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1.
Nanomaterials (Basel) ; 11(9)2021 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-34578594

RESUMO

In this study, we accentuate the facile and green synthesis of ecologically viable silver nanoparticles (AgNPs) using aqueous (A-BGE) and ethanolic (E-BGE) dried bitter gourd (Momordica charantia) fruit extract as reducing and capping agents. Although AgNPs synthesized using BGEs have been reported earlier in fundamental antimicrobial studies, the possible antioxidant activity, antibacterial efficacy against superbugs, and a potential antimicrobial mechanism are still lacking. The characterization of as-prepared AgNPs was studied through UV-vis, TEM, Zeta-potential, FT-IR, XRD, and XPS analysis. The antioxidant ability of BG-AgNPs was extensively evaluated through DPPH and FRAP assays, which showed that A-BG-AgNPs possessed higher scavenging ability and superior reducing power due to the high phenolic content present in the BG extract. Furthermore, A-BG-AgNPs were highly stable in various physiological media and displayed excellent antibacterial activity against drug-resistant bacterial strains (i.e., MIC value of 4 µg/mL). The generation of reactive oxygen species evidenced that the possible antimicrobial mechanism was induced by BG-AgNPs, resulting in bacterial cell damage. Within the minimal hemolysis, the BG-mediated AgNPs possessed synergistic antioxidant and antibacterial agents and open another avenue for the inhibition of the growth of pathogens.

2.
Int J Mol Sci ; 22(12)2021 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-34207373

RESUMO

A nucleic acid aptamer that specifically recognizes methicillin-resistant Staphylococcus aureus (MRSA) has been immobilized on magnetic nanoparticles to capture the target bacteria prior to mass spectrometry analysis. After the MRSA species were captured, they were further eluted from the nanoparticles and identified using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). The combination of aptamer-based capture/enrichment and MS analysis of microorganisms took advantage of the selectivity of both techniques and should enhance the accuracy of MRSA identification. The capture and elution efficiencies for MRSA were optimized by examining factors such as incubation time, temperature, and elution solvents. The aptamer-modified magnetic nanoparticles showed a capture rate of more than 90% under the optimized condition, whereas the capture rates were less than 11% for non-target bacteria. The as-prepared nanoparticles exhibited only a 5% decrease in the capture rate and a 9% decrease in the elution rate after 10 successive cycles of utilization. Most importantly, the aptamer-modified nanoparticles revealed an excellent selectivity towards MRSA in bacterial mixtures. The capture of MRSA at a concentration of 102 CFU/mL remained at a good percentage of 82% even when the other two species were at 104 times higher concentration (106 CFU/mL). Further, the eluted MRSA bacteria were successfully identified using MALDI mass spectrometry.


Assuntos
Aptâmeros de Nucleotídeos/química , Nanopartículas de Magnetita/química , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Staphylococcus aureus Resistente à Meticilina/citologia , Técnica de Seleção de Aptâmeros/métodos
3.
Cell Rep ; 26(12): 3191-3202.e8, 2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30893593

RESUMO

Clock neurons within the mammalian suprachiasmatic nuclei (SCN) encode circadian time using interlocked transcription-translation feedback loops (TTFLs) that drive rhythmic gene expression. However, the contributions of other transcription factors outside of the circadian TTFLs to the functionality of the SCN remain obscure. Here, we report that the stem and progenitor cell transcription factor, sex-determining region Y-box 2 (SOX2), is expressed in adult SCN neurons and positively regulates transcription of the core clock gene, Period2. Mice lacking SOX2 selectively in SCN neurons display imprecise, poorly consolidated behavioral rhythms that do not entrain efficiently to environmental light cycles and that are highly susceptible to constant light-induced arrhythmicity. RNA sequencing revealed that Sox2 deficiency alters the SCN transcriptome, reducing the expression of core clock genes and neuropeptide-receptor systems. By defining the transcriptional landscape within SCN neurons, SOX2 enables the generation of robust, entrainable circadian rhythms that accurately reflect environmental time.


Assuntos
Relógios Circadianos/fisiologia , Proteínas Circadianas Period/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Núcleo Supraquiasmático/metabolismo , Transcrição Genética , Animais , Camundongos , Camundongos Transgênicos , Proteínas Circadianas Period/genética , Fatores de Transcrição SOXB1/genética , Núcleo Supraquiasmático/citologia
4.
Front Aging Neurosci ; 11: 368, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-32009941

RESUMO

Aging is associated with cognitive decline and dysregulation of the circadian system, which modulates hippocampal-dependent memory as well as biological processes underlying hippocampal function. While circadian dysfunction and memory impairment are common features of aging and several neurodegenerative brain disorders, how aging impacts the circadian expression patterns of proteins involved in processes that underlie hippocampal-dependent memory is not well understood. In this study, we profiled the hippocampal proteomes of young and middle-aged mice across two circadian cycles using quantitative mass spectrometry in order to explore aging-associated changes in the temporal orchestration of biological pathways. Of the ∼1,420 proteins that were accurately quantified, 15% (214 proteins) displayed circadian rhythms in abundance in the hippocampus of young mice, while only 1.6% (23 proteins) were rhythmic in middle-aged mice. Remarkably, aging disrupted the circadian regulation of proteins involved in cellular functions critical for hippocampal function and memory, including dozens of proteins participating in pathways of energy metabolism, neurotransmission, and synaptic plasticity. These included processes such as glycolysis, the tricarboxylic acid cycle, synaptic vesicle cycling, long-term potentiation, and cytoskeletal organization. Moreover, aging altered the daily expression rhythms of proteins implicated in hallmarks of aging and the pathogenesis of several age-related neurodegenerative brain disorders affecting the hippocampus. Notably, we identified age-related alterations in the rhythmicity of proteins involved in mitochondrial dysfunction and loss of proteostasis, as well as proteins involved in the pathogenesis of disorders such as Alzheimer's disease and Parkinson's disease. These insights into aging-induced changes in the hippocampal proteome provide a framework for understanding how the age-dependent circadian decline may contribute to cognitive impairment and the development of neurodegenerative diseases during aging.

5.
Anal Chim Acta ; 1031: 128-133, 2018 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-30119730

RESUMO

A forced dried droplet method (FDD) is developed to overcome the drawbacks of the conventional dried-droplet (DD) method for matrix assisted laser desorption/ionization (MALDI) sample preparation. The crystals produced by the DD method are heterogeneous and irregularly distributed, and thus many methods have tried to solve the problems. However, most of them spend more time or need additional instruments to generate homogeneous microcrystals. The FDD sample preparation method can produce uniform microcrystals with homogeneous size distribution in few minutes without additional instruments. Stirring the sample spot solution (an agitation process) with a pipette tip can change the crystal size distribution which is observed by the microscope. Mass spectrometric analysis shows that the smaller the crystal size is, the better the ion signal intensity is. The formation of microcrystals can be explained with the effective rate of secondary nucleation. The relative standard deviation (RSD) of the FDD method is ∼16% which is comparable to the two-layer (TL) method and is better than the DD method.

6.
Am J Gastroenterol ; 113(5): 713-724, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29531307

RESUMO

OBJECTIVE: Improved biomarkers are an unmet clinical need for suspected inflammatory bowel disease (IBD). Need is greatest for children, since current biomarkers suffers from low specificity, particularly in this population; thus, invasive testing methods, with the accompanying risk of complications, are necessary. Additionally, current biomarkers do not delineate disease extent assessment for ulcerative colitis (UC), a factor involved in therapeutic decisions. METHODS: Intestinal mucosal-luminal interface (MLI) aspirates from the ascending colon (AC) and descending colon (DC) were collected during diagnostic colonoscopy from treatment-naïve children. The MLI proteomes of 18 non-IBD and 42 IBD patients were analyzed by liquid chromatography mass spectrometry. Analyses of proteomic data generated protein panels distinguishing IBD from non-IBD and pancolitis from non-pancolitis (UC disease extent). Select protein biomarkers were evaluated in stool samples by enzyme-linked immunosorbent assay (n = 24). RESULTS: A panel of four proteins discriminated active IBD from non-IBD (discovery cohort) with a sensitivity of 0.954 (95% confidence interval (CI): 0.772-0.999) and >0.999 (95% CI: 0.824-1.00) for the AC and DC, respectively, and a specificity of >0.999 (AC, 95% CI: 0.815-1.00; DC, 95% CI:0.692-1.00) for both the AC and DC. A separate panel of four proteins distinguished pancolitis from non-pancolitis in UC patients with sensitivity >0.999 (95% CI: 0.590-1.00) and specificity >0.999 (95% CI: 0.715-1.00). Catalase (p < 0.0001) and LTA4H (p = 0.0002) were elevated in IBD stool samples compared to non-IBD stool samples. CONCLUSION: This study identified panels of proteins that have significantly different expression levels and contribute to accurate IBD diagnosis and disease extent characterization in children with UC. Biomarkers identified from the MLI demonstrate transferable results in stool samples.


Assuntos
Colite Ulcerativa/diagnóstico , Mucosa Intestinal/patologia , Adolescente , Biomarcadores/metabolismo , Catalase/metabolismo , Criança , Colite Ulcerativa/patologia , Colo Ascendente/patologia , Colo Descendente/patologia , Colonoscopia , Ensaio de Imunoadsorção Enzimática , Epóxido Hidrolases/metabolismo , Fezes/química , Feminino , Humanos , Masculino , Proteômica/métodos , Sensibilidade e Especificidade
7.
J Proteome Res ; 17(1): 154-163, 2018 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-29130306

RESUMO

In vitro culture based approaches are time- and cost-effective solutions for rapidly evaluating the effects of drugs or natural compounds against microbiomes. The nutritional composition of the culture medium is an important determinant for effectively maintaining the gut microbiome in vitro. This study combines orthogonal experimental design and a metaproteomics approach to obtaining functional insights into the effects of different medium components on the microbiome. Our results show that the metaproteomic profile respond differently to medium components, including inorganic salts, bile salts, mucin, and short-chain fatty acids. Multifactor analysis of variance further revealed significant main and interaction effects of inorganic salts, bile salts, and mucin on the different functional groups of gut microbial proteins. While a broad regulating effect was observed on basic metabolic pathways, different medium components also showed significant modulations on cell wall, membrane, and envelope biogenesis and cell motility related functions. In particular, flagellar assembly related proteins were significantly responsive to the presence of mucin. This study provides information on the functional influences of medium components on the in vitro growth of microbiome communities and gives insight on the key components that must be considered when selecting and optimizing media for culturing ex vivo microbiotas.


Assuntos
Meios de Cultura/química , Microbioma Gastrointestinal/efeitos dos fármacos , Proteômica/métodos , Projetos de Pesquisa , Técnicas de Cultura de Células , Humanos
8.
J Biol Chem ; 292(48): 19826-19839, 2017 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-28978645

RESUMO

One of the major biological functions of the retinal pigmented epithelium (RPE) is the clearance of shed photoreceptor outer segments (POS) through a multistep process resembling phagocytosis. RPE phagocytosis helps maintain the viability of photoreceptors that otherwise could succumb to the high metabolic flux and photo-oxidative stress associated with visual processing. The regulatory mechanisms underlying phagocytosis in the RPE are not fully understood, although dysfunction of this process contributes to the pathogenesis of multiple human retinal degenerative disorders, including age-related macular degeneration. Here, we present an integrated transcriptomic, proteomic, and phosphoproteomic analysis of phagocytosing RPE cells, utilizing three different experimental models: the human-derived RPE-like cell line ARPE-19, cultured murine primary RPE cells, and RPE samples from live mice. Our combined results indicated that early stages of phagocytosis in the RPE are mainly characterized by pronounced changes in the protein phosphorylation level. Global phosphoprotein enrichment analysis revealed involvement of PI3K/Akt, mechanistic target of rapamycin (mTOR), and MEK/ERK pathways in the regulation of RPE phagocytosis, confirmed by immunoblot analyses and in vitro phagocytosis assays. Most strikingly, phagocytosis of POS by cultured RPE cells was almost completely blocked by pharmacological inhibition of phosphorylation of Akt. Our findings, along with those of previous studies, indicate that these phosphorylation events allow the RPE to integrate multiple signals instigated by shed POS at different stages of the phagocytic process.


Assuntos
Fagocitose , Fosfoproteínas/metabolismo , Proteômica , Epitélio Pigmentado da Retina/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Quinases/metabolismo , Epitélio Pigmentado da Retina/citologia , Serina-Treonina Quinases TOR/metabolismo , Transcriptoma
9.
Cell Rep ; 19(3): 505-520, 2017 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-28423315

RESUMO

The central circadian pacemaker, the suprachiasmatic nucleus (SCN), encodes day length information by mechanisms that are not well understood. Here, we report that genetic ablation of miR-132/212 alters entrainment to different day lengths and non-24 hr day-night cycles, as well as photoperiodic regulation of Period2 expression in the SCN. SCN neurons from miR-132/212-deficient mice have significantly reduced dendritic spine density, along with altered methyl CpG-binding protein (MeCP2) rhythms. In Syrian hamsters, a model seasonal rodent, day length regulates spine density on SCN neurons in a melatonin-independent manner, as well as expression of miR-132, miR-212, and their direct target, MeCP2. Genetic disruption of Mecp2 fully restores the level of dendritic spines of miR-132/212-deficient SCN neurons. Our results reveal that, by regulating the dendritic structure of SCN neurons through a MeCP2-dependent mechanism, miR-132/212 affects the capacity of the SCN to encode seasonal time.


Assuntos
Adaptação Fisiológica/genética , Relógios Circadianos/genética , Dendritos/metabolismo , MicroRNAs/metabolismo , Estações do Ano , Adaptação Fisiológica/efeitos da radiação , Animais , Comportamento Animal , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Relógios Circadianos/efeitos da radiação , Dendritos/efeitos da radiação , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/efeitos da radiação , Feminino , Deleção de Genes , Regulação da Expressão Gênica/efeitos da radiação , Luz , Masculino , Mesocricetus , Proteína 2 de Ligação a Metil-CpG/genética , Proteína 2 de Ligação a Metil-CpG/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/genética , Neurônios/metabolismo , Fotoperíodo , Proteoma/metabolismo , Transdução de Sinais/efeitos da radiação , Núcleo Supraquiasmático/metabolismo , Núcleo Supraquiasmático/efeitos da radiação , Serina-Treonina Quinases TOR/metabolismo , Fatores de Tempo
10.
Front Neurol ; 8: 110, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28382018

RESUMO

The circadian clock is an endogenous oscillator that drives daily rhythms in physiology, behavior, and gene expression. The underlying mechanisms of circadian timekeeping are cell-autonomous and involve oscillatory expression of core clock genes that is driven by interconnecting transcription-translation feedback loops (TTFLs). Circadian clock TTFLs are further regulated by posttranslational modifications, in particular, phosphorylation. The hippocampus plays an important role in spatial memory and the conversion of short- to long-term memory. Several studies have reported the presence of a peripheral oscillator in the hippocampus and have highlighted the importance of circadian regulation in memory formation. Given the general importance of phosphorylation in circadian clock regulation, we performed global quantitative proteome and phosphoproteome analyses of the murine hippocampus across the circadian cycle, applying spiked-in labeled reference and high accuracy mass spectrometry (MS). Of the 3,052 proteins and 2,868 phosphosites on 1,368 proteins that were accurately quantified, 1.7% of proteins and 5.2% of phosphorylation events exhibited time-of-day-dependent expression profiles. The majority of circadian phosphopeptides displayed abrupt fluctuations at mid-to-late day without underlying rhythms of protein abundance. Bioinformatic analysis of cyclic phosphorylation events revealed their diverse distribution in different biological pathways, most notably, cytoskeletal organization and neuronal morphogenesis. This study provides the first large-scale, quantitative MS analysis of the circadian phosphoproteome and proteome of the murine hippocampus and highlights the significance of rhythmic regulation at the posttranslational level in this peripheral oscillator. In addition to providing molecular insights into the hippocampal circadian clock, our results will assist in the understanding of genetic factors that underlie rhythms-associated pathological states of the hippocampus.

11.
Gut ; 66(9): 1573-1583, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-27216938

RESUMO

OBJECTIVE: Accurate differentiation between Crohn's disease (CD) and UC is important to ensure early and appropriate therapeutic intervention. We sought to identify proteins that enable differentiation between CD and UC in children with new onset IBD. DESIGN: Mucosal biopsies were obtained from children undergoing baseline diagnostic endoscopy prior to therapeutic interventions. Using a super-stable isotope labeling with amino acids in cell culture (SILAC)-based approach, the proteomes of 99 paediatric control and biopsies of patients with CD and UC were compared. Multivariate analysis of a subset of these (n=50) was applied to identify novel biomarkers, which were validated in a second subset (n=49). RESULTS: In the discovery cohort, a panel of five proteins was sufficient to distinguish control from IBD-affected tissue biopsies with an AUC of 1.0 (95% CI 0.99 to 1.0); a second panel of 12 proteins segregated inflamed CD from UC within an AUC of 0.95 (95% CI 0.86 to 1.0). Application of the two panels to the validation cohort resulted in accurate classification of 95.9% (IBD from control) and 80% (CD from UC) of patients. 116 proteins were identified to have correlation with the severity of disease, four of which were components of the two panels, including visfatin and metallothionein-2. CONCLUSIONS: This study has identified two panels of candidate biomarkers for the diagnosis of IBD and the differentiation of IBD subtypes to guide appropriate therapeutic interventions in paediatric patients.


Assuntos
Colite Ulcerativa , Colo Ascendente , Doença de Crohn , Subunidade beta da Proteína Mitocondrial Trifuncional/análise , Nicotinamida Fosforribosiltransferase/análise , Proteômica/métodos , Adolescente , Biomarcadores/análise , Biópsia/métodos , Canadá , Colite Ulcerativa/diagnóstico , Colite Ulcerativa/metabolismo , Colite Ulcerativa/patologia , Colo Ascendente/metabolismo , Colo Ascendente/patologia , Doença de Crohn/diagnóstico , Doença de Crohn/metabolismo , Doença de Crohn/patologia , Estudos Transversais , Diagnóstico Diferencial , Intervenção Médica Precoce , Feminino , Humanos , Masculino , Seleção de Pacientes
12.
Nat Commun ; 7: 13419, 2016 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-27876802

RESUMO

Intestinal microbial dysbiosis is associated with Crohn's disease (CD). However, the mechanisms leading to the chronic mucosal inflammation that characterizes this disease remain unclear. In this report, we use systems-level approaches to study the interactions between the gut microbiota and host in new-onset paediatric patients to evaluate causality and mechanisms of disease. We report an altered host proteome in CD patients indicative of impaired mitochondrial functions. In particular, mitochondrial proteins implicated in H2S detoxification are downregulated, while the relative abundance of H2S microbial producers is increased. Network correlation analysis reveals that Atopobium parvulum controls the central hub of H2S producers. A. parvulum induces pancolitis in colitis-susceptible interleukin-10-deficient mice and this phenotype requires the presence of the intestinal microbiota. Administrating the H2S scavenger bismuth mitigates A. parvulum-induced colitis in vivo. This study reveals that host-microbiota interactions are disturbed in CD and thus provides mechanistic insights into CD pathogenesis.


Assuntos
Bactérias/genética , Doença de Crohn/microbiologia , Microbioma Gastrointestinal , Adolescente , Animais , Bactérias/classificação , Bactérias/isolamento & purificação , Criança , Pré-Escolar , Feminino , Vida Livre de Germes , Humanos , Interleucina-10/genética , Interleucina-10/metabolismo , Masculino , Camundongos , Camundongos Knockout , Filogenia
13.
Microbiome ; 4(1): 31, 2016 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-27343061

RESUMO

BACKGROUND: The gut microbiota has been shown to be closely associated with human health and disease. While next-generation sequencing can be readily used to profile the microbiota taxonomy and metabolic potential, metaproteomics is better suited for deciphering microbial biological activities. However, the application of gut metaproteomics has largely been limited due to the low efficiency of protein identification. Thus, a high-performance and easy-to-implement gut metaproteomic approach is required. RESULTS: In this study, we developed a high-performance and universal workflow for gut metaproteome identification and quantification (named MetaPro-IQ) by using the close-to-complete human or mouse gut microbial gene catalog as database and an iterative database search strategy. An average of 38 and 33 % of the acquired tandem mass spectrometry (MS) spectra was confidently identified for the studied mouse stool and human mucosal-luminal interface samples, respectively. In total, we accurately quantified 30,749 protein groups for the mouse metaproteome and 19,011 protein groups for the human metaproteome. Moreover, the MetaPro-IQ approach enabled comparable identifications with the matched metagenome database search strategy that is widely used but needs prior metagenomic sequencing. The response of gut microbiota to high-fat diet in mice was then assessed, which showed distinct metaproteome patterns for high-fat-fed mice and identified 849 proteins as significant responders to high-fat feeding in comparison to low-fat feeding. CONCLUSIONS: We present MetaPro-IQ, a metaproteomic approach for highly efficient intestinal microbial protein identification and quantification, which functions as a universal workflow for metaproteomic studies, and will thus facilitate the application of metaproteomics for better understanding the functions of gut microbiota in health and disease.


Assuntos
Bactérias/classificação , Microbioma Gastrointestinal , Metagenômica/métodos , Proteômica/métodos , Animais , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Biologia Computacional/métodos , Bases de Dados Genéticas , Dieta Hiperlipídica , Microbioma Gastrointestinal/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Espectrometria de Massas em Tandem
15.
Cell Rep ; 12(8): 1272-88, 2015 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-26279567

RESUMO

The pacemaker properties of the suprachiasmatic nucleus (SCN) circadian clock are shaped by mechanisms that influence the expression and behavior of clock proteins. Here, we reveal that G-protein-coupled receptor kinase 2 (GRK2) modulates the period, amplitude, and entrainment characteristics of the SCN. Grk2-deficient mice show phase-dependent alterations in light-induced entrainment, slower recovery from jetlag, and longer behavioral rhythms. Grk2 ablation perturbs intrinsic rhythmic properties of the SCN, increasing amplitude and decreasing period. At the cellular level, GRK2 suppresses the transcription of the mPeriod1 gene and the trafficking of PERIOD1 and PERIOD2 proteins to the nucleus. Moreover, GRK2 can physically interact with PERIOD1/2 and promote PERIOD2 phosphorylation at Ser545, effects that may underlie its ability to regulate PERIOD1/2 trafficking. Together, our findings identify GRK2 as an important modulator of circadian clock speed, amplitude, and entrainment by controlling PERIOD at the transcriptional and post-translational levels.


Assuntos
Núcleo Celular/metabolismo , Relógios Circadianos/genética , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Proteínas Circadianas Period/metabolismo , Processamento de Proteína Pós-Traducional , Transporte Ativo do Núcleo Celular , Animais , Linhagem Celular , Células Cultivadas , Quinase 2 de Receptor Acoplado a Proteína G/genética , Masculino , Camundongos , Proteínas Circadianas Period/genética , Fosforilação , Ligação Proteica
16.
PLoS Genet ; 10(10): e1004695, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25330117

RESUMO

The suprachiasmatic nucleus (SCN) acts as the central clock to coordinate circadian oscillations in mammalian behavior, physiology and gene expression. Despite our knowledge of the circadian transcriptome of the SCN, how it impacts genome-wide protein expression is not well understood. Here, we interrogated the murine SCN proteome across the circadian cycle using SILAC-based quantitative mass spectrometry. Of the 2112 proteins that were accurately quantified, 20% (421 proteins) displayed a time-of-day-dependent expression profile. Within this time-of-day proteome, 11% (48 proteins) were further defined as circadian based on a sinusoidal expression pattern with a ∼24 h period. Nine circadianly expressed proteins exhibited 24 h rhythms at the transcript level, with an average time lag that exceeded 8 h. A substantial proportion of the time-of-day proteome exhibited abrupt fluctuations at the anticipated light-to-dark and dark-to-light transitions, and was enriched for proteins involved in several key biological pathways, most notably, mitochondrial oxidative phosphorylation. Additionally, predicted targets of miR-133ab were enriched in specific hierarchical clusters and were inversely correlated with miR133ab expression in the SCN. These insights into the proteomic landscape of the SCN will facilitate a more integrative understanding of cellular control within the SCN clock.


Assuntos
Ritmo Circadiano/fisiologia , Proteoma/metabolismo , Núcleo Supraquiasmático/metabolismo , Animais , Regulação da Expressão Gênica , Luz , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Proteínas Mitocondriais/metabolismo , Fosforilação Oxidativa , Mapas de Interação de Proteínas , Proteoma/análise , Proteômica/instrumentação , Proteômica/métodos , Transcriptoma
17.
PLoS One ; 9(8): e103103, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25084275

RESUMO

Circadian rhythms of behavior and physiology are driven by the biological clock that operates endogenously but can also be entrained to the light-dark cycle of the environment. In mammals, the master circadian pacemaker is located in the suprachiasmatic nucleus (SCN), which is composed of individual cellular oscillators that are driven by a set of core clock genes interacting in transcriptional/translational feedback loops. Light signals can trigger molecular events in the SCN that ultimately impact on the phase of expression of core clock genes to reset the master pacemaker. While transcriptional regulation has received much attention in the field of circadian biology in the past, other mechanisms including targeted protein degradation likely contribute to the clock timing and entrainment process. In the present study, proteome-wide screens of the murine SCN led to the identification of ubiquitin protein ligase E3 component N-recognin 4 (UBR4), a novel E3 ubiquitin ligase component of the N-end rule pathway, as a time-of-day-dependent and light-inducible protein. The spatial and temporal expression pattern of UBR4 in the SCN was subsequently characterized by immunofluorescence microscopy. UBR4 is expressed across the entire rostrocaudal extent of the SCN in a time-of-day-dependent fashion. UBR4 is localized exclusively to arginine vasopressin (AVP)-expressing neurons of the SCN shell. Upon photic stimulation in the early subjective night, the number of UBR4-expressing cells within the SCN increases. This study is the first to identify a novel E3 ubiquitin ligase component, UBR4, in the murine SCN and to implicate the N-end rule degradation pathway as a potential player in regulating core clock mechanisms and photic entrainment.


Assuntos
Relógios Circadianos/genética , Regulação da Expressão Gênica , Luz , Proteínas Associadas aos Microtúbulos/genética , Núcleo Supraquiasmático/metabolismo , Ubiquitina-Proteína Ligases/genética , Animais , Proteínas de Ligação a Calmodulina , Linhagem Celular , Ritmo Circadiano/genética , Masculino , Camundongos , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Fotoperíodo , Ligação Proteica , Transporte Proteico , Proteoma , Proteômica/métodos , Reprodutibilidade dos Testes , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo
18.
Methods Mol Biol ; 1164: 1-13, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24927830

RESUMO

Proteins play important roles in biochemical processes. Most biological functions are realized through protein-protein interactions (PPI). Co-immunoprecipitation is the most straightforward method to detect PPI. With the development of modern mass spectrometry (MS), throughput, sensitivity, and confidence for the detection of PPI can be readily achieved by scaling up traditional antibody-based strategies. Herein, we describe a typical workflow for general PPI detection using mass spectrometry coupled techniques, covering from Co-immunoprecipitation (Co-IP), to gel display, in-gel digestion, liquid chromatography mass spectrometry (LC-MS) analysis, as well as result interpretation and statistic filtering. This protocol provides an overview of the technique as well as practical tips.


Assuntos
Espectrometria de Massas/métodos , Mapeamento de Interação de Proteínas/métodos , Proteínas/metabolismo , Animais , Cromatografia Líquida/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Humanos , Imunoprecipitação/métodos , Mapas de Interação de Proteínas , Proteínas/análise , Proteômica/métodos , Software
20.
J Proteome Res ; 12(3): 1512-9, 2013 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-23394071

RESUMO

In proteomics, detergents and chaotropes are indispensable for proteome analysis, not only for protein extraction, but also for protein digestion. To increase the protein extraction efficiency, detergents are usually added in the lysis buffer to extract membrane proteins out of membrane structure and to maintain protein in solutions. In general, these detergents need to be removed prior to protein digestion, usually by precipitation or ultrafiltration. Digestion often takes place in the presence of chaotropic reagents, such as urea, which often need to be removed prior to mass spectrometry. The addition and removal of detergents and chaotropes require multiple steps that are time-consuming and can cause sample losses. Amphipols (APols) are a different class of detergents that have physical and solubilization properties that are distinct from conventional detergents. They have primarily been used in protein structure analysis for membrane protein trapping and stabilization. Here, we demonstrate a simple and rapid protocol for total and membrane proteome preparation using APols. We demonstrate that APols added for cell lysis help maintain the proteome in solution, are compatible with protein digestion using trypsin, and can readily be removed prior to mass spectrometry by a one-step acidification and centrifugation. This protocol is much faster, can be performed in a single tube, and can readily replace the conventional detergent/chaotrope approaches for total and membrane proteome analysis.


Assuntos
Peptídeos/metabolismo , Proteômica , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Humanos , Espectrometria de Massas por Ionização por Electrospray
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