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Molecular motion regulates the activity of the mitochondrial serine protease HtrA2
Publication . Merski, Matthew; Moreira, Cátia; Abreu, Rui M.V.; Ramos, Maria João; Fernandes, Pedro Alexandrino; Martins, L. Miguel; Pereira, Pedro José Barbosa; Macedo-Ribeiro, Sandra; Merski, Matthew
HtrA2 (high-temperature requirement 2) is a human mitochondrial protease that has a role in apoptosis and Parkinson's disease. The structure of HtrA2 with an intact catalytic triad was determined, revealing a conformational change in the active site loops, involving mainly the regulatory LD loop, which resulted in burial of the catalytic serine relative to the previously reported structure of the proteolytically inactive mutant. Mutations in the loops surrounding the active site that significantly restricted their mobility, reduced proteolytic activity both in vitro and in cells, suggesting that regulation of HtrA2 activity cannot be explained by a simple transition to an activated conformational state with enhanced active site accessibility. Manipulation of solvent viscosity highlighted an unusual bi-phasic behavior of the enzymatic activity, which together with MD calculations supports the importance of motion in the regulation of the activity of HtrA2. HtrA2 is an unusually thermostable enzyme (TM=97.3 °C), a trait often associated with structural rigidity, not dynamic motion. We suggest that this thermostability functions to provide a stable scaffold for the observed loop motions, allowing them a relatively free conformational search within a rather restricted volume.
A microfluidic deformability assessment of pathological red blood cells flowing in a hyperbolic converging microchannel
Publication . Faustino, Vera; Rodrigues, Raquel Oliveira; Pinho, Diana; Costa, Elísio; Santos-Silva, Alice; Miranda, Vasco; Amaral, Joana S.; Lima, Rui A.
The loss of the red blood cells (RBCs) deformability is related with many human diseases,
such as malaria, hereditary spherocytosis, sickle cell disease, or renal diseases. Hence, during the
last years, a variety of technologies have been proposed to gain insights into the factors a ecting the
RBCs deformability and their possible direct association with several blood pathologies. In this work,
we present a simple microfluidic tool that provides the assessment of motions and deformations of
RBCs of end-stage kidney disease (ESKD) patients, under a well-controlled microenvironment. All of
the flow studies were performed within a hyperbolic converging microchannels where single-cell
deformability was assessed under a controlled homogeneous extensional flow field. By using a
passive microfluidic device, RBCs passing through a hyperbolic-shaped contraction were measured
by a high-speed video microscopy system, and the velocities and deformability ratios (DR) calculated.
Blood samples from 27 individuals, including seven healthy controls and 20 having ESKD with or
without diabetes, were analysed. The obtained data indicates that the proposed device is able to
detect changes in DR of the RBCs, allowing for distinguishing the samples from the healthy controls
and the patients. Overall, the deformability of ESKD patients with and without diabetes type II
is lower in comparison with the RBCs from the healthy controls, with this di erence being more
evident for the group of ESKD patients with diabetes. RBCs from ESKD patients without diabetes
elongate on average 8% less, within the hyperbolic contraction, as compared to healthy controls;
whereas, RBCs from ESKD patients with diabetes elongate on average 14% less than the healthy
controls. The proposed strategy can be easily transformed into a simple and inexpensive diagnostic
microfluidic system to assess blood cells deformability due to the huge progress in image processing
and high-speed microvisualization technology.
Phenolic composition and antioxidant activity assessment of southeastern and south Brazilian propolis
Publication . Coelho, Joana C.M.M.; Falcão, Soraia; Vale, Nuno; Almeida-Muradian, Ligia Bicudo; Vilas-Boas, Miguel
Propolis is a resinous substance collected by honey bees Apis mellifera from several plant sources and used in the hive to seal the walls, to strengthen the ends of the honey comb or embalm dead invaders. The chemical specificity of propolis is directly determined by the variability of the plant origins and by geographical and climatic features of the collection site. The aim of this work was the quality assessment of 16 south and southeast Brazilian propolis samples through the identification and quantification of phenolic compounds using chromatographic and spectroscopic techniques such as HPLC and LC/DAD/ESI-MS n . Generally, the samples presented a phenolic profile related to Brazilian green propolis with origin in Baccharis spp. leaves, where the caffeoylquinic ac id derivatives as well as dihydrokaempferide and artepillin C were the main compounds. Moreover, DPPH • free radical-scavenging activity, reducing power and differential pulse voltammetry were applied to evaluate the antioxidant activity. Differential pulse voltammetry proved to be a rapid and easy tool for the quantification of the total electroactive species present in the samples. The results revealed a richer phenolic composition and higher bioactivity in Minas Gerais samples rather than the southern ones.
Arylxanthones with anti-inflammator potential in cellular systems
Publication . Santos, Clementina M.M.; Ribeiro, Daniela; Silva, Artur; Fernandes, Eduarda
Xanthones are a family of naturally-occurring oxygenated heterocyclic compounds. A wide range of
natural and synthetic analogues are known to possess diverse biological activities including antifungal,
antimalarial, antioxidant and antitumor, among others [1]. There are only a few publications related to
arylxanthones [2], most of them focused on arylxanthones synthetic strategies and their biomedical
potential, mainly highlighting them as effective scavengers of reactive oxygen species (ROS) and
reactive nitrogen species (RNS) [3,4]. As far as we know, the anti-inflammatory potential of xanthones
bearing an aryl group has not been studied so far. With this idea in mind, our purpose was to evaluate
the putative anti-inflammatory effects of several arylxanthones bearing hydroxyl groups in certain
positions of their main core, namely through their ability to inhibit 5-lipoxygenase (5-LOX) and
cyclooxygenase 1 (COX-1) and 2 (COX-2), both enzymes involved in the arachidonic acid metabolism
[5,6].
Preliminary results showed that some of the studied arylxanthones were able to prevent leukotriene B4
production in human neutrophils, the xanthone with a catechol group at position 2 being the most active
one. The inhibition of prostaglandin E2 production was assessed in human whole blood and the majority
of the tested compounds were able to inhibit COX-1 while being completely ineffective in COX-2.
Design, synthesis and preliminar antioxidant evaluation of new hydroxy- chromone and xanthone derivatives
Publication . Albuquerque, Hélio; Proença, Carina; Ribeiro, Daniela; Freitas, Marisa; Santos, Clementina M.M.; Fernandes, Eduarda; Silva, Artur
Chromone and xanthone derivativas are well-known for their outstanding antioxidant
properties. ln an effort to develop new antioxidants with improved efficacy, here we
cteveloped a new synthetic strategy to prepare hydroxylated chromones 3 and xanthones 4
with extended conjugated rr-systems. The synthetic strategy involved the aldolcondensation
of 2-methylchromones 1 with cinnamaldehyde 2 to give chromones 3.
Subsequent electrocyclization and oxidation of chromones 3 afforded xanthones 4 (Fig. 1 ).
The scavenging activities of both derivativas 3 and 4 were addressed against both reactive
oxygen species (ROS) and reactive nitrogen species (RNS). Ali tested compounds exhibited
scavenger effects dependent on the concentration, with ICso values found in the micromolar
range [1].
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Funding agency
Fundação para a Ciência e a Tecnologia
Funding programme
5876
Funding Award Number
UID/Multi/04378/2013