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1.
Cell Syst ; 12(8): 780-794.e7, 2021 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-34139154

RESUMO

COVID-19 is highly variable in its clinical presentation, ranging from asymptomatic infection to severe organ damage and death. We characterized the time-dependent progression of the disease in 139 COVID-19 inpatients by measuring 86 accredited diagnostic parameters, such as blood cell counts and enzyme activities, as well as untargeted plasma proteomes at 687 sampling points. We report an initial spike in a systemic inflammatory response, which is gradually alleviated and followed by a protein signature indicative of tissue repair, metabolic reconstitution, and immunomodulation. We identify prognostic marker signatures for devising risk-adapted treatment strategies and use machine learning to classify therapeutic needs. We show that the machine learning models based on the proteome are transferable to an independent cohort. Our study presents a map linking routinely used clinical diagnostic parameters to plasma proteomes and their dynamics in an infectious disease.


Assuntos
Biomarcadores/análise , COVID-19/patologia , Progressão da Doença , Proteoma/fisiologia , Fatores Etários , Contagem de Células Sanguíneas , Gasometria , Ativação Enzimática , Humanos , Inflamação/patologia , Aprendizado de Máquina , Prognóstico , Proteômica , SARS-CoV-2/imunologia
2.
Nat Biotechnol ; 39(7): 846-854, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33767396

RESUMO

Accurate quantification of the proteome remains challenging for large sample series and longitudinal experiments. We report a data-independent acquisition method, Scanning SWATH, that accelerates mass spectrometric (MS) duty cycles, yielding quantitative proteomes in combination with short gradients and high-flow (800 µl min-1) chromatography. Exploiting a continuous movement of the precursor isolation window to assign precursor masses to tandem mass spectrometry (MS/MS) fragment traces, Scanning SWATH increases precursor identifications by ~70% compared to conventional data-independent acquisition (DIA) methods on 0.5-5-min chromatographic gradients. We demonstrate the application of ultra-fast proteomics in drug mode-of-action screening and plasma proteomics. Scanning SWATH proteomes capture the mode of action of fungistatic azoles and statins. Moreover, we confirm 43 and identify 11 new plasma proteome biomarkers of COVID-19 severity, advancing patient classification and biomarker discovery. Thus, our results demonstrate a substantial acceleration and increased depth in fast proteomic experiments that facilitate proteomic drug screens and clinical studies.


Assuntos
Proteômica/métodos , Espectrometria de Massas em Tandem , Arabidopsis/metabolismo , Biomarcadores/metabolismo , COVID-19/sangue , COVID-19/diagnóstico , Linhagem Celular , Humanos , Peptídeos/análise , Proteoma/análise , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Índice de Gravidade de Doença
3.
Autophagy ; 17(3): 779-795, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-32079454

RESUMO

Viral infections are often accompanied by the induction of autophagy as an intrinsic cellular defense mechanism. Herpesviruses have developed strategies to evade autophagic degradation and to manipulate autophagy of the host cells to their benefit. Here we addressed the role of macroautophagy/autophagy in human cytomegalovirus replication and for particle morphogenesis. We found that proteins of the autophagy machinery localize to cytoplasmic viral assembly compartments and enveloped virions in the cytoplasm. Surprisingly, the autophagy receptor SQSTM1/p62 was also found to colocalize with HCMV capsids in the nucleus of infected cells. This finding indicates that the autophagy machinery interacts with HCMV already at the early nuclear stages of particle morphogenesis. The membrane-bound form of LC3 and several autophagy receptors were packaged into extracellular HCMV virions. This suggested that autophagic membranes were included during secondary envelopment of HCMV virions. To further address the importance of autophagy in HCMV infection, we generated an HCMV mutant that expressed a dominant-negative version of the protease ATG4B (BAD-ATG4BC74A). The proteolytic activity of ATG4B is required for LC3 cleavage, priming it for membrane conjugation. Surprisingly, both genome replication and virus release were enhanced in cells infected with BAD-ATG4BC74A, compared to control strains. These results show that autophagy operates as an antiviral process during HCMV infection but is dispensable for secondary HCMV particle envelopment.Abbreviations: ATG: autophagy-related; BAC: bacterial artificial chromosome; BECN1: beclin 1; CPE: cytopathic effect; cVACs: cytoplasmic viral assembly compartments; d.p.i.: days post-infection; DB: dense body; EBV: Epstein-Barr virus; galK: galactokinase; HCMV: human cytomegalovirus; HFF: human foreskin fibroblasts; IE: immediate-early; IRS: internal repeat short; LC3: MAP1LC3A/B; m.o.i.; multiplicity of infection; MCP: major capsid protein; Pp: phosphoprotein; sCP/UL48a: smallest capsid protein; TRS: terminal repeat short; UL: unique long; US: unique short.

4.
Nat Metab ; 2(9): 918-933, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32778834

RESUMO

Regeneration after injury occurs in axons that lie in the peripheral nervous system but fails in the central nervous system, thereby limiting functional recovery. Differences in axonal signalling in response to injury that might underpin this differential regenerative ability are poorly characterized. Combining axoplasmic proteomics from peripheral sciatic or central projecting dorsal root ganglion (DRG) axons with cell body RNA-seq, we uncover injury-dependent signalling pathways that are uniquely represented in peripheral versus central projecting sciatic DRG axons. We identify AMPK as a crucial regulator of axonal regenerative signalling that is specifically downregulated in injured peripheral, but not central, axons. We find that AMPK in DRG interacts with the 26S proteasome and its CaMKIIα-dependent regulatory subunit PSMC5 to promote AMPKα proteasomal degradation following sciatic axotomy. Conditional deletion of AMPKα1 promotes multiple regenerative signalling pathways after central axonal injury and stimulates robust axonal growth across the spinal cord injury site, suggesting inhibition of AMPK as a therapeutic strategy to enhance regeneration following spinal cord injury.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Axônios , Gânglios Espinais/metabolismo , Regeneração Nervosa , Células Receptoras Sensoriais/metabolismo , Traumatismos da Medula Espinal/metabolismo , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Animais , Transporte Axonal , Axotomia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Feminino , Gânglios Espinais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteômica , Nervo Isquiático/metabolismo , Nervo Isquiático/patologia , Células Receptoras Sensoriais/patologia , Traumatismos da Medula Espinal/patologia
5.
Cell Syst ; 11(1): 11-24.e4, 2020 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-32619549

RESUMO

The COVID-19 pandemic is an unprecedented global challenge, and point-of-care diagnostic classifiers are urgently required. Here, we present a platform for ultra-high-throughput serum and plasma proteomics that builds on ISO13485 standardization to facilitate simple implementation in regulated clinical laboratories. Our low-cost workflow handles up to 180 samples per day, enables high precision quantification, and reduces batch effects for large-scale and longitudinal studies. We use our platform on samples collected from a cohort of early hospitalized cases of the SARS-CoV-2 pandemic and identify 27 potential biomarkers that are differentially expressed depending on the WHO severity grade of COVID-19. They include complement factors, the coagulation system, inflammation modulators, and pro-inflammatory factors upstream and downstream of interleukin 6. All protocols and software for implementing our approach are freely available. In total, this work supports the development of routine proteomic assays to aid clinical decision making and generate hypotheses about potential COVID-19 therapeutic targets.


Assuntos
Proteínas Sanguíneas/metabolismo , Infecções por Coronavirus/sangue , Pneumonia Viral/sangue , Proteômica/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Betacoronavirus/isolamento & purificação , Biomarcadores/sangue , Proteínas Sanguíneas/análise , COVID-19 , Infecções por Coronavirus/classificação , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias/classificação , Pneumonia Viral/classificação , Pneumonia Viral/patologia , Pneumonia Viral/virologia , SARS-CoV-2 , Adulto Jovem
6.
Angew Chem Int Ed Engl ; 57(26): 7893-7897, 2018 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-29624844

RESUMO

Recently discovered new chemical entities in RNA modifications have involved surprising functional groups that enlarge the chemical space of RNA. Using LC-MS, we found over 100 signals of RNA constituents that contained a ribose moiety in tRNAs from E. coli. Feeding experiments with variegated stable isotope labeled compounds identified 37 compounds that are new structures of RNA modifications. One structure was elucidated by deuterium exchange and high-resolution mass spectrometry. The structure of msms2 i6 A (2-methylthiomethylenethio-N6-isopentenyl-adenosine) was confirmed by methione-D3 feeding experiments and by synthesis of the nucleobase. The msms2 i6 A contains a thioacetal, shown in vitro to be biosynthetically derived from ms2 i6 A by the radical-SAM enzyme MiaB. This enzyme performs thiomethylation, forming ms2 i6 A from i6 A in a first turnover. The new thioacetal is formed by a second turnover. Along with the pool of 36 new modifications, this work describes a new layer of RNA modification chemistry.


Assuntos
Acetais/química , RNA Bacteriano/química , Compostos de Sulfidrila/química , Cromatografia Líquida , Escherichia coli/genética , Conformação de Ácido Nucleico , Espectrometria de Massas em Tandem
7.
PLoS Pathog ; 12(2): e1005439, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26910529

RESUMO

Developmental differentiation is a universal biological process that allows cells to adapt to different environments to perform specific functions. African trypanosomes progress through a tightly regulated life cycle in order to survive in different host environments when they shuttle between an insect vector and a vertebrate host. Transcriptomics has been useful to gain insight into RNA changes during stage transitions; however, RNA levels are only a moderate proxy for protein abundance in trypanosomes. We quantified 4270 protein groups during stage differentiation from the mammalian-infective to the insect form and provide classification for their expression profiles during development. Our label-free quantitative proteomics study revealed previously unknown components of the differentiation machinery that are involved in essential biological processes such as signaling, posttranslational protein modifications, trafficking and nuclear transport. Furthermore, guided by our proteomic survey, we identified the cause of the previously observed differentiation impairment in the histone methyltransferase DOT1B knock-out strain as it is required for accurate karyokinesis in the first cell division during differentiation. This epigenetic regulator is likely involved in essential chromatin restructuring during developmental differentiation, which might also be important for differentiation in higher eukaryotic cells. Our proteome dataset will serve as a resource for detailed investigations of cell differentiation to shed more light on the molecular mechanisms of this process in trypanosomes and other eukaryotes.


Assuntos
Proteoma/genética , Proteômica , Trypanosoma brucei brucei/metabolismo , Sequência de Aminoácidos , Animais , Diferenciação Celular , Cromatina/metabolismo , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Estágios do Ciclo de Vida/genética , Proteômica/métodos , Trypanosoma brucei brucei/genética
8.
Proteomics ; 16(3): 402-16, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26572502

RESUMO

The slowing down or stalling of replication forks is commonly known as replication stress and arises from multiple causes such as DNA lesions, nucleotide depletion, RNA-DNA hybrids, and oncogene activation. The ataxia telangiectasia and Rad3-related kinase (ATR) plays an essential role in the cellular response to replication stress and inhibition of ATR has emerged as therapeutic strategy for the treatment of cancers that exhibit high levels of replication stress. However, the cellular signaling induced by replication stress and the substrate spectrum of ATR has not been systematically investigated. In this study, we employed quantitative MS-based proteomics to define the cellular signaling after nucleotide depletion-induced replication stress and replication fork collapse following ATR inhibition. We demonstrate that replication stress results in increased phosphorylation of a subset of proteins, many of which are involved in RNA splicing and transcription and have previously not been associated with the cellular replication stress response. Furthermore, our data reveal the ATR-dependent phosphorylation following replication stress and discover novel putative ATR target sites on MCM6, TOPBP1, RAD51AP1, and PSMD4. We establish that ATR inhibition rewires cellular signaling networks induced by replication stress and leads to the activation of the ATM-driven double-strand break repair signaling.


Assuntos
Antineoplásicos/farmacologia , Replicação do DNA/efeitos dos fármacos , Redes Reguladoras de Genes , Hidroxiureia/farmacologia , Transdução de Sinais/efeitos dos fármacos , Sequência de Aminoácidos , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Componente 6 do Complexo de Manutenção de Minicromossomo/genética , Componente 6 do Complexo de Manutenção de Minicromossomo/metabolismo , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoblastos/patologia , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Mapeamento de Interação de Proteínas , Splicing de RNA , Proteínas de Ligação a RNA , Estresse Fisiológico , Transcrição Genética
9.
BMC Genomics ; 16: 965, 2015 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-26577093

RESUMO

BACKGROUND: To date, telomere research in fungi has mainly focused on Saccharomyces cerevisiae and Schizosaccharomyces pombe, despite the fact that both yeasts have degenerated telomeric repeats in contrast to the canonical TTAGGG motif found in vertebrates and also several other fungi. RESULTS: Using label-free quantitative proteomics, we here investigate the telosome of Neurospora crassa, a fungus with canonical telomeric repeats. We show that at least six of the candidates detected in our screen are direct TTAGGG-repeat binding proteins. While three of the direct interactors (NCU03416 [ncTbf1], NCU01991 [ncTbf2] and NCU02182 [ncTay1]) feature the known myb/homeobox DNA interaction domain also found in the vertebrate telomeric factors, we additionally show that a zinc-finger protein (NCU07846) and two proteins without any annotated DNA-binding domain (NCU02644 and NCU05718) are also direct double-strand TTAGGG binders. We further find two single-strand binders (NCU02404 [ncGbp2] and NCU07735 [ncTcg1]). CONCLUSION: By quantitative label-free interactomics we identify TTAGGG-binding proteins in Neurospora crassa, suggesting candidates for telomeric factors that are supported by phylogenomic comparison with yeast species. Intriguingly, homologs in yeast species with degenerated telomeric repeats are also TTAGGG-binding proteins, e.g. in S. cerevisiae Tbf1 recognizes the TTAGGG motif found in its subtelomeres. However, there is also a subset of proteins that is not conserved. While a rudimentary core TTAGGG-recognition machinery may be conserved across yeast species, our data suggests Neurospora as an emerging model organism with unique features.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/metabolismo , Neurospora crassa/metabolismo , Motivos de Nucleotídeos , Proteômica , Sequências Repetitivas de Ácido Nucleico , Telômero/genética , Animais , Sequência de Bases , Sequência Conservada , Evolução Molecular , Neurospora crassa/genética , Vertebrados/genética
10.
Proteomics ; 14(16): 1882-9, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24920314

RESUMO

Phytoplasmas are pathogenic bacteria within the class of Mollicutes, which are associated with more than 1000 plant diseases. In this study, we applied quantitative mass spectrometry to analyse affected pathways of the model plant tobacco (Nicotiana occidentalis) upon Candidatus Phytoplasma mali strain AT infection. Using tissue obtained from leaf midribs, 1466 plant-assigned proteins were identified. For 1019 of these proteins, we could reproducibly quantify the expression changes of infected versus noninfected plants, of which 157 proteins were up- and 173 proteins were downregulated. Differential expression took place in a number of pathways, among others strong downregulation of porphyrin and chlorophyll metabolism and upregulation of alpha-linolenic acid metabolism, which was consistent with observed increased levels of jasmonic acid, a key signal molecule of plant defence. Our data shed light on the molecular networks that are involved in defence of plants against phytoplasma infection and provide a resource for further studies.


Assuntos
Interações Hospedeiro-Patógeno , Phytoplasma/fisiologia , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Proteômica/métodos , Tabaco/metabolismo , Tabaco/microbiologia , Ciclopentanos/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica de Plantas , Oxilipinas/metabolismo , Doenças das Plantas/genética , Proteínas de Plantas/genética , Transdução de Sinais , Tabaco/genética
11.
Mol Nutr Food Res ; 58(4): 903-7, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24272914

RESUMO

Over the last decades polyetiological metabolic diseases such as obesity and type 2 diabetes have emerged as a global epidemic. Efficient strategies for prevention and treatment include dietary intervention and the development of validated nutraceuticals. Safe extracts of edible plants provide a resource of structurally diverse molecules that can effectively interfere with multifactorial diseases. In this study, we describe the application of ethanolic lemon balm (Melissa officinalis) leaves extract for the treatment of insulin-resistance and dyslipidemia in mice. We show that lemon balm extract (LBE) activates the peroxisome proliferator-activated receptors (PPARs), which have key roles in the regulation of whole body glucose and lipid metabolism. Application of LBE (0.6 mg/mL) to human primary adipocytes resulted in specific peroxisome proliferator-activated receptor target gene expression. LBE treatment of insulin-resistant high-fat diet-fed C57BL/6 mice (200 mg/kg/day) for 6 weeks considerably reduced hyperglycemia and insulin resistance, plasma triacylglycerol, nonesterified fatty acids and LDL/VLDL cholesterol levels. Taken together, ethanolic lemon balm extract can potentially be used to prevent or concomitantly treat type 2 diabetes and associated disorders such as dyslipidemia and hypercholesterolemia.


Assuntos
Hipoglicemiantes/farmacologia , Hipolipemiantes/farmacologia , Melissa/química , Extratos Vegetais/farmacologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Células Cultivadas , Dislipidemias/prevenção & controle , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Resistência à Insulina , Camundongos Endogâmicos C57BL , Camundongos Obesos , PPAR alfa/metabolismo , PPAR gama/metabolismo
12.
Proteomics ; 13(23-24): 3424-8, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24174276

RESUMO

Cellular communication is a fundamental process in biology. The interaction of adipocytes with macrophages is a key event in the development of common diseases such as type 2 diabetes. We applied an established bilayer cell co-culture system and comprehensive mass spectrometry analysis to detect proteome-wide the paracrine interaction of murine adipocytes and macrophages. Altogether, we identified 4486 proteins with at least two unique peptides of which 2392 proteins were informative for 3T3-L1 adipocytes and 2957 proteins for RAW 264.7 macrophages. Further, we observed over 12,000 phosphorylation sites of which we could assign 3,200 informative phosphopeptides with a single phosphosite for adipocytes and 4,514 for macrophages. Using protein set enrichment and phosphosite analyses, we deciphered regulatory protein pathways involved in cellular stress and inflammation, which can contribute to metabolic impairment of cells including insulin resistance and other disorders. The generated data sets provide a holistic, molecular pathway-centric view on the interplay of adipocytes and macrophages in disease processes and a resource for further studies.


Assuntos
Adipócitos/metabolismo , Comunicação Celular , Macrófagos/metabolismo , Proteoma/metabolismo , Animais , Linhagem Celular , Técnicas de Cocultura , Camundongos , Fosfopeptídeos/metabolismo , Fosforilação , Transdução de Sinais , Transcriptoma
13.
PLoS One ; 8(11): e80335, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24265809

RESUMO

Given the significant increases in the incidence of metabolic diseases, efficient strategies for preventing and treating of these common disorders are urgently needed. This includes the development of phytopharmaceutical products or functional foods to prevent or cure metabolic diseases. Plant extracts from edible biomaterial provide a potential resource of structurally diverse molecules that can synergistically interfere with complex disorders. In this study we describe the safe application of ethanolic chamomile (Matricaria recutita) flowers extract (CFE) for the treatment and prevention of type 2 diabetes and associated disorders. We show in vitro that this extract activates in particular nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) and its isotypes. In a cellular context, in human primary adipocytes CFE administration (300 µg/ml) led to specific expression of target genes of PPARγ, whereas in human hepatocytes CFE-induced we detected expression changes of genes that were regulated by PPARα. In vivo treatment of insulin-resistant high-fat diet (HFD)-fed C57BL/6 mice with CFE (200 mg/kg/d) for 6 weeks considerably reduced insulin resistance, glucose intolerance, plasma triacylglycerol, non-esterified fatty acids (NEFA) and LDL/VLDL cholesterol. Co-feeding of lean C57BL/6 mice a HFD with 200 mg/kg/d CFE for 20 weeks showed effective prevention of fatty liver formation and hepatic inflammation, indicating additionally hepatoprotective effects of the extract. Moreover, CFE treatment did not reveal side effects, which have otherwise been associated with strong synthetic PPAR-targeting molecules, such as weight gain, liver disorders, hemodilution or bone cell turnover. Taken together, modulation of PPARs and other factors by chamomile flowers extract has the potential to prevent or treat type 2 diabetes and related disorders.


Assuntos
Camomila/química , Flores/química , Regulação da Expressão Gênica/efeitos dos fármacos , Hipoglicemiantes/farmacologia , Receptores Ativados por Proliferador de Peroxissomo/genética , Extratos Vegetais/farmacologia , Transcrição Genética/efeitos dos fármacos , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Dieta Hiperlipídica , Relação Dose-Resposta a Droga , Dislipidemias/tratamento farmacológico , Dislipidemias/genética , Dislipidemias/metabolismo , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Fígado Gorduroso/prevenção & controle , Técnicas de Silenciamento de Genes , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Hipoglicemiantes/administração & dosagem , Hipoglicemiantes/efeitos adversos , Resistência à Insulina/genética , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Obesos , Hepatopatia Gordurosa não Alcoólica , PPAR gama/agonistas , PPAR gama/genética , PPAR gama/metabolismo , Receptores Ativados por Proliferador de Peroxissomo/agonistas , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Extratos Vegetais/administração & dosagem , Extratos Vegetais/efeitos adversos , Ativação Transcricional
15.
Proc Natl Acad Sci U S A ; 109(19): 7257-62, 2012 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-22509006

RESUMO

Given worldwide increases in the incidence of obesity and type 2 diabetes, new strategies for preventing and treating metabolic diseases are needed. The nuclear receptor PPARγ (peroxisome proliferator-activated receptor gamma) plays a central role in lipid and glucose metabolism; however, current PPARγ-targeting drugs are characterized by undesirable side effects. Natural products from edible biomaterial provide a structurally diverse resource to alleviate complex disorders via tailored nutritional intervention. We identified a family of natural products, the amorfrutins, from edible parts of two legumes, Glycyrrhiza foetida and Amorpha fruticosa, as structurally new and powerful antidiabetics with unprecedented effects for a dietary molecule. Amorfrutins bind to and activate PPARγ, which results in selective gene expression and physiological profiles markedly different from activation by current synthetic PPARγ drugs. In diet-induced obese and db/db mice, amorfrutin treatment strongly improves insulin resistance and other metabolic and inflammatory parameters without concomitant increase of fat storage or other unwanted side effects such as hepatoxicity. These results show that selective PPARγ-activation by diet-derived ligands may constitute a promising approach to combat metabolic disease.


Assuntos
Produtos Biológicos/farmacologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Fabaceae/química , Hipoglicemiantes/farmacologia , Salicilatos/farmacologia , Células 3T3-L1 , Animais , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Western Blotting , Células CHO , Cricetinae , Cricetulus , Cristalografia por Raios X , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/etiologia , Dieta Hiperlipídica/efeitos adversos , Suplementos Nutricionais , Expressão Gênica/efeitos dos fármacos , Glycyrrhiza/química , Humanos , Hipoglicemiantes/química , Hipoglicemiantes/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Obesidade/complicações , Obesidade/tratamento farmacológico , Obesidade/etiologia , PPAR gama/genética , PPAR gama/metabolismo , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Salicilatos/química , Salicilatos/metabolismo
17.
Nat Protoc ; 4(5): 732-42, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19390529

RESUMO

Bacteria are a convenient source of intrinsic marker proteins, which can be detected efficiently by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The patterns of protein masses observed can be used for accurate classification and identification of bacteria. Key to the reliability of the method is a robust and standardized procedure for sample preparations, including bacterial culturing, chemical treatment for bacterial cell wall disruption and for protein extraction, and mass spectrometry analysis. The protocol is an excellent alternative to classical microbiological classification and identification procedures, requiring minimal sample preparation efforts and costs. Without cell culturing, the protocol takes in general <1 h.


Assuntos
Proteínas de Bactérias/química , Enterobacteriaceae/classificação , Espectrometria de Massas/métodos , Filogenia , Classificação/métodos , Enterobacteriaceae/crescimento & desenvolvimento
18.
PLoS One ; 3(7): e2843, 2008 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-18665227

RESUMO

BACKGROUND: In general, the definite determination of bacterial species is a tedious process and requires extensive manual labour. Novel technologies for bacterial detection and analysis can therefore help microbiologists in minimising their efforts in developing a number of microbiological applications. METHODOLOGY: We present a robust, standardized procedure for automated bacterial analysis that is based on the detection of patterns of protein masses by MALDI mass spectrometry. We particularly applied the approach for classifying and identifying strains in species of the genus Erwinia. Many species of this genus are associated with disastrous plant diseases such as fire blight. Using our experimental procedure, we created a general bacterial mass spectra database that currently contains 2800 entries of bacteria of different genera. This database will be steadily expanded. To support users with a feasible analytical method, we developed and tested comprehensive software tools that are demonstrated herein. Furthermore, to gain additional analytical accuracy and reliability in the analysis we used genotyping of single nucleotide polymorphisms by mass spectrometry to unambiguously determine closely related strains that are difficult to distinguish by only relying on protein mass pattern detection. CONCLUSIONS: With the method for bacterial analysis, we could identify fire blight pathogens from a variety of biological sources. The method can be used for a number of additional bacterial genera. Moreover, the mass spectrometry approach presented allows the integration of data from different biological levels such as the genome and the proteome.


Assuntos
Bactérias/metabolismo , Biologia Computacional/métodos , Regulação Bacteriana da Expressão Gênica , Espectrometria de Massas/métodos , Algoritmos , Bactérias/classificação , Bactérias/genética , Bases de Dados de Proteínas , Erwinia/metabolismo , Genótipo , Microbiologia , Modelos Biológicos , Filogenia , Proteômica/métodos , Análise de Sequência de DNA , Software , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
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