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1.
Sci Rep ; 11(1): 16430, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34385527

RESUMO

Until there is an effective implementation of COVID-19 vaccination program, a robust testing strategy, along with prevention measures, will continue to be the most viable way to control disease spread. Such a strategy should rely on disparate diagnostic tests to prevent a slowdown in testing due to lack of materials and reagents imposed by supply chain problems, which happened at the beginning of the pandemic. In this study, we have established a single-tube test based on RT-LAMP that enables the visual detection of less than 100 viral genome copies of SARS-CoV-2 within 30 min. We benchmarked the assay against the gold standard test for COVID-19 diagnosis, RT-PCR, using 177 nasopharyngeal RNA samples. For viral loads above 100 copies, the RT-LAMP assay had a sensitivity of 100% and a specificity of 96.1%. Additionally, we set up a RNA extraction-free RT-LAMP test capable of detecting SARS-CoV-2 directly from saliva samples, albeit with lower sensitivity. The saliva was self-collected and the collection tube remained closed until inactivation, thereby ensuring the protection of the testing personnel. As expected, RNA extraction from saliva samples increased the sensitivity of the test. To lower the costs associated with RNA extraction, we performed this step using an alternative protocol that uses plasmid DNA extraction columns. We also produced the enzymes needed for the assay and established an in-house-made RT-LAMP test independent of specific distribution channels. Finally, we developed a new colorimetric method that allowed the detection of LAMP products by the visualization of an evident color shift, regardless of the reaction pH.


Assuntos
Teste para COVID-19/métodos , COVID-19/virologia , Colorimetria/métodos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , SARS-CoV-2/isolamento & purificação , COVID-19/diagnóstico , Humanos , Pandemias , Portugal/epidemiologia , RNA Viral/genética , SARS-CoV-2/genética , Saliva/química , Saliva/virologia , Sensibilidade e Especificidade
2.
F1000Res ; 9: 317, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32595956

RESUMO

Invertebrate glia performs most of the key functions controlled by mammalian glia in the nervous system and provides an ideal model for genetic studies of glial functions. To study the influence of adult glial cells in ageing we have performed a genetic screen in Drosophila using a collection of transgenic lines providing conditional expression of micro-RNAs (miRNAs). Here, we describe a methodological algorithm to identify and rank genes that are candidate to be targeted by miRNAs that shorten lifespan when expressed in adult glia. We have used four different databases for miRNA target prediction in Drosophila but find little agreement between them, overall. However, top candidate gene analysis shows potential to identify essential genes involved in adult glial functions. One example from our top candidates' analysis is gartenzwerg ( garz). We establish that garz is necessary in many glial cell types, that it affects motor behaviour and, at the sub-cellular level, is responsible for defects in cellular membranes, autophagy and mitochondria quality control. We also verify the remarkable conservation of functions between garz and its mammalian orthologue, GBF1, validating the use of Drosophila as an alternative 3Rs-beneficial model to knock-out mice for studying the biology of GBF1, potentially involved in human neurodegenerative diseases.


Assuntos
Proteínas de Drosophila/genética , Drosophila , Fatores de Troca do Nucleotídeo Guanina/genética , MicroRNAs , Neuroglia/fisiologia , Animais , Animais Geneticamente Modificados , Drosophila/genética , Camundongos Knockout , MicroRNAs/genética
3.
Plant Cell Rep ; 39(1): 89-100, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31583429

RESUMO

KEY MESSAGE: Extracts from hairy root cultures of Cynara cardunculus L. contain proteases and show milk-clotting activity. Cynara cardunculus L. or cardoon is often used as rennet in traditional cheese manufacturing, due to the presence of specific proteases in the flower. However, the flower extracts are variable depending on the provenance and quality of the flowers as well as high genetic variability among cardoon populations, and this affects the quality of the final product. In search for alternative sources of milk-clotting enzymes, hairy root cultures from cardoon were obtained and characterized regarding their protease content and proteolytic activity toward milk proteins. Aspartic, serine and cysteine proteases were identified in hairy roots by mass spectrometry analysis and an azocasein assay combined with specific inhibitors. RT-PCR analysis revealed the expression of cardosin A and D, and immunoblotting analysis suggested the presence of cardosin A or cardosin A-like enzyme in its mature form, supporting this system as an alternative source of cardosins. Hairy root protein extracts showed activity over caseins, supporting its use as milk coagulant, which was further tested by milk-clotting assays. This is also the first report on the establishment of hairy root cultures from cardoon, which paves the way for future work on controlled platforms for production of valuable metabolites which are known to be present in this species.


Assuntos
Cynara/enzimologia , Cynara/microbiologia , Hipocótilo/enzimologia , Raízes de Plantas/enzimologia , Agrobacterium , Animais , Ácido Aspártico Endopeptidases/metabolismo , Ácido Aspártico Proteases/metabolismo , Caseínas/metabolismo , Queijo/microbiologia , Cynara/química , Cynara/metabolismo , Cisteína Proteases/metabolismo , Flores/enzimologia , Hipocótilo/crescimento & desenvolvimento , Hipocótilo/microbiologia , Leite , Proteínas de Plantas/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Proteólise , Proteoma/metabolismo , Serina Proteases/metabolismo
4.
Microb Cell ; 6(6): 267-285, 2019 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-31172012

RESUMO

Yeast adaptation to stress has been extensively studied. It involves large reprogramming of genome expression operated by many, more or less specific, transcription factors. Here, we review our current knowledge on the function of the eight Yap transcription factors (Yap1 to Yap8) in Saccharomyces cerevisiae, which were shown to be involved in various stress responses. More precisely, Yap1 is activated under oxidative stress, Yap2/Cad1 under cadmium, Yap4/Cin5 and Yap6 under osmotic shock, Yap5 under iron overload and Yap8/Arr1 by arsenic compounds. Yap3 and Yap7 seem to be involved in hydroquinone and nitrosative stresses, respectively. The data presented in this article illustrate how much knowledge on the function of these Yap transcription factors is advanced. The evolution of the Yap family and its roles in various pathogenic and non-pathogenic fungal species is discussed in the last section.

5.
J Neurosci ; 39(27): 5269-5283, 2019 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-31064860

RESUMO

The fruitfly Drosophila melanogaster has been extensively used as a genetic model for the maintenance of nervous system's functions. Glial cells are of utmost importance in regulating the neuronal functions in the adult organism and in the progression of neurological pathologies. Through a microRNA-based screen in adult Drosophila glia, we uncovered the essential role of a major glia developmental determinant, repo, in the adult fly. Here, we report that Repo expression is continuously required in adult glia to transcriptionally regulate the highly conserved function of neurotransmitter recycling in both males and females. Transient loss of Repo dramatically shortens fly lifespan, triggers motor deficits, and increases the sensibility to seizures, partly due to the impairment of the glutamate/GABA/glutamine cycle. Our findings highlight the pivotal role of transcriptional regulation of genes involved in the glutamate/GABA/glutamine cycle in glia to control neurotransmitter levels in neurons and their behavioral output. The mechanism identified here in Drosophila exemplifies how adult functions can be modulated at the transcriptional level and suggest an active synchronized regulation of genes involved in the same pathway. The process of neurotransmitter recycling is of essential importance in human epileptic and psychiatric disorders and our findings may thus have important consequences for the understanding of the role that transcriptional regulation of neurotransmitter recycling in astrocytes has in human disease.SIGNIFICANCE STATEMENT Glial cells are an essential support to neurons in adult life and have been involved in a number of neurological disorders. What controls the maintenance and modulation of glial functions in adult life is not fully characterized. Through a miR overexpression screen in adult glia in Drosophila, we identify an essential role in adult glia of repo, which directs glial differentiation during embryonic development. Repo levels modulate, via transcriptional regulation, the ability of glial cells to support neurons in the glutamate/GABA/glutamine cycle. This leads to significant abnormalities in motor behavior as assessed through a novel automated paradigm. Our work points to the importance of transcriptional regulation in adult glia for neurotransmitter recycling, a key process in several human neurological disorders.


Assuntos
Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica , Ácido Glutâmico/metabolismo , Glutamina/metabolismo , Proteínas de Homeodomínio/metabolismo , Atividade Motora , Neuroglia/metabolismo , Convulsões/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Drosophila melanogaster , Feminino , Masculino , MicroRNAs/metabolismo
6.
J Mater Sci Mater Med ; 29(9): 139, 2018 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-30120571

RESUMO

Bacteria-synthesized polysaccharides have attracted interest for biomedical applications as promising biomaterials to be used as implants and scaffolds. The present study tested the hypothesis that cellulose exopolysaccharide (CEC) produced from sugarcane molasses of low cost and adequate purity would be suitable as a template for 2D and 3D neuron and/or astrocyte primary cultures, considering its low toxicity. CEC biocompatibility in these primary cultures was evaluated with respect to cell viability, adhesion, growth and cell function (calcium imaging). Polystyrene or Matrigel® matrix were used as comparative controls. We demonstrated that the properties of this CEC in the 2D or 3D configurations are suitable for differentiation of cortical astrocytes and neurons in single or mixed cultures. No toxicity was detected in neurons that showed NMDA-induced Ca2+ influx. Unlike other polysaccharides of bacterial synthesis, the CEC was efficient as a support even in the absence of surface conjugation with extracellular matrix proteins, maintaining physiological characteristics of cultured neural cells. These observations open up the perspective for development of a novel 3D biofunctional scaffold produced from bacterial cellulose and obtained from renewable sources whose residues are not pollutants. Its low cost and possibility to be manufactured in scale are also suitable for potential applications in regenerative medicine.


Assuntos
Astrócitos/citologia , Neurônios/patologia , Polissacarídeos/química , Cultura Primária de Células , Saccharum/química , Animais , Materiais Biocompatíveis , Cálcio/química , Adesão Celular , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Coloides/química , Matriz Extracelular/metabolismo , Feminino , Hidrogéis/química , Imageamento Tridimensional , Imuno-Histoquímica , Melaço , N-Metilaspartato/química , Neurônios/metabolismo , Ratos , Ratos Wistar , Estresse Mecânico , Engenharia Tecidual/métodos
7.
Biochim Biophys Acta Gen Subj ; 1862(10): 2152-2161, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30025855

RESUMO

In the eukaryotic model yeast Saccharomyces cerevisiae, arsenic (As) detoxification is regulated by two transcriptional factors, Yap8 and Yap1. Yap8 specifically controls As extrusion from the cell, whether Yap1 avoids arsenic-induced oxidative damages. Accordingly, cells lacking both Yap1 and Yap8 are more sensitive to arsenate than cells lacking each regulator individually. Strikingly enough, the same sensitivity pattern was observed under anoxia, suggesting that Yap1 role in As detoxification might not be restricted to the regulation of the oxidative stress response. This finding prompted us to study the transcriptomic profile of wild-type and yap1 mutant cells exposed to arsenate. Interestingly, we found that, under such conditions, several genes involved in the biogenesis of FeS proteins were upregulated in a Yap1-dependent way. In line with this observation, arsenate treatment decreases the activity of the mitochondrial aconitase, Aco1, an FeS cluster-containing enzyme, this effect being even more pronounced in the yap1 mutant. Reinforcing the relevance of FeS cluster biogenesis in arsenate detoxification, the overexpression of several ISC and CIA machinery genes alleviates the deleterious effect of arsenate caused by the absence of Yap1 and Yap8. Altogether our data suggest that the upregulation of FeS biogenesis genes regulated by Yap1 might work as a cellular shield against arsenate toxicity.


Assuntos
Arseniatos/toxicidade , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Proteínas Ferro-Enxofre/biossíntese , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional/efeitos dos fármacos , Proteínas Ferro-Enxofre/efeitos dos fármacos , Proteínas Ferro-Enxofre/genética , Estresse Oxidativo/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética
8.
FEBS J ; 285(10): 1861-1872, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29604179

RESUMO

In the yeast Saccharomyces cerevisiae Aft1, the low iron-sensing transcription factor is known to regulate the expression of the FET3 gene. However, we found that a strain-lacking FET3 is more sensitive to copper excess than a strain-lacking AFT1, and accordingly, FET3 expression is not fully compromised in the latter. These findings suggest that, under such conditions, another regulator comes into play and controls FET3 expression. In this work, we identify Ace1, the regulator of copper detoxification genes, as a regulator of FET3. We suggest that the activation of FET3 by Ace1 prevents the hyper activation of Aft1, possibly by assuring the adequate functioning of mitochondrial iron-sulfur cluster biogenesis. While reinforcing the link between iron and copper homeostasis, this work unveils a novel protection mechanism against copper toxicity mediated by Ace1, which relies in the activation of FET3 and results in the restriction of Aft1 activity as a means to prevent excessive copper accumulation.


Assuntos
Ceruloplasmina/metabolismo , Cobre/metabolismo , Proteínas de Ligação a DNA/fisiologia , Inativação Metabólica/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia , Ceruloplasmina/genética , Cobre/toxicidade , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Homeostase , Ferro/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
9.
J Nutr Biochem ; 55: 229-242, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29573696

RESUMO

Omega-3 (n-3) fatty acids modulate epigenetic changes critical to genesis and differentiation of neural cells. Conversely, maternal protein-malnutrition can negatively modify these changes. This study investigated whether a low n-6/n-3 ratio in a maternal diet could favor histone-3 (H3) modifications, gene transcription and differentiation in the offspring neural cells even under protein-deficiency. Female rats fed a control (Ct), or 3 types of multideficient diets differing in protein levels or linoleic/alpha-linolenic fatty acid ratios (RBD, RBD-C, RBD-SO) from 30 days prior to mating and during pregnancy. Cerebral cortex tissue and cortical cultures of progeny embryonic neurons and postnatal astrocytes were analyzed. H3K9 acetylation and H3K27 or H3K4 di-methylation levels were assessed by flow cytometry and/or immunocytochemistry. In astrocyte cultures and cortical tissue, the GFAP protein levels were assessed. Glial derived neurotrophic factor (GDNF) and leukemia inhibitory factor (LIF) gene expression were evaluated in the cortical tissue. GFAP levels were similar in astrocytes of Ct, RBD and RBD-C, but 65% lower in RBD-SO group. Higher levels of H3K9Ac were found in the neurons and H3K4Me2 in the astrocytes of the RBD group. No intergroup difference in the cortical GDNF mRNA expression or the H3K27Me2 levels in astrocytes was detected. LIF mRNA levels were higher in the RDB (P=.002) or RBD-C (P=.004) groups than in the control. The findings indicate the importance of dietary n-3 availability for the brain, even under a protein-deficient condition, inducing Histone modifications and increasing LIF gene transcription, involved in neural cell differentiation and reactivity.


Assuntos
Astrócitos/efeitos dos fármacos , Ácidos Graxos Ômega-3/farmacologia , Ácidos Graxos Ômega-6/farmacologia , Histonas/metabolismo , Fator Inibidor de Leucemia/genética , Animais , Animais Recém-Nascidos , Astrócitos/metabolismo , Proteínas na Dieta/administração & dosagem , Epigênese Genética , Ácidos Graxos/análise , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Proteína Glial Fibrilar Ácida/metabolismo , Histonas/efeitos dos fármacos , Fenômenos Fisiológicos da Nutrição Materna , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Gravidez , Ratos
10.
Biochim Biophys Acta Gene Regul Mech ; 1860(4): 472-481, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28188921

RESUMO

Response to arsenic stress in Saccharomyces cerevisiae is orchestrated by the regulatory protein Yap8, which mediates transcriptional activation of ACR2 and ACR3. This study contributes to the state of art knowledge of the molecular mechanisms underlying yeast stress response to arsenate as it provides the genetic and biochemical evidences that Yap8, through cysteine residues 132, 137, and 274, is the sensor of presence of arsenate in the cytosol. Moreover, it is here reported for the first time the essential role of the Mediator complex in the transcriptional activation of ACR2 by Yap8. Based on our data, we propose an order-of-function map to recapitulate the sequence of events taking place in cells injured with arsenate. Modification of the sulfhydryl state of these cysteines converts Yap8 in its activated form, triggering the recruitment of the Mediator complex to the ACR2/ACR3 promoter, through the interaction with the tail subunit Med2. The Mediator complex then transfers the regulatory signals conveyed by Yap8 to the core transcriptional machinery, which culminates with TBP occupancy, ACR2 upregulation and cell adaptation to arsenate stress. Additional co-factors are required for the transcriptional activation of ACR2 by Yap8, particularly the nucleosome remodeling activity of SWI/SNF and SAGA complexes.


Assuntos
Arseniato Redutases/genética , Arseniatos/toxicidade , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Complexo Mediador/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ativação Transcricional/genética , Arseniato Redutases/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/química , Cisteína/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Subunidades Proteicas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Estresse Fisiológico/efeitos dos fármacos
11.
Cerebellum ; 16(1): 103-117, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27003678

RESUMO

The cerebellum is vulnerable to malnutrition effects. Notwithstanding, it is able to incorporate higher amount of docosahexaenoic acid (DHA) than the cerebral cortex (Cx) when low n-6/n-3 fatty acid ratio is present in a multideficient diet. Considering importance of DHA for brain redox balance, we hypothesize that this cerebellum feature improves its antioxidant status compared to the Cx. A chronic malnutrition status was induced on dams before mating and kept until weaning or adulthood (offspring). A group nutritionally rehabilitated from weaning was also analyzed. Morphometric parameters, total-superoxide dismutase (t-SOD) and catalase activities, lipoperoxidation (LP), nitric oxide (NO), reduced (GSH) and oxidized (GSSG) glutathione, reactive oxygen species (ROS), and reduced nicotinamide adenine dinucleotide/phosphate levels were assessed. Both ROS and LP levels were increased (∼53 %) in the Cx of malnourished young animals while the opposite was seen in the cerebellum (72 and 20 % of the control, respectively). Consistently, lower (∼35 %) and higher t-SOD (∼153 %) and catalase (CAT) (∼38 %) activities were respectively detected in the Cx and cerebellum compared to the control. In malnourished adult animals, redox balance was maintained in the cerebellum and recovered in the Cx (lower ROS and LP levels and higher GSH/GSSG ratio). NO production was impaired by malnutrition at either age, mainly in the cerebellum. The findings suggest that despite a multinutrient deficiency and a modified structural development, a low dietary n-6/n-3 ratio favors early antioxidant resources in the male cerebellum and indicates an important role of astrocytes in the redox balance recovery of Cx in adulthood.


Assuntos
Cerebelo/crescimento & desenvolvimento , Dieta com Restrição de Proteínas , Ácidos Graxos Ômega-3 , Ácidos Graxos Ômega-6/deficiência , Desnutrição/metabolismo , Estresse Oxidativo/fisiologia , Ração Animal , Animais , Antioxidantes/metabolismo , Cerebelo/metabolismo , Cerebelo/patologia , Doença Crônica , Modelos Animais de Doenças , Feminino , Peroxidação de Lipídeos/fisiologia , Masculino , Desnutrição/patologia , Gravidez , Efeitos Tardios da Exposição Pré-Natal , Distribuição Aleatória , Ratos , Desmame
12.
Biochim Biophys Acta ; 1849(12): 1385-97, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26470684

RESUMO

Response to hyperosmotic stress in the yeast Saccharomyces cerevisiae involves the participation of the general stress response mediated by Msn2/4 transcription factors and the HOG pathway. One of the transcription factors activated through this pathway is Hot1, which contributes to the control of the expression of several genes involved in glycerol synthesis and flux, or in other functions related to adaptation to adverse conditions. This work provides new data about the interaction mechanism of this transcription factor with DNA. By means of one-hybrid and electrophoretic mobility assays, we demonstrate that the C-terminal region, which corresponds to amino acids 610-719, is the DNA-binding domain of Hot1. We also describe how this domain recognizes sequence 5'-GGGACAAA-3' located in the promoter of gene STL1. The bioinformatics analysis carried out in this work allowed the identification of identical or similar sequences (with up to two mismatches) in the promoter of other Hot1 targets, where central element GGACA was quite conserved among them. Finally, we found that small variations in the sequence recognized by Hot1 may influence its ability to recognize its targets in vivo.


Assuntos
DNA Fúngico/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas de Membrana Transportadoras/genética , Regiões Promotoras Genéticas/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Simulação por Computador , Sequência Conservada , DNA Fúngico/genética , Genes Fúngicos , Dados de Sequência Molecular , Mutação , Osmorregulação/genética , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Deleção de Sequência , Homologia de Sequência de Aminoácidos
13.
Curr Biol ; 25(19): 2479-92, 2015 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-26387716

RESUMO

Autophagy plays key roles in development, oncogenesis, cardiovascular, metabolic, and neurodegenerative diseases. Hence, understanding how autophagy is regulated can reveal opportunities to modify autophagy in a disease-relevant manner. Ideally, one would want to functionally define autophagy regulators whose enzymatic activity can potentially be modulated. Here, we describe the STK38 protein kinase (also termed NDR1) as a conserved regulator of autophagy. Using STK38 as bait in yeast-two-hybrid screens, we discovered STK38 as a novel binding partner of Beclin1, a key regulator of autophagy. By combining molecular, cell biological, and genetic approaches, we show that STK38 promotes autophagosome formation in human cells and in Drosophila. Upon autophagy induction, STK38-depleted cells display impaired LC3B-II conversion; reduced ATG14L, ATG12, and WIPI-1 puncta formation; and significantly decreased Vps34 activity, as judged by PI3P formation. Furthermore, we observed that STK38 supports the interaction of the exocyst component Exo84 with Beclin1 and RalB, which is required to initiate autophagosome formation. Upon studying the activation of STK38 during autophagy induction, we found that STK38 is stimulated in a MOB1- and exocyst-dependent manner. In contrast, RalB depletion triggers hyperactivation of STK38, resulting in STK38-dependent apoptosis under prolonged autophagy conditions. Together, our data establish STK38 as a conserved regulator of autophagy in human cells and flies. We also provide evidence demonstrating that STK38 and RalB assist the coordination between autophagic and apoptotic events upon autophagy induction, hence further proposing a role for STK38 in determining cellular fate in response to autophagic conditions.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Autofagia/fisiologia , Proteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Apoptose/fisiologia , Proteína Beclina-1 , Linhagem Celular Tumoral , Células Cultivadas , Drosophila , Células HEK293 , Células HeLa , Humanos , Imunoprecipitação , Ligação Proteica , Técnicas do Sistema de Duplo-Híbrido
14.
FEBS Open Bio ; 5: 594-604, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26273559

RESUMO

Desulfovibrio gigas belongs to the group of sulfate reducing bacteria (SRB). These ubiquitous and metabolically versatile microorganisms are often exposed to reactive nitrogen species (RNS). Nonetheless, the mechanisms and regulatory elements involved in nitrosative stress protection are still poorly understood. The transcription factor HcpR has emerged as a putative regulator of nitrosative stress response among anaerobic bacteria. HcpR is known to orchestrate the expression of the hybrid cluster protein gene, hcp, proposed to be involved in cellular defense against RNS. According to phylogenetic analyses, the occurrence of hcpR paralog genes is a common feature among several Desulfovibrio species. Within the D. gigas genome we have identified two HcpR-related sequences. One of these sequences, hcpR1, was found in the close vicinity of the hcp gene and this finding prompted us to proceed with its functional characterization. We observed that the growth of a D. gigas strain lacking hcpR1 is severely impaired under nitrosative stress. An in silico search revealed several putative targets of HcpR1 that were experimentally validated. The fact that HcpR1 regulates several genes encoding proteins involved in nitrite and nitrate metabolism, together with the sensitive growth phenotype to NO displayed by an hcpR1 mutant strain, strongly supports a relevant role of this factor under nitrosative stress. Moreover, the finding that several Desulfovibrio species possess HcpR paralogs, which have been transmitted vertically in the evolution and diversification of the genus, suggests that these sequences may confer adaptive or survival advantage to these organisms, possibly by increasing their tolerance to nitrosative stress.

15.
FEBS Lett ; 589(19 Pt B): 2841-9, 2015 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-26296316

RESUMO

Yap2 is a cadmium responsive transcription factor that interacts with MAPK-activated protein (MAPKAP) kinase Rck1. We show that Rck1 deletion confers protection against cadmium toxicity and that the mechanism underlying this observation relies on Yap2. Rck1 removal from the yeast genome potentiates Yap2 activity by increasing protein half-life and delaying its nuclear export. As a consequence, several Yap2 antioxidant targets are over-activated by a mechanism that also requires Yap1. Several genes of the cell wall integrity (CWI) pathway are upregulated under cadmium stress in a Yap2 dependent way. We showed that deletion of CWI genes renders yeast cells more sensitive to cadmium. These findings led us to suggest that in response to cadmium stress Yap2 may serve a dual purpose: oxidative stress attenuation and cell wall maintenance.


Assuntos
Cádmio/toxicidade , Poluentes Ambientais/toxicidade , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Antioxidantes/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Parede Celular/efeitos dos fármacos , Parede Celular/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Mutação , Estresse Oxidativo/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/genética
16.
J Biol Chem ; 290(30): 18584-95, 2015 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-26063801

RESUMO

Cadmium is a well known mutagenic metal that can enter cells via nonspecific metal transporters, causing several cellular damages and eventually leading to death. In the yeast Saccharomyces cerevisiae, the transcription factor Yap1 plays a key role in the regulation of several genes involved in metal stress response. We have previously shown that Yap1 represses the expression of FET4, a gene encoding a low affinity iron transporter able to transport metals other than iron. Here, we have studied the relevance of this repression in cell tolerance to cadmium. Our results indicate that genomic deletion of Yap1 increases FET4 transcript and protein levels. In addition, the cadmium toxicity exhibited by this strain is completely reversed by co-deletion of FET4 gene. These data correlate well with the increased intracellular levels of cadmium observed in the mutant yap1. Rox1, a well known aerobic repressor of hypoxic genes, conveys the Yap1-mediated repression of FET4. We further show that, in a scenario where the activity of Yap1 or Rox1 is compromised, cells activate post-transcriptional mechanisms, involving the exoribonuclease Xrn1, to compensate the derepression of FET4. Our data thus reveal a novel protection mechanism against cadmium toxicity mediated by Yap1 that relies on the aerobic repression of FET4 and results in the impairment of cadmium uptake.


Assuntos
Cádmio/metabolismo , Proteínas de Transporte de Cátions/biossíntese , Proteínas de Ligação ao Ferro/biossíntese , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/metabolismo , Estresse Fisiológico/genética , Fatores de Transcrição/metabolismo , Transporte Biológico/genética , Cádmio/toxicidade , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Proteínas de Transporte de Cobre , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Exorribonucleases/metabolismo , Regulação Fúngica da Expressão Gênica , Ferro/metabolismo , Proteínas de Ligação ao Ferro/genética , Proteínas de Ligação ao Ferro/metabolismo , Mutação , Proteínas Repressoras/genética , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética
17.
Microbiologyopen ; 3(4): 513-30, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25055974

RESUMO

Desulfovibrio gigas is a model organism of sulfate-reducing bacteria of which energy metabolism and stress response have been extensively studied. The complete genomic context of this organism was however, not yet available. The sequencing of the D. gigas genome provides insights into the integrated network of energy conserving complexes and structures present in this bacterium. Comparison with genomes of other Desulfovibrio spp. reveals the presence of two different CRISPR/Cas systems in D. gigas. Phylogenetic analysis using conserved protein sequences (encoded by rpoB and gyrB) indicates two main groups of Desulfovibrio spp, being D. gigas more closely related to D. vulgaris and D. desulfuricans strains. Gene duplications were found such as those encoding fumarate reductase, formate dehydrogenase, and superoxide dismutase. Complexes not yet described within Desulfovibrio genus were identified: Mnh complex, a v-type ATP-synthase as well as genes encoding the MinCDE system that could be responsible for the larger size of D. gigas when compared to other members of the genus. A low number of hydrogenases and the absence of the codh/acs and pfl genes, both present in D. vulgaris strains, indicate that intermediate cycling mechanisms may contribute substantially less to the energy gain in D. gigas compared to other Desulfovibrio spp. This might be compensated by the presence of other unique genomic arrangements of complexes such as the Rnf and the Hdr/Flox, or by the presence of NAD(P)H related complexes, like the Nuo, NfnAB or Mnh.


Assuntos
DNA Bacteriano/química , DNA Bacteriano/genética , Desulfovibrio gigas/genética , Genoma Bacteriano , Análise de Sequência de DNA , Proteínas de Bactérias/genética , Análise por Conglomerados , Sequência Conservada , Variação Genética , Dados de Sequência Molecular , Filogenia
18.
Biochim Biophys Acta ; 1840(6): 1977-86, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24486411

RESUMO

BACKGROUND: Cobalt has a rare occurrence in nature, but may accumulate in cells to toxic levels. In the present study, we have investigated how the transcription factor Yap1 mediates tolerance to cobalt toxicity. METHODS: Fluorescence microscopy was used to address how cobalt activates Yap1. Using microarray analysis, we compared the transcriptional profile of a strain lacking Yap1 to that of its parental strain. To evaluate the extent of the oxidative damage caused by cobalt, GSH was quantified by HPLC and protein carbonylation levels were assessed. RESULTS: Cobalt activates Yap1 under aerobiosis and anaerobiosis growth conditions. This metal generates a severe oxidative damage in the absence of Yap1. However, when challenged with high concentrations of cobalt, yap1 mutant cells accumulate lower levels of this metal. Accordingly, microarray analysis revealed that the expression of the high affinity phosphate transporter, PHO84, a well-known cobalt transporter, is compromised in the yap1 mutant. Moreover, we show that Yap1 is a repressor of the low affinity iron transporter, FET4, which is also known to transport cobalt. CONCLUSIONS: Cobalt activates Yap1 that alleviates the oxidative damage caused by this metal. Yap1 partially controls cobalt cellular uptake via the regulation of PHO84. Although FET4 repression by Yap1 has no effect on cobalt uptake, it may be its first line of defense against other toxic metals. GENERAL SIGNIFICANCE: Our results emphasize the important role of Yap1 in mediating cobalt-induced oxidative damages and reveal new routes for cell protection provided by this regulator.


Assuntos
Cobalto/toxicidade , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/efeitos dos fármacos , Fatores de Transcrição/fisiologia , Proteínas de Transporte de Cátions/fisiologia , Cobalto/metabolismo , Proteínas de Transporte de Cobre , Proteínas de Ligação ao Ferro/fisiologia , Fosfatos/metabolismo , Simportadores de Próton-Fosfato/fisiologia , Saccharomyces cerevisiae/metabolismo , Superóxidos/metabolismo
19.
Biochim Biophys Acta ; 1840(6): 1902-12, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24361617

RESUMO

BACKGROUND: Our previous study demonstrated that essential fatty acid (EFA) dietary restriction over two generations induced midbrain dopaminergic cell loss and oxidative stress in the substantia nigra (SN) but not in the striatum of young rats. In the present study we hypothesized that omega-3 deficiency until adulthood would reduce striatum's resilience, increase nitric oxide (NO) levels and the number of BDNF-expressing neurons, both potential mechanisms involved in SN neurodegeneration. METHODS: Second generation rats were raised from gestation on control or EFA-restricted diets until young or adulthood. Lipoperoxidation, NO content, total superoxide dismutase (t-SOD) and catalase enzymatic activities were assessed in the SN and striatum. The number of tyrosine hydroxylase (TH)- and BDNF-expressing neurons was analyzed in the SN. RESULTS: Increased NO levels were observed in the striatum of both young and adult EFA-deficient animals but not in the SN, despite a similar omega-3 depletion (~65%) in these regions. Increased lipoperoxidation and decreased catalase activity were found in both regions, while lower tSOD activity was observed only in the striatum. Fewer TH- (~40%) and BDNF-positive cells (~20%) were detected at the SN compared to the control. CONCLUSION: The present findings demonstrate a differential effect of omega-3 deficiency on NO production in the rat's nigrostriatal system. Prolonging omega-3 depletion until adulthood impaired striatum's anti-oxidant resources and BDNF distribution in the SN, worsening dopaminergic cell degeneration. GENERAL SIGNIFICANCE: Omega-3 deficiency can reduce the nigrostriatal system's ability to maintain homeostasis under oxidative conditions, which may enhance the risk of Parkinson's disease.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/fisiologia , Ácidos Graxos Ômega-3/fisiologia , Óxido Nítrico/biossíntese , Doença de Parkinson/etiologia , Substância Negra/fisiologia , Animais , Fator Neurotrófico Derivado do Encéfalo/análise , Catalase/metabolismo , Feminino , Peroxidação de Lipídeos , Masculino , Estresse Oxidativo , Ratos , Ratos Wistar , Superóxido Dismutase/metabolismo , Tirosina 3-Mono-Oxigenase/análise
20.
PLoS One ; 8(12): e83328, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24358276

RESUMO

In Saccharomyces cerevisiae, the transcription factor Yap8 is a key determinant in arsenic stress response. Contrary to Yap1, another basic region-leucine zipper (bZIP) yeast regulator, Yap8 has a very restricted DNA-binding specificity and only orchestrates the expression of ACR2 and ACR3 genes. In the DNA-binding basic region, Yap8 has three distinct amino acids residues, Leu26, Ser29 and Asn31, at sites of highly conserved positions in the other Yap family of transcriptional regulators and Pap1 of Schizosaccharomyces pombe. To evaluate whether these residues are relevant to Yap8 specificity, we first built a homology model of the complex Yap8bZIP-DNA based on Pap1-DNA crystal structure. Several Yap8 mutants were then generated in order to confirm the contribution of the residues predicted to interact with DNA. Using bioinformatics analysis together with in vivo and in vitro approaches, we have identified several conserved residues critical for Yap8-DNA binding. Moreover, our data suggest that Leu26 is required for Yap8 binding to DNA and that this residue together with Asn31, hinder Yap1 response element recognition by Yap8, thus narrowing its DNA-binding specificity. Furthermore our results point to a role of these two amino acids in the stability of the Yap8-DNA complex.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/química , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , DNA/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Fatores de Transcrição de Zíper de Leucina Básica/genética , Sequência Conservada , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Associadas a Pancreatite , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Elementos de Resposta , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Homologia de Sequência , Fatores de Transcrição/genética
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