RESUMO
The Polycomb repressor complex 2 (PRC2) is composed of the core subunits Ezh1/2, Suz12, and Eed, and it mediates all di- and tri-methylation of histone H3 at lysine 27 in higher eukaryotes. However, little is known about how the catalytic activity of PRC2 is regulated to demarcate H3K27me2 and H3K27me3 domains across the genome. To address this, we mapped the endogenous interactomes of Ezh2 and Suz12 in embryonic stem cells (ESCs), and we combined this with a functional screen for H3K27 methylation marks. We found that Nsd1-mediated H3K36me2 co-locates with H3K27me2, and its loss leads to genome-wide expansion of H3K27me3. These increases in H3K27me3 occurred at PRC2/PRC1 target genes and as de novo accumulation within what were previously broad H3K27me2 domains. Our data support a model in which Nsd1 is a key modulator of PRC2 function required for regulating the demarcation of genome-wide H3K27me2 and H3K27me3 domains in ESCs.
Assuntos
Proteínas de Transporte/metabolismo , Montagem e Desmontagem da Cromatina , Histonas/metabolismo , Células-Tronco Embrionárias Murinas/enzimologia , Proteínas Nucleares/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Animais , Proteínas de Transporte/genética , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Histona-Lisina N-Metiltransferase , Humanos , Metilação , Camundongos , Proteínas Nucleares/genética , Complexo Repressor Polycomb 2/genética , Processamento de Proteína Pós-TraducionalRESUMO
Pseudomonas putida KT2440 is an important bioplastic-producing industrial microorganism capable of synthesizing the polymeric carbon-rich storage material, polyhydroxyalkanoate (PHA). PHA is sequestered in discrete PHA granules, or carbonosomes, and accumulates under conditions of stress, for example, low levels of available nitrogen. The pha locus responsible for PHA metabolism encodes both anabolic and catabolic enzymes, a transcription factor, and carbonosome-localized proteins termed phasins. The functions of phasins are incompletely understood but genetic disruption of their function causes PHA-related phenotypes. To improve our understanding of these proteins, we investigated the PHA pathways of P.putida KT2440 using three types of experiments. First, we profiled cells grown in nitrogen-limited and nitrogen-excess media using global expression proteomics, identifying sets of proteins found to coordinately increase or decrease within clustered pathways. Next, we analyzed the protein composition of isolated carbonosomes, identifying two new putative components. We carried out physical interaction screens focused on PHA-related proteins, generating a protein-protein network comprising 434 connected proteins. Finally, we confirmed that the outer membrane protein OprL (the Pal component of the Pal-Tol system) localizes to the carbonosome and shows a PHA-related phenotype and therefore is a novel phasin. The combined datasets represent a valuable overview of the protein components of the PHA system in P.putida highlighting the complex nature of regulatory interactions responsive to nutrient stress.
Assuntos
Lipoproteínas , Poli-Hidroxialcanoatos , Proteômica , Pseudomonas putida , Poli-Hidroxialcanoatos/metabolismo , Pseudomonas putida/metabolismo , Pseudomonas putida/genética , Proteômica/métodos , Lipoproteínas/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/metabolismo , Nitrogênio/metabolismo , Lectinas de PlantasRESUMO
The metabolic adaptation of eukaryotic cells to hypoxia involves increasing dependence upon glycolytic adenosine triphosphate (ATP) production, an event with consequences for cellular bioenergetics and cell fate. This response is regulated at the transcriptional level by the hypoxia-inducible factor-1(HIF-1)-dependent transcriptional upregulation of glycolytic enzymes (GEs) and glucose transporters. However, this transcriptional upregulation alone is unlikely to account fully for the levels of glycolytic ATP produced during hypoxia. Here, we investigated additional mechanisms regulating glycolysis in hypoxia. We observed that intestinal epithelial cells treated with inhibitors of transcription or translation and human platelets (which lack nuclei and the capacity for canonical transcriptional activity) maintained the capacity for hypoxia-induced glycolysis, a finding which suggests the involvement of a nontranscriptional component to the hypoxia-induced metabolic switch to a highly glycolytic phenotype. In our investigations into potential nontranscriptional mechanisms for glycolytic induction, we identified a hypoxia-sensitive formation of complexes comprising GEs and glucose transporters in intestinal epithelial cells. Surprisingly, the formation of such glycolytic complexes occurs independent of HIF-1-driven transcription. Finally, we provide evidence for the presence of HIF-1α in cytosolic fractions of hypoxic cells which physically interacts with the glucose transporter GLUT1 and the GEs in a hypoxia-sensitive manner. In conclusion, we provide insights into the nontranscriptional regulation of hypoxia-induced glycolysis in intestinal epithelial cells.
Assuntos
Células Epiteliais , Glicólise , Humanos , Glicólise/genética , Trifosfato de Adenosina , Expressão Gênica , GlucoseRESUMO
Marked sexual dimorphism is displayed in the onset and progression of pulmonary hypertension (PH). Females more commonly develop pulmonary arterial hypertension, yet females with pulmonary arterial hypertension and other types of PH have better survival than males. Pulmonary microvascular endothelial cells play a crucial role in pulmonary vascular remodeling and increased pulmonary vascular resistance in PH. Given this background, we hypothesized that there are sex differences in the pulmonary microvascular endothelium basally and in response to hypoxia that are independent of the sex hormone environment. Human pulmonary microvascular endothelial cells (HPMECs) from healthy male and female donors, cultured under physiological shear stress, were analyzed using RNA sequencing and label-free quantitative proteomics. Gene set enrichment analysis identified a number of sex-different pathways in both normoxia and hypoxia, including pathways that regulate cell proliferation. In vitro, the rate of proliferation in female HPMECs was lower than in male HPMECs, a finding that supports the omics results. Interestingly, thrombospondin-1, an inhibitor of proliferation, was more highly expressed in female cells than in male cells. These results demonstrate, for the first time, important differences between female and male HPMECs that persist in the absence of sex hormone differences and identify novel pathways for further investigation that may contribute to sexual dimorphism in pulmonary hypertensive diseases.NEW & NOTEWORTHY There is marked sexual dimorphism in the development and progression of pulmonary hypertension. We show differences in RNA and protein expression between female and male human pulmonary microvascular endothelial cells grown under conditions of physiological shear stress, which identify sex-different cellular pathways both in normoxia and hypoxia. Importantly, these differences were detected in the absence of sex hormone differences. The pathways identified may provide novel targets for the development of sex-specific therapies.
Assuntos
Hipertensão Pulmonar , Hipertensão Arterial Pulmonar , Humanos , Masculino , Feminino , Células Endoteliais/metabolismo , Caracteres Sexuais , Hipertensão Pulmonar/metabolismo , Hipertensão Arterial Pulmonar/metabolismo , Proteômica , Hipóxia/metabolismo , Células Cultivadas , Endotélio/metabolismo , Perfilação da Expressão Gênica , Hormônios Esteroides Gonadais/metabolismoRESUMO
Blood flow produces shear stress that homeostatically regulates the phenotype of pulmonary endothelial cells, exerting antiinflammatory and antithrombotic actions and maintaining normal barrier function. Hypoxia due to diseases, such as chronic obstructive pulmonary disease (COPD), causes vasoconstriction, increased vascular resistance, and pulmonary hypertension. Hypoxia-induced changes in endothelial function play a central role in the development of pulmonary hypertension. However, the interactive effects of hypoxia and shear stress on the pulmonary endothelial phenotype have not been studied. Human pulmonary microvascular endothelial cells were cultured in normoxia or hypoxia while subjected to physiological shear stress or in static conditions. Unbiased proteomics was used to identify hypoxia-induced changes in protein expression. Using publicly available single-cell RNA sequencing datasets, differences in gene expression between the alveolar endothelial cells from COPD and healthy lungs were identified. Sixty proteins were identified whose expression changed in response to hypoxia in conditions of physiological shear stress but not in static conditions. These included proteins that are crucial for endothelial homeostasis (e.g., JAM-A [junctional adhesion molecule A], ERG [ETS transcription factor ERG]) or implicated in pulmonary hypertension (e.g., thrombospondin-1). Fifty-five of these 60 have not been previously implicated in the development of hypoxic lung diseases. mRNA for 5 of the 60 (ERG, MCRIP1 [MAPK regulated corepressor interacting protein 1], EIF4A2 [eukaryotic translation initiation factor 4A2], HSP90AA1 [heat shock protein 90 alpha family class A member 1], and DNAJA1 [DnaJ Hsp40 (heat shock protein family) member A1]) showed similar changes in the alveolar endothelial cells of COPD compared with healthy lungs in females but not in males. These data show that the proteomic responses of the pulmonary microvascular endothelium to hypoxia are significantly altered by shear stress and suggest that these shear-hypoxia interactions are important in the development of hypoxic pulmonary vascular disease.
Assuntos
Hipertensão Pulmonar , Doença Pulmonar Obstrutiva Crônica , Masculino , Feminino , Humanos , Hipertensão Pulmonar/metabolismo , Células Endoteliais/metabolismo , Proteômica , Pulmão/metabolismo , Hipóxia/metabolismo , Endotélio Vascular/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Células CultivadasRESUMO
RAB28 is a farnesylated, ciliary G-protein. Patient variants in RAB28 are causative of autosomal recessive cone-rod dystrophy (CRD), an inherited human blindness. In rodent and zebrafish models, the absence of Rab28 results in diminished dawn, photoreceptor, outer segment phagocytosis (OSP). Here, we demonstrate that Rab28 is also required for dusk peaks of OSP, but not for basal OSP levels. This study further elucidated the molecular mechanisms by which Rab28 controls OSP and inherited blindness. Proteomic profiling identified factors whose expression in the eye or whose expression at dawn and dusk peaks of OSP is dysregulated by loss of Rab28. Notably, transgenic overexpression of Rab28, solely in zebrafish cones, rescues the OSP defect in rab28 KO fish, suggesting rab28 gene replacement in cone photoreceptors is sufficient to regulate Rab28-OSP. Rab28 loss also perturbs function of the visual cycle as retinoid levels of 11-cRAL, 11cRP, and atRP are significantly reduced in larval and adult rab28 KO retinae (p < .05). These data give further understanding on the molecular mechanisms of RAB28-associated CRD, highlighting roles of Rab28 in both peaks of OSP, in vitamin A metabolism and in retinoid recycling.
Assuntos
Proteômica , Peixe-Zebra , Animais , Cegueira/metabolismo , Humanos , Fagocitose , Células Fotorreceptoras Retinianas Cones/metabolismo , Retinoides/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Sin3a is the central scaffold protein of the prototypical Hdac1/2 chromatin repressor complex, crucially required during early embryonic development for the growth of pluripotent cells of the inner cell mass. Here, we compare the composition of the Sin3a-Hdac complex between pluripotent embryonic stem (ES) and differentiated cells by establishing a method that couples two independent endogenous immunoprecipitations with quantitative mass spectrometry. We define the precise composition of the Sin3a complex in multiple cell types and identify the Fam60a subunit as a key defining feature of a variant Sin3a complex present in ES cells, which also contains Ogt and Tet1. Fam60a binds on H3K4me3-positive promoters in ES cells, together with Ogt, Tet1 and Sin3a, and is essential to maintain the complex on chromatin. Finally, we show that depletion of Fam60a phenocopies the loss of Sin3a, leading to reduced proliferation, an extended G1-phase and the deregulation of lineage genes. Taken together, Fam60a is an essential core subunit of a variant Sin3a complex in ES cells that is required to promote rapid proliferation and prevent unscheduled differentiation.
Assuntos
Proliferação de Células , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/fisiologia , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/metabolismo , Animais , Diferenciação Celular , Imunoprecipitação , Espectrometria de Massas , Camundongos , Ligação ProteicaRESUMO
SETD1A is a SET domain-containing methyltransferase involved in epigenetic regulation of transcription. It is the main catalytic component of a multiprotein complex that methylates lysine 4 of histone H3, a histone mark associated with gene activation. In humans, six related protein complexes with partly nonredundant cellular functions share several protein subunits but are distinguished by unique catalytic SET-domain proteins. We surveyed physical interactions of the SETD1A-complex using endogenous immunoprecipitation followed by label-free quantitative proteomics on three subunits: SETD1A, RBBP5, and ASH2L. Surprisingly, SETD1A, but not RBBP5 or ASH2L, was found to interact with the DNA damage repair protein RAD18. Reciprocal RAD18 immunoprecipitation experiments confirmed the interaction with SETD1A, whereas size exclusion and protein network analysis suggested an interaction independent of the main SETD1A complex. We found evidence of SETD1A and RAD18 influence on mutual gene expression levels. Further, knockdown of the genes individually showed a DNA damage repair phenotype, whereas simultaneous knockdown resulted in an epistatic effect. This adds to a growing body of work linking epigenetic enzymes to processes involved in genome stability.
Assuntos
Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Regulação para Baixo , Células HEK293 , Histonas/metabolismo , Humanos , Lisina/metabolismo , Metilação , Fenótipo , Ligação Proteica , Mapas de Interação de Proteínas , Subunidades Proteicas/metabolismo , Proteômica , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
RATIONALE: Mutations in the cystic fibrosis transmembrane regulator (CFTR) gene form the basis of cystic fibrosis (CF). There remains an important knowledge gap in CF as to how diminished CFTR activity leads to the dominant inflammatory response within CF airways. OBJECTIVES: To investigate if extracellular vesicles (EVs) contribute to inflammatory signalling in CF. METHODS: EVs released from CFBE41o-, CuFi-5, 16HBE14o- and NuLi-1 cells were characterised by nanoparticle tracking analysis (NTA). EVs isolated from bronchoalveolar lavage fluid (BALF) from 30 people with CF (PWCF) were analysed by NTA and mass spectrometry and compared with controls. Neutrophils were isolated from the blood of 8 PWCF to examine neutrophil migration in the presence of CFBE41o- EVs. RESULTS: A significantly higher level of EVs were released from CFBE41o- (p<0.0001) and CuFi-5 (p=0.0209) relative to control cell lines. A significantly higher level of EVs were detected in BALF of PWCF, in three different age groups relative to controls (p=0.01, 0.001, 0.002). A significantly lower level of EVs were released from CFBE41o- (p<0.001) and CuFi-5 (p=0.0002) cell lines treated with CFTR modulators. Significant changes in the protein expression of 126 unique proteins was determined in EVs obtained from the BALF of PWCF of different age groups (p<0.001-0.05). A significant increase in chemotaxis of neutrophils derived from PWCF was observed in the presence of CFBE41o EVs (p=0.0024) compared with controls. CONCLUSION: This study demonstrates that EVs are produced in CF airway cells, have differential protein expression at different ages and drive neutrophil recruitment in CF.
Assuntos
Fibrose Cística/metabolismo , Vesículas Extracelulares/metabolismo , Adolescente , Adulto , Fatores Etários , Líquido da Lavagem Broncoalveolar/química , Linhagem Celular , Movimento Celular , Células Cultivadas , Quimiotaxia , Criança , Pré-Escolar , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Feminino , Humanos , Lactente , Masculino , Espectrometria de Massas , Nanopartículas , Neutrófilos/metabolismo , Projetos Piloto , Transdução de Sinais , TransfecçãoRESUMO
Monocytes/macrophages drive progression and regression of atherosclerosis. Conjugated linoleic acid (CLA), an anti-inflammatory lipid, mediates atheroprotective effects. We investigated how CLA alters monocyte/macrophage phenotype during attenuated progression and regression of atherosclerosis. Apolipoprotein E knockout (ApoE-/-) mice were fed a high-fat (60%) high-cholesterol (1%) diet (HFHCD) for 2 wk, followed by 6-wk 1% CLA 80:20 supplementation to investigate disease progression. Simultaneously, ApoE-/- mice were fed a 12-wk HFHCD with/without CLA for the final 4 wk to investigate regression. Aortic lesions were quantified by en face staining. Proteomic analysis, real-time quantitative PCR and flow cytometry were used to interrogate monocyte/macrophage phenotypes. CLA supplementation inhibited atherosclerosis progression coincident with decreased proinflammatory and increased anti-inflammatory macrophages. However, CLA-induced regression was associated with increased proinflammatory monocytes resulting in increased proresolving M2 bone marrow-derived macrophages, splenic macrophages, and dendritic cells in lesion-draining lymph nodes. Proteomic analysis confirmed regulation of a proinflammatory bone marrow response, which was abolished upon macrophage differentiation. Thus, in attenuation and regression of atherosclerosis, regardless of the monocyte signature, during monocyte to macrophage differentiation, proresolving macrophages prevail, mediating vascular repair. This study provides novel mechanistic insight into the monocyte/macrophage phenotypes in halted atherosclerosis progression and regression of atherosclerosis.-Bruen, R., Curley, S., Kajani, S., Lynch, G., O'Reilly, M. E., Dillon, E. T., Fitzsimons, S., Mthunzi, L., McGillicuddy, F. C., Belton, O. Different monocyte phenotypes result in proresolving macrophages in conjugated linoleic acid-induced attenuated progression and regression of atherosclerosis.
Assuntos
Aterosclerose/tratamento farmacológico , Diferenciação Celular , Ácidos Linoleicos Conjugados/farmacologia , Fenótipo , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Apolipoproteínas E/genética , Aterosclerose/etiologia , Aterosclerose/metabolismo , Células Cultivadas , Dieta Hiperlipídica/efeitos adversos , Ácidos Linoleicos Conjugados/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células Precursoras de Monócitos e Macrófagos/citologia , Células Precursoras de Monócitos e Macrófagos/efeitos dos fármacos , Células Precursoras de Monócitos e Macrófagos/metabolismo , Proteoma/genética , Proteoma/metabolismoRESUMO
We have shown that the glucagon-like peptide-1 receptor agonist (GLP-1RA) liraglutide (Lir) inhibits development of early atherosclerosis in vivo by modulating immune cell function. We hypothesized that Lir could attenuate pre-established disease by modulating monocyte or macrophage phenotype to induce atheroprotective responses. Human atherosclerotic plaques obtained postendarterectomy and human peripheral blood macrophages were treated ex vivo with Lir. In parallel, apolipoprotein E-deficient (ApoE-/-) mice received a high-fat, high-cholesterol diet to induce atherosclerosis for 8 weeks, after which ApoE-/- mice received 300 µg/kg of Lir daily or vehicle control for a further 4 weeks to investigate the attenuation of atherosclerosis. Lir inhibited proinflammatory monocyte chemoattractant protein-1 secretion from human endarterectomy samples and monocyte chemoattractant protein-1, tumor necrosis factor-α, and interleukin (IL)-1ß secretion from human macrophages after ex vivo treatment. An increase in CD206 mRNA and IL-10 secretion was also detected, which implies resolution of inflammation. Importantly, Lir significantly attenuated pre-established atherosclerosis in ApoE-/- mice in the whole aorta and aortic root. Proteomic analysis of ApoE-/- bone marrow cells showed that Lir upregulated the proinflammatory cathepsin protein family, which was abolished in differentiated macrophages. In addition, flow cytometry analysis of bone marrow cells induced a shift toward reduced proinflammatory and increased anti-inflammatory macrophages. We concluded that Lir attenuates pre-established atherosclerosis in vivo by altering proinflammatory mediators. This is the first study to describe a mechanism through which Lir attenuates atherosclerosis by increasing bone marrow proinflammatory protein expression, which is lost in differentiated bone marrow-derived macrophages. This study contributes to our understanding of the anti-inflammatory and cardioprotective role of GLP-1RAs. SIGNIFICANCE STATEMENT: It is critical to understand the mechanisms through which liraglutide (Lir) mediates a cardioprotective effect as many type 2 diabetic medications increase the risk of myocardial infarction and stroke. We have identified that Lir reduces proinflammatory immune cell populations and mediators from plaque-burdened murine aortas in vivo and augments proresolving bone marrow-derived macrophages in attenuation of atherosclerotic disease, which provides further insight into the atheroprotective effect of Lir.
Assuntos
Apolipoproteínas E/deficiência , Mediadores da Inflamação/metabolismo , Liraglutida/farmacologia , Fenótipo , Placa Aterosclerótica/imunologia , Placa Aterosclerótica/metabolismo , Animais , Quimiocinas/metabolismo , Progressão da Doença , Feminino , Humanos , Liraglutida/uso terapêutico , Masculino , Camundongos , Placa Aterosclerótica/tratamento farmacológicoRESUMO
It was previously demonstrated that the WNT/ß-catenin pathway is present and active in platelets and established that the canonical WNT ligand, WNT-3a, suppresses platelet adhesion and activation. In nucleated cells, ß-catenin, the key downstream effector of this pathway, is a dual function protein, regulating the coordination of gene transcription and cell-cell adhesion. The specific role of ß-catenin in the anucleate platelet however remains elusive. Here, a label-free quantitative proteomic analysis of ß-catenin immunoprecipitates from human platelets is performed and nine co-immunoprecipitating proteins are identified. Three of the co-immunoprecipitating proteins (α-catenin-1, cadherin-6, and ß-catenin-interacting protein 1) are common to both resting and activated conditions. Bioinformatics analysis of proteomics data reveal a strong association of the dataset with both cadherin adherens junctions and regulators of WNT signaling. It is then verified that platelet ß-catenin and cadherin-6 interact and that this interaction is regulated by the activation state of the platelet. Taken together, this proteomics study suggests a novel role for ß-catenin in human platelets where it interacts with platelet cadherins and associated junctional proteins.
Assuntos
Junções Aderentes/metabolismo , Plaquetas/metabolismo , Caderinas/metabolismo , Proteoma/análise , beta Catenina/metabolismo , Adesão Celular , Humanos , Via de Sinalização WntRESUMO
Excess blood vessel growth contributes to the pathology of metastatic cancers and age-related retinopathies. Despite development of improved treatments, these conditions are associated with high economic costs and drug resistance. Bevacizumab (Avastin®), a monoclonal antibody against vascular endothelial growth factor (VEGF), is used clinically to treat certain types of metastatic cancers. Unfortunately, many patients do not respond or inevitably become resistant to bevacizumab, highlighting the need for more effective antiangiogenic drugs with novel mechanisms of action. Previous studies discovered quininib, an antiangiogenic small molecule antagonist of cysteinyl leukotriene receptors 1 and 2 (CysLT1 and CysLT2). Here, we screened a series of quininib analogues and identified a more potent antiangiogenic novel chemical entity (IUPAC name (E)-2-(2-quinolin-2-yl-vinyl)-benzene-1,4-diol HCl) hereafter designated Q8. Q8 inhibits developmental angiogenesis in Tg(fli1:EGFP) zebrafish and inhibits human microvascular endothelial cell (HMEC-1) proliferation, tubule formation, and migration. Q8 elicits antiangiogenic effects in a VEGF-independent in vitro model of angiogenesis and exerts an additive antiangiogenic response with the anti-VEGF biologic bevacizumab. Cell-based receptor binding assays confirm that Q8 is a CysLT1 antagonist and is sufficient to reduce cellular levels of NF-κB and calpain-2 and secreted levels of the proangiogenic proteins intercellular adhesion molecule-1, vascular cell adhesion protein-1, and VEGF. Distinct reductions of VEGF by bevacizumab explain the additive antiangiogenic effects observed in combination with Q8. In summary, Q8 is a more effective antiangiogenic drug compared with quininib. The VEGF-independent activity coupled with the additive antiangiogenic response observed in combination with bevacizumab demonstrates that Q8 offers an alternative therapeutic strategy to combat resistance associated with conventional anti-VEGF therapies.
Assuntos
Inibidores da Angiogênese/farmacologia , Derivados de Benzeno/farmacologia , Bevacizumab/farmacologia , Cisteína/química , Antagonistas de Leucotrienos/farmacologia , Neovascularização Patológica/metabolismo , Fenóis/farmacologia , Quinolinas/farmacologia , Animais , Animais Geneticamente Modificados , Linhagem Celular , Movimento Celular , Sobrevivência Celular , Relação Dose-Resposta a Droga , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Proteínas de Fluorescência Verde/metabolismo , Humanos , Microscopia de Fluorescência , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Peixe-ZebraRESUMO
Tandem mass spectrometry (MS/MS) techniques, developed for protein identification, are increasingly being applied in the field of peptidomics. Using this approach, the set of protein fragments observed in a sample of interest can be determined to gain insights into important biological processes such as signaling and other bioactivities. As the peptidomics era progresses, there is a need for robust and convenient methods to inspect and analyze MS/MS derived data. Here, we present Peptigram, a novel tool dedicated to the visualization and comparison of peptides detected by MS/MS. The principal advantage of Peptigram is that it provides visualizations at both the protein and peptide level, allowing users to simultaneously visualize the peptide distributions of one or more samples of interest, mapped to their parent proteins. In this way rapid comparisons between samples can be made in terms of their peptide coverage and abundance. Moreover, Peptigram integrates and displays key sequence features from external databases and links with peptide analysis tools to offer the user a comprehensive peptide discovery resource. Here, we illustrate the use of Peptigram on a data set of milk hydrolysates. For convenience, Peptigram is implemented as a web application, and is freely available for academic use at http://bioware.ucd.ie/peptigram .
Assuntos
Peptídeos/genética , Proteômica/métodos , Software , Bases de Dados de Proteínas , Internet , Peptídeos/classificação , Espectrometria de Massas em TandemRESUMO
BACKGROUND: Acute inflammation impairs reverse cholesterol transport (RCT) and reduces high-density lipoprotein (HDL) function in vivo. This study hypothesized that obesity-induced inflammation impedes RCT and alters HDL composition, and investigated if dietary replacement of saturated (SFA) for monounsaturated (MUFA) fatty acids modulates RCT. METHODS AND RESULTS: Macrophage-to-feces RCT, HDL efflux capacity, and HDL proteomic profiling was determined in C57BL/6j mice following 24 weeks on SFA- or MUFA-enriched high-fat diets (HFDs) or low-fat diet. The impact of dietary SFA consumption and insulin resistance on HDL efflux function was also assessed in humans. Both HFDs increased plasma (3)H-cholesterol counts during RCT in vivo and ATP-binding cassette, subfamily A, member 1-independent efflux to plasma ex vivo, effects that were attributable to elevated HDL cholesterol. By contrast, ATP-binding cassette, subfamily A, member 1-dependent efflux was reduced after both HFDs, an effect that was also observed with insulin resistance and high SFA consumption in humans. SFA-HFD impaired liver-to-feces RCT, increased hepatic inflammation, and reduced ABC subfamily G member 5/8 and ABC subfamily B member 11 transporter expression in comparison with low-fat diet, whereas liver-to-feces RCT was preserved after MUFA-HFD. HDL particles were enriched with acute-phase proteins (serum amyloid A, haptoglobin, and hemopexin) and depleted of paraoxonase-1 after SFA-HFD in comparison with MUFA-HFD. CONCLUSIONS: Ex vivo efflux assays validated increased macrophage-to-plasma RCT in vivo after both HFDs but failed to capture differential modulation of hepatic cholesterol trafficking. By contrast, proteomics revealed the association of hepatic-derived inflammatory proteins on HDL after SFA-HFD in comparison with MUFA-HFD, which reflected differential hepatic cholesterol trafficking between groups. Acute-phase protein levels on HDL may serve as novel biomarkers of impaired liver-to-feces RCT in vivo.
Assuntos
Colesterol/metabolismo , Dieta Hiperlipídica/efeitos adversos , Ácidos Graxos Monoinsaturados/administração & dosagem , Ácidos Graxos/administração & dosagem , Lipoproteínas HDL/genética , Proteômica/métodos , Adolescente , Adulto , Animais , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Gorduras na Dieta/administração & dosagem , Gorduras na Dieta/efeitos adversos , Ácidos Graxos/efeitos adversos , Ácidos Graxos Monoinsaturados/efeitos adversos , Feminino , Humanos , Lipoproteínas HDL/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Obesidade/etiologia , Obesidade/metabolismo , Adulto JovemRESUMO
Copper-chelated chitosan microgels were investigated as an immobilized metal affinity chromatography (IMAC) phase for peptide separation. The copper-crosslinked chitosan beads were shown to strongly interact with a range of amino acids, in a wide range of pH and saline conditions. The beads exhibited an affinity that seemed to depend on the isoelectric point of the amino acid, with the extent of uptake increasing with decreasing isoelectric point. This selective interaction with anionic amino acids resulted in a significant relative enrichment of the supernatant solution in cationic amino acids. The beads were then studied as a novel fractionation system for complex milk hydrolysates. The copper chitosan beads selectively removed larger peptides from the hydrolysate aqueous solution, yielding a solution relatively enriched in medium and smaller peptides, which was characterized both quantitatively and qualitatively by size exclusion chromatography (SEC). Liquid chromatography-mass spectrometry (LCMS) work provided comprehensive data on a peptide sequence level and showed that a depletion of the anionic peptides by the beads resulted in a relative enrichment of the cationic peptides in the supernatant solution. It could be concluded that after fractionation a dramatic relative enrichment in respect to small- and medium-sized cationic peptides in the solution, characteristics that have been linked to bioactivities, such as anti-microbial and cell-penetrating properties. The results demonstrate the use of the chitosan copper gel bead system in lab scale fractionation of complex hydrolysate mixtures, with the potential to enhance milk hydrolysate bioactivity.
RESUMO
In the yeast genera Saccharomycopsis and Ascoidea, which comprise the taxonomic order Ascoideales, nuclear genes use a nonstandard genetic code in which CUG codons are translated as serine instead of leucine, due to a tRNA-Ser with the unusual anticodon CAG. However, some species in this clade also retain an ancestral tRNA-Leu gene with the same anticodon. One of these species, Ascoidea asiatica, has been shown to have a stochastic proteome in which proteins contain â¼50% Ser and 50% Leu at CUG codon sites, whereas previously examined Saccharomycopsis species translate CUG only as Ser. Here, we investigated the presence, conservation, and possible functionality of the tRNA-Leu(CAG) gene in the genus Saccharomycopsis. We sequenced the genomes of 23 strains that, together with previously available data, include almost every known species of this genus. We found that most Saccharomycopsis species have genes for both tRNA-Leu(CAG) and tRNA-Ser(CAG). However, tRNA-Leu(CAG) has been lost in Saccharomycopsis synnaedendra and Saccharomycopsis microspora, and its predicted cloverleaf structure is aberrant in all the other Saccharomycopsis species. We deleted the tRNA-Leu(CAG) gene of Saccharomycopsis capsularis and found that it is not essential. Proteomic analyses in vegetative and sporulating cultures of S. capsularis and Saccharomycopsis fermentans showed only translation of CUG as Ser. Despite its unusual structure, the tRNA-Leu(CAG) gene shows evidence of sequence conservation among Saccharomycopsis species, particularly in its acceptor stem and leucine identity elements, which suggests that it may have been retained in order to carry out an unknown nontranslational function.
Assuntos
Evolução Molecular , Código Genético , Saccharomycopsis/genética , Biossíntese de Proteínas , Filogenia , RNA de Transferência de Leucina/genética , Anticódon/genética , RNA de Transferência de Serina/genéticaRESUMO
The study investigates the effect of conventional and novel extraction techniques on the protein extraction yield from bitter gourd seeds (Momordica charantia). Ultrasound assisted-extraction (UAE) treatment for 30 min at 4 °C using a 20 kHz ultrasound probe resulted in the highest extraction yield of crude proteins. After purification, 9.08 ± 0.23 g of protein with 82.69 ± 0.78% purity was obtained from 100 g of M. charantia seeds on a dry basis. Mass spectrometry identified proteins with reported antidiabetic activity. Antidiabetic assays showed significantly higher antidiabetic activity for the purified protein (81.10 ± 2.64%) compared to the crude protein (32.59 ± 2.76%). In vitro cytotoxicity analysis showed minimal cytotoxicity levels at concentrations <200 µg.mL-1. Overall, UAE was effective to obtain crude protein from M. charantia seeds and a subsequent purification step enhanced antidiabetic activity. However, further research is required to demonstrate in-vivo antidiabetic activity.
Assuntos
Hipoglicemiantes , Momordica charantia , Extratos Vegetais , Proteínas de Plantas , Sementes , Momordica charantia/química , Hipoglicemiantes/química , Hipoglicemiantes/farmacologia , Hipoglicemiantes/isolamento & purificação , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/química , Proteínas de Plantas/farmacologia , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Sementes/química , Humanos , Fracionamento Químico/métodos , AnimaisRESUMO
Uveal melanoma (UM) is an ocular cancer, with propensity for lethal liver metastases. When metastatic UM (MUM) occurs, as few as 8% of patients survive beyond two years. Efficacious treatments for MUM are urgently needed. 1,4-dihydroxy quininib, a cysteinyl leukotriene receptor 1 (CysLT1) antagonist, alters UM cancer hallmarks in vitro, ex vivo and in vivo. Here, we investigated the 1,4-dihydroxy quininib mechanism of action and its translational potential in MUM. Proteomic profiling of OMM2.5 cells identified proteins differentially expressed after 1,4-dihydroxy quininib treatment. Glutathione peroxidase 4 (GPX4), glutamate-cysteine ligase modifier subunit (GCLM), heme oxygenase 1 (HO-1) and 4 hydroxynonenal (4-HNE) expression were assessed by immunoblots. Biliverdin, glutathione and lipid hydroperoxide were measured biochemically. Association between the expression of a specific ferroptosis signature and UM patient survival was performed using public databases. Our data revealed that 1,4-dihydroxy quininib modulates the expression of ferroptosis markers in OMM2.5 cells. Biochemical assays validated that GPX4, biliverdin, GCLM, glutathione and lipid hydroperoxide were significantly altered. HO-1 and 4-HNE levels were significantly increased in MUM tumor explants from orthotopic patient-derived xenografts (OPDX). Expression of genes inhibiting ferroptosis is significantly increased in UM patients with chromosome 3 monosomy. We identified IFerr, a novel ferroptosis signature correlating with UM patient survival. Altogether, we demontrated that in MUM cells and tissues, 1,4-dihydroxy quininib modulates key markers that induce ferroptosis, a relatively new type of cell death driven by iron-dependent peroxidation of phospholipids. Furthermore, we showed that high expression of specific genes inhibiting ferroptosis is associated with a worse UM prognosis, thus, the IFerr signature is a potential prognosticator for which patients develop MUM. All in all, ferroptosis has potential as a clinical biomarker and therapeutic target for MUM.
RESUMO
OBJECTIVE: Sporadic and familial amyotrophic lateral sclerosis (ALS) is a fatal progressive neurodegenerative disease that results in loss of motor neurons and, in some patients, associates with frontotemporal dementia (FTD). Apart from the accumulation of proteinaceous deposits, emerging literature indicates that aberrant mitochondrial bioenergetics may contribute to the onset and progression of ALS/FTD. Here we sought to investigate the pathophysiological signatures of mitochondrial dysfunction associated with ALS/FTD. METHODS: By means of label-free mass spectrometry (MS) and mRNA sequencing (mRNA-seq), we report pre-symptomatic changes in the cortices of TDP-43 and FUS mutant mouse models. Using tissues from transgenic mouse models of mitochondrial diseases as a reference, we performed comparative analyses and extracted unique and common mitochondrial signatures that revealed neuroprotective compensatory mechanisms in response to early damage. RESULTS: In this regard, upregulation of both Acyl-CoA Synthetase Long-Chain Family Member 3 (ACSL3) and mitochondrial tyrosyl-tRNA synthetase 2 (YARS2) were the most representative change in pre-symptomatic ALS/FTD tissues, suggesting that fatty acid beta-oxidation and mitochondrial protein translation are mechanisms of adaptation in response to ALS/FTD pathology. CONCLUSIONS: Together, our unbiased integrative analyses unveil novel molecular components that may influence mitochondrial homeostasis in the earliest phase of ALS.