suitable for biomedical applications. FCNG’s anti-inflammatory activity was studied in macrophage-differentiated THP1 cells, and in primary vascular smooth muscle cells and chondrocytes, inflamed with LPS, TNFα and IL-1β, respectively. In all these in vitro human cell systems, FCNG treatments resulted in increased intra and extracellular GRP levels, and decreased pro-inflammatory responses of target cells, by decreasing pro-inflammatory cytokines and inflammation mediators. These results suggest the retained anti-inflammatory bioactivity of ucGRP in FCNG, strengthening the potential use of ucGRP as an anti-inflammatory agent with a wide spectrum of application, and opening up perspectives for its therapeutic application in CIDs.
- Redox thermodynamics of low-potential iron-sulfur proteinsPublication . Battistuzzi, Gianantonio; D'Onofrio, Mariapina; Borsari, Marco; Sola, Marco; Macedo, Anjos L.; Moura, José J. G.; Rodrigues, PedroThe enthalpy and entropy changes associated with protein reduction ( ) were determined for a number of low-potential iron-sulfur proteins through variable temperature direct electrochemical experiments. These data add to previous estimates making available, overall, the reduction thermodynamics for twenty species from various sources containing all the different types of metal centers. These parameters are discussed with reference to structural data and calculated electrostatic metal-environment interaction energies, and redox properties of model complexes. This work, which is the first systematic investigation on the reduction thermodynamics of Fe-S proteins, contributes to the comprehension of the determinants of the differences in reduction potential among different protein families within a novel perspective. Moreover, comparison with analogous data obtained previously for electron transport (ET) metalloproteins with positive reduction potentials, i. e., cytochromes c, blue copper proteins, and HiPIPs, helps our understanding of the factors controlling the reduction potential in ET species containing different metal cofactors. The main result of this work can be summarized as follows. HiPIPs as compares to the low-potential 4Fe Fds is entirely due to enthalpic effects related to the electostatic interactions of the cluster with the proteins matrix and the solvent.
- Spectroscopic characterization of a novel 2×[4Fe–4S] ferredoxin isolated from Desulfovibrio desulfuricans ATCC 27774Publication . Rodrigues, Pedro; Moura, Isabel; Macedo, Anjos L.; Moura, José J. G.A novel iron /sulfur containing protein, a ferredoxin (Fd), was purified to homogeneity from the extract of Desulfovibrio desulfuricans American type culture collection (ATCC) 27774. The purified protein is a 13.4 kDa homodimer with a polypeptide chain of 60 amino acids residues, containing eight cysteines that coordinate two [4Fe /4S] clusters. The protein is shown to be air sensitive and cluster conversions take place. We structurally characterize a redox state that contains two [4Fe /4S] cores. 1D and 2D 1H NMR studies are reported on form containing the clusters in the oxidized state. Based on the nuclear Overhauser effect (NOE), relaxation measurements and comparison of the present data with the available spectra of the analogous 8Fe Fds, the cluster ligands were specifically assigned to the eight-cysteinyl residues.
- The 3Fe containing ferredoxin from desulfovibrio gigas: an NMR characterization of the oxidised and intermediate statesPublication . Macedo, Anjos L.; Rodrigues, Pedro; Goodfellow, B. J.Ferredoxin II (FdII), isolated from Desulfo6ibrio gigas, is a small electron transfer protein that contains one [3Fe–4S] cluster per monomer (6 kDa). The characterization of the oxidized and intermediate forms of FdII was carried out by NMR spectroscopy, in conjunction with other spectroscopic techniques, such as EPR, UV–vis and Mo¨ssbauer spectroscopy, in order to fully understand the redox and electronic properties of the protein. The native oxidized state of FdII has been studied from the point of view of the spin coupling between the iron atoms, via the 1H-NMR temperature dependence of the b-CH2 proton resonances of the cysteinyl cluster ligands (Cys8, Cys14 and Cys50). The assignment of the 2D-NOESY spectrum has also been carried out: distances have been obtained for the diamagnetic region and 1D-NOE experiments were performed in order to detect NOEs for protons in the vicinity of the cluster, allowing the determination of the protein structure in solution. An intermediate state, FdIIint, detected by NMR spectroscopy, was attributed to the opening of the S–S bridge between Cys18 and Cys42, in the potential range of cluster reduction. This state has been characterized by several spectroscopic techniques, showing that the protein can transfer three electrons in one redox step, that involves the [3Fe–4S] cluster and the disulfide bridge. The temperature behavior of the hyperfine shifts, when compared with FdIIox, lead to the conclusion that the spin coupling of the cluster changes upon reduction of the S–S bond. 2D-NOESY spectra of the diamagnetic region were also collected for FdIIint, and compared with the native state, in order to clarify the structural changes occurring in the protein on cleavage of the S–S bridge.
- The solution structure of a [3Fe-4S] ferredoxin: oxidised ferredoxin II from desulfovibrio gigasPublication . Goodfellow, B. J.; Macedo, Anjos L.; Rodrigues, Pedro; Moura, Isabel; Wray, Victor; Moura, José J. G.The use of standard 2D NMR experiments in combination with 1D NOE experiments allowed the assignment of 51 of the 58 spin systems of oxidised [3Fe-4S] ferredoxin isolated from Desulfovibrio gigas. The NMR solution structure was determined using data from 1D NOE and 2D NOESY spectra, as distance constraints, and information from the X-ray structure for the spin systems not detected by NMR in torsion angle dynamics calculations to produce a family of 15 low target function structures. The quality of the NMR family, as judged by the backbone r.m.s.d. values, was good (0.80 Å), with the majority of f/c angles falling within the allowed region of the Ramachandran plot. A comparison with the X-ray structure indicated that the overall global fold is very similar in solution and in the solid state. The determination of the solution structure of ferredoxin II (FdII) in the oxidised state (FdIIox) opens the way for the determination of the solution structure of the redox intermediate state of FdII (FdIIint), for which no X-ray structure is available.