RESUMO
Owing to its roles in cellular signal transduction, protein phosphorylation plays critical roles in myriad cell processes. That said, detecting and quantifying protein phosphorylation has remained a challenge. We describe the use of a novel mass spectrometer (Orbitrap Astral) coupled with data-independent acquisition (DIA) to achieve rapid and deep analysis of human and mouse phosphoproteomes. With this method, we map approximately 30,000 unique human phosphorylation sites within a half-hour of data collection. The technology is benchmarked to other state-of-the-art MS platforms using both synthetic peptide standards and with EGF-stimulated HeLa cells. We apply this approach to generate a phosphoproteome multi-tissue atlas of the mouse. Altogether, we detect 81,120 unique phosphorylation sites within 12 hours of measurement. With this unique dataset, we examine the sequence, structural, and kinase specificity context of protein phosphorylation. Finally, we highlight the discovery potential of this resource with multiple examples of phosphorylation events relevant to mitochondrial and brain biology.
Assuntos
Espectrometria de Massas , Fosfoproteínas , Proteoma , Proteômica , Humanos , Fosfoproteínas/metabolismo , Fosfoproteínas/análise , Animais , Células HeLa , Fosforilação , Camundongos , Proteoma/metabolismo , Espectrometria de Massas/métodos , Proteômica/métodosRESUMO
Owing to its roles in cellular signal transduction, protein phosphorylation plays critical roles in myriad cell processes. That said, detecting and quantifying protein phosphorylation has remained a challenge. We describe the use of a novel mass spectrometer (Orbitrap Astral) coupled with data-independent acquisition (DIA) to achieve rapid and deep analysis of human and mouse phosphoproteomes. With this method we map approximately 30,000 unique human phosphorylation sites within a half-hour of data collection. We applied this approach to generate a phosphoproteome multi-tissue atlas of the mouse. Altogether, we detected 81,120 unique phosphorylation sites within 12 hours of measurement. With this unique dataset, we examine the sequence and structural context of protein phosphorylation. Finally, we highlight the discovery potential of this resource with multiple examples of novel phosphorylation events relevant to mitochondrial and brain biology.
RESUMO
With age, people tend to accumulate body fat and reduce energy expenditure 1 . Brown (BAT) and beige adipose tissue dissipate heat and increase energy expenditure via the activity of the uncoupling protein UCP1 and other thermogenic futile cycles 2,3 . The activity of brown and beige depots inversely correlates with BMI and age 4-11 , suggesting that promoting thermogenesis may be an effective approach for combating age-related metabolic disease 12-15 . Heme is an enzyme cofactor and signaling molecule that we recently showed to regulate BAT function 16 . Here, we show that heme biosynthesis is the primary contributor to intracellular heme levels in brown adipocytes. Inhibition of heme biosynthesis leads to mitochondrial dysfunction and reduction in UCP1. Although supplementing heme can restore mitochondrial function in heme-synthesis-deficient cells, the downregulation of UCP1 persists due to the accumulation of the heme precursors, particularly propionyl-CoA, which is a product of branched-chain amino acids (BCAA) catabolism. Cold exposure promotes BCAA uptake in BAT, and defects in BCAA catabolism in this tissue hinder thermogenesis 17 . However, BCAAs' contribution to the TCA cycle in BAT and WAT never exceeds 2% of total TCA flux 18 . Our work offers a way to integrate current literature by describing heme biosynthesis as an important metabolic sink for BCAAs.
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As a central coordinator of physiologic metabolism, adipose tissue has long been appreciated as a highly plastic organ that dynamically responds to environmental cues. Once thought of as a homogenous storage depot, recent advances have enabled deep characterizations of the underlying structure and composition of adipose tissue depots. As the obesity and metabolic disease epidemics continue to accelerate due to modern lifestyles and an aging population, elucidation of the underlying mechanisms that control adipose and systemic homeostasis are of critical importance. Within the past decade, the emergence of deep cell profiling at tissue- and, recently, single-cell level has furthered our understanding of the complex dynamics that contribute to tissue function and their implications in disease development. Although many paradigm-shifting findings may lie ahead, profound advances have been made to forward our understanding of the adipose tissue niche in both health and disease. Now widely accepted as a highly heterogenous organ with major roles in metabolic homeostasis, endocrine signaling, and immune function, the study of adipose tissue dynamics has reached a new frontier. In this review, we will provide a synthesis of the latest advances in adipose tissue biology made possible by the use of single-cell technologies, the impact of epigenetic mechanisms on adipose function, and suggest what next steps will further our understanding of the role that adipose tissue plays in systemic physiology.
Assuntos
Adipócitos , Termogênese , Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Idoso , Comunicação Celular , Humanos , Obesidade/metabolismo , Termogênese/fisiologiaRESUMO
In obesity, signaling through the IRE1 arm of the unfolded protein response exerts both protective and harmful effects. Overexpression of the IRE1-regulated transcription factor XBP1s in liver or fat protects against obesity-linked metabolic deterioration. However, hyperactivation of IRE1 engages regulated IRE1-dependent decay (RIDD) and TRAF2/JNK pro-inflammatory signaling, which accelerate metabolic dysfunction. These pathologic IRE1-regulated processes have hindered efforts to pharmacologically harness the protective benefits of IRE1/XBP1s signaling in obesity-linked conditions. Here, we report the effects of a XBP1s-selective pharmacological IRE1 activator, IXA4, in diet-induced obese (DIO) mice. IXA4 transiently activates protective IRE1/XBP1s signaling in liver without inducing RIDD or TRAF2/JNK signaling. IXA4 treatment improves systemic glucose metabolism and liver insulin action through IRE1-dependent remodeling of the hepatic transcriptome that reduces glucose production and steatosis. IXA4-stimulated IRE1 activation also enhances pancreatic function. Our findings indicate that systemic, transient activation of IRE1/XBP1s signaling engenders multi-tissue benefits that integrate to mitigate obesity-driven metabolic dysfunction.
Assuntos
Proteínas de Membrana/metabolismo , Proteínas de Membrana/farmacologia , Obesidade/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/farmacologia , Proteína 1 de Ligação a X-Box/metabolismo , Animais , Fígado Gorduroso/metabolismo , Regulação da Expressão Gênica , Glucose/metabolismo , Homeostase , Fígado/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Obesos , Medicina Molecular , Obesidade/genética , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Fatores de Transcrição/metabolismo , Resposta a Proteínas não Dobradas , Proteína 1 de Ligação a X-Box/genéticaRESUMO
Untargeted metabolomics of disease-associated intestinal microbiota can detect quantitative changes in metabolite profiles and complement other methodologies to reveal the full effect of intestinal dysbiosis. Here, we used the T cell transfer mouse model of colitis to identify small-molecule metabolites with altered abundance due to intestinal inflammation. We applied untargeted metabolomics to detect metabolite signatures in cecal, colonic, and fecal samples from healthy and colitic mice and to uncover differences that would aid in the identification of colitis-associated metabolic processes. We provided an unbiased spatial survey of the GI tract for small molecules, and we identified the likely source of metabolites and biotransformations. Several prioritized metabolites that we detected as being altered in colitis were evaluated for their ability to induce inflammatory signaling in cultured macrophages, such as NF-κB signaling and the expression of cytokines and chemokines upon LPS stimulation. Multiple previously uncharacterized anti-inflammatory and inflammation-augmenting metabolites were thus identified, with phytosphingosine showing the most effective anti-inflammatory activity in vitro. We further demonstrated that oral administration of phytosphingosine decreased inflammation in a mouse model of colitis induced by the compound TNBS. The collection of distinct metabolites we identified and characterized, many of which have not been previously associated with colitis, may offer new biological insight into IBD-associated inflammation and disease pathogenesis.
Assuntos
Colite , Linfócitos T , Anti-Inflamatórios , Humanos , MetabolômicaRESUMO
The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white preadipocyte cell lines. These cultured cell lines, however, have limitations. They do not fully capture the diverse functional spectrum of the heterogenous adipocyte populations that are now known to exist within white adipose depots. To provide a more physiologically relevant model to study the complexity of white adipose tissue, a protocol has been developed and optimized to enable simultaneous isolation of primary white and brown adipocyte progenitors from newborn mice, their rapid expansion in culture, and their differentiation in vitro into mature, fully functional adipocytes. The primary advantage of isolating primary cells from newborn, rather than adult mice, is that the adipose depots are actively developing and are, therefore, a rich source of proliferating preadipocytes. Primary preadipocytes isolated using this protocol differentiate rapidly upon reaching confluence and become fully mature in 4-5 days, a temporal window that accurately reflects the appearance of developed fat pads in newborn mice. Primary cultures prepared using this strategy can be expanded and studied with high reproducibility, making them suitable for genetic and phenotypic screens and enabling the study of the cell-autonomous adipocyte phenotypes of genetic mouse models. This protocol offers a simple, rapid, and inexpensive approach to study the complexity of adipose tissue in vitro.
Assuntos
Adipócitos Marrons/citologia , Adipócitos Brancos/citologia , Diferenciação Celular , Separação Celular/métodos , Animais , Animais Recém-Nascidos , Células Cultivadas , Metabolismo Energético , Camundongos , Reprodutibilidade dos TestesRESUMO
Activity-based protein profiling (ABPP) has been used extensively to discover and optimize selective inhibitors of enzymes. Here, we show that ABPP can also be implemented to identify the converse-small-molecule enzyme activators. Using a kinetically controlled, fluorescence polarization-ABPP assay, we identify compounds that stimulate the activity of LYPLAL1-a poorly characterized serine hydrolase with complex genetic links to human metabolic traits. We apply ABPP-guided medicinal chemistry to advance a lead into a selective LYPLAL1 activator suitable for use in vivo. Structural simulations coupled to mutational, biochemical and biophysical analyses indicate that this compound increases LYPLAL1's catalytic activity likely by enhancing the efficiency of the catalytic triad charge-relay system. Treatment with this LYPLAL1 activator confers beneficial effects in a mouse model of diet-induced obesity. These findings reveal a new mode of pharmacological regulation for this large enzyme family and suggest that ABPP may aid discovery of activators for additional enzyme classes.
Assuntos
Ativadores de Enzimas/química , Ativadores de Enzimas/farmacologia , Lisofosfolipase/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Descoberta de Drogas , Ativadores de Enzimas/farmacocinética , Polarização de Fluorescência , Células HEK293 , Ensaios de Triagem em Larga Escala/métodos , Humanos , Resistência à Insulina , Lisofosfolipase/química , Lisofosfolipase/genética , Masculino , Síndrome Metabólica/tratamento farmacológico , Síndrome Metabólica/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Obesos , Simulação de Dinâmica Molecular , Estrutura Molecular , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacocinética , Relação Estrutura-AtividadeRESUMO
Mammals maintain a nearly constant core body temperature (Tb) by balancing heat production and heat dissipation. This comes at a high metabolic cost that is sustainable if adequate calorie intake is maintained. When nutrients are scarce or experimentally reduced such as during calorie restriction (CR), endotherms can reduce energy expenditure by lowering Tb [1-6]. This adaptive response conserves energy, limiting the loss of body weight due to low calorie intake [7-10]. Here we show that this response is regulated by the kappa opioid receptor (KOR). CR is associated with increased hypothalamic levels of the endogenous opioid Leu-enkephalin, which is derived from the KOR agonist precursor dynorphin [11]. Pharmacological inhibition of KOR, but not of the delta or the mu opioid receptor subtypes, fully blocked CR-induced hypothermia and increased weight loss during CR independent of calorie intake. Similar results were seen with DIO mice subjected to CR. In contrast, inhibiting KOR did not change Tb in animals fed ad libitum (AL). Chemogenetic inhibition of KOR neurons in the hypothalamic preoptic area reduced the CR-induced hypothermia, whereas chemogenetic activation of prodynorphin-expressing neurons in the arcuate or the parabrachial nucleus lowered Tb. These data indicate that KOR signaling is a pivotal regulator of energy homeostasis and can affect body weight during dieting by modulating Tb and energy expenditure.
Assuntos
Regulação da Temperatura Corporal/genética , Regulação da Temperatura Corporal/fisiologia , Receptores Opioides kappa/metabolismo , Analgésicos Opioides/metabolismo , Animais , Peso Corporal/fisiologia , Encéfalo/metabolismo , Restrição Calórica/métodos , Ingestão de Energia/fisiologia , Metabolismo Energético/fisiologia , Feminino , Hipotálamo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Receptores Opioides kappa/genética , Receptores Opioides mu/metabolismo , Receptores Opioides mu/fisiologia , Redução de Peso/fisiologiaRESUMO
Haem is an essential prosthetic group of numerous proteins and a central signalling molecule in many physiologic processes1,2. The chemical reactivity of haem means that a network of intracellular chaperone proteins is required to avert the cytotoxic effects of free haem, but the constituents of such trafficking pathways are unknown3,4. Haem synthesis is completed in mitochondria, with ferrochelatase adding iron to protoporphyrin IX. How this vital but highly reactive metabolite is delivered from mitochondria to haemoproteins throughout the cell remains poorly defined3,4. Here we show that progesterone receptor membrane component 2 (PGRMC2) is required for delivery of labile, or signalling haem, to the nucleus. Deletion of PGMRC2 in brown fat, which has a high demand for haem, reduced labile haem in the nucleus and increased stability of the haem-responsive transcriptional repressors Rev-Erbα and BACH1. Ensuing alterations in gene expression caused severe mitochondrial defects that rendered adipose-specific PGRMC2-null mice unable to activate adaptive thermogenesis and prone to greater metabolic deterioration when fed a high-fat diet. By contrast, obese-diabetic mice treated with a small-molecule PGRMC2 activator showed substantial improvement of diabetic features. These studies uncover a role for PGRMC2 in intracellular haem transport, reveal the influence of adipose tissue haem dynamics on physiology and suggest that modulation of PGRMC2 may revert obesity-linked defects in adipocytes.
Assuntos
Adipócitos/metabolismo , Heme/metabolismo , Proteínas de Membrana/metabolismo , Receptores de Progesterona/metabolismo , Animais , Homeostase , Humanos , Espaço Intracelular/metabolismo , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Chaperonas Moleculares/metabolismo , Receptores de Progesterona/deficiência , Receptores de Progesterona/genética , Transcrição GênicaRESUMO
OBJECTIVES: Extracts of the hops plant have been shown to reduce weight and insulin resistance in rodents and humans, but elucidation of the mechanisms responsible for these benefits has been hindered by the use of heterogeneous hops-derived mixtures. Because hop extracts are used as flavoring agents for their bitter properties, we hypothesized that bitter taste receptors (Tas2rs) could be mediating their beneficial effects in metabolic disease. Studies have shown that exposure of cultured enteroendocrine cells to bitter tastants can stimulate release of hormones, including glucagon-like peptide 1 (GLP-1). These findings have led to the suggestion that activation of Tas2rs may be of benefit in diabetes, but this tenet has not been tested. Here, we have assessed the ability of a pure derivative of a hops isohumulone with anti-diabetic properties, KDT501, to signal through Tas2rs. We have further used this compound as a tool to systematically assess the impact of bitter taste receptor activation in obesity-diabetes. METHODS: KDT501 was tested in a panel of bitter taste receptor signaling assays. Diet-induced obese mice (DIO) were dosed orally with KDT501 and acute effects on glucose homeostasis determined. A wide range of metabolic parameters were evaluated in DIO mice chronically treated with KDT501 to establish the full impact of activating gut bitter taste signaling. RESULTS: We show that KDT501 signals through Tas2r108, one of 35 mouse Tas2rs. In DIO mice, acute treatment stimulated GLP-1 secretion and enhanced glucose tolerance. Chronic treatment caused weight and fat mass loss, increased energy expenditure, enhanced glucose tolerance and insulin sensitivity, normalized plasma lipids, and induced broad suppression of inflammatory markers. Chronic KDT501 treatment altered enteroendocrine hormone levels and bile acid homeostasis and stimulated sustained GLP-1 release. Combined treatment with a dipeptidyl peptidase IV inhibitor amplified the incretin-based benefits of this pure isohumulone. CONCLUSIONS: Activation of Tas2r108 in the gut results in a remodeling of enteroendocrine hormone release and bile acid metabolism that ameliorates multiple features of metabolic syndrome. Targeting extraoral bitter taste receptors may be useful in metabolic disease.
Assuntos
Ciclopentanos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/fisiologia , Animais , Peso Corporal/efeitos dos fármacos , Ciclopentanos/farmacologia , Diabetes Mellitus Tipo 2/metabolismo , Modelos Animais de Doenças , Células Enteroendócrinas/metabolismo , Peptídeo 1 Semelhante ao Glucagon/efeitos dos fármacos , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Humulus/metabolismo , Hipoglicemiantes/farmacologia , Resistência à Insulina/fisiologia , Mucosa Intestinal/metabolismo , Intestinos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
Defects in adipocyte function associated with obesity drive the development of systemic insulin resistance and type 2 diabetes. Agents that correct obesity-linked adipocyte dysfunction serve as useful insulin sensitizers in humans, as is exemplified by the thiazolidinediones (TZDs). We have developed a new platform that integrates advanced chemoproteomics with phenotypic screening to identify small molecules that promote differentiation and lipid storage in adipocytes, and, in tandem, their molecular target(s). These molecules mimic the activity of TZDs in culture and thus may also serve as insulin sensitizers in vivo. Central to this platform is the use of fully functionalized fragment (FFF) probes that consist of a variable, fragment-like recognition element linked to an alkyne-diazirine group that enables the photoactivated capture of probe-bound proteins directly in living cells and subsequent copper-catalyzed azide-alkyne cycloaddition to reporter tags for enrichment and identification of these probe-bound proteins by mass spectrometry. This platform, which can be adapted to diverse screens and cell types beyond adipocytes, has the potential to uncover new biological pathways amenable to pharmacological modulation that may impact human disease.
Assuntos
Adipócitos/efeitos dos fármacos , Adipócitos/fisiologia , Descoberta de Drogas , Células 3T3-L1 , Adipogenia/efeitos dos fármacos , Animais , Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos , Variação Genética , Humanos , Espectrometria de Massas , Camundongos , Fenótipo , Proteoma , Proteômica/métodos , Bibliotecas de Moléculas Pequenas , Tiazolidinedionas/farmacologiaRESUMO
Advances in the synthesis and screening of small-molecule libraries have accelerated the discovery of chemical probes for studying biological processes. Still, only a small fraction of the human proteome has chemical ligands. Here, we describe a platform that marries fragment-based ligand discovery with quantitative chemical proteomics to map thousands of reversible small molecule-protein interactions directly in human cells, many of which can be site-specifically determined. We show that fragment hits can be advanced to furnish selective ligands that affect the activity of proteins heretofore lacking chemical probes. We further combine fragment-based chemical proteomics with phenotypic screening to identify small molecules that promote adipocyte differentiation by engaging the poorly characterized membrane protein PGRMC2. Fragment-based screening in human cells thus provides an extensive proteome-wide map of protein ligandability and facilitates the coordinated discovery of bioactive small molecules and their molecular targets.
Assuntos
Descoberta de Drogas/métodos , Proteômica/métodos , Adipócitos/citologia , Diferenciação Celular , Cristalografia por Raios X , Ensaios de Triagem em Larga Escala , Humanos , Hidrolases/química , Ligantes , Proteínas de Membrana/antagonistas & inibidores , Oxirredutases/química , Ligação Proteica , Receptores de Progesterona/antagonistas & inibidores , Bibliotecas de Moléculas PequenasRESUMO
The diterpenoid ester ingenol mebutate (IngMeb) is the active ingredient in the topical drug Picato, a first-in-class treatment for the precancerous skin condition actinic keratosis. IngMeb is proposed to exert its therapeutic effects through a dual mode of action involving (i) induction of cell death that is associated with mitochondrial dysfunction followed by (ii) stimulation of a local inflammatory response, at least partially driven by protein kinase C (PKC) activation. Although this therapeutic model has been well characterized, the complete set of molecular targets responsible for mediating IngMeb activity remains ill-defined. Here, we have synthesized a photoreactive, clickable analogue of IngMeb and used this probe in quantitative proteomic experiments to map several protein targets of IngMeb in human cancer cell lines and primary human keratinocytes. Prominent among these targets was the mitochondrial carnitine-acylcarnitine translocase SLC25A20, which we show is inhibited in cells by IngMeb and the more stable analogue ingenol disoxate (IngDsx), but not by the canonical PKC agonist 12-O-tetradecanoylphorbol-13-acetate (TPA). SLC25A20 blockade by IngMeb and IngDsx leads to a buildup of cellular acylcarnitines and blockade of fatty acid oxidation (FAO), pointing to a possible mechanism for IngMeb-mediated perturbations in mitochondrial function.
RESUMO
Arylfluorosulfates have appeared only rarely in the literature and have not been explored as probes for covalent conjugation to proteins, possibly because they were assumed to possess high reactivity, as with other sulfur(VI) halides. However, we find that arylfluorosulfates become reactive only under certain circumstances, e.g., when fluoride displacement by a nucleophile is facilitated. Herein, we explore the reactivity of structurally simple arylfluorosulfates toward the proteome of human cells. We demonstrate that the protein reactivity of arylfluorosulfates is lower than that of the corresponding aryl sulfonyl fluorides, which are better characterized with regard to proteome reactivity. We discovered that simple hydrophobic arylfluorosulfates selectively react with a few members of the intracellular lipid binding protein (iLBP) family. A central function of iLBPs is to deliver small-molecule ligands to nuclear hormone receptors. Arylfluorosulfate probe 1 reacts with a conserved tyrosine residue in the ligand-binding site of a subset of iLBPs. Arylfluorosulfate probes 3 and 4, featuring a biphenyl core, very selectively and efficiently modify cellular retinoic acid binding protein 2 (CRABP2), both in vitro and in living cells. The X-ray crystal structure of the CRABP2-4 conjugate, when considered together with binding site mutagenesis experiments, provides insight into how CRABP2 might activate arylfluorosulfates toward site-specific reaction. Treatment of breast cancer cells with probe 4 attenuates nuclear hormone receptor activity mediated by retinoic acid, an endogenous client lipid of CRABP2. Our findings demonstrate that arylfluorosulfates can selectively target single iLBPs, making them useful for understanding iLBP function.
Assuntos
Proteínas de Ligação a Ácido Graxo/metabolismo , Receptores do Ácido Retinoico/metabolismo , Ácidos Sulfúricos/metabolismo , Tirosina/química , Sítios de Ligação , Técnicas de Cultura de Células , Cristalografia por Raios X , Proteínas de Ligação a Ácido Graxo/química , Flúor , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Células MCF-7 , Receptores do Ácido Retinoico/química , Ácidos Sulfúricos/químicaRESUMO
Obesity develops when energy intake chronically exceeds energy expenditure. Because brown adipose tissue (BAT) dissipates energy in the form of heat, increasing energy expenditure by augmenting BAT-mediated thermogenesis may represent an approach to counter obesity and its complications. The ability of BAT to dissipate energy is dependent on expression of mitochondrial uncoupling protein 1 (UCP1). To facilitate the identification of pharmacological modulators of BAT UCP1 levels, which may have potential as antiobesity medications, we developed a transgenic model in which luciferase activity faithfully mimics endogenous UCP1 expression and its response to physiologic stimuli. Phenotypic screening of a library using cells derived from this model yielded a small molecule that increases UCP1 expression in brown fat cells and mice. Upon adrenergic stimulation, compound-treated mice showed increased energy expenditure. These tools offer an opportunity to identify pharmacologic modulators of UCP1 expression and uncover regulatory pathways that impact BAT-mediated thermogenesis.
Assuntos
Tecido Adiposo Marrom/metabolismo , Canais Iônicos/metabolismo , Proteínas Mitocondriais/metabolismo , Ativação Transcricional/efeitos dos fármacos , Animais , Fármacos Antiobesidade/farmacologia , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Metabolismo Energético , Expressão Gênica , Canais Iônicos/genética , Masculino , Camundongos Transgênicos , Proteínas Mitocondriais/genética , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Transdução de Sinais , Termogênese , Proteína Desacopladora 1RESUMO
Activity-based protein profiling (ABPP) is a chemical proteomics approach that utilizes small-molecule probes to determine the functional state of enzymes directly in native systems. ABPP probes selectively label active enzymes, but not their inactive forms, facilitating the characterization of changes in enzyme activity that occur without alterations in protein levels. ABPP can be a tool superior to conventional gene expression and proteomic profiling methods to discover new enzymes active in adipocytes and to detect differences in the activity of characterized enzymes that may be associated with disorders of adipose tissue function. ABPP probes have been developed that react selectively with most members of specific enzyme classes. Here, using as an example the serine hydrolase family that includes many enzymes with critical roles in adipocyte physiology, we describe methods to apply ABPP analysis to the study of adipocyte enzymatic pathways.
Assuntos
Adipócitos/enzimologia , Ensaios Enzimáticos/métodos , Proteômica/métodos , Serina Proteases/metabolismo , Animais , Humanos , Técnicas de Sonda Molecular , Proteoma/metabolismoRESUMO
Phenotypic screening is making a comeback in drug discovery as the maturation of chemical proteomics methods has facilitated target identification for bioactive small molecules. A limitation of these approaches is that time-consuming genetic methods or other means are often required to determine the biologically relevant target (or targets) from among multiple protein-compound interactions that are typically detected. Here, we have combined phenotypic screening of a directed small-molecule library with competitive activity-based protein profiling to map and functionally characterize the targets of screening hits. Using this approach, we identify carboxylesterase 3 (Ces3, also known as Ces1d) as a primary molecular target of bioactive compounds that promote lipid storage in adipocytes. We further show that Ces3 activity is markedly elevated during adipocyte differentiation. Treatment of two mouse models of obesity-diabetes with a Ces3 inhibitor ameliorates multiple features of metabolic syndrome, illustrating the power of the described strategy to accelerate the identification and pharmacologic validation of new therapeutic targets.
Assuntos
Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Diabetes Mellitus/genética , Obesidade/genética , Fenótipo , Bibliotecas de Moléculas Pequenas , Animais , Células Cultivadas , Modelos Animais de Doenças , Sistemas de Liberação de Medicamentos , Descoberta de Drogas , Camundongos , Análise Serial de Proteínas , ProteômicaRESUMO
Hyperthermia is a potentially lethal side effect of Methamphetamine (Meth) abuse, which involves the participation of peripheral thermogenic sites such as the Brown Adipose Tissue (BAT). In a previous study we found that the anti-oxidant N-acetyl cysteine (NAC) can prevent the high increase in temperature in a mouse model of Meth-hyperthermia. Here, we have further explored the ability of NAC to modulate Meth-induced hyperthermia in correlation with changes in BAT. We found that NAC treatment in controls causes hypothermia, and, when administered prior or upon the onset of Meth-induced hyperthermia, can ameliorate the temperature increase and preserve mitochondrial numbers and integrity, without affecting locomotor activity. This was different from Dantrolene, which decreased motor activity without affecting temperature. The effects of NAC were seen in spite of its inability to recover the decrease of mitochondrial superoxide induced in BAT by Meth. In addition, NAC did not prevent the Meth-induced decrease of BAT glutathione. Treatment with S-adenosyl-L-methionine, which improves glutathione activity, had an effect in ameliorating Meth-induced hyperthermia, but also modulated motor activity. This suggests a role for the remaining glutathione for controlling temperature. However, the mechanism by which NAC operates is independent of glutathione levels in BAT and specific to temperature. Our results show that, in spite of the absence of a clear mechanism of action, NAC is a pharmacological tool to examine the dissociation between Meth-induced hyperthermia and motor activity, and a drug of potential utility in treating the hyperthermia associated with Meth-abuse.
RESUMO
Adipose tissue renewal and obesity-driven expansion of fat cell number are dependent on proliferation and differentiation of adipose progenitors that reside in the vasculature that develops in coordination with adipose depots. The transcriptional events that regulate commitment of progenitors to the adipose lineage are poorly understood. Because expression of the nuclear receptor PPARγ defines the adipose lineage, isolation of elements that control PPARγ expression in adipose precursors may lead to discovery of transcriptional regulators of early adipocyte determination. Here, we describe the identification and validation in transgenic mice of 5 highly conserved non-coding sequences from the PPARγ locus that can drive expression of a reporter gene in a manner that recapitulates the tissue-specific pattern of PPARγ expression. Surprisingly, these 5 elements appear to control PPARγ expression in adipocyte precursors that are associated with the vasculature of adipose depots, but not in mature adipocytes. Characterization of these five PPARγ regulatory sequences may enable isolation of the transcription factors that bind these cis elements and provide insight into the molecular regulation of adipose tissue expansion in normal and pathological states.