Defective protein folding and function in metabolic disorders: studies on the mitochondrial flavoenzyme ETF

Dissertation presented to obtain the PhD degree in Biochemistry at the Instituto de Tecnologia Química e Biológica, Universidade Nova de LisboaThe work presented in this dissertation concerns the study of the electron transfer flavoprotein (ETF), a protein involved in mitochondrial β-oxidation whose deficiency is associated to multiple acyl-CoA dehydrogenase deficiency (MADD). The thesis will focus on establishing the functional, cellular and molecular consequences of the genetic variability in ETF, and in particular it aims to clarify the basis for the effect of heat stress on disease progression. Moreover, the beneficial effects of vitamin B2 supplementation will be addressed.(...

Tropicalization of fish assemblages in temperate biogeographic transition zones

Biogeographic transition zones in marine temperate systems are often hotspots of biodiversity, with high levels of resilience to short-term climate shifts due to naturally occurring cyclic oscillations of oceanographic conditions. However, these environments are likely vulnerable to a steady global warming scenario in which these cyclical conditions could be disrupted. Here, we evaluate how changes in local oceanography affect the structure of rocky reef fish assemblages over a period of 50 yr in a biogeographic transition zone. Using a 12 yr time series of rocky reef fish assemblage structure, we identified the set of oceanographic variables that most influenced assemblage dynamics. Descriptive and predictive models (multivariate regression trees) were compared to observed data using the area under the curve. Winter northward wind stress and sea surface temperature (SST) were the most important drivers of change in assemblage structure. Only warmer years had indicator species with warm-temperate or tropical affinities. A fish assemblage 'tropicalization' index was developed in response to both local-spatial resolution and short-term environmental variation (1993-2011), and to regional spatial resolution and long-term SST (1960-2012). Predictive modelling for the last 50 yr revealed that species with tropical affinities have increased in frequency compared to cold-temperate species, coinciding with the trend of increasing mean winter SST. Since the mid-1980s, warm-temperate and tropical species have responded rapidly to more frequent warm winters, suggesting that species distributions are shifting polewards. Our results support a hypothesis that cold species retreat more slowly than the advance of warm species. We discuss the importance of transition zones as ` barometers' of climate change

Mutations at the flavin binding site of ETF:QO yield a MADD-like severe phenotype in Drosophila

Following a screening on EMS-induced Drosophila mutants defective for formation and morphogenesis of epithelial cells, we have identified three lethal mutants defective for the production of embryonic cuticle. The mutants are allelic to the CG12140 gene, the fly homologue of electron transfer flavoprotein:ubiquinone oxidoreductase (ETF:QO). In humans, inherited defects in this inner membrane protein account for multiple acyl-CoA dehydrogenase deficiency (MADD), a metabolic disease of beta-oxidation, with a broad range of clinical phenotypes, varying from embryonic lethal to mild forms. The three mutant alleles carried distinct missense mutations in ETF:QO (G65E, A68V and S104F) and maternal mutant embryos for ETF:QO showed lethal morphogenetic defects and a significant induction of apoptosis following germ-band elongation. This phenotype is accompanied by an embryonic accumulation of short- and medium-chain acylcarnitines (C4. C8 and 02) as well as long-chain acylcarnitines (C14 and C16:1), whose elevation is also found in severe MADD forms in humans under intense metabolic decompensation. In agreement the ETF:QO activity in the mutant embryos is markedly decreased in relation to wild type activity. Amino acid sequence analysis and structural mapping into a molecular model of ETF:QO show that all mutations map at FAD interacting residues, two of which at the nucleotide-binding Rossmann fold. This structural domain is composed by a beta-strand connected by a short loop to an alpha-helix, and its perturbation results in impaired cofactor association via structural destabilisation and consequently enzymatic inactivation. This work thus pinpoints the molecular origins of a severe MADD-like phenotype in the fruit fly and establishes the proof of concept concerning the suitability of this organism as,a potential model organism for MADD. (C) 2012 Elsevier B.V. All rights reserved.Fundacao para a Ciencia e Tecnologia (FCT/MCTES, Portugal) [PTDC/SAU-GMG/70033/2006, PTDC/QUI-BIQ/113027/2009, PTDC/BIA-BCM/111822/2009, PTDC/SAU-BID/111796/2009, SFRH/BPD/41609/2007, SFRH/BPD/74475/2010, SFRH/BPD/34763/2007]; CLIMB UK; [PEst-OE/EQB/LA0004/2011]info:eu-repo/semantics/publishedVersio

Deglutarylation of glutaryl-CoA dehydrogenase by deacylating enzyme SIRT5 promotes lysine oxidation in mice

A wide range of protein acyl modifications has been identified on enzymes across various metabolic processes; however, the impact of these modifications remains poorly understood. Protein glutarylation is a recently identified modification that can be nonenzymatically driven by glutaryl-CoA. In mammalian systems, this unique metabolite is only produced in the lysine and tryptophan oxidative pathways. To better understand the biology of protein glutarylation, we studied the relationship between enzymes within the lysine/tryptophan catabolic pathways, protein glutarylation, and regulation by the deglutarylating enzyme sirtuin 5 (SIRT5). Here, we identify glutarylation on the lysine oxidation pathway enzyme glutaryl-CoA dehydrogenase (GCDH) and show increased GCDH glutarylation when glutaryl-CoA production is stimulated by lysine catabolism. Our data reveal that glutarylation of GCDH impacts its function, ultimately decreasing lysine oxidation. We also demonstrate the ability of SIRT5 to deglutarylate GCDH, restoring its enzymatic activity. Finally, metabolomic and bioinformatic analyses indicate an expanded role for SIRT5 in regulating amino acid metabolism. Together, these data support a feedback loop model within the lysine/tryptophan oxidation pathway in which glutaryl-CoA is produced, in turn inhibiting GCDH function via glutaryl modification of GCDH lysine residues and can be relieved by SIRT5 deacylation activity

Is the proteome of bronchoalveolar lavage extracellular vesicles a marker of advanced lung cancer?

Acellular bronchoalveolar lavage (BAL) proteomics can partially separate lung cancer from non-lung cancer patients based on principal component analysis and multivariate analysis. Furthermore, the variance in the proteomics data sets is correlated mainly with lung cancer status and, to a lesser extent, smoking status and gender. Despite these advances BAL small and large extracellular vehicles (EVs) proteomes reveal aberrant protein expression in paracrine signaling mechanisms in cancer initiation and progression. We consequently present a case-control study of 24 bronchoalveolar lavage extracellular vesicle samples which were analyzed by state-of-the-art liquid chromatography-mass spectrometry (LC-MS). We obtained evidence that BAL EVs proteome complexity correlated with lung cancer stage 4 and mortality within two years´ follow-up (p value = 0.006). The potential therapeutic target DNMT3B complex is significantly up-regulated in tumor tissue and BAL EVs. The computational analysis of the immune and fibroblast cell markers in EVs suggests that patients who deceased within the follow-up period display higher marker expression indicative of innate immune and fibroblast cells (four out of five cases). This study provides insights into the proteome content of BAL EVs and their correlation to clinical outcomes.R.M. is supported by Fundação para a Ciência e a Tecnologia (CEEC position, 2019–2025 investigator). This article is a result of the projects (iNOVA4Health—UID/Multi/04462/2013), supported by Lisboa Portugal Regional Operational Programme (Lisboa2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). This work is also funded by FEDER funds through the COMPETE 2020 Programme and National Funds through FCT—Portuguese Foundation for Science and Technology under the projects number PTDC/BTM-TEC/30087/2017 and PTDC/BTM-TEC/30088/2017. This work was supported by the Wellcome Trust/DBT India Alliance Margdarshi Fellowship (grant number IA/M/15/1/502023) awarded to A.P. B.C.-S., M.C.S.C. and C.B. are supported by the Champalimaud Foundation and the EMBO Installation Grant 3921

Outcomes from elective colorectal cancer surgery during the SARS-CoV-2 pandemic

This study aimed to describe the change in surgical practice and the impact of SARS-CoV-2 on mortality after surgical resection of colorectal cancer during the initial phases of the SARS-CoV-2 pandemic

Functional Recovery of a GCDH Variant Associated to Severe Deflavinylation—Molecular Insights into Potential Beneficial Effects of Riboflavin Supplementation in Glutaric Aciduria-Type I Patients

Riboflavin is the biological precursor of two important flavin cofactors—flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN)—that are critical prosthetic groups in several redox enzymes. While dietary supplementation with riboflavin is a recognized support therapy in several inborn errors of metabolism, it has yet unproven benefits in several other pathologies affecting flavoproteins. This is the case for glutaric aciduria type I (GA-I), a rare neurometabolic disorder associated with mutations in the GCDH gene, which encodes for glutaryl-coenzyme A (CoA) dehydrogenase (GCDH). Although there are a few reported clinical cases that have responded to riboflavin intake, there is still not enough molecular evidence supporting therapeutic recommendation. Hence, it is necessary to elucidate the molecular basis in favor of riboflavin supplementation in GA-I patients. Here, using a combination of biochemical and biophysical methodologies, we investigate the clinical variant GCDH-p.Val400Met as a model for a phenotype associated with severe deflavinylation. Through a systematic analysis, we establish that recombinant human GCDH-p.Val400Met is expressed in a nonfunctional apo form, which is mainly monomeric rather than tetrameric. However, we show that exogenous FAD is a driver for structural reorganization of the mutant enzyme with concomitant functional recovery, improved thermolability, and resistance to trypsin digestion. Overall, these results establish proof of principle for the beneficial effects of riboflavin supplementation in GA-I patients

A polymorphic position in electron transfer flavoprotein modulates kinetic stability as evidenced by thermal stress

AbstractThe electron transfer flavoprotein (ETF) is a hub interacting with at least 11 mitochondrial flavoenzymes and linking them to the respiratory chain. Here we report the effect of the ETFα-T/I171 polymorphism on protein conformation and kinetic stability under thermal stress. Although variants have comparable thermodynamic stabilities, kinetically their behavior is rather distinct as ETFα-T171 displays increased susceptibility to cofactor flavin adenine dinucleotide (FAD) loss and enhanced kinetics of inactivation during thermal stress. Mimicking a fever episode yields substantial activity loss. However, the presence of substoichiometric concentrations of GroEL is sufficient to act as an effective buffer against long-term thermal denaturation. Our investigations are compatible with the notion that the ETFα-T171 variant displays an altered conformational landscape that results in reduced protein function under thermal stress

Assessment of catches, landings and fishing effort as useful tools for MPA management

Marine protected areas (MPAs) have been widely recognized as a tool to achieve both fisheries management and conservation goals. Simultaneously achieving these multiple goals is difficult due to conflicts between conservation (often long-term) and economic (often short-term) objectives. MPA implementation often includes additional control measures on fisheries (e.g. vessel size restrictions, gear exclusion, catch controls) that in the short-term may have impacts on local fishers' communities. Thus, monitoring fisheries catches before, during and after MPA implementation is essential to document changes in fisheries activities and to evaluate the impact of MPAs in fishers' communities. Remarkably, in contrast with standard fisheries-independent biological surveys, these data are rarely measured at appropriate spatial scales following MPA implementation. Here, the effects of MPA implementation on local fisheries are assessed in a temperate MPA (Arrabida Marine Park, Portugal), using fisheries monitoring methods combining spatial distribution of fishing effort, on-board observations and official landings statistics at scales appropriate to the Marine Park. Fisheries spatial distribution, fishing effort, on-board data collection and official landings registered for the same vessels over time were analysed between 2004 and 2010. The applicability and reliability of using landings statistics alone was tested (i.e. when no sampling data are available) and we conclude that landings data alone only allow the identification of general patterns. The combination of landings information (which is known to be unreliable in many coastal communities) with other methods, provides an effective tool to evaluate fisheries dynamics in response to MPA implementation. As resources for monitoring socio-ecological responses to MPAs are frequently scarce, the use of landings data calibrated with fisheries information (from vessels, gear distribution and on-board data) is a valuable tool applicable to many worldwide coastal small-scale fisheries. (C) 2015 Elsevier B.V. All rights reserved