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BACKGROUND: Environmental pollutants, including polychlorinated biphenyls (PCBs) have been implicated in the pathogenesis of liver disease. Our group recently demonstrated that PCB126 promoted steatosis, hepatomegaly, and modulated intermediary metabolism in a rodent model of alcohol-associated liver disease (ALD). OBJECTIVE: To better understand how PCB126 promoted ALD in our previous model, the current study adopts multiple omics approaches to elucidate potential mechanistic hypotheses. METHODS: Briefly, male C57BL/6J mice were exposed to 0.2mg/kg polychlorinated biphenyl (PCB) 126 or corn oil vehicle prior to ethanol (EtOH) or control diet feeding in the chronic-binge alcohol feeding model. Liver tissues were collected and prepared for mRNA sequencing, phosphoproteomics, and inductively coupled plasma mass spectrometry for metals quantification. RESULTS: Principal component analysis showed that PCB126 uniquely modified the transcriptome in EtOH-fed mice. EtOH feeding alone resulted in >4,000 differentially expressed genes (DEGs), and PCB126 exposure resulted in more DEGs in the EtOH-fed group (907 DEGs) in comparison with the pair-fed group (503 DEGs). Top 20 significant gene ontology (GO) biological processes included "peptidyl tyrosine modifications," whereas top 25 significantly decreasing GO molecular functions included "metal/ion/zinc binding." Quantitative, label-free phosphoproteomics and western blot analysis revealed no major significant PCB126 effects on total phosphorylated tyrosine residues in EtOH-fed mice. Quantified hepatic essential metal levels were primarily significantly lower in EtOH-fed mice. PCB126-exposed mice had significantly lower magnesium, cobalt, and zinc levels in EtOH-fed mice. DISCUSSION: Previous work has demonstrated that PCB126 is a modifying factor in metabolic dysfunction-associated steatotic liver disease (MASLD), and our current work suggests that pollutants also modify ALD. PCB126 may, in part, be contributing to the malnutrition aspect of ALD, where metal deficiency is known to contribute and worsen prognosis. https://doi.org/10.1289/EHP14132.
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Poluentes Ambientais , Fígado Gorduroso , Hepatopatias Alcoólicas , Bifenilos Policlorados , Masculino , Camundongos , Animais , Multiômica , Camundongos Endogâmicos C57BL , Etanol/toxicidade , Etanol/metabolismo , Fígado/metabolismo , Bifenilos Policlorados/toxicidade , Bifenilos Policlorados/metabolismo , Hepatopatias Alcoólicas/etiologia , Hepatopatias Alcoólicas/metabolismo , Hepatopatias Alcoólicas/patologia , Poluentes Ambientais/toxicidade , Poluentes Ambientais/metabolismo , Zinco/metabolismo , Tirosina/metabolismoRESUMO
Introduction: Nephrotic syndrome (NS) occurs commonly in children with glomerular disease and glucocorticoids (GCs) are the mainstay treatment. Steroid resistant NS (SRNS) develops in 15% to 20% of children, increasing the risk of chronic kidney disease compared to steroid sensitive NS (SSNS). NS pathogenesis is unclear in most children, and no biomarkers exist that predict the development of pediatric SRNS. Methods: We studied a unique patient cohort with plasma specimens collected before GC treatment, yielding a disease-only sample not confounded by steroid-induced gene expression changes (SSNS n = 8; SRNS n = 7). A novel "patient-specific" bioinformatic approach merged paired pretreatment and posttreatment proteomic and metabolomic data and identified candidate SRNS biomarkers and altered molecular pathways in SRNS versus SSNS. Results: Joint pathway analyses revealed perturbations in nicotinate or nicotinamide and butanoate metabolic pathways in patients with SRNS. Patients with SSNS had perturbations of lysine degradation, mucin type O-glycan biosynthesis, and glycolysis or gluconeogenesis pathways. Molecular analyses revealed frequent alteration of molecules within these pathways that had not been observed by separate proteomic and metabolomic studies. We observed upregulation of NAMPT, NMNAT1, and SETMAR in patients with SRNS, in contrast to upregulation of ALDH1B1, ACAT1, AASS, ENPP1, and pyruvate in patients with SSNS. Pyruvate regulation was the change seen in our previous analysis; all other targets were novel. Immunoblotting confirmed increased NAMPT expression in SRNS and increased ALDH1B1 and ACAT1 expression in SSNS, following GC treatment. Conclusion: These studies confirmed that a novel "patient-specific" bioinformatic approach can integrate disparate omics datasets and identify candidate SRNS biomarkers not observed by separate proteomic or metabolomic analysis.
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Chronic kidney disease (CKD) affects 30 million adults, costs ~$79 billion dollars (2016) in Medicare expenditures, and is the ninth leading cause of death in the United States. The disease is silent or undiagnosed in almost half of people with severely reduced kidney function. Urine provides an ideal biofluid that is accessible to high-sensitivity mass spectrometry-based proteomic interrogation and is an indicator of renal homeostasis. While the accurate and precise diagnosis and better disease management of CKD can be aided using urine biomarkers, their discovery in excessive protein or nephrotic urine samples can present challenges. In this work we present a mass spectrometry-based method utilizing multiplex tandem mass tag (TMT) quantification and improved protein quantification using reporter ion normalization to urinary creatinine to analyze urinary proteins from patients with a form of nephrotic syndrome (FSGS). A comparative analysis was performed for urine from patients in remission versus active disease flare. Two-dimensional LC-MS/MS TMT quantitative analysis identified over 1058 urine proteins, 580 proteins with 2 peptides or greater and quantifiable. Normalization of TMT abundance values to creatinine per ml of urine concentrated reduced variability in 2D-TMT-LC-MS/MS experiments. Univariate and multivariate analyses showed that 27 proteins were significantly increased in proteinuric disease flare. Hierarchical heatmap clustering showed that SERPINA1 and ORM1 were >1.5 fold increased in active disease versus remission urine samples. ELISA validation of SERPINA1 and ORM1 abundance agreed with our quantitative TMT proteomics analysis. These findings provide support for the utility of this method for identification of novel diagnostic markers of CKD and identify SERPINA1 and ORM1 as promising candidate diagnostic markers for FSGS.
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Chronic kidney disease (CKD) can progress to kidney failure and require dialysis or transplantation, while early diagnosis can alter the course of disease and lead to better outcomes in both pediatric and adult patients. Significant CKD comorbidities include the manifestation of cardiovascular disease, heart failure, coronary disease, and hypertension. The pathogenesis of chronic kidney diseases can present as subtle and especially difficult to distinguish between different glomerular pathologies. Early detection of adult and pediatric CKD and detailed mechanistic understanding of the kidney damage can be helpful in delaying or curtailing disease progression via precise intervention toward diagnosis and prognosis. Clinically, serum creatinine and albumin levels can be indicative of CKD, but often are a lagging indicator only significantly affected once kidney function has severely diminished. The evolution of proteomics and mass spectrometry technologies has begun to provide a powerful research tool in defining these mechanisms and identifying novel biomarkers of CKD. Many of the same challenges and advances in proteomics apply to adult and pediatric patient populations. Additionally, proteomic analysis of adult CKD patients can be transferred directly toward advancing our knowledge of pediatric CKD as well. In this review, we highlight applications of proteomics that have yielded such biomarkers as PLA2R, SEMA3B, and other markers of membranous nephropathy as well as KIM-1, MCP-1, and NGAL in lupus nephritis among other potential diagnostic and prognostic markers. The potential for improving the clinical toolkit toward better treatment of pediatric kidney diseases is significantly aided by current and future development of proteomic applications.
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Nefropatias , Insuficiência Renal Crônica , Adulto , Biomarcadores , Criança , Taxa de Filtração Glomerular , Humanos , Nefropatias/diagnóstico , Proteômica , Diálise RenalRESUMO
Glucose metabolism comprises numerous amphibolic metabolites that provide precursors for not only the synthesis of cellular building blocks but also for ATP production. In this study, we tested how phosphofructokinase-1 (PFK1) activity controls the fate of glucose-derived carbon in murine hearts in vivo. PFK1 activity was regulated by cardiac-specific overexpression of kinase- or phosphatase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase transgenes in mice (termed GlycoLo or GlycoHi mice, respectively). Dietary delivery of 13C6-glucose to these mice, followed by deep network metabolic tracing, revealed that low rates of PFK1 activity promote selective routing of glucose-derived carbon to the purine synthesis pathway to form 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR). Consistent with a mechanism of physical channeling, we found multimeric protein complexes that contained phosphoribosylaminoimidazole carboxylase (PAICS)-an enzyme important for AICAR biosynthesis, as well as chaperone proteins such as Hsp90 and other metabolic enzymes. We also observed that PFK1 influenced glucose-derived carbon deposition in glycogen, but did not affect hexosamine biosynthetic pathway activity. These studies demonstrate the utility of deep network tracing to identify metabolic channeling and changes in biosynthetic pathway activity in the heart in vivo and present new potential mechanisms by which metabolic branchpoint reactions modulate biosynthetic pathways.
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Vias Biossintéticas , Fosfofrutoquinase-2 , Animais , Glucose/metabolismo , Glicólise , Camundongos , Miocárdio/metabolismo , Fosfofrutoquinase-1/metabolismo , Fosfofrutoquinase-2/metabolismo , Fosfofrutoquinases/metabolismoRESUMO
Pathological cardiac remodeling during heart failure is associated with higher levels of lipid peroxidation products and lower abundance of several aldehyde detoxification enzymes, including aldehyde dehydrogenase 2 (ALDH2). An emerging idea that could explain these findings concerns the role of electrophilic species in redox signaling, which may be important for adaptive responses to stress or injury. The purpose of this study was to determine whether genetically increasing ALDH2 activity affects pressure overload-induced cardiac dysfunction. Mice subjected to transverse aortic constriction (TAC) for 12 weeks developed myocardial hypertrophy and cardiac dysfunction, which were associated with diminished ALDH2 expression and activity. Cardiac-specific expression of the human ALDH2 gene in mice augmented myocardial ALDH2 activity but did not improve cardiac function in response to pressure overload. After 12 weeks of TAC, ALDH2 transgenic mice had larger hearts than their wild-type littermates and lower capillary density. These findings show that overexpression of ALDH2 augments the hypertrophic response to pressure overload and imply that downregulation of ALDH2 may be an adaptive response to certain forms of cardiac pathology.
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Aldeído-Desidrogenase Mitocondrial/genética , Insuficiência Cardíaca/genética , Estresse Oxidativo/genética , Remodelação Ventricular/genética , Animais , Aorta/metabolismo , Regulação da Expressão Gênica , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Humanos , Camundongos , Camundongos Transgênicos , Mitocôndrias/metabolismo , Miocárdio/metabolismo , Miocárdio/patologia , Oxirredução , Pressão , Transdução de Sinais/genéticaRESUMO
Members of the casein kinase 1 (CK1) family of serine-threonine protein kinases are implicated in the regulation of many cellular processes, including the cell cycle, circadian rhythms, and Wnt and Hedgehog signaling. Because these kinases exhibit constitutive activity in biochemical assays, it is likely that their activity in cells is controlled by subcellular localization, interactions with inhibitory proteins, targeted degradation, or combinations of these mechanisms. We identified members of the FAM83 family of proteins as partners of CK1 in cells. All eight members of the FAM83 family (FAM83A to FAM83H) interacted with the α and α-like isoforms of CK1; FAM83A, FAM83B, FAM83E, and FAM83H also interacted with the δ and ε isoforms of CK1. We detected no interaction between any FAM83 member and the related CK1γ1, CK1γ2, and CK1γ3 isoforms. Each FAM83 protein exhibited a distinct pattern of subcellular distribution and colocalized with the CK1 isoform(s) to which it bound. The interaction of FAM83 proteins with CK1 isoforms was mediated by the conserved domain of unknown function 1669 (DUF1669) that characterizes the FAM83 family. Mutations in FAM83 proteins that prevented them from binding to CK1 interfered with the proper subcellular localization and cellular functions of both the FAM83 proteins and their CK1 binding partners. On the basis of its function, we propose that DUF1669 be renamed the polypeptide anchor of CK1 domain.
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Caseína Quinase I/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteínas de Neoplasias/química , Domínios Proteicos , Caseína Quinase I/química , Caseína Quinase I/genética , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Isoformas de Proteínas , Transdução de SinaisRESUMO
The BMP and Wnt signalling pathways determine axis specification during embryonic development. Our previous work has shown that PAWS1 (also known as FAM83G) interacts with SMAD1 and modulates BMP signalling. Here, surprisingly, we show that overexpression of PAWS1 in Xenopus embryos activates Wnt signalling and causes complete axis duplication. Consistent with these observations in Xenopus, Wnt signalling is diminished in U2OS osteosarcoma cells lacking PAWS1, while BMP signalling is unaffected. We show that PAWS1 interacts and co-localises with the α isoform of casein kinase 1 (CK1), and that PAWS1 mutations incapable of binding CK1 fail both to activate Wnt signalling and to elicit axis duplication in Xenopus embryos.
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Caseína Quinase Ialfa/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Via de Sinalização Wnt , Animais , Proteína Axina/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Linhagem Celular Tumoral , Núcleo Celular , Expressão Ectópica do Gene , Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Complexos Multiproteicos/metabolismo , Fosforilação , Ligação Proteica , Transporte Proteico , Xenopus , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , beta Catenina/metabolismoRESUMO
Identification and characterization of large protein complexes is a mainstay of biochemical toolboxes. Utilization of cross-linking chemicals can facilitate the capture and identification of transient or weak interactions of a transient nature (Huang and Kim, PloS One 8:e61430, 2013; Gao et al., J Vis Exp doi: 10.3791/51387, 2014). Here we describe a detailed methodology for a cell culture-based proteomic approach. We describe the generation of cells stably expressing green fluorescent protein (GFP)-tagged proteins under the tetracycline-inducible promoter and subsequent proteomic analysis of GFP-interacting proteins. We include a list of proteins that were identified as interactors of GFP.
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Mapeamento de Interação de Proteínas/métodos , Proteínas/metabolismo , Proteômica/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Sequência de Aminoácidos , Animais , Linhagem Celular , Reagentes de Ligações Cruzadas/química , Expressão Gênica , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Mapas de Interação de Proteínas , Proteínas/análise , Proteínas/genética , Transfecção/métodosRESUMO
Our previous studies of PAWS1 (protein associated with SMAD1; also known as FAM83G) have suggested that this molecule has roles beyond BMP signalling. To investigate these roles, we have used CRISPR/Cas9 to generate PAWS1-knockout U2OS osteosarcoma cells. Here, we show that PAWS1 plays a role in the regulation of the cytoskeletal machinery, including actin and focal adhesion dynamics, and cell migration. Confocal microscopy and live cell imaging of actin in U2OS cells indicate that PAWS1 is also involved in cytoskeletal dynamics and organization. Loss of PAWS1 causes severe defects in F-actin organization and distribution as well as in lamellipodial organization, resulting in impaired cell migration. PAWS1 interacts in a dynamic fashion with the actin/cytoskeletal regulator CD2AP at lamellae, suggesting that its association with CD2AP controls actin organization and cellular migration. Genetic ablation of CD2AP from U2OS cells instigates actin and cell migration defects reminiscent of those seen in PAWS1-knockout cells.This article has an associated First Person interview with the first authors of the paper.
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Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Movimento Celular , Proteínas do Citoesqueleto/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Proteínas do Citoesqueleto/genética , Adesões Focais/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Transdução de SinaisRESUMO
Adipose tissue metabolism is a critical regulator of adiposity and whole body energy expenditure; however, metabolic changes that occur in white adipose tissue (WAT) with obesity remain unclear. The purpose of this study was to understand the metabolic and bioenergetic changes occurring in WAT with obesity. Wild-type (C57BL/6J) mice fed a high-fat diet (HFD) showed significant increases in whole body adiposity, had significantly lower VÌ(O2), VÌ(CO2), and respiratory exchange ratios, and demonstrated worsened glucose and insulin tolerance compared with low-fat-fed mice. Metabolomic analysis of WAT showed marked changes in lipid, amino acid, carbohydrate, nucleotide, and energy metabolism. Tissue levels of succinate and malate were elevated, and metabolites that could enter the Krebs cycle via anaplerosis were mostly diminished in high-fat-fed mice, suggesting altered mitochondrial metabolism. Despite no change in basal oxygen consumption or mitochondrial DNA abundance, citrate synthase activity was decreased by more than 50%, and responses to FCCP were increased in WAT from mice fed a high-fat diet. Moreover, Pgc1a was downregulated and Cox7a1 upregulated after 6 wk of HFD. After 12 wk of high-fat diet, the abundance of several proteins in the mitochondrial respiratory chain or matrix was diminished. These changes were accompanied by increased Parkin and Pink1, decreased p62 and LC3-I, and ultrastructural changes suggestive of autophagy and mitochondrial remodeling. These studies demonstrate coordinated restructuring of metabolism and autophagy that could contribute to the hypertrophy and whitening of adipose tissue in obesity.
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Gordura Abdominal/metabolismo , Adiposidade , Autofagia , Metabolismo Energético , Regulação Enzimológica da Expressão Gênica , Dinâmica Mitocondrial , Obesidade/metabolismo , Gordura Abdominal/patologia , Gordura Abdominal/ultraestrutura , Animais , Tamanho Celular , Citrato (si)-Sintase/metabolismo , Ciclo do Ácido Cítrico , Dieta Hiperlipídica/efeitos adversos , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Hipertrofia , Metabolismo dos Lipídeos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/etiologia , Obesidade/patologia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Protein kinase ALK3/BMPR1A mediates bone morphogenetic protein (BMP) signalling through phosphorylation and activation of SMADs 1/5/8. SMAD6, a transcriptional target of BMP, negatively regulates the BMP pathway by recruiting E3 ubiquitin ligases and targeting ALK3 for ubiquitin-mediated degradation. Here, we identify a deubiquitylating enzyme USP15 as an interactor of SMAD6 and ALK3. We show that USP15 enhances BMP-induced phosphorylation of SMAD1 by interacting with and deubiquitylating ALK3. RNAi-mediated depletion of USP15 increases ALK3 K48-linked polyubiquitylation, and reduces both BMP-induced SMAD1 phosphorylation and transcription of BMP target genes. We also show that loss of USP15 expression from mouse myoblast cells inhibits BMP-induced osteoblast differentiation. Furthermore, USP15 modulates BMP-induced phosphorylation of SMAD1 and transcription during Xenopus embryogenesis.
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Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Proteína Smad1/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Animais , Ácidos Borônicos/farmacologia , Bortezomib , Linhagem Celular , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Células HeLa , Humanos , Camundongos , Fosforilação , Pirazinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , UbiquitinaçãoRESUMO
BACKGROUND: Cardiac hypertrophy and heart failure are associated with metabolic dysregulation and a state of chronic energy deficiency. Although several disparate changes in individual metabolic pathways have been described, there has been no global assessment of metabolomic changes in hypertrophic and failing hearts in vivo. Hence, we investigated the impact of pressure overload and infarction on myocardial metabolism. METHODS AND RESULTS: Male C57BL/6J mice were subjected to transverse aortic constriction or permanent coronary occlusion (myocardial infarction [MI]). A combination of LC/MS/MS and GC/MS techniques was used to measure 288 metabolites in these hearts. Both transverse aortic constriction and MI were associated with profound changes in myocardial metabolism affecting up to 40% of all metabolites measured. Prominent changes in branched-chain amino acids were observed after 1 week of transverse aortic constriction and 5 days after MI. Changes in branched-chain amino acids after MI were associated with myocardial insulin resistance. Longer duration of transverse aortic constriction and MI led to a decrease in purines, acylcarnitines, fatty acids, and several lysolipid and sphingolipid species but a marked increase in pyrimidines as well as ascorbate, heme, and other indices of oxidative stress. Cardiac remodeling and contractile dysfunction in hypertrophied hearts were associated with large increases in myocardial, but not plasma, levels of the polyamines putrescine and spermidine as well as the collagen breakdown product prolylhydroxyproline. CONCLUSIONS: These findings reveal extensive metabolic remodeling common to both hypertrophic and failing hearts that are indicative of extracellular matrix remodeling, insulin resistance and perturbations in amino acid, and lipid and nucleotide metabolism.
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Cardiomegalia/metabolismo , Metabolismo Energético/fisiologia , Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo , Estresse Oxidativo , Animais , Cardiomegalia/diagnóstico , Cardiomegalia/fisiopatologia , Modelos Animais de Doenças , Ecocardiografia , Insuficiência Cardíaca/diagnóstico , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/diagnóstico , Infarto do Miocárdio/fisiopatologia , Miocárdio/patologia , Volume Sistólico , Espectrometria de Massas em TandemRESUMO
The singly coded gene O-linked-ß-N-acetylglucosamine (O-GlcNAc) transferase (Ogt) resides on the X chromosome and is necessary for embryonic stem cell viability during embryogenesis. In mature cells, this enzyme catalyzes the posttranslational modification known as O-GlcNAc to various cellular proteins. Several groups, including our own, have shown that acute increases in protein O-GlcNAcylation are cardioprotective both in vitro and in vivo. Yet, little is known about how OGT affects cardiac function because total body knockout (KO) animals are not viable. Presently, we sought to establish the potential involvement of cardiomyocyte Ogt in cardiac maturation. Initially, we characterized a constitutive cardiomyocyte-specific (cm)OGT KO (c-cmOGT KO) mouse and found that only 12% of the c-cmOGT KO mice survived to weaning age (4 wk old); the surviving animals were smaller than their wild-type littermates, had dilated hearts, and showed overt signs of heart failure. Dysfunctional c-cmOGT KO hearts were more fibrotic, apoptotic, and hypertrophic. Several glycolytic genes were also upregulated; however, there were no gross changes in mitochondrial O2 consumption. Histopathology of the KO hearts indicated the potential involvement of endoplasmic reticulum stress, directing us to evaluate expression of 78-kDa glucose-regulated protein and protein disulfide isomerase, which were elevated. Additional groups of mice were subjected to inducible deletion of cmOGT, which did not produce overt dysfunction within the first couple of weeks of deletion. Yet, long-term loss (via inducible deletion) of cmOGT produced gradual and progressive cardiomyopathy. Thus, cardiomyocyte Ogt is necessary for maturation of the mammalian heart, and inducible deletion of cmOGT in the adult mouse produces progressive ventricular dysfunction.
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Miócitos Cardíacos/metabolismo , N-Acetilglucosaminiltransferases/genética , Animais , Apoptose , Cardiomiopatia Dilatada/congênito , Cardiomiopatia Dilatada/patologia , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático , Fibrose/congênito , Fibrose/patologia , Deleção de Genes , Glicólise , Insuficiência Cardíaca/congênito , Insuficiência Cardíaca/patologia , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Camundongos , Camundongos Knockout , Miócitos Cardíacos/patologia , N-Acetilglucosaminiltransferases/metabolismo , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismoRESUMO
BACKGROUND: While investigating estrogen response element (ERE) binding properties of human estrogen receptor-α (hERα) in breast cancer cytosols, other ERE-binding proteins (ERE-BP) were observed. DESIGN AND METHODS: Recognition properties of ERE-BP were evaluated by electrophoretic mobility shift assays (EMSA) with ERE sequences of the 5'-flanking region of the estrogen responsive gene vitellogenin A2 (VitA2). Cytosols were incubated 16 h, 4 °C with [32P]ERE sequences and separated by EMSA. A method of estimating ERE-BP levels was developed by measuring band intensity from EMSA profiles, expressed in digital light units (DLU)/µg protein and normalized to total DLU. ERE-BP were purified by affinity chromatography and EMSA, and then identified by mass spectrometry. RESULTS: ERE-BP in cytosols did not supershift in the presence of anti-hERα or anti-hERß antibodies recognizing different ER epitopes suggesting that they are not fragments of either receptor isoform. ERE-BP competed with hERα for binding to VitA2-ERE. Increased levels of ERE-BP DNA-binding activities measured in 310 cytosols prepared from breast cancer biopsies correlated with decreased patient survival. Strikingly, breast cancer patients with ER negative status and high ERE-BP expression exhibited the poorest disease-free and overall survival. After purification, ERE-BP were identified as Ku70 (XRCC6) and Ku80 (XRCC5) using mass spectrometry. ERE-BP were confirmed to be Ku70/80 by supershift assay. CONCLUSION: Presence of these novel ERE-binding proteins in a breast carcinoma is a strong predictor of poor prognosis. Our results suggest that ERE-BP, identified as Ku70/Ku80, in cytosols prepared from breast carcinoma biopsies are useful biomarkers for assessing risk of breast cancer recurrence.
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Biomarcadores Tumorais/metabolismo , Estrogênios/metabolismo , Proteínas de Neoplasias/metabolismo , Elementos de Resposta/genética , Anticorpos Antineoplásicos/imunologia , Antígenos Nucleares/metabolismo , Ligação Competitiva , Citosol/metabolismo , DNA de Neoplasias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Intervalo Livre de Doença , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Feminino , Humanos , Autoantígeno Ku , Espectrometria de Massas , Ligação Proteica , Extratos de TecidosRESUMO
Vascular injury and chronic arterial diseases result in exposure of VSMCs (vascular smooth muscle cells) to increased concentrations of growth factors. The mechanisms by which growth factors trigger VSMC phenotype transitions remain unclear. Because cellular reprogramming initiated by growth factors requires not only the induction of genes involved in cell proliferation, but also the removal of contractile proteins, we hypothesized that autophagy is an essential modulator of VSMC phenotype. Treatment of VSMCs with PDGF (platelet-derived growth factor)-BB resulted in decreased expression of the contractile phenotype markers calponin and α-smooth muscle actin and up-regulation of the synthetic phenotype markers osteopontin and vimentin. Autophagy, as assessed by LC3 (microtubule-associated protein light chain 3 α; also known as MAP1LC3A)-II abundance, LC3 puncta formation and electron microscopy, was activated by PDGF exposure. Inhibition of autophagy with 3-methyladenine, spautin-1 or bafilomycin stabilized the contractile phenotype. In particular, spautin-1 stabilized α-smooth muscle cell actin and calponin in PDGF-treated cells and prevented actin filament disorganization, diminished production of extracellular matrix, and abrogated VSMC hyperproliferation and migration. Treatment of cells with PDGF prevented protein damage and cell death caused by exposure to the lipid peroxidation product 4-hydroxynonenal. The results of the present study demonstrate a distinct form of autophagy induced by PDGF that is essential for attaining the synthetic phenotype and for survival under the conditions of high oxidative stress found to occur in vascular lesions.
Assuntos
Aorta/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Actinas/genética , Actinas/metabolismo , Adenina/análogos & derivados , Adenina/farmacologia , Aldeídos/farmacologia , Animais , Aorta/citologia , Aorta/metabolismo , Autofagia/genética , Biomarcadores/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Macrolídeos/farmacologia , Masculino , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Osteopontina/genética , Osteopontina/metabolismo , Estresse Oxidativo , Fenótipo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Vimentina/genética , Vimentina/metabolismo , CalponinasRESUMO
Clinical trials demonstrate the regenerative potential of cardiac stem cell (CSC) therapy in the postinfarcted heart. Despite these encouraging preliminary clinical findings, the basic biology of these cells remains largely unexplored. The principal requirement for cell transplantation is to effectively prime them for survival within the unfavorable environment of the infarcted myocardium. In the adult mammalian heart, the ß-O-linkage of N-acetylglucosamine (i.e., O-GlcNAc) to proteins is a unique post-translational modification that confers cardioprotection from various otherwise lethal stressors. It is not known whether this signaling system exists in CSCs. In this study, we demonstrate that protein O-GlcNAcylation is an inducible stress response in adult murine Sca-1(+) /lin(-) CSCs and exerts an essential prosurvival role. Posthypoxic CSCs responded by time-dependently increasing protein O-GlcNAcylation upon reoxygenation. We used pharmacological interventions for loss- and gain-of-function, that is, enzymatic inhibition of O-GlcNAc transferase (OGT) (adds the O-GlcNAc modification to proteins) by TT04, or inhibition of OGA (removes O-GlcNAc) by thiamet-G (ThG). Reduction in the O-GlcNAc signal (via TT04, or OGT gene deletion using Cre-mediated recombination) significantly sensitized CSCs to posthypoxic injury, whereas augmenting O-GlcNAc levels (via ThG) enhanced cell survival. Diminished O-GlcNAc levels render CSCs more susceptible to the onset of posthypoxic apoptotic processes via elevated poly(ADP-ribose) polymerase cleavage due to enhanced caspase-3/7 activation, whereas promoting O-GlcNAcylation can serve as a pre-emptive antiapoptotic signal regulating the survival of CSCs. Thus, we report the primary demonstration of protein O-GlcNAcylation as an important prosurvival signal in CSCs, which could enhance CSC survival prior to in vivo autologous transfer.
Assuntos
Miócitos Cardíacos/metabolismo , Transdução de Sinais/fisiologia , Células-Tronco/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Hipóxia Celular/genética , Hipóxia Celular/fisiologia , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Células Cultivadas , Masculino , Camundongos , Microscopia Confocal , Miócitos Cardíacos/citologia , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Transdução de Sinais/genética , Células-Tronco/citologiaRESUMO
Oxidative modifications to cellular proteins are critical in mediating redox-sensitive processes such as autophagy, the antioxidant response, and apoptosis. The proteins that become modified by reactive species are often compartmentalized to specific organelles or regions of the cell. Here, we detail protocols for identifying the subcellular protein targets of lipid oxidation and for linking protein modifications with biological responses such as autophagy. Fluorophores such as BODIPY-labeled arachidonic acid or BODIPY-conjugated electrophiles can be paired with organelle-specific probes to identify specific biological processes and signaling pathways activated in response to oxidative stress. In particular, we demonstrate "negative" and "positive" labeling methods using BODIPY-tagged reagents for examining oxidative modifications to protein nucleophiles. The protocol describes the use of these probes in slot immunoblotting, quantitative Western blotting, in-gel fluorescence, and confocal microscopy techniques. In particular, the use of the BODIPY fluorophore with organelle- or biological process-specific dyes and chromophores is highlighted. These methods can be used in multiple cell types as well as isolated organelles to interrogate the role of oxidative modifications in regulating biological responses to oxidative stress.
Assuntos
Corantes Fluorescentes/análise , Peroxidação de Lipídeos , Proteoma/análise , Corantes Fluorescentes/química , Estresse OxidativoRESUMO
RATIONALE: Endothelial dysfunction is a characteristic feature of diabetes and obesity in animal models and humans. Deficits in nitric oxide production by endothelial nitric oxide synthase (eNOS) are associated with insulin resistance, which is exacerbated by high-fat diet. Nevertheless, the metabolic effects of increasing eNOS levels have not been studied. OBJECTIVE: The current study was designed to test whether overexpression of eNOS would prevent diet-induced obesity and insulin resistance. METHODS AND RESULTS: In db/db mice and in high-fat diet-fed wild-type C57BL/6J mice, the abundance of eNOS protein in adipose tissue was decreased without significant changes in eNOS levels in skeletal muscle or aorta. Mice overexpressing eNOS (eNOS transgenic mice) were resistant to diet-induced obesity and hyperinsulinemia, although systemic glucose intolerance remained largely unaffected. In comparison with wild-type mice, high-fat diet-fed eNOS transgenic mice displayed a higher metabolic rate and attenuated hypertrophy of white adipocytes. Overexpression of eNOS did not affect food consumption or diet-induced changes in plasma cholesterol or leptin levels, yet plasma triglycerides and fatty acids were decreased. Metabolomic analysis of adipose tissue indicated that eNOS overexpression primarily affected amino acid and lipid metabolism; subpathway analysis suggested changes in fatty acid oxidation. In agreement with these findings, adipose tissue from eNOS transgenic mice showed higher levels of PPAR-α and PPAR-γ gene expression, elevated abundance of mitochondrial proteins, and a higher rate of oxygen consumption. CONCLUSIONS: These findings demonstrate that increased eNOS activity prevents the obesogenic effects of high-fat diet without affecting systemic insulin resistance, in part, by stimulating metabolic activity in adipose tissue.