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- 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.
- 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.
- Preserving and hydrogel-matrixing the bioactive properties of aromatic medicinal halophytic herbs from the coastline of the Iberian PeninsulaPublication . Parracho, Tiago; Cruz, Pedro F.; Peralta, Claúdia C.; Silva, Cândida G.; Campos, Maria Jorge; Neves, Marta; Cordeiro, Rachel; Trindade, Daniela; Moura, Carla; Almeida, Zaida L.; Pereira, Cidália D.; Guimarães, Carla; Brito, Rui M. M.; Guerra, Mauro; Reboredo, Fernando; Veríssimo, Paula; Vaz, Daniela C.; Santos Ribeiro, VâniaEdible wild plants are part of the ethnobotanic heritage of a certain geographical area and are important sources of essential oils, antioxidants, minerals, and special flavours. Corema album (Portuguese crowberry), Crithmum maritimum (sea fennel), Eryngium maritimum (sea holly), Helichrysum italicum (curry plant) and Otanthus maritimus (cottonweed) wildly flourish along the sandy dunes of the coast of the Iberian Peninsula. These plants are locally known for their beneficial properties, with important value for food, cosmetics and/or medicinal applications. Hence, leaves of these endemic species were collected at four different locations and submitted to different preserving treatments (oven-drying, freezing, and freeze-drying). Acetonic extracts of the different plants submitted to the different post-harvesting treatments were analysed regarding their antioxidant capacities and phenolic contents. Plant extracts were also analysed by diffusion-ordered nuclear magnetic resonance spectroscopy (DOSY-NMR). In general, freeze-drying was the best method of preserving plant minerals, antioxidants (~4 mgVCEAC/g fw) and polyphenols (~5 mgGAE/g fw). Minerals were quantified via energy-dispersive X-ray fluorescence spectrometry, and despite their location, all plants were rich in Ca, Cl, K, S and P. Hierarchical clustering and principal component analyses (PCA) pointed towards chemical/metabolic proximity between taxonomic families. Alginate hydrogels loaded with 0.1 % and 0.2 % (w/v) of extracts presented homogenous surface properties by scanning electron microscopy, good mechanical tensile strength (~30 MPa) and antibacterial activity against S. aureus. Edible alginate hydrogels enriched with plant extracts hold great nutraceutical potential to be used as natural preservatives for food coating and packaging or as sources of bioactive compounds for biomedical applications.