Protein folding, metal ions and conformational states: the case of a di-cluster ferredoxin

Dissertation presented to obtain the PhD degree in Biochemistry at the Instituto de Tecnologia Química e Biológica, Universidade Nova de LisboaMetal ions are present in over thirty percent of known proteins. Apart from a well established function in catalysis and electron transfer, metals and metal centres are also important structural elements which may as well play a key role in modulating protein folding and stability. In this respect, cofactors can act not only as local structural stabilizing elements in the native state, contributing to the maintenance of a given specific structural fold, but may also function as potential nucleation points during the protein folding process...Fundação para a Ciência e Tecnologia is acknowledged for financial support, by awarding a PhD Grant SFRH/BD/18653/2004. This work has been funded by the projects POCTI/QUI/37521; POCTI/QUI/45758 and PTDC/QUI/70101 all to Cláudio M. Gomes

S100A6 Amyloid Fibril Formation Is Calcium-modulated and Enhances Superoxide Dismutase-1 (SOD1) Aggregation

S100A6 is a small EF-hand calcium- and zinc-binding protein involved in the regulation of cell proliferation and cytoskeletal dynamics. It is overexpressed in neurodegenerative disorders and a proposed marker for Amyotrophic Lateral Sclerosis (ALS). Following recent reports of amyloid formation by S100 proteins, we investigated the aggregation properties of S100A6. Computational analysis using aggregation predictors Waltz and Zyggregator revealed increased propensity within S100A6 helices HI and HIV. Subsequent analysis of Thioflavin-T binding kinetics under acidic conditions elicited a very fast process with no lag phase and extensive formation of aggregates and stacked fibrils as observed by electron microscopy. Ca2+ exerted an inhibitory effect on the aggregation kinetics, which could be reverted upon chelation. An FT-IR investigation of the early conformational changes occurring under these conditions showed that Ca2+ promotes anti-parallel β-sheet conformations that repress fibrillation. At pH 7, Ca2+ rendered the fibril formation kinetics slower: time-resolved imaging showed that fibril formation is highly suppressed, with aggregates forming instead. In the absence of metals an extensive network of fibrils is formed. S100A6 oligomers, but not fibrils, were found to be cytotoxic, decreasing cell viability by up to 40%. This effect was not observed when the aggregates were formed in the presence of Ca2+. Interestingly, native S1006 seeds SOD1 aggregation, shortening its nucleation process. This suggests a cross-talk between these two proteins involved in ALS. Overall, these results put forward novel roles for S100 proteins, whose metal-modulated aggregation propensity may be a key aspect in their physiology and function

A protocol to evaluate retinal vascular response using optical coherence tomography angiography

Copyright © 2019 Sousa, Leal, Moreira, do Vale, Silva-Herdade, Aguiar, Dionísio, Abegão Pinto, Castanho and Marques-Neves. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY).The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these termsIntroduction: Optical coherence tomography angiography (OCT-A) is a novel diagnostic tool with increasing applications in ophthalmology clinics that provides non-invasive high-resolution imaging of the retinal microvasculature. Our aim is to report in detail an experimental protocol for analyzing both vasodilatory and vasoconstriction retinal vascular responses with the available OCT-A technology. Methods: A commercial OCT-A device was used (AngioVue®, Optovue, CA, United States), and all examinations were performed by an experienced technician using the standard protocol for macular examination. Two standardized tests were applied: (i) the hypoxia challenge test (HCT) and (ii) the handgrip test, in order to induce a vasodilatory and vasoconstriction response, respectively. OCT-A was performed at baseline conditions and during the stress test. Macular parafoveal vessel density of the superficial and deep plexuses was assessed from the en face angiograms. Statistical analysis was performed using STATA v14.1 and p < 0.05 was considered for statistical significance. Results: Twenty-four eyes of 24 healthy subjects (10 male) were studied. Mean age was 31.8 ± 8.2 years (range, 18–57 years). Mean parafoveal vessel density in the superficial plexus increased from 54.7 ± 2.6 in baseline conditions to 56.0 ± 2.0 in hypoxia (p < 0.01). Mean parafoveal vessel density in the deep plexuses also increased, from 60.4 ± 2.2 at baseline to 61.5 ± 2.1 during hypoxia (p < 0.01). The OCT-A during the handgrip test revealed a decrease in vessel density in both superficial (55.5 ± 2.6 to 53.7 ± 2.9, p < 0.001) and deep (60.2 ± 1.8 to 56.7 ± 2.8, p < 0.001) parafoveal plexuses. Discussion: In this work, we detail a simple, non-invasive, safe, and non-costly protocol to assess a central nervous system vascular response (i.e., the retinal circulation) using OCT-A technology. A vasodilatory response and a vasoconstriction response were observed in two physiologic conditions—mild hypoxia and isometric exercise, respectively. This protocol constitutes a new way of studying retinal vascular changes that may be applied in health and disease of multiple medical fields.This study was supported by the Faculty of Medicine of the University of Lisbon, AstraZeneca Foundation – 14th Grant.info:eu-repo/semantics/publishedVersio

Retinal vascular reactivity in type 1 diabetes patients without retinopathy using optical coherence tomography angiography

Copyright © 2020 The Authors. This work is licensed under a Creative Commons Attribution-Non-Commercial-No-Derivatives 4.0 International License.Purpose: We hypothesize that patients with type 1 diabetes (T1D) may have abnormal retinal vascular responses before diabetic retinopathy (DR) is clinically evident. Optical coherence tomography angiography (OCTA) was used to dynamically assess the retinal microvasculature of diabetic patients with no clinically visible retinopathy. Methods: Controlled nonrandomized interventional study. The studied population included 48 eyes of 24 T1D patients and 24 demographically similar healthy volunteers. A commercial OCTA device (AngioVue) was used, and two tests were applied: (1) the hypoxia challenge test (HCT) and (2) the handgrip test to induce a vasodilatory or vasoconstrictive response, respectively. The HCT is a standardized test that creates a mild hypoxic environment equivalent to a flight cabin. The handgrip test (i.e., isometric exercise) induces a sympathetic autonomic response. Changes in the parafoveal superficial and deep capillary plexuses in both tests were compared in each group. Systemic cardiovascular responses were also comparatively evaluated. Results: In the control cohort, the vessel density of the median parafoveal superficial and deep plexuses increased during hypoxia (F1,23 = 15.69, P < 0.001 and F1,23 = 16.26, P < 0.001, respectively). In the T1D group, this physiological response was not observed in either the superficial or the deep retinal plexuses. Isometric exercise elicited a significant decrease in vessel density in both superficial and deep plexuses in the control group (F1,23 = 27.37, P < 0.0001 and F1,23 = 27.90, P < 0.0001, respectively). In the T1D group, this response was noted only in the deep plexus (F1,23 = 11.04, P < 0.01). Conclusions: Our work suggests there is an early impairment of the physiological retinal vascular response in patients with T1D without clinical diabetic retinopathy.info:eu-repo/semantics/publishedVersio

Characterization of Plasma Labile Heme in Hemolytic Conditions

The deposited article is the accepted manuscript (post-print version) posted online 7 August 2017 and provided by The Febs Journal. This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. The deposited article version contains attached the supplementary materials within the pdf. This publication hasn't any creative commons license associated, although it is in open access.Extracellular hemoglobin (Hb), a byproduct of hemolysis, can release its prosthetic heme groups upon oxidation. This produces metabolically active heme that is exchangeable between acceptor proteins, macromolecules and low molecular weight ligands, termed here labile heme. As it accumulates in plasma labile heme acts in a pro-oxidant manner and regulates cellular metabolism while exerting pro-inflammatory and cytotoxic effects that foster the pathogenesis of hemolytic diseases. Here we developed and characterized a panel of heme-specific single domain antibodies (sdAbs) that together with a cellular-based heme reporter assay, allow for quantification and characterization of labile heme in plasma during hemolytic conditions. Using these approaches we demonstrate that labile heme generated during hemolytic conditions is bound to plasma molecules with an affinity higher than 10(-7) M and that 2-8% (~2-5 μM) of the total amount of heme detected in plasma can be internalized by bystander cells, i.e. bioavailable heme. Acute, but not chronic, hemolysis is associated with transient reduction of plasma heme binding capacity (HBC1/2 ), that is, the ability of plasma molecules to bind labile heme with an affinity higher than 10(-7) M. The heme-specific sdAbs neutralize the pro-oxidant activity of soluble heme in vitro, suggesting that these maybe used to counter the pathologic effects of labile heme during hemolytic conditions. Finally, we show that heme-specific sdAbs can be used to visualize cellular heme. In conclusion, we describe a panel of heme-specific sdAbs that when used with other approaches provide novel insights to the pathophysiology of heme. This article is protected by copyright. All rights reserved.Fundação para a Ciência e Tecnologia grants: (RECI-IMI-IMU-0038-2012, PTDC/SAU-TOX/116627/2010, HMSP-ICT/0018/2011, SFRH/BD/44828/2008, SFRH/BPD/47477/2008, PTDC/SAU-FAR/119173/2010, IF/01010/2013/CP1183/CT0001); ERC grants: (ERC-2011-AdG 294709-DAMAGECONTROL); NHMRC Senior Principal Research Fellowship: (1003484).info:eu-repo/semantics/acceptedVersio

Avaliação da Vergonha em Adolescentes: ‘The Other as Shamer Scale’

Shame, as a self-conscious, multidimensional and socially focused emotion, plays a central role in the mental health of individuals. In adolescents, shame is also a frequent experience and its assessment is important for research and clinical practice. This study aims to validate a brief measure of external shame (Other as Shamer Scale – brief version for adolescents: (OASB-A). The participants were 834 adolescents with a mean age of 15 years. The final model of the OASB-A (8 items), obtained through CFA, presents a good fit to the data. The OASB-A shows a good internal consistency and an adequate temporal reliability. The OASB-A also reveals significant correlations with traumatic shame experiences (IES-R) and psychopathological symptoms (DASS-21). The OASB-A is an economic and reliable measure to assess external shame in adolescents

A comprehensive assessment of the transcriptome of cork oak (Quercus suber) through EST sequencing

Background: Cork oak (Quercus suber) is one of the rare trees with the ability to produce cork, a material widely used to make wine bottle stoppers, flooring and insulation materials, among many other uses. The molecular mechanisms of cork formation are still poorly understood, in great part due to the difficulty in studying a species with a long life-cycle and for which there is scarce molecular/genomic information. Cork oak forests are of great ecological importance and represent a major economic and social resource in Southern Europe and Northern Africa. However, global warming is threatening the cork oak forests by imposing thermal, hydric and many types of novel biotic stresses. Despite the economic and social value of the Q. suber species, few genomic resources have been developed, useful for biotechnological applications and improved forest management. Results: We generated in excess of 7 million sequence reads, by pyrosequencing 21 normalized cDNA libraries derived from multiple Q. suber tissues and organs, developmental stages and physiological conditions. We deployed a stringent sequence processing and assembly pipeline that resulted in the identification of ~159,000 unigenes. These were annotated according to their similarity to known plant genes, to known Interpro domains, GO classes and E.C. numbers. The phylogenetic extent of this ESTs set was investigated, and we found that cork oak revealed a significant new gene space that is not covered by other model species or EST sequencing projects. The raw data, as well as the full annotated assembly, are now available to the community in a dedicated web portal at http://www.corkoakdb.org. Conclusions: This genomic resource represents the first trancriptome study in a cork producing species. It can be explored to develop new tools and approaches to understand stress responses and developmental processes in forest trees, as well as the molecular cascades underlying cork differentiation and disease response.Peer Reviewe

Calcium dysregulation links ALS defective proteins and motor neuron selective vulnerability

More than 20 distinct gene loci have so far been implicated in amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder characterized by progressive neurodegeneration of motor neurons (MN) and death. Most of this distinct set of ALS-related proteins undergoes toxic deposition specifically in MN for reasons which remain unclear. Here we overview a recent body of evidence indicative that mutations in ALS-related proteins can disrupt fundamental Ca2+ signalling pathways in MN, and that Ca2+ itself impacts both directly or indirectly in many ALS critical proteins and cellular processes that result in MN neurodegeneration. We argue that the inherent vulnerability of MN to dysregulation of intracellular Ca2+ is deeply associated with discriminating pathogenicity and aberrant crosstalk of most of the critical proteins involved in ALS. Overall, Ca2+ deregulation in MN is at the cornerstone of different ALS processes and is likely one of the factors contributing to the selective susceptibility of these cells to this particular neurodegenerative disease

Small Molecules Present in the Cerebrospinal Fluid Metabolome Influence Superoxide Dismutase 1 Aggregation

Superoxide dismutase 1 (SOD1) aggregation is one of the pathological markers of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder. The underlying molecular grounds of SOD1 pathologic aggregation remains obscure as mutations alone are not exclusively the cause for the formation of protein inclusions. Thus, other components in the cell environment likely play a key role in triggering SOD1 toxic aggregation in ALS. Recently, it was found that ALS patients present a specific altered metabolomic profile in the cerebrospinal fluid (CSF) where SOD1 is also present and potentially interacts with metabolites. Here we have investigated how some of these small molecules affect apoSOD1 structure and aggregation propensity. Our results show that as co-solvents, the tested small molecules do not affect apoSOD1 thermal stability but do influence its tertiary interactions and dynamics, as evidenced by combined biophysical analysis and proteolytic susceptibility. Moreover, these compounds influence apoSOD1 aggregation, decreasing nucleation time and promoting the formation of larger and less soluble aggregates, and in some cases polymeric assemblies apparently composed by spherical species resembling the soluble native protein. We conclude that some components of the ALS metabolome that shape the chemical environment in the CSF may influence apoSOD1 conformers and aggregation

Role of a novel disulfide bridge within the all-beta fold of soluble Rieske proteins.

Rieske proteins and Rieske ferredoxins are present in the three domains of life and are involved in a variety of cellular processes. Despite their functional diversity, these small Fe-S proteins contain a highly conserved all-beta fold, which harbors a [2Fe-2S] Rieske center. We have identified a novel subtype of Rieske ferredoxins present in hyperthermophilic archaea, in which a two-cysteine conserved SKTPCX((2-3))C motif is found at the C-terminus. We establish that in the Acidianus ambivalens representative, Rieske ferredoxin 2 (RFd2), these cysteines form a novel disulfide bond within the Rieske fold, which can be selectively broken under mild reducing conditions insufficient to reduce the [2Fe-2S] cluster or affect the secondary structure of the protein, as shown by visible circular dichroism, absorption, and attenuated total reflection Fourier transform IR spectroscopies. RFd2 presents all the EPR, visible absorption, and visible circular dichroism spectroscopic features of the [2Fe-2S] Rieske center. The cluster has a redox potential of +48 mV (25 degrees C and pH 7) and a pK (a) of 10.1 +/- 0.2. These shift to +77 mV and 8.9 +/- 0.3, respectively, upon reduction of the disulfide. RFd2 has a melting temperature near the boiling point of water (T(m) = 99 degrees C, pH 7.0), but it becomes destabilized upon disulfide reduction (DeltaT(m) = -9 degrees C, DeltaC(m) = -0.7 M guanidinium hydrochloride). This example illustrates how the incorporation of an additional structural element such as a disulfide bond in a highly conserved fold such as that of the Rieske domain may fine-tune the protein for a particular function or for increased stability