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Hormone absence or inactivity is common in congenital disease, but hormone antagonism remains controversial. Here, we characterize two novel homozygous leptin variants that yielded antagonistic proteins in two unrelated children with intense hyperphagia, severe obesity, and high circulating levels of leptin. Both variants bind to the leptin receptor but trigger marginal, if any, signaling. In the presence of nonvariant leptin, the variants act as competitive antagonists. Thus, treatment with recombinant leptin was initiated at high doses, which were gradually lowered. Both patients eventually attained near-normal weight. Antidrug antibodies developed in the patients, although they had no apparent effect on efficacy. No severe adverse events were observed. (Funded by the German Research Foundation and others.).
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Leptina , Obesidade Mórbida , Criança , Humanos , Anticorpos , Homozigoto , Leptina/genética , Obesidade Mórbida/genética , Transdução de SinaisRESUMO
SIGNIFICANCE STATEMENT: G protein-coupled receptor kinase 4 (GRK4) regulates renal sodium and water reabsorption. Although GRK4 variants with elevated kinase activity have been associated with salt-sensitive or essential hypertension, this association has been inconsistent among different study populations. In addition, studies elucidating how GRK4 may modulate cellular signaling are sparse. In an analysis of how GRK4 affects the developing kidney, the authors found that GRK4 modulates mammalian target of rapamycin (mTOR) signaling. Loss of GRK4 in embryonic zebrafish causes kidney dysfunction and glomerular cysts. Moreover, GRK4 depletion in zebrafish and cellular mammalian models results in elongated cilia. Rescue experiments suggest that hypertension in carriers of GRK4 variants may not be explained solely by kinase hyperactivity; instead, elevated mTOR signaling may be the underlying cause. BACKGROUND: G protein-coupled receptor kinase 4 (GRK4) is considered a central regulator of blood pressure through phosphorylation of renal dopaminergic receptors and subsequent modulation of sodium excretion. Several nonsynonymous genetic variants of GRK4 have been only partially linked to hypertension, although these variants demonstrate elevated kinase activity. However, some evidence suggests that function of GRK4 variants may involve more than regulation of dopaminergic receptors alone. Little is known about the effects of GRK4 on cellular signaling, and it is also unclear whether or how altered GRK4 function might affect kidney development. METHODS: To better understand the effect of GRK4 variants on the functionality of GRK4 and GRK4's actions in cellular signaling during kidney development, we studied zebrafish, human cells, and a murine kidney spheroid model. RESULTS: Zebrafish depleted of Grk4 develop impaired glomerular filtration, generalized edema, glomerular cysts, pronephric dilatation, and expansion of kidney cilia. In human fibroblasts and in a kidney spheroid model, GRK4 knockdown produced elongated primary cilia. Reconstitution with human wild-type GRK4 partially rescues these phenotypes. We found that kinase activity is dispensable because kinase-dead GRK4 (altered GRK4 that cannot result in phosphorylation of the targeted protein) prevented cyst formation and restored normal ciliogenesis in all tested models. Hypertension-associated genetic variants of GRK4 fail to rescue any of the observed phenotypes, suggesting a receptor-independent mechanism. Instead, we discovered unrestrained mammalian target of rapamycin signaling as an underlying cause. CONCLUSIONS: These findings identify GRK4 as novel regulator of cilia and of kidney development independent of GRK4's kinase function and provide evidence that the GRK4 variants believed to act as hyperactive kinases are dysfunctional for normal ciliogenesis.
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Cistos , Hipertensão , Humanos , Animais , Camundongos , Fosforilação , Cílios/metabolismo , Peixe-Zebra/metabolismo , Rim/metabolismo , Sódio/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Cistos/metabolismo , Mamíferos/metabolismoRESUMO
Mutations in the gene encoding leptin (LEP) typically lead to an absence of circulating leptin and to extreme obesity. We describe a 2-year-old boy with early-onset extreme obesity due to a novel homozygous transversion (c.298GâT) in LEP, leading to a change from aspartic acid to tyrosine at amino acid position 100 (p.D100Y) and high immunoreactive levels of leptin. Overexpression studies confirmed that the mutant protein is secreted but neither binds to nor activates the leptin receptor. The mutant protein failed to reduce food intake and body weight in leptin-deficient ob/ob mice. Treatment of the patient with recombinant human leptin (metreleptin) rapidly normalized eating behavior and resulted in weight loss.
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Leptina/análogos & derivados , Leptina/genética , Mutação , Obesidade/genética , Idade de Início , Animais , Índice de Massa Corporal , Células Cultivadas , Pré-Escolar , Comportamento Alimentar/efeitos dos fármacos , Feminino , Humanos , Leptina/deficiência , Leptina/metabolismo , Leptina/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos , Obesidade/tratamento farmacológico , Receptores para Leptina/metabolismo , Análise de Sequência de DNARESUMO
In multiple sclerosis (MS), neurons in addition to inflammatory cells are now considered to mediate disease origin and progression. So far, molecular and cellular mechanisms of neuronal MS contributions are poorly understood. Herein we analyzed whether neuron-restricted signaling by the neuroprotective transcription factor serum response factor (SRF) modulates de- and remyelination in a rodent MS model. In the mouse cuprizone model, neuron- (Srf (flox/flox;CaMKCreERT2)) but not glia-specific (Srf (flox/flox;PlpCreERT2)) SRF depletion impaired demyelination suggesting impaired debris clearance by astrocytes and microglia. This supports an important role of SRF expression in neurons but not oligodendrocytes in de- and remyelination. During remyelination, NG2- and OLIG2-positive cells of the oligodendrocyte lineage as well as de novo mRNA synthesis of myelin genes were also reduced in neuron-specific Srf mutants. Using the stripe assay, we demonstrate that cortices of cuprizone-fed wild-type mice elicited astrocyte and microglia activation whereas this was abrogated in cuprizone-fed neuron-specific Srf mutants. We identified CCL chemokines (e.g. CCL2) as neuron-derived SRF-regulated paracrine signals rescuing immune cell activation upon neuronal SRF deletion. In summary, we uncovered important roles of neurons and neuronally expressed SRF in MS associated de- and remyelination.
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Esclerose Múltipla/fisiopatologia , Bainha de Mielina/fisiologia , Neurônios/metabolismo , Fator de Resposta Sérica/metabolismo , Animais , Astrócitos/patologia , Astrócitos/fisiologia , Células Cultivadas , Quimiocina CCL2/metabolismo , Cuprizona , Modelos Animais de Doenças , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/patologia , Microglia/fisiologia , Esclerose Múltipla/patologia , Bainha de Mielina/metabolismo , Bainha de Mielina/patologia , Neurônios/patologia , Oligodendroglia/patologia , Oligodendroglia/fisiologia , RNA Mensageiro/metabolismo , Fator de Resposta Sérica/genéticaRESUMO
PURPOSE: Bi-allelic pathogenic leptin gene variants cause severe early onset obesity usually associated with low or undetectable circulating leptin levels. Recently, variants have been described resulting in secreted mutant forms of the hormone leptin with either biologically inactive or antagonistic properties. METHODS: We conducted a systematic literature research supplemented by unpublished data from patients at our center as well as new in vitro analyses to provide a systematic classification of congenital leptin deficiency based on the molecular and functional characteristics of the underlying leptin variants and investigated the correlation of disease subtype with severity of the clinical phenotype. RESULTS: A total of 28 distinct homozygous leptin variants were identified in 148 patients. The identified variants can be divided into three different subtypes of congenital leptin deficiency: classical hormone deficiency (21 variants in 128 patients), biologically inactive hormone (3 variants in 12 patients) and antagonistic hormone (3 variants in 7 patients). Only 1 variant (n=1 patient) remained unclassified. Patients with biological inactive leptin have a higher percentage of 95th BMI percentile (%BMIp95) compared to patients with classical hormone deficiency. While patients with both classical hormone deficiency and biological inactive hormone can be treated with the same starting dose of metreleptin, patients with antagonistic hormone need a variant-tailored treatment approach to overcome the antagonistic properties of the variant leptin. MAIN CONCLUSIONS: Categorization of leptin variants based on molecular and functional characteristics helps to determine the most adequate approach to treatment of patients with congenital leptin deficiency.
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Cystic fibrosis (CF) lung disease severity is largely independent on the CF transmembrane conductance regulator (CFTR) genotype, indicating the contribution of genetic modifiers. The chemokine receptors CXCR1 and CXCR2 have been found to play essential roles in the pathogenesis of CF lung disease. Here, we determine whether genetic variation of CXCR1 and CXCR2 influences CF lung disease severity. Genomic DNA of CF patients in Germany (n = 442) was analysed for common variations in CXCR1 and CXCR2 using a single-nucleotide polymorphism (SNP) tagging approach. Associations of CXCR1 and CXCR2 SNPs and haplotypes with CF lung disease severity, CXCR1 and CXCR2 expression, and neutrophil effector functions were assessed. Four SNPs in CXCR1 and three in CXCR2 strongly correlated with age-adjusted lung function in CF patients. SNPs comprising haplotypes CXCR1_Ha and CXCR2_Ha were in high linkage disequilibrium and patients heterozygous for the CXCR1-2 haplotype cluster (CXCR1-2_Ha) had lower lung function compared with patients with homozygous wild-type alleles (forced expiratory volume in 1 s ≤ 70% predicted, OR 7.24; p = 2.30 × 10(-5)). CF patients carrying CXCR1-2_Ha showed decreased CXCR1 combined with increased CXCR2 mRNA and protein expression, and displayed disturbed antibacterial effector functions. CXCR1 and CXCR2 genotypes modulate lung function and antibacterial host defence in CF lung disease.
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Fibrose Cística/imunologia , Haplótipos/genética , Receptores de Interleucina-8A/genética , Receptores de Interleucina-8B/genética , Adolescente , Adulto , Criança , Fibrose Cística/microbiologia , Feminino , Variação Genética , Alemanha , Humanos , Desequilíbrio de Ligação/genética , Pulmão/imunologia , Pulmão/fisiologia , Masculino , Neutrófilos/imunologia , Neutrófilos/microbiologia , Pneumonia Bacteriana/genética , Pneumonia Bacteriana/imunologia , Polimorfismo de Nucleotídeo Único , Receptores de Interleucina-8A/biossíntese , Receptores de Interleucina-8A/imunologia , Receptores de Interleucina-8B/biossíntese , Receptores de Interleucina-8B/imunologia , Índice de Gravidade de Doença , Adulto JovemRESUMO
Autoinflammatory diseases are a heterogenous group of disorders defined by fever and systemic inflammation suggesting involvement of genes regulating innate immune responses. Patients with homozygous loss-of-function variants in the OTU-deubiquitinase OTULIN suffer from neonatal-onset OTULIN-related autoinflammatory syndrome (ORAS) characterized by fever, panniculitis, diarrhea, and arthritis. Here, we describe an atypical form of ORAS with distinct clinical manifestation of the disease caused by two new compound heterozygous variants (c.258G>A (p.M86I)/c.500G>C (p.W167S)) in the OTULIN gene in a 7-year-old affected by a life-threatening autoinflammatory episode with sterile abscess formation. On the molecular level, we find binding of OTULIN to linear ubiquitin to be compromised by both variants; however, protein stability and catalytic activity is most affected by OTULIN variant p.W167S. These molecular changes together lead to increased levels of linear ubiquitin linkages in patient-derived cells triggering the disease. Our data indicate that the spectrum of ORAS patients is more diverse than previously thought and, thus, supposedly asymptomatic individuals might also be affected. Based on our results, we propose to subdivide the ORAS into classical and atypical entities.
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Endopeptidases , Doenças Hereditárias Autoinflamatórias/genética , Ubiquitina , Criança , Endopeptidases/genética , Humanos , Recém-Nascido , Inflamação/genética , Ubiquitina/metabolismoRESUMO
We performed in vitro co-cultivation experiments with primary human nasal epithelial cells (HNEC) and isolated lymphocytes to investigate whether reactive formaldehyde (FA) can be passed on from nasal epithelial cells (site of first contact) to lymphocytes located in close proximity and induce DNA damage in these cells. A modified comet assay was used as a sensitive method for the detection of FA-induced DNA-protein cross links (DPX) because DPX are the most relevant type of FA-induced DNA damage. Our results clearly indicate that co-cultivation of lymphocytes with HNEC exposed to FA for 1 h causes a concentration-related induction of DPX in lymphocytes when co-cultivation takes place in the exposure medium. However, when the exposure medium is changed after FA treatment of HNEC and before lymphocytes are added, no induction of DPX is measured in lymphocytes even after exposure of HNEC to high FA concentrations (300 microM) and extended co-cultivation (4 h). Direct measurement of FA in the cell culture medium by a sensitive fluorescent detection kit indicated that FA is actually not released even from highly exposed cells into the cell culture medium. These results suggest that FA that has entered nasal epithelial cells is not released and does not damage other cells in close proximity to the epithelial cells. If these results also apply to the in vivo situation, FA would only be genotoxic towards directly exposed cells (site of first contact) and there should be no significant delivery of inhaled FA to other cells and distant sites. Our results do not support a recently proposed hypothetic mechanism for FA-induced leukaemia by damaging circulating haematopoietic stem cells or haematopoietic progenitor cells in nasal passages, which then travel to the bone marrow and become initiated leukaemic stem cells.
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Dano ao DNA , Células Epiteliais/efeitos dos fármacos , Formaldeído/toxicidade , Linfócitos/efeitos dos fármacos , Mutagênicos/toxicidade , Mucosa Nasal/citologia , Linhagem Celular , Técnicas de Cocultura , Ensaio Cometa , Células Epiteliais/metabolismo , Humanos , Linfócitos/metabolismo , Mucosa Nasal/efeitos dos fármacosRESUMO
Expression of the human cytomegalovirus (HCMV)-encoded chemokine receptor homologue pUS28 in mammalian cells results in ligand-dependent and -independent changes in the activity of multiple cellular signal transduction pathways. The ligand-dependent signalling activity of pUS28 has been shown to be predominantly mediated by heterotrimeric G proteins of the G(i/o) and G(12/13) subfamilies. Ligand-independent constitutive activity of pUS28 causing stimulation of inositol phosphate formation has been correlated with the coupling of pUS28 to G proteins of the G(q) family. It is well known that activation of G(q) proteins by cell surface receptors is coupled to activation of the Rho GTPase RhoA. Activated RhoA regulates numerous cellular functions, including the activity of the transcription factor serum response factor (SRF). The marked activation of G(q) proteins by pUS28 in transfected and HCMV-infected cells prompted us to investigate its effect on SRF activity. The results presented herein demonstrate that expression of pUS28 in COS-7 cells caused a vigorous induction of SRF activity. This effect was observed in the absence of chemokines known to interact with pUS28, and was specifically mediated by endogenous G(q) and/or G(11) as well as RhoA and/or a closely related Rho GTPase. The stimulatory effect of pUS28 and Galpha(q/11) was independent of phospholipase C-beta (PLCbeta) activation and was markedly sensitive to inhibition by wild-type, but not by constitutively active Galpha(16), thus identifying Galpha(16) as a modulator of Galpha(q/11) function likely to act by competing with Galpha(q/11) for and thus uncoupling Galpha(q/11) from activation by pUS28.
Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Receptores de Quimiocinas/metabolismo , Fator de Resposta Sérica/metabolismo , Transdução de Sinais , Proteínas Virais/metabolismo , Animais , Células COS , Chlorocebus aethiops , Fosfolipase C beta/metabolismo , Ativação Transcricional , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
In man, two CC chemokine receptor isoforms, CCR2a and CCR2b, are present that belong to the rhodopsin-like G protein-coupled receptor family, and couple to Gi and Gq family members. The CCR2 receptors are known to regulate canonical functions of chemokines such as directed migration of leukocytes, and to potentially control non-canonical functions such as differentiation, proliferation, and gene transcription of immune and non-immune cells. We recently reported on the activation of phospholipase C isoenzymes and RhoA GTPases by coupling of the two CCR2 receptors to members of the Gq family, in particular Gαq and Gα14. So far little is known about the structural requirements for the CCR2/Gq/14 interaction. Interestingly, the CCR2 receptor isoforms are identical up to arginine 313 (R313) that is part of the putative 8th helix in CCR2 receptors, and the 8th helix has been implicated in the interaction of rhodopsin-like G protein-coupled receptors with Gαq. In the present work we describe that the 8th helix of both CCR2a and CCR2b is critically involved in selectively activating Gαq/14-regulated signaling. Refined analysis using various CCR2a and CCR2b mutants and analyzing their cellular signaling, e.g. ligand-dependent (i) activation of phospholipase C isoenzymes, (ii) stimulation of serum response factor-mediated gene transcription, (iii) activation of mitogen-activated protein kinases, (iv) internalization, and (v) changes in intracellular calcium concentrations, identified arginine 313 within the amino terminal portion of helix 8 to play a role for the agonist-mediated conformational changes and the formation of a Gαq/14 binding surface. We show that R313 determines Gαq/14 protein-dependent but not Gi protein-dependent cellular signaling, and plays no role in Gq/Gi-independent receptor internalization, indicating a role of R313 in biased signaling of CCR2 receptors.
Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Receptores CCR2/metabolismo , Animais , Arginina/análise , Arginina/metabolismo , Células COS , Chlorocebus aethiops , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/química , Células HEK293 , Humanos , Conformação Proteica em alfa-Hélice , Mapas de Interação de Proteínas , Receptores CCR2/química , Transdução de SinaisRESUMO
Several case series of extreme early-onset obesity due to mutations in the human leptin receptor (LEPR) gene have been reported. In this review we summarize published functional and phenotypic data on mutations in the human LEPR gene causing severe early-onset obesity. Additionally, we included data on six new cases from our obesity center. Literature research was performed using PubMed and OMIM. Functional relevance of mutations was estimated based on reported functional analysis, mutation size, and location, as well as phenotypic characteristics of affected patients. We identified 57 cases with 38 distinct LEPR mutations. We found severe early-onset obesity, hyperphagia, and hypogonadotropic hypogonadism as cardinal features of a complete loss of LEPR function. Other features, for example, metabolic disorders and recurring infections, were variable in manifestation. Obesity degree or other manifestations did not aggregate by genotype. Few patients underwent bariatric surgery with variable success. Most mutations occurred in the fibronectin III and cytokine receptor homology II domains, whereas none was found in cytoplasmic domain. In silico data were available for 25 mutations and in vitro data were available for four mutations, revealing residual activity in one case. By assessing provided information on the clinical phenotype, functional analysis, and character of the 38 mutations, we assume residual LEPR activity for five additional mutations. Functional in vitro analysis is necessary to confirm this assumption.
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The endocannabinoid system (ECS) consists particularly of cannabinoid receptors 1 and 2 (CB1 and CB2), their endogenous ligands, and enzymes that synthesize and degrade their ligands. It acts in a variety of organs and disease states ranging from cancer progression over neuropathic pain to neurodegeneration. Protein components engaged in the signaling, trafficking, and homeostasis machinery of the G-protein coupled CB2, are however largely unknown. It is therefore important to identify further interaction partners to better understand CB2 receptor functions in physiology and pathophysiology. For this purpose, we used an affinity purification and mass spectrometry-based proteomics approach of Strep-HA-CB2 receptor in HEK293 cells. After subtraction of background interactions and protein frequency library assessment we could identify 83 proteins that were classified by the identification of minimally 2 unique peptides as highly probable interactors. A functional protein association network analysis obtained an interaction network with a significant enrichment of proteins functionally involved in protein metabolic process, in endoplasmic reticulum, response to stress but also in lipid metabolism and membrane organization. The network especially contains proteins involved in biosynthesis and trafficking like calnexin, Sec61A, tubulin chains TUBA1C and TUBB2B, TMED2, and TMED10. Six proteins that were only expressed in stable CB2 expressing cells were DHC24, DHRS7, GGT7, HECD3, KIAA2013, and PLS1. To exemplify the validity of our approach, we chose a candidate having a relatively low number of edges in the network to increase the likelihood of a direct protein interaction with CB2 and focused on the scaffold/phagosomal protein p62/SQSTM1. Indeed, we independently confirmed the interaction by co-immunoprecipitation and immunocytochemical colocalization studies. 3D reconstruction of confocal images furthermore showed CB2 localization in close proximity to p62 positive vesicles at the cell membrane. In summary, we provide a comprehensive repository of the CB2 interactome in HEK293 cells identified by a systematic unbiased approach, which can be used in future experiments to decipher the signaling and trafficking complex of this cannabinoid receptor. Future studies will have to analyze the exact mechanism of the p62-CB2 interaction as well as its putative role in disease pathophysiology.
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Most cortical interneurons are generated in the subpallial ganglionic eminences and migrate tangentially to their final destinations in the neocortex. Within the cortex, interneurons follow mainly stereotype routes in the subventricular zone/intermediate zone (SVZ/IZ) and in the marginal zone. It has been suggested that interactions between invading interneurons and locally generated projection neurons are implicated in the temporal and spatial regulation of the invasion process. However, so far experimental evidence for such interactions is lacking. We show here that the chemokine stromal-derived factor 1 (SDF-1; CXCL12) is expressed in the main invasion route for cortical interneurons in the SVZ/IZ. Most SDF-1-positive cells are proliferating and express the homeodomain transcription factors Cux1 and Cux2. Using MASH-1 mutant mice in concert with the interneuron marker DLX, we exclude that interneurons themselves produce the chemokine in an autocrine manner. We conclude that the SDF-1-expressing cell population represents the precursors of projection neurons during their transition and amplification in the SVZ/IZ. Using mice lacking the SDF-1 receptor CXCR4 or Pax6, we demonstrate that SDF-1 expression in the cortical SVZ/IZ is essential for recognition of this pathway by interneurons. These results represent the first evidence for a molecular interaction between precursors of projection neurons and invading interneurons during corticogenesis.
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Comunicação Celular/fisiologia , Córtex Cerebral/metabolismo , Ventrículos Cerebrais/metabolismo , Quimiocinas CXC/fisiologia , Interneurônios/metabolismo , Neurônios/metabolismo , Receptores CXCR4/fisiologia , Transdução de Sinais/fisiologia , Animais , Movimento Celular/fisiologia , Córtex Cerebral/citologia , Córtex Cerebral/fisiologia , Ventrículos Cerebrais/citologia , Ventrículos Cerebrais/fisiologia , Quimiocina CXCL12 , Quimiocinas CXC/deficiência , Quimiocinas CXC/genética , Interneurônios/citologia , Interneurônios/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Vias Neurais/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Receptores CXCR4/deficiência , Receptores CXCR4/genética , Células-Tronco/citologia , Células-Tronco/metabolismo , Células-Tronco/fisiologiaRESUMO
Two distinct genes are present in the human genome encoding receptors for human interleukin-8 (hCXCL8), referred to as hCXCR1 and hCXCR2. While it seems clear that orthologous genes are present in the genomes of several mammals, the existence of a gene encoding an ortholog of hCXCR1 in the mouse has thus far been controversial. We have isolated a cDNA that is highly similar to the cDNAs of hCXCR1 and hCXCR2, but is clearly distinct from the cDNA encoding mouse CXCR2 (mCXCR2). The encoded protein, designated mouse CXCR1-like (mCXCR1-like), shares 64, 57, 57, and 89% identical amino acids with hCXCR1, hCXCR2, mCXCR2, and rCXCR1-like, respectively. The gene encoding mCXCR1-like was mapped to mouse chromosome 1 and its genomic organization was determined to be very similar to the organization of the gene encoding hCXCR1. Like hCXCR1, mCXCR1-like was found to be expressed at the mRNA level in neutrophils. In addition, mRNA encoding mCXCR1-like was detected in liver, kidney, and spleen. In spleen, mCXCR1-like transcripts were predominantly found in CD4+ T cells. In liver, mCXCR1-like transcripts were identified in residual CD3+ T cells and macrophages, suggesting that mCXCR1-like may regulate inflammatory and immunological processes in the liver. When expressed as a recombinant protein, mCXCR1-like was not activated by a large panel of known CXC chemokines of human and murine origin. These findings suggest that a homolog or ortholog of hCXCR1 is expressed in the mouse to be activated by a hitherto unknown CXC chemokine of the mouse.
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Receptores de Interleucina-8A/química , Receptores de Interleucina-8A/genética , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Mapeamento Cromossômico , Cromossomos/genética , DNA Complementar/genética , Dimerização , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/química , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/genética , Humanos , Rim/química , Rim/metabolismo , Fígado/química , Fígado/metabolismo , Linfócitos/metabolismo , Camundongos , Dados de Sequência Molecular , Receptores de Interleucina-8A/análise , Receptores de Interleucina-8B/química , Receptores de Interleucina-8B/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Alinhamento de Sequência , Baço/química , Baço/metabolismo , Distribuição TecidualRESUMO
BACKGROUND: Mutations in the leptin gene (LEP) can alter the secretion or interaction of leptin with its receptor, leading to extreme early-onset obesity. The purpose of this work was to estimate the prevalence of heterozygous and homozygous mutations in the leptin gene with the help of the Exome Aggregation Consortium (ExAC) database ( http://exac.broadinstitute.org/about ). RESULTS: The ExAC database encompasses exome sequencing data from 60,706 individuals. We searched for listed leptin variants and identified 36 missense, 1 in-frame deletion, and 3 loss-of-function variants. The functional relevance of these variants was assessed by the in silico prediction tools PolyPhen-2, Sorting Intolerant from Tolerant (SIFT), and Loss-Of-Function Transcript Effect Estimator (LOFTEE). PolyPhen-2 predicted 7 of the missense variants to be probably damaging and 10 to be possibly damaging. SIFT predicted 7 of the missense variants to be deleterious. Three loss-of-function variants were predicted by LOFTEE. Excluding double counts, we can summarize 21 variants as potentially damaging. Considering the allele count, we identified 31 heterozygous but no homozygous subjects with at least probably damaging variants. In the ExAC population, the estimated prevalence of heterozygous carriers of these potentially damaging variants was 1:2000. The probability of homozygosity was 1:15,000,000. We furthermore tried to assess the functionality of ExAC-listed leptin variants by applying a knowledge-driven approach. By this approach, additional 6 of the ExAC-listed variants were considered potentially damaging, increasing the number of heterozygous subjects to 58, the prevalence of heterozygosity to 1:1050, and the probability of homozygosity to 1:4,400,000. CONCLUSION: Using exome sequencing data from ExAC, in silico prediction tools and by applying a knowledge-driven approach, we identified 27 probably damaging variants in the leptin gene of 58 heterozygous subjects. With this information, we estimate the prevalence for heterozygosity at 1:1050 corresponding to an incidence of homozygosity of 1:4,400,000 in this large pluriethnic cohort.
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Glioblastomas (GBM) are the most malignant brain tumors in humans and have a very poor prognosis. New therapeutic options are urgently needed. A novel drug, Vacquinol-1 (Vac), a quinolone derivative, displays promising properties by inducing rapid cell death in GBM but not in non-transformed tissues. Features of this type of cell death are compatible with a process termed methuosis. Here we tested Vac on a highly malignant glioma cell line observed by long-term video microscopy. Human dental-pulp stem cells (DPSCs) served as controls. A major finding was that an exogenous ATP concentration of as little as 1 µM counter regulated the Vac-induced cell death. Studies using carvacrol, an inhibitor of transient receptor potential cation channel, subfamily M, member 7 (TRPM7), demonstrated that the ATP-inducible inhibitory effect is likely to be via TRPM7. Exogenous ATP is of relevance in GBM with large necrotic areas. Our results support the use of GBM cultures with different grades of malignancy to address their sensitivity to methuosis. The video-microscopy approach presented here allows decoding of signaling pathways as well as mechanisms of chemotherapeutic resistance by long-term observation. Before implementing Vac as a novel therapeutic drug in GBM, cells from each individual patient need to be assessed for their ATP sensitivity. In summary, the current investigation supports the concept of methuosis, described as non-apoptotic cell death and a promising approach for GBM treatment. Tissue-resident ATP/necrosis may interfere with this cell-death pathway but can be overcome by a natural compound, carvacrol that even penetrates the blood-brain barrier.
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Trifosfato de Adenosina/farmacologia , Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Piperidinas/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Quinolinas/farmacologia , Canais de Cátion TRPM/metabolismo , Neoplasias Encefálicas/tratamento farmacológico , Caspase 3/metabolismo , Caspase 7/metabolismo , Morte Celular , Linhagem Celular Tumoral , Cimenos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Humanos , Monoterpenos/farmacologiaRESUMO
CONTEXT AND AIMS: Functional leptin deficiency is characterized by high levels of circulating immunoreactive leptin (irLep), but a reduced bioactivity of the hormone due to defective receptor binding. As a result of the fact that affected patients can be successfully treated with metreleptin, it was aimed to develop and validate a diagnostic tool to detect functional leptin deficiency. METHODS: An immunoassay capable of recognizing the functionally relevant receptor-binding complex with leptin was developed (bioLep). The analytical quality of bioLep was validated and compared to a conventional assay for immune-reactive leptin (irLep). Its clinical relevance was evaluated in a cohort of lean and obese children and adults as well as in children diagnosed with functional leptin deficiency and their parents. RESULTS: In the clinical cohort, a bioLep/irLep ratio of 1.07 (range: 0.80-1.41) was observed. Serum of patients with non-functional leptin due to homozygous amino acid exchanges (D100Y or N103K) revealed high irLep but non-detectable bioLep levels. Upon treatment of these patients with metreleptin, irLep levels decreased, whereas levels of bioLep increased continuously. In patient relatives with heterozygous amino acid exchanges, a bioLep/irLep ratio of 0.52 (range: 0.48-0.55) being distinct from normal was observed. CONCLUSIONS: The new bioLep assay is able to diagnose impaired leptin bioactivity in severely obese patients with a homozygous gene defect and in heterozygous carriers of such mutations. The assay serves as a diagnostic tool to monitor leptin bioactivity during treatment of these patients.
Assuntos
Imunoensaio/métodos , Leptina/sangue , Leptina/deficiência , Adolescente , Criança , Feminino , Humanos , MasculinoRESUMO
Among all chemokine receptors CXCR4 possesses a unique response profile and distinguishes itself through a prolonged signaling capacity. Here, we investigated the signaling capacity of CXCR4 to its so far known unique ligand CXCL12 in B cell lines and primary CD19(+) B lymphocytes. During lymphopoiesis, CXCR4 is continuously expressed on the surface of B cells. However, its signaling profile changes inasmuch preB and proB cells migrate towards CXCL12, mobilize intracellular calcium and activate the small GTPases Rac1 and Cdc42, whereas mature B cells do not show these responses, albeit the cells retain the capability to migrate in response to CXCL13 and CCL21. By contrast, stimulation of B cells with CXCL12 at all stages of development results in the activation of the MAP-kinase cascade and in rapid CXCR4 internalization. The pathways leading to ERK1/2 activation are different in preB and mature B cell lines. In either case, ERK1/2 activation is pertussis toxin sensitive, but only in mature B-cells inhibition of PI3-kinase causes an almost complete block of ERK1/2 activation. Taken together, the results show that CXCR4 changes its coupling to downstream signal-transduction pathways in B cells, suggesting that receptor activity may depend on accessory proteins.
Assuntos
Linfócitos B/metabolismo , Receptores CXCR4/metabolismo , Transdução de Sinais , Antígenos CD19/sangue , Linfócitos B/imunologia , Sinalização do Cálcio , Quimiocina CXCL12 , Quimiocinas CXC/metabolismo , Quimiotaxia de Leucócito , Humanos , Linfopoese , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismoRESUMO
Migration and positioning of cells is fundamental for complex functioning of multicellular organisms. During an immune response, cells are recruited from remote distances to a distinct location. Cells that are passively transported leave the circulation stimulated by locally produced signals and follow chemotactic cues to reach specific destinations. Such gradients are short (<150 µm) and require a source of production where the concentration is the highest and a sink in apposition where the attractant dissipates and the concentration is the lowest. Several straight forward methods exist to identify in vitro and in vivo cells producing chemoattractants. This can be achieved at the transcriptional level and by measuring secreted proteins. However, to demonstrate the activity of sinks in vitro and in vivo is more challenging. Cell-mediated dissipation of an attractant must be revealed by measuring its uptake and subsequent destruction. Elimination of chemoattractants such as chemokines can be monitored in vitro using radiolabeled ligands or more elegantly with fluorescent-labeled chemoattractants. The latter method can also be used in vivo and enables to monitor the process in real time using time-lapse video microscopy. In this chapter, we describe methods to produce fluorescently labeled chemokines either as fusion proteins secreted from insect cells or as recombinant bacterial proteins that can enzymatically be labeled. We discuss methods that were successfully used to demonstrate sink activities of scavenger receptors. Moreover, fluorescent chemokines can be used to noninvasively analyze receptor expression and activity in living cells.
Assuntos
Quimiocinas/metabolismo , Biologia Molecular/métodos , Receptores de Quimiocinas/análise , Receptores de Quimiocinas/metabolismo , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Quimiocinas/análise , Quimiocinas/genética , Fatores Quimiotáticos/metabolismo , Clonagem Molecular , Escherichia coli/genética , Citometria de Fluxo , Corantes Fluorescentes/química , Humanos , Insetos/citologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
The activator of G protein signalling AGS2 (Tctex-1) forms protein complexes with Gßγ, and controls cell proliferation by regulating cell cycle progression. A direct interaction of Tctex-1 with various G protein-coupled receptors has been reported. Since the carboxyl terminal portion of CB2 carries a putative Tctex-1 binding motif, we investigated the potential interplay of CB2 and Tctex-1 in the absence and presence of Gßγ. The supposed interaction of cannabinoid receptor CB2 with Tctex-1 and the influence of CB2 on the formation of Tctex-1-Gßγ-complexes were studied by co- and/or immunoprecipitation experiments in transiently transfected HEK293 cells. The analysis on Tctex-1 protein was performed in the absence and presence of the ligands JWH 133, 2-AG, and AM 630, the protein biosynthesis inhibitor cycloheximide or the protein degradation blockers MG132, NH4Cl/leupeptin or bafilomycin. Our results show that CB2 neither directly nor indirectly via Gßγ interacts with Tctex-1, but competes with Tctex-1 in binding to Gßγ. The Tctex-1-Gßγ protein interaction was disrupted by CB2 receptor expression resulting in a release of Tctex-1 from the complex, and its degradation by the proteasome and partly by lysosomes. The decrease in Tctex-1 protein levels is induced by CB2 expression "dose-dependently" and is independent of stimulation by agonist or blocking by an inverse agonist treatment. The results suggest that CB2 receptor expression independent of its activation by agonists is sufficient to competitively disrupt Gßγ-Tctex-1 complexes, and to initiate Tctex-1 degradation. These findings implicate that CB2 receptor expression modifies the stability of intracellular protein complexes by a non-canonical pathway.