RESUMO
Abnormally high serum homocysteine levels have been associated with several disorders, including obesity, cardiovascular diseases or neurological diseases. Leptin is an anti-obesity protein and its action is mainly mediated by the activation of its Ob-R receptor in neuronal cells. The inability of leptin to induce activation of its specific signaling pathways, especially under endoplasmic reticulum stress, leads to the leptin resistance observed in obesity. The present study examined the effect of homocysteine on leptin signaling in SH-SY5Y neuroblastoma cells expressing the leptin receptor Ob-Rb. Phosphorylation of the signal transducer and activator of transcription (STAT3) and leptin-induced STAT3 transcriptional activity were significantly inhibited by homocysteine treatment. These effects may be specific to homocysteine and to the leptin pathway, as other homocysteine-related compounds, namely methionine and cysteine, have weak effect on leptin-induced inhibition of STAT3 phosphorylation, and homocysteine has no impact on IL-6-induced activation of STAT3. The direct effect of homocysteine on leptin-induced Ob-R activation, analyzed by Ob-R BRET biosensor to monitor Ob-R oligomerization and conformational change, suggested that homocysteine treatment does not affect early events of leptin-induced Ob-R activation. Instead, we found that, unlike methionine or cysteine, homocysteine increases the expression of the endoplasmic reticulum (ER) stress response gene, a homocysteine-sensitive ER resident protein. These results suggest that homocysteine may induce neuronal resistance to leptin by suppressing STAT3 phosphorylation downstream of the leptin receptor via ER stress.
Assuntos
Leptina , Neuroblastoma , Humanos , Leptina/metabolismo , Receptores para Leptina/genética , Homocisteína/farmacologia , Cisteína/farmacologia , Estresse do Retículo Endoplasmático , Fator de Transcrição STAT3/metabolismo , Obesidade/metabolismo , Metionina/farmacologiaRESUMO
Metabolic health depends on the brain's ability to control food intake and nutrient use versus storage, processes that require peripheral signals such as the adipocyte-derived hormone, leptin, to cross brain barriers and mobilize regulatory circuits. We have previously shown that hypothalamic tanycytes shuttle leptin into the brain to reach target neurons. Here, using multiple complementary models, we show that tanycytes express functional leptin receptor (LepR), respond to leptin by triggering Ca2+ waves and target protein phosphorylation, and that their transcytotic transport of leptin requires the activation of a LepR-EGFR complex by leptin and EGF sequentially. Selective deletion of LepR in tanycytes blocks leptin entry into the brain, inducing not only increased food intake and lipogenesis but also glucose intolerance through attenuated insulin secretion by pancreatic ß-cells, possibly via altered sympathetic nervous tone. Tanycytic LepRb-EGFR-mediated transport of leptin could thus be crucial to the pathophysiology of diabetes in addition to obesity, with therapeutic implications.
Assuntos
Encéfalo/metabolismo , Células Ependimogliais/metabolismo , Receptores ErbB/metabolismo , Leptina/metabolismo , Metabolismo dos Lipídeos , Pâncreas/metabolismo , Receptores para Leptina/metabolismo , Diabetes Mellitus/etiologia , Diabetes Mellitus/metabolismo , Metabolismo Energético , Células Secretoras de Insulina/metabolismo , FosforilaçãoRESUMO
Leptin links body energy stores to high energy demanding processes like reproduction and immunity. Based on leptin's role in autoimmune diseases and cancer, several leptin and leptin receptor (LR) antagonists have been developed, but these intrinsically lead to unwanted weight gain. Here, we report on the uncoupling of leptin's metabolic and immune functions based on the cross talk with the epidermal growth factor receptor (EGFR). We show that both receptors spontaneously interact and, remarkably, that this complex can partially overrule the lack of LR activation by a leptin antagonistic mutein. Moreover, this leptin mutant induces EGFR phosphorylation comparable to wild-type leptin. Exploiting this non-canonical leptin signalling pathway, we identified a camelid single-domain antibody that selectively inhibits this LR-EGFR cross talk without interfering with homotypic LR signalling. Administration in vivo showed that this single-domain antibody did not interfere with leptin's metabolic functions, but could reverse the leptin-driven protection against starvation-induced thymic and splenic atrophy. These findings offer new opportunities for the design and clinical application of selective leptin and LR antagonists that avoid unwanted metabolic side effects.
Assuntos
Leptina/imunologia , Leptina/metabolismo , Receptores para Leptina/antagonistas & inibidores , Receptores para Leptina/metabolismo , Anticorpos de Domínio Único/farmacologia , Animais , Camelídeos Americanos/imunologia , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Células HEK293 , Humanos , Leptina/genética , Ligantes , Camundongos Endogâmicos C57BL , Mutação , Ligação Proteica/efeitos dos fármacos , Receptor Cross-Talk/efeitos dos fármacos , Receptores para Leptina/genética , Transdução de SinaisRESUMO
Neuroplin 1 (NRP1), a transmembrane protein interacting with Vascular Endothelial Growth Factor VEGF-A165 (called here VEGF165) and the tyrosine kinase Receptor 2 (VEGFR2) promote angiogenesis and vascular homeostasis. In a pathophysiological context, several studies suggested that VEGFR2 and NRP1 mediate tumor development and progression. Given the involvement of the VEGF165 network in promoting tumor angiogenesis, NRP1, VEGFR2 and VEGF165 have been identified as targets for anti-angiogenic therapy. No binding assay exists to monitor specifically the binding of VEGF165 to the VEGFR2/NRP1 complex in intact cells. We established a binding assay based on the homogenous time-resolved fluorescence (HTRF®) technology. This unique binding assay enables to assess the interaction of VEGF165 with VEGFR2 or NRP1 within the VEGFR2/NRP1 complex. Ligand binding saturation experiments revealed that VEGF165 binds the VEGFR2/NRP1 complex at the cell surface with a ten to twenty-fold higher affinity compared to SNAP-VEGFR2 or SNAP-NRP1 receptors alone not engaged in the heteromeric complex. The assay allows characterizing the impact of NRP1 ligands on VEGF165 to the complex. It shows high specificity, reproducibility and robustness, making it compatible with high throughput screening (HTS) applications for identifying new VEGF165 antagonists selective for NRP1 or the VEGFR2/NRP1 complex.
Assuntos
Neuropilina-1/metabolismo , Proteínas Semelhantes à Proteína de Ligação a TATA-Box/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células HEK293 , Células HeLa , Humanos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Transforming growth factor-ß (TGFß) signaling is initiated by the type I, II TGFß receptor (TßRI/TßRII) complex. Here we report the formation of an alternative complex between TßRI and the orphan GPR50, belonging to the G protein-coupled receptor super-family. The interaction of GPR50 with TßRI induces spontaneous TßRI-dependent Smad and non-Smad signaling by stabilizing the active TßRI conformation and competing for the binding of the negative regulator FKBP12 to TßRI. GPR50 overexpression in MDA-MB-231 cells mimics the anti-proliferative effect of TßRI and decreases tumor growth in a xenograft mouse model. Inversely, targeted deletion of GPR50 in the MMTV/Neu spontaneous mammary cancer model shows decreased survival after tumor onset and increased tumor growth. Low GPR50 expression is associated with poor survival prognosis in human breast cancer irrespective of the breast cancer subtype. This describes a previously unappreciated spontaneous TGFß-independent activation mode of TßRI and identifies GPR50 as a TßRI co-receptor with potential impact on cancer development.
Assuntos
Neoplasias Mamárias Animais/prevenção & controle , Proteínas do Tecido Nervoso/fisiologia , Receptor do Fator de Crescimento Transformador beta Tipo I/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Endossomos/metabolismo , Feminino , Perfilação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Neoplasias Mamárias Animais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Proteínas do Tecido Nervoso/genética , Análise de Sequência com Séries de Oligonucleotídeos , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais , Proteínas Smad/metabolismo , Proteína 1A de Ligação a Tacrolimo/metabolismoRESUMO
The hypothalamic arcuate nucleus (ARC) is a major integration center for energy and glucose homeostasis that responds to leptin. Resistance to leptin in the ARC is an important component of the development of obesity and type 2 diabetes. Recently, we showed that Endospanin1 (Endo1) is a negative regulator of the leptin receptor (OBR) that interacts with OBR and retains the receptor inside the cell, leading to a decreased activation of the anorectic STAT3 pathway. Endo1 is up-regulated in the ARC of high fat diet (HFD)-fed mice, and its silencing in the ARC of lean and obese mice prevents and reverses the development of obesity. OBJECTIVE: Herein we investigated whether decreased Endo1 expression in the hypothalamic ARC, associated with reduced obesity, could also ameliorate glucose homeostasis accordingly. METHODS: We studied glucose homeostasis in lean or obese mice silenced for Endo1 in the ARC via stereotactic injection of shRNA-expressing lentiviral vectors. RESULTS: We observed that despite being leaner, Endo1-silenced mice showed impaired glucose homeostasis on HFD. Mechanistically, we show that Endo1 interacts with p85, the regulatory subunit of PI3K, and mediates leptin-induced PI3K activation. CONCLUSIONS: Our results thus define Endo1 as an important hypothalamic integrator of leptin signaling, and its silencing differentially regulates the OBR-dependent functions.
Assuntos
Proteínas de Transporte/metabolismo , Obesidade/metabolismo , Receptores para Leptina/metabolismo , Animais , Núcleo Arqueado do Hipotálamo/metabolismo , Peso Corporal/fisiologia , Proteínas de Transporte/fisiologia , Diabetes Mellitus Tipo 2/metabolismo , Dieta Hiperlipídica/efeitos adversos , Glucose/metabolismo , Homeostase/efeitos dos fármacos , Hipotálamo/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Leptina/metabolismo , Leptina/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Receptores para Leptina/fisiologia , Fator de Transcrição STAT3/efeitos dos fármacos , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Recent evidence has highlighted the role of histamine in inflammation. Since this monoamine has also been strongly implicated in the pathogenesis of type-1 diabetes, we assessed its effect in the nonobese diabetic (NOD) mouse model. To this end, we used mice (inactivated) knocked out for the gene encoding histidine decarboxylase, the unique histamine-forming enzyme, backcrossed on a NOD genetic background. We found that the lack of endogenous histamine in NOD HDC(-/-) mice decreased the incidence of diabetes in relation to their wild-type counterpart. Whereas the proportion of regulatory T and myeloid-derived suppressive cells was similar in both strains, histamine deficiency was associated with increased levels of immature macrophages, as compared with wild-type NOD mice. Concerning the cytokine pattern, we found a decrease in circulating IL-12 and IFN-γ in HDC(-/-) mice, while IL-6 or leptin remained unchanged, suggesting that histamine primarily modulates the inflammatory environment. Paradoxically, exogenous histamine given to NOD HDC(-/-) mice provided also protection against T1D. Our study supports the notion that histamine is involved in the pathogenesis of diabetes, thus providing additional evidence for its role in the regulation of the immune response.
Assuntos
Citocinas/imunologia , Diabetes Mellitus Tipo 1/genética , Histamina/imunologia , Histidina Descarboxilase/genética , Camundongos Endogâmicos NOD/genética , Animais , Diabetes Mellitus Tipo 1/imunologia , Interferon gama/imunologia , Interleucina-12/imunologia , Interleucina-6/imunologia , Leptina/imunologia , Camundongos , Camundongos Endogâmicos NOD/imunologia , Camundongos Knockout , Linfócitos T Reguladores/imunologiaRESUMO
The pleiotropic cytokine hormone leptin, by activating its receptor OB-R, plays a major role in many biological processes, including energy homeostasis, immune function, and cell survival and proliferation. Abnormal leptin action is associated with obesity, autoimmune diseases, and cancer. The pharmacological characterization of OB-R and the development of synthetic OB-R ligands are still in their infancy because currently available binding assays are not compatible with ligand saturation binding experiments and high-throughput screening (HTS) approaches. We have developed here a novel homogeneous time-resolved fluorescence-based binding assay that overcomes these limitations. In this assay, fluorescently labeled leptin or leptin antagonist binds to the SNAP-tagged OB-R covalently labeled with terbium cryptate (Tb). Successful binding is monitored by measuring the energy transfer between the Tb energy donor and the fluorescently labeled leptin energy acceptor. Ligand binding saturation experiments revealed high-affinity dissociation constants in the subnanomolar range with an excellent signal-to-noise ratio. The assay performed in a 384-well format shows high specificity and reproducibility, making it perfectly compatible with HTS applications to identify new OB-R agonists or antagonists. In addition, fluorescently labeled leptin and SNAP-tagged OB-R will be valuable tools for monitoring leptin and OB-R trafficking in cells and tissues.
Assuntos
Fluorescência , Receptores para Leptina/metabolismo , Fosfatase Alcalina/química , Fosfatase Alcalina/metabolismo , Sítios de Ligação/efeitos dos fármacos , Células Cultivadas , Células HEK293 , Ensaios de Triagem em Larga Escala , Antagonistas de Hormônios/química , Antagonistas de Hormônios/metabolismo , Antagonistas de Hormônios/farmacologia , Humanos , Leptina/antagonistas & inibidores , Leptina/química , Leptina/metabolismo , Ligantes , Ligação Proteica , Receptores para Leptina/análise , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
Natural killer (NK) cells play a vital role in the detection and destruction of virally infected and tumor cells during innate immune responses. The highly polymorphic Ly49 family of NK receptors regulates NK cell function by sensing major histocompatibility complex class I (MHC-I) molecules on target cells. Despite the determination of two Ly49-MHC-I complex structures, the molecular features of Ly49 receptors that confer specificity for particular MHC-I alleles have not been identified. To understand the functional architecture of Ly49-binding sites, we determined the crystal structures of Ly49C and Ly49G and completed refinement of the Ly49C-H-2K(b) complex. This information, combined with mutational analysis of Ly49A, permitted a structure-based classification of Ly49s that we used to dissect the binding site into three distinct regions, each having different roles in MHC recognition. One region, located at the center of the binding site, has a similar structure across the Ly49 family and mediates conserved interactions with MHC-I that contribute most to binding. However, the preference of individual Ly49s for particular MHC-I molecules is governed by two regions that flank the central region and are structurally more variable. One of the flanking regions divides Ly49s into those that recognize both H-2D and H-2K versus only H-2D ligands, whereas the other discriminates among H-2D or H-2K alleles. The modular design of Ly49-binding sites provides a framework for predicting the MHC-binding specificity of Ly49s that have not been characterized experimentally.
Assuntos
Antígenos Ly/química , Regulação da Expressão Gênica , Antígenos de Histocompatibilidade Classe I/química , Células Matadoras Naturais/metabolismo , Lectinas Tipo C/química , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Dimerização , Humanos , Cinética , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , Ligação Proteica , Receptores Semelhantes a Lectina de Células NK , Homologia de Sequência de AminoácidosRESUMO
Natural killer (NK) cells play a vital role in the detection and elimination of virally infected and tumor cells. The Ly49 family of NK receptors regulates NK cell function by sensing major histocompatibility complex (MHC) class I molecules on target cells. Previous crystal studies revealed that the Ly49A homodimer binds one MHC molecule in an asymmetric interaction, whereas the Ly49C homodimer binds two MHC in a symmetrical fashion. Moreover, the bound receptors adopt distinctly different homodimeric forms: a "closed state" for Ly49A and an "open state" for Ly49C. Steric clashes between MHC molecules would preclude the closed Ly49A dimer from engaging two MHC in the manner of the open Ly49C dimer. To determine whether individual Ly49 receptors can undergo a conformational switch enabling them to bind MHC in different ways, we carried out a solution NMR study of unbound Ly49A, aided by dipolar coupling technology. This study reveals that, in solution, unligated Ly49A adopts a symmetric, open-state, homodimer conformation similar to that seen previously for Ly49C. Hence, Ly49A can assume both closed and open states. To address whether the Ly49A dimer can bind two MHC molecules in solution, besides the binding of one MHC observed in the crystal, we carried out analytical ultracentrifugation experiments. Velocity sedimentation demonstrates that the Ly49A dimer can engage two MHC molecules in solution, in agreement with NMR results showing that unbound Ly49A exists predominantly in the open state.
Assuntos
Antígenos Ly/química , Genes MHC Classe I , Lectinas Tipo C/química , Estrutura Quaternária de Proteína , Animais , Antígenos Ly/genética , Antígenos Ly/metabolismo , Dimerização , Ligação de Hidrogênio , Células Matadoras Naturais/imunologia , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Camundongos , Modelos Moleculares , Subfamília A de Receptores Semelhantes a Lectina de Células NK , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína , Receptores Semelhantes a Lectina de Células NKRESUMO
The structure of the natural cytotoxicity receptor NKp44, described in this issue of Structure, adds to our rapidly expanding knowledge of the structure of natural killer cell receptors, which play a key role in the elimination of virally infected and tumor cells during innate immune responses.