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1.
Nature ; 572(7768): 249-253, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31367038

RESUMO

Both single and multicellular organisms depend on anti-stress mechanisms that enable them to deal with sudden changes in the environment, including exposure to heat and oxidants. Central to the stress response are dynamic changes in metabolism, such as the transition from the glycolysis to the pentose phosphate pathway-a conserved first-line response to oxidative insults1,2. Here we report a second metabolic adaptation that protects microbial cells in stress situations. The role of the yeast polyamine transporter Tpo1p3-5 in maintaining oxidant resistance is unknown6. However, a proteomic time-course experiment suggests a link to lysine metabolism. We reveal a connection between polyamine and lysine metabolism during stress situations, in the form of a promiscuous enzymatic reaction in which the first enzyme of the polyamine pathway, Spe1p, decarboxylates lysine and forms an alternative polyamine, cadaverine. The reaction proceeds in the presence of extracellular lysine, which is taken up by cells to reach concentrations up to one hundred times higher than those required for growth. Such extensive harvest is not observed for the other amino acids, is dependent on the polyamine pathway and triggers a reprogramming of redox metabolism. As a result, NADPH-which would otherwise be required for lysine biosynthesis-is channelled into glutathione metabolism, leading to a large increase in glutathione concentrations, lower levels of reactive oxygen species and increased oxidant tolerance. Our results show that nutrient uptake occurs not only to enable cell growth, but when the nutrient availability is favourable it also enables cells to reconfigure their metabolism to preventatively mount stress protection.


Assuntos
Antioxidantes/metabolismo , Lisina/metabolismo , Poliaminas/metabolismo , Saccharomyces cerevisiae/metabolismo , Antiporters/metabolismo , Cadaverina/metabolismo , Glutamina/metabolismo , Glutationa/metabolismo , NADP/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Ornitina Descarboxilase/metabolismo , Oxidantes/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
2.
Cell ; 175(5): 1418-1429.e9, 2018 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-30454649

RESUMO

We report here a simple and global strategy to map out gene functions and target pathways of drugs, toxins, or other small molecules based on "homomer dynamics" protein-fragment complementation assays (hdPCA). hdPCA measures changes in self-association (homomerization) of over 3,500 yeast proteins in yeast grown under different conditions. hdPCA complements genetic interaction measurements while eliminating the confounding effects of gene ablation. We demonstrate that hdPCA accurately predicts the effects of two longevity and health span-affecting drugs, the immunosuppressant rapamycin and the type 2 diabetes drug metformin, on cellular pathways. We also discovered an unsuspected global cellular response to metformin that resembles iron deficiency and includes a change in protein-bound iron levels. This discovery opens a new avenue to investigate molecular mechanisms for the prevention or treatment of diabetes, cancers, and other chronic diseases of aging.


Assuntos
Ferro/metabolismo , Metaloproteínas/metabolismo , Metformina/farmacologia , Saccharomyces cerevisiae/metabolismo , Sirolimo/farmacologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Teste de Complementação Genética , Humanos , Metaloproteínas/genética , Saccharomyces cerevisiae/genética
3.
Elife ; 42015 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-26499891

RESUMO

Metabolite exchange among co-growing cells is frequent by nature, however, is not necessarily occurring at growth-relevant quantities indicative of non-cell-autonomous metabolic function. Complementary auxotrophs of Saccharomyces cerevisiae amino acid and nucleotide metabolism regularly fail to compensate for each other's deficiencies upon co-culturing, a situation which implied the absence of growth-relevant metabolite exchange interactions. Contrastingly, we find that yeast colonies maintain a rich exometabolome and that cells prefer the uptake of extracellular metabolites over self-synthesis, indicators of ongoing metabolite exchange. We conceived a system that circumvents co-culturing and begins with a self-supporting cell that grows autonomously into a heterogeneous community, only able to survive by exchanging histidine, leucine, uracil, and methionine. Compensating for the progressive loss of prototrophy, self-establishing communities successfully obtained an auxotrophic composition in a nutrition-dependent manner, maintaining a wild-type like exometabolome, growth parameters, and cell viability. Yeast, as a eukaryotic model, thus possesses extensive capacity for growth-relevant metabolite exchange and readily cooperates in metabolism within progressively establishing communities.


Assuntos
Aminoácidos/metabolismo , Interações Microbianas , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Técnicas de Cocultura , Metaboloma , Viabilidade Microbiana
4.
J Mol Biol ; 427(21): 3389-406, 2015 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-26156863

RESUMO

The polyamines (PAs) spermidine, spermine, putrescine and cadaverine are an essential class of metabolites found throughout all kingdoms of life. In this comprehensive review, we discuss their metabolism, their various intracellular functions and their unusual and conserved regulatory features. These include the regulation of translation via upstream open reading frames, the over-reading of stop codons via ribosomal frameshifting, the existence of an antizyme and an antizyme inhibitor, ubiquitin-independent proteasomal degradation, a complex bi-directional membrane transport system and a unique posttranslational modification-hypusination-that is believed to occur on a single protein only (eIF-5A). Many of these features are broadly conserved indicating that PA metabolism is both concentration critical and evolutionary ancient. When PA metabolism is disrupted, a plethora of cellular processes are affected, including transcription, translation, gene expression regulation, autophagy and stress resistance. As a result, the role of PAs has been associated with cell growth, aging, memory performance, neurodegenerative diseases, metabolic disorders and cancer. Despite comprehensive studies addressing PAs, a unifying concept to interpret their molecular role is missing. The precise biochemical function of polyamines is thus one of the remaining mysteries of molecular cell biology.


Assuntos
Neoplasias/metabolismo , Doenças Neurodegenerativas/metabolismo , Poliaminas/metabolismo , Envelhecimento , Animais , Vias Biossintéticas , Proliferação de Células , Regulação da Expressão Gênica , Humanos , Neoplasias/genética , Doenças Neurodegenerativas/genética , Ornitina Descarboxilase/genética , Ornitina Descarboxilase/metabolismo , Poliaminas/análise
5.
Cell Mol Life Sci ; 71(20): 3969-85, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25008043

RESUMO

Microglial cells contribute to normal function of the central nervous system (CNS). Besides playing a role in the innate immunity, they are also involved in neuronal plasticity and homeostasis of the CNS. While microglial cells get activated and undergo phenotypic changes in different disease contexts, they are far from being the "villains" in the CNS. Mounting evidence indicates that microglial dysfunction can exacerbate the pathogenesis of several diseases in the CNS. Several molecular mechanisms tightly regulate the production of inflammatory and toxic factors released by microglia. These mechanisms involve the interaction with other glial cells and neurons and the fine regulation of signaling and transcription activation pathways. The purpose of this review is to discuss microglia activation and to highlight the molecular pathways that can counteract the detrimental role of microglia in several neurologic diseases. Recent work presented in this review support that the understanding of microglial responses can pave the way to design new therapies for inflammatory diseases of the CNS.


Assuntos
Inflamação/metabolismo , Microglia/metabolismo , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Anti-Inflamatórios não Esteroides/uso terapêutico , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Doenças do Sistema Nervoso Central/tratamento farmacológico , Doenças do Sistema Nervoso Central/metabolismo , Doenças do Sistema Nervoso Central/patologia , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Plasticidade Neuronal/fisiologia , Transdução de Sinais/fisiologia , Ativação Transcricional
6.
Proc Natl Acad Sci U S A ; 111(19): 7012-7, 2014 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-24706893

RESUMO

The yeast Hsp31 minifamily proteins (Hsp31, Hsp32, Hsp33, Hsp34) belong to the highly conserved DJ-1 superfamily. The human DJ-1 protein is associated with cancer and neurodegenerative disorders, such as Parkinson disease. However, the precise function of human and yeast DJ-1 proteins is unclear. Here we show that the yeast DJ-1 homologs have a role in diauxic-shift (DS), characterized by metabolic reprogramming because of glucose limitation. We find that the Hsp31 genes are strongly induced in DS and in stationary phase (SP), and that deletion of these genes reduces chronological lifespan, impairs transcriptional reprogramming at DS, and impairs the acquisition of several typical characteristics of SP, including autophagy induction. In addition, under carbon starvation, the HSP31 family gene-deletion strains display impaired autophagy, disrupted target of rapamycin complex 1 (TORC1) localization to P-bodies, and caused abnormal TORC1-mediated Atg13 phosphorylation. Repression of TORC1 by rapamycin in the gene-deletion strains completely reversed their sensitivity to heat shock. Taken together, our data indicate that Hsp31 minifamily is required for DS reprogramming and cell survival in SP, and plays a role upstream of TORC1. The enhanced understanding of the cellular function of these genes sheds light into the biological role of other members of the superfamily, including DJ-1, which is an attractive target for therapeutic intervention in cancer and in Parkinson disease.


Assuntos
Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Aminopeptidases/genética , Aminopeptidases/metabolismo , Autofagia/genética , Carbono/metabolismo , Técnicas de Silenciamento de Genes , Glucose/metabolismo , Humanos , Dados de Sequência Molecular , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Estresse Oxidativo/fisiologia , Saccharomyces cerevisiae/citologia , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo
7.
Autophagy ; 10(3): 514-7, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24430166

RESUMO

The proteins that comprise the Atg1 kinase complex constitute a key set of components that participate in macroautophagy (hereafter autophagy). Among these proteins, Atg13 plays a particularly important, although as yet undefined role, in that it is critical for the proper localization of Atg1 to the phagophore assembly site (PAS) and its efficient kinase activity. Atg13 is hyperphosphorylated in vegetative conditions when autophagy occurs at a basal level, and is largely dephosphorylated upon the induction of autophagy. Inhibitory phosphorylation of Atg13 reflects the activity of TOR complex 1 (TORC1) and protein kinase A. Accordingly, monitoring the phosphorylation state of Atg13 provides a convenient way to follow early steps of autophagy induction as well as the activity of some of the upstream nutrient-sensing kinases. However, the detection of Atg13 by western blot can be problematic. Here, we present a detailed protocol for sample preparation and detection of the Atg13 protein from yeast.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autofagia/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Relacionadas à Autofagia , Western Blotting , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Fosforilação/fisiologia , Saccharomyces cerevisiae
8.
Hum Mol Genet ; 23(3): 755-66, 2014 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-24070869

RESUMO

The oxidation-sensitive chaperone protein DJ-1 has been implicated in several human disorders including cancer and neurodegenerative diseases. During neurodegeneration associated with protein misfolding, such as that observed in Alzheimer's disease and Huntington's disease (HD), both oxidative stress and protein chaperones have been shown to modulate disease pathways. Therefore, we set out to investigate whether DJ-1 plays a role in HD. We found that DJ-1 expression and its oxidation state are abnormally increased in the human HD brain, as well as in mouse and cell models of HD. Furthermore, overexpression of DJ-1 conferred protection in vivo against neurodegeneration in yeast and Drosophila. Importantly, the DJ-1 protein directly interacted with an expanded fragment of huntingtin Exon 1 (httEx1) in test tube experiments and in cell models and accelerated polyglutamine aggregation and toxicity in an oxidation-sensitive manner. Our findings clearly establish DJ-1 as a potential therapeutic target for HD and provide the basis for further studies into the role of DJ-1 in protein misfolding diseases.


Assuntos
Encéfalo/metabolismo , Doença de Huntington/patologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Oncogênicas/metabolismo , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Encéfalo/patologia , Estudos de Casos e Controles , Modelos Animais de Doenças , Drosophila/genética , Humanos , Proteína Huntingtina , Doença de Huntington/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Transgênicos , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Oncogênicas/genética , Oxirredução , Peptídeos/metabolismo , Peroxirredoxinas , Proteína Desglicase DJ-1 , Leveduras/genética
9.
EMBO J ; 32(19): 2603-16, 2013 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-24013120

RESUMO

Deleterious sustained inflammation mediated by activated microglia is common to most of neurologic disorders. Here, we identified sirtuin 2 (SIRT2), an abundant deacetylase in the brain, as a major inhibitor of microglia-mediated inflammation and neurotoxicity. SIRT2-deficient mice (SIRT2(-/-)) showed morphological changes in microglia and an increase in pro-inflammatory cytokines upon intracortical injection of lipopolysaccharide (LPS). This response was associated with increased nitrotyrosination and neuronal cell death. Interestingly, manipulation of SIRT2 levels in microglia determined the response to Toll-like receptor (TLR) activation. SIRT2 overexpression inhibited microglia activation in a process dependent on serine 331 (S331) phosphorylation. Conversely, reduction of SIRT2 in microglia dramatically increased the expression of inflammatory markers, the production of free radicals, and neurotoxicity. Consistent with increased NF-κB-dependent transcription of inflammatory genes, NF-κB was found hyperacetylated in the absence of SIRT2, and became hypoacetylated in the presence of S331A mutant SIRT2. This finding indicates that SIRT2 functions as a 'gatekeeper', preventing excessive microglial activation through NF-κB deacetylation. Our data uncover a novel role for SIRT2 opening new perspectives for therapeutic intervention in neuroinflammatory disorders.


Assuntos
Encefalite/imunologia , Microglia/imunologia , Sirtuína 2/imunologia , Animais , Animais Recém-Nascidos , Linhagem Celular , Células Cultivadas , Interleucina-6/imunologia , Lipopolissacarídeos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/imunologia , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores Toll-Like/imunologia
10.
JAMA Neurol ; 70(4): 481-7, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23400676

RESUMO

IMPORTANCE: Hereditary spastic paraplegias (HSPs) are a group of diseases caused by corticospinal tract degeneration. Mutations in 3 genes (SPG4, SPG3, and SPG31) are said to be the cause in half of the autosomal dominant HSPs (AD-HSPs). This study is a systematic review of families with HSP resulting from a population-based survey. Novel genotype-phenotype correlations were established. OBJECTIVE: To describe the clinical, genetic, and epidemiological features of Portuguese AD-HSP families. DESIGN: Retrospective medical record review. SETTING: A population-based systematic survey of hereditary ataxias and spastic paraplegias conducted in Portugal from 1993 to 2004. PARTICIPANTS: Families with AD-HSP. MAIN OUTCOME MEASURE: Mutation detection in the most prevalent genes. RESULTS: We identified 239 patients belonging to 89 AD-HSP families. The prevalence was 2.4 in 100 000. Thirty-one distinct mutations (26 in SPG4, 4 in SPG3, and 1 in SPG31) segregated in 41% of the families (33.7%, 6.2%, and 1.2% had SPG4, SPG3 and SPG31 mutations, respectively). Seven of the SPG4 mutations were novel, and 7% of all SPG4 mutations were deletions. When disease onset was before the first decade, 31% had SPG4 mutations and 27% had SPG3 mutations. In patients with SPG4 mutations, those with large deletions had the earliest disease onset, followed by those with missense, frameshift, nonsense, and alternative-splicing mutations. Rate of disease progression was not significantly different among patients with SPG3 and SPG4 mutations in a multivariate analysis. For patients with SPG4 mutations, disease progression was worst in patients with later-onset disease. CONCLUSIONS AND RELEVANCE: The prevalence of AD-HSP and frequency of SPG3 and SPG4 mutations in the current study were similar to what has been described in other studies except that the frequency of SPG4 deletions was lower. In contrast, the frequency of SPG31 mutations in the current study was rare compared with other studies. The most interesting aspects of this study are that even in patients with early-onset disease the probability of finding a SPG4 mutation was higher than for patients with SPG3 mutations; there was no difference in disease progression with genotype but an association with the age at onset; 7 new SPG4 mutations were identified; and for the first time, to our knowledge, the nature of the SPG4 mutations was found to predict the age at onset.


Assuntos
Adenosina Trifosfatases/genética , Catarata/epidemiologia , Catarata/genética , Saúde da Família , Transtornos do Crescimento/epidemiologia , Transtornos do Crescimento/genética , Mutação/genética , Paraplegia Espástica Hereditária/epidemiologia , Paraplegia Espástica Hereditária/genética , Adulto , Idade de Início , Osso e Ossos/anormalidades , Análise Mutacional de DNA , Avaliação da Deficiência , Progressão da Doença , Feminino , Proteínas de Ligação ao GTP/genética , Genes Dominantes/genética , Genótipo , Inquéritos Epidemiológicos , Humanos , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Exame Neurológico , Fenótipo , Portugal/epidemiologia , Estudos Retrospectivos , Índice de Gravidade de Doença , Espastina , Estatística como Assunto
11.
PLoS Curr ; 3: RRN1210, 2011 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-21331289

RESUMO

We developed a new cell model for the visualization of toxic huntingtin oligomers in living cells. Huntingtin exon 1 (25Q or 103Q) was fused to non-fluorescent halves of the Venus protein. When huntingtin dimerizes inside the cells, Venus becomes functionally reconstituted and emits fluorescence. Oligomerization, aggregation and toxicity of mutant huntingtin were assessed by several procedures. We also present evidence that the transmission of huntingtin between cells can be determined in a quantitative manner with our model. Thus, this model can be a powerful screening tool for the identification of modifiers of oligomerization and cell-to-cell traffic of mutant huntingtin.

12.
J Biol Chem ; 286(1): 410-9, 2011 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-21044956

RESUMO

Huntington disease (HD) is a neurodegenerative disorder caused by the expansion of a polyglutamine tract in the huntingtin (htt) protein. To uncover candidate therapeutic targets and networks involved in pathogenesis, we integrated gene expression profiling and functional genetic screening to identify genes critical for mutant htt toxicity in yeast. Using mRNA profiling, we have identified genes differentially expressed in wild-type yeast in response to mutant htt toxicity as well as in three toxicity suppressor strains: bna4Δ, mbf1Δ, and ume1Δ. BNA4 encodes the yeast homolog of kynurenine 3-monooxygenase, a promising drug target for HD. Intriguingly, despite playing diverse cellular roles, these three suppressors share common differentially expressed genes involved in stress response, translation elongation, and mitochondrial transport. We then systematically tested the ability of the differentially expressed genes to suppress mutant htt toxicity when overexpressed and have thereby identified 12 novel suppressors, including genes that play a role in stress response, Golgi to endosome transport, and rRNA processing. Integrating the mRNA profiling data and the genetic screening data, we have generated a robust network that shows enrichment in genes involved in rRNA processing and ribosome biogenesis. Strikingly, these observations implicate dysfunction of translation in the pathology of HD. Recent work has shown that regulation of translation is critical for life span extension in Drosophila and that manipulation of this process is protective in Parkinson disease models. In total, these observations suggest that pharmacological manipulation of translation may have therapeutic value in HD.


Assuntos
Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Perfilação da Expressão Gênica , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/toxicidade , Proteínas Nucleares/metabolismo , Proteínas Nucleares/toxicidade , Biossíntese de Proteínas , Saccharomyces cerevisiae/genética , Sequência de Bases , Deleção de Genes , Genômica , Humanos , Proteína Huntingtina , Doença de Huntington/genética , Proteínas Mutantes/biossíntese , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Mutantes/toxicidade , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Análise de Sequência com Séries de Oligonucleotídeos , Fases de Leitura Aberta/genética , RNA Ribossômico/genética , Sequências Reguladoras de Ácido Nucleico/genética , Ribossomos/metabolismo , Saccharomyces cerevisiae/citologia , Transgenes/genética
13.
Biotechnol J ; 3(3): 325-38, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18228539

RESUMO

Protein misfolding and aggregation are central events in many disorders including several neurodegenerative diseases. This suggests that alterations in normal protein homeostasis may contribute to pathogenesis, but the exact molecular mechanisms involved are still poorly understood. The budding yeast Saccharomyces cerevisiae is one of the model systems of choice for studies in molecular medicine. Modeling human neurodegenerative diseases in this simple organism has already shown the incredible power of yeast to unravel the complex mechanisms and pathways underlying these pathologies. Indeed, this work has led to the identification of several potential therapeutic targets and drugs for many diseases, including the neurodegenerative diseases. Several features associated with these diseases, such as formation of protein aggregates, cellular toxicity mediated by misfolded proteins, oxidative stress and hallmarks of apoptosis have been faithfully recapitulated in yeast, enabling researchers to take advantage of this powerful model to rapidly perform genetic and compound screens with the aim of identifying novel candidate therapeutic targets and drugs. Here we review the work undertaken to model human brain disorders in yeast, and how these models provide insight into novel therapeutic approaches for these diseases.


Assuntos
Encéfalo/metabolismo , Modelos Biológicos , Proteínas do Tecido Nervoso/metabolismo , Doenças Neurodegenerativas/metabolismo , Neurônios/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Humanos
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