The involvement of sphingolipids in apoptosis induced by acetic acid in yeast

This work was funded by FEDER through the program “Programa Operacional Factores de Competitividade-COMPETE” and by FCT (Fundação para a Ciência e Tecnologia) through the projects Pest-C/BIA/UI4050/2011 and FCOMP-01-0124-FEDER-007047. A.R. was supported by a FCT fellowship (FCOMP-01-0124-FEDER-007047)

Yeast as a tool to explore cathepsin D function

Cathepsin D has garnered increased attention in recent years, mainly since it has been associated with several human pathologies. In particular, cathepsin D is often overexpressed and hypersecreted in cancer cells, implying it may constitute a therapeutic target. However, cathepsin D can have both anti- and pro-survival functions depending on its proteolytic activity, cellular context and stress stimulus. Therefore, a more detailed understanding of cathepsin D regulation and how to modulate its apoptotic functions is clearly needed. In this review, we provide an overview of the role of cathepsin D in physiological and pathological scenarios. We then focus on the opposing functions of cathepsin D in apoptosis, particularly relevant in cancer research. Emphasis is given to the role of the yeast protease Pep4p, the vacuolar counterpart of cathepsin D, in life and death. Finally, we discuss how insights from yeast cathepsin D and its role in regulated cell death can unveil novel functions of mammalian cathepsin D in apoptosis and cancer.FEDER through POFC – COMPETE and by Fundação para a Ciência e Tecnologia through projects PEst-OE/BIA/UI4050/2014 and FCTANR/BEX-BCM/0175/2012, as well as fellowships to H. Pereira (SFRH/BD/73139/2010), C.S.F. Oliveira (SFRH/BD/77449/2011), L. Castro (SFRH/BD/93589/2013) and S. Chaves (SFRH/ BPD/89980/2012).info:eu-repo/semantics/publishedVersio

Genome-wide identification of genes involved in the positive and negative regulation of acetic acid-induced programmed cell death in Saccharomyces cerevisiae

Background: Acetic acid is mostly known as a toxic by-product of alcoholic fermentation carried out by Saccharomyces cerevisiae, which it frequently impairs. The more recent finding that acetic acid triggers apoptotic programmed cell death (PCD) in yeast sparked an interest to develop strategies to modulate this process, to improve several biotechnological applications, but also for biomedical research. Indeed, acetate can trigger apoptosis in cancer cells, suggesting its exploitation as an anticancer compound. Therefore, we aimed to identify genes involved in the positive and negative regulation of acetic acid-induced PCD by optimizing a functional analysis of a yeast Euroscarf knock-out mutant collection. Results: The screen consisted of exposing the mutant strains to acetic acid in YPD medium, pH 3.0, in 96-well plates, and subsequently evaluating the presence of culturable cells at different time points. Several functional categories emerged as greatly relevant for modulation of acetic acid-induced PCD (e.g.: mitochondrial function, transcription of glucose-repressed genes, protein synthesis and modifications, and vesicular traffic for protection, or amino acid transport and biosynthesis, oxidative stress response, cell growth and differentiation, protein phosphorylation and histone deacetylation for its execution). Known pro-apoptotic and anti-apoptotic genes were found, validating the approach developed. Metabolism stood out as a main regulator of this process, since impairment of major carbohydrate metabolic pathways conferred resistance to acetic acid-induced PCD. Among these, lipid catabolism arose as one of the most significant new functions identified. The results also showed that many of the cellular and metabolic features that constitute hallmarks of tumour cells (such as higher glycolytic energetic dependence, lower mitochondrial functionality, increased cell division and metabolite synthesis) confer sensitivity to acetic acid-induced PCD, potentially explaining why tumour cells are more susceptible to acetate than untransformed cells and reinforcing the interest in exploiting this acid in cancer therapy. Furthermore, our results clearly establish a connection between cell proliferation and cell death regulation, evidencing a conserved developmental role of programmed cell death in unicellular eukaryotes. Conclusions: This work advanced the characterization of acetic acid-induced PCD, providing a wealth of new information on putative molecular targets for its control with impact both in biotechnology and biomedicine.This work was supported by FEDER through POFC-COMPETE and national funds from FCT PEst-C/BIA/UI4050/2011 and PTDC/AGR-ALI/102608/2008. A. Pacheco was the recipient of a FCT fellowship (SFRH/BPD/65003)

Asymmetric introgression between sympatric molestus and pipiens forms of Culex pipiens (Diptera: Culicidae) in the Comporta region, Portugal

<p>Abstract</p> <p>Background</p> <p><it>Culex pipiens </it>L. is the most widespread mosquito vector in temperate regions. This species consists of two forms, denoted molestus and pipiens, that exhibit important behavioural and physiological differences. The evolutionary relationships and taxonomic status of these forms remain unclear. In northern European latitudes molestus and pipiens populations occupy different habitats (underground <it>vs</it>. aboveground), a separation that most likely promotes genetic isolation between forms. However, the same does not hold in southern Europe where both forms occur aboveground in sympatry. In these southern habitats, the extent of hybridisation and its impact on the extent of genetic divergence between forms under sympatric conditions has not been clarified. For this purpose, we have used phenotypic and genetic data to characterise <it>Cx. pipiens </it>collected aboveground in Portugal. Our aims were to determine levels of genetic differentiation and the degree of hybridisation between forms occurring in sympatry, and to relate these with both evolutionary and epidemiological tenets of this biological group.</p> <p>Results</p> <p>Autogeny and stenogamy was evaluated in the F1 progeny of 145 individual <it>Cx. pipiens </it>females. Bayesian clustering analysis based on the genotypes of 13 microsatellites revealed two distinct genetic clusters that were highly correlated with the alternative traits that define pipiens and molestus. Admixture analysis yielded hybrid rate estimates of 8-10%. Higher proportions of admixture were observed in pipiens individuals suggesting that more molestus genes are being introgressed into the pipiens form than the opposite.</p> <p>Conclusion</p> <p>Both physiological/behavioural and genetic data provide evidence for the sympatric occurrence of molestus and pipiens forms of <it>Cx. pipiens </it>in the study area. In spite of the significant genetic differentiation between forms, hybridisation occurs at considerable levels. The observed pattern of asymmetric introgression probably relates to the different mating strategies adopted by each form. Furthermore, the differential introgression of molestus genes into the pipiens form may induce a more opportunistic biting behaviour in the latter thus potentiating its capacity to act as a bridge-vector for the transmission of arboviral infections.</p

Vacuole-mitochondrial crosstalk during apoptosis induced by acetic acid in yeast

This work was supported by the FCT project PTDC/BIA-BCM/69448/200

What yeast can tell us about how cells commit suicide?

Multicellular organisms developed a complex system to balance cell proliferation and cell death in order to guarantee correct embryonic development and tissue homeostasis. Failure of cells to undergo programmed cell death (PCD) can potentially lead to severe diseases, including neural degeneration, autoimmunity and cancer. Identifying the molecules involved in PCD and understanding the regulation of the process are crucial for prevention and management of these diseases. Evidence of the enormous impact of PCD, of which apoptosis is the most frequent morphological phenotype, on human health makes it one of the today’s main research topics. Since PCD was initially considered specific of metazoans, biological models were first restricted to animal cells. Actually, based on the absence of known crucial PCD regulators, as indicated by plain homologies searches, as well as on the difficulty to explain the sense of cell suicide in a unicellular organism, it was not accepted that these organisms could possess a PDC mechanism. However, evidence has been reported in the last decade indicating that the process of self-destruction in different unicellular organisms, namely in yeast, can also take place. In the present communication, I will present the research we have been developing on PCD, based on the exploration/exploitation of yeast as a simple eukaryotic unicellular model system. Particular focus will be given to our more recent studies suggesting a complex regulation and interplay between mitochondria and the vacuole in acetic acid induced PCD. The validation in mammalian cell lines of the hypothesis postulated with the yeast model will be also discussed.Fundação para a Ciência e a Tecnologia (FCT

SARS-CoV-2 introductions and early dynamics of the epidemic in Portugal

Genomic surveillance of SARS-CoV-2 in Portugal was rapidly implemented by the National Institute of Health in the early stages of the COVID-19 epidemic, in collaboration with more than 50 laboratories distributed nationwide. Methods By applying recent phylodynamic models that allow integration of individual-based travel history, we reconstructed and characterized the spatio-temporal dynamics of SARSCoV-2 introductions and early dissemination in Portugal. Results We detected at least 277 independent SARS-CoV-2 introductions, mostly from European countries (namely the United Kingdom, Spain, France, Italy, and Switzerland), which were consistent with the countries with the highest connectivity with Portugal. Although most introductions were estimated to have occurred during early March 2020, it is likely that SARS-CoV-2 was silently circulating in Portugal throughout February, before the first cases were confirmed. Conclusions Here we conclude that the earlier implementation of measures could have minimized the number of introductions and subsequent virus expansion in Portugal. This study lays the foundation for genomic epidemiology of SARS-CoV-2 in Portugal, and highlights the need for systematic and geographically-representative genomic surveillance.We gratefully acknowledge to Sara Hill and Nuno Faria (University of Oxford) and Joshua Quick and Nick Loman (University of Birmingham) for kindly providing us with the initial sets of Artic Network primers for NGS; Rafael Mamede (MRamirez team, IMM, Lisbon) for developing and sharing a bioinformatics script for sequence curation (https://github.com/rfm-targa/BioinfUtils); Philippe Lemey (KU Leuven) for providing guidance on the implementation of the phylodynamic models; Joshua L. Cherry (National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health) for providing guidance with the subsampling strategies; and all authors, originating and submitting laboratories who have contributed genome data on GISAID (https://www.gisaid.org/) on which part of this research is based. The opinions expressed in this article are those of the authors and do not reflect the view of the National Institutes of Health, the Department of Health and Human Services, or the United States government. This study is co-funded by Fundação para a Ciência e Tecnologia and Agência de Investigação Clínica e Inovação Biomédica (234_596874175) on behalf of the Research 4 COVID-19 call. Some infrastructural resources used in this study come from the GenomePT project (POCI-01-0145-FEDER-022184), supported by COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation (POCI), Lisboa Portugal Regional Operational Programme (Lisboa2020), Algarve Portugal Regional Operational Programme (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and by Fundação para a Ciência e a Tecnologia (FCT).info:eu-repo/semantics/publishedVersio

Vacuole-mitochondrial cross-talk during apoptosis in yeast: a model for understanding lysosome-mitochondria-mediated apoptosis in mammals

The yeast apoptosis field emerged with the finding that key components of the apoptotic machinery are conserved in these simple eukaryotes. Thus it became possible to exploit these genetically tractable organisms to improve our understanding of the intricate mechanisms of cell death in higher eukaryotes and of severe human diseases associated with apoptosis dysfunctions. Early on, it was recognized that a mitochondria-mediated apoptotic pathway showing similarities to the mammalian intrinsic pathway was conserved in yeast. Recently, lysosomes have also emerged as central players in mammalian apoptosis. Following LMP (lysosomal membrane permeabilization), lysosomal proteases such as cathepsins B, D and L are released into the cytosol and can trigger a mitochondrial apoptotic cascade. CatD (cathepsin D) can also have anti-apoptotic effects in some cellular types and specific contexts. Nonetheless, the mechanisms underlying LMP and the specific role of cathepsins after their release into the cytosol remain poorly understood. We have recently shown that yeast vacuoles, membrane-bound acidic organelles, which share many similarities to plant vacuoles and mammalian lysosomes, are also involved in the regulation of apoptosis and that the vacuolar protease Pep4p, orthologue of the human CatD, is released from the vacuole into the cytosol in response to acetic acid. Here, we discuss how the conservation of cell-death regulation mechanisms in yeast by the lysosome-like organelle and mitochondria may provide new insights into the understanding of the complex interplay between themitochondria and lysosome-mediated signalling routes during mammalian apoptosis.This work was supported by the ‘Fundação para a Ciência e Tecnologia’ project [grant numbers FCOMP-01-0124-FEDER-007047 and PTDC/AGR-ALI/100636/2008]

The role of diet related short-chain fatty acids in colorectal cancer metabolism and survival: prevention and therapeutic implications

Colorectal cancer (CRC) is a major cause of cancer-related death worldwide. CRC increased risk has been associated with alterations in the intestinal microbiota, with decreased production of short chain fatty acids (SCFAs). SCFAs produced in the human colon are the major products of bacterial fermentation of undigested dietary fiber and starch. While colonocytes use the three major SCFAs, namely acetate, propionate and butyrate, as energy sources, transformed CRC cells primarily undergo aerobic glycolysis. Compared to normal colonocytes, CRC cells exhibit increased sensitivity to SCFAs, thus indicating they play an important role in cell homeostasis. Manipulation of SCFA levels in the intestine, through changes in microbiota, has therefore emerged as a potential preventive/therapeutic strategy for CRC. Interest in understanding SCFAs mechanism of action in CRC cells has increased in the last years. Several SCFA transporters like SMCT-1, MCT-1 and aquaporins have been identified as the main transmembrane transporters in intestinal cells. Recently, it was shown that acetate promotes plasma membrane re-localization of MCT-1 and triggers changes in the glucose metabolism. SCFAs induce apoptotic cell death in CRC cells, and further mechanisms have been discovered, including the involvement of lysosomal membrane permeabilization, associated with mitochondria dysfunction and degradation. In this review, we will cover the current knowledge on the transport of SCFAs by CRC cells and their effects on CRC metabolism and survival. The impact of increasing SCFA production by manipulation of colon microbiota on the prevention/therapy of CRC will also be addressed.This article is a result of the project EcoAgriFood NORTE-01-0145-FEDER-00009, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF)It counts also with the support of the strategic programme UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) funded by national funds through the FCT I.P. and by the ERDF through the COMPETE2020 -Programa Operacional Competitividade e Internacionalizacao (POCI).It has also been supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of the UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01- 0145-FEDER-006684).info:eu-repo/semantics/acceptedVersio