RESUMO
Lon is an ATP-dependent protease belonging to the "ATPase associated with diverse cellular activities" (AAA+) protein family. In humans, Lon is translated as a precursor and imported into the mitochondria matrix through deletion of the first 114 amino acid residues. In mice, embryonic knockout of lon is lethal. In humans, some dysfunctional lon mutations are tolerated but they cause a developmental disorder known as the CODAS syndrome. To gain a better understanding on the enzymology of human mitochondrial Lon, this study compares the structure-function relationship of the WT versus one of the CODAS mutants R721G to identify the mechanistic features in Lon catalysis that are affected. To this end, steady-state kinetics were used to quantify the difference in ATPase and ATP-dependent peptidase activities between WT and R721G. The Km values for the intrinsic as well as protein-stimulated ATPase were increased whereas the kcat value for ATP-dependent peptidase activity was decreased in the R721G mutant. The mutant protease also displayed substrate inhibition kinetics. In vitro studies revealed that R721G did not degrade the endogenous mitochondrial Lon substrate pyruvate dehydrogenase kinase isoform 4 (PDK4) effectively like WT hLon. Furthermore, the pyruvate dehydrogenase complex (PDH) protected PDK4 from hLon degradation. Using hydrogen deuterium exchange/mass spectrometry and negative stain electron microscopy, structural perturbations associated with the R721G mutation were identified. To validate the in vitro findings under a physiologically relevant condition, the intrinsic stability as well as proteolytic activity of WT versus R721G mutant towards PDK 4 were compared in cell lysates prepared from immortalized B lymphocytes expressing the respective protease. The lifetime of PDK4 is longer in the mutant cells, but the lifetime of Lon protein is longer in the WT cells, which corroborate the in vitro structure-functional relationship findings.
Assuntos
Mitocôndrias/enzimologia , Protease La/química , Protease La/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Linfócitos B/enzimologia , Biocatálise , Anormalidades Craniofaciais/enzimologia , Anormalidades Craniofaciais/genética , Estabilidade Enzimática/genética , Anormalidades do Olho/enzimologia , Anormalidades do Olho/genética , Transtornos do Crescimento/enzimologia , Transtornos do Crescimento/genética , Luxação Congênita de Quadril/enzimologia , Luxação Congênita de Quadril/genética , Humanos , Cinética , Camundongos , Modelos Moleculares , Simulação de Dinâmica Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Osteocondrodisplasias/enzimologia , Osteocondrodisplasias/genética , Protease La/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato , Anormalidades Dentárias/enzimologia , Anormalidades Dentárias/genéticaRESUMO
Peters Plus Syndrome (PTRPLS OMIM #261540) is a severe congenital disorder of glycosylation where patients have multiple structural anomalies, including Peters anomaly of the eye (anterior segment dysgenesis), disproportionate short stature, brachydactyly, dysmorphic facial features, developmental delay, and variable additional abnormalities. PTRPLS patients and some Peters Plus-like (PTRPLS-like) patients (who only have a subset of PTRPLS phenotypes) have mutations in the gene encoding ß1,3-glucosyltransferase (B3GLCT). B3GLCT catalyzes the transfer of glucose to O-linked fucose on thrombospondin type-1 repeats. Most B3GLCT substrate proteins belong to the ADAMTS superfamily and play critical roles in extracellular matrix. We sought to determine whether the PTRPLS or PTRPLS-like mutations abrogated B3GLCT activity. B3GLCT has two putative active sites, one in the N-terminal region and the other in the C-terminal glycosyltransferase domain. Using sequence analysis and in vitro activity assays, we demonstrated that the C-terminal domain catalyzes transfer of glucose to O-linked fucose. We also generated a homology model of B3GLCT and identified D421 as the catalytic base. PTRPLS and PTRPLS-like mutations were individually introduced into B3GLCT, and the mutated enzymes were evaluated using in vitro enzyme assays and cell-based functional assays. Our results demonstrated that PTRPLS mutations caused loss of B3GLCT enzymatic activity and/or significantly reduced protein stability. In contrast, B3GLCT with PTRPLS-like mutations retained enzymatic activity, although some showed a minor destabilizing effect. Overall, our data supports the hypothesis that loss of glucose from B3GLCT substrate proteins is responsible for the defects observed in PTRPLS patients, but not for those observed in PTRPLS-like patients.
Assuntos
Fenda Labial/enzimologia , Fenda Labial/genética , Córnea/anormalidades , Galactosiltransferases/genética , Galactosiltransferases/metabolismo , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Transtornos do Crescimento/enzimologia , Transtornos do Crescimento/genética , Deformidades Congênitas dos Membros/enzimologia , Deformidades Congênitas dos Membros/genética , Mutação/genética , Proteínas ADAMTS/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Biocatálise , Córnea/enzimologia , Estabilidade Enzimática , Fucose/metabolismo , Galactosiltransferases/química , Glucose/metabolismo , Glucosiltransferases/química , Células HEK293 , Humanos , Cinética , Modelos Moleculares , Domínios Proteicos , Sequências Repetitivas de Aminoácidos , Homologia Estrutural de ProteínaAssuntos
Classe Ia de Fosfatidilinositol 3-Quinase/genética , Mutação em Linhagem Germinativa , Transtornos do Crescimento/genética , Hipercalcemia/genética , Doenças Metabólicas/genética , Nefrocalcinose/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Adolescente , Transtornos do Crescimento/enzimologia , Humanos , Hipercalcemia/enzimologia , Masculino , Doenças Metabólicas/enzimologia , Nefrocalcinose/enzimologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/enzimologiaRESUMO
Carboxyl ester lipase (Cel), is a lipolytic enzyme secreted by the pancreas, which hydrolyzes various species of lipids in the gut. Cel is also secreted by mammary gland during lactation and exists in breast milk. It facilitates dietary fat digestion and absorption, thus contributing to normal infant development. This study aimed to examine whether the Cel in zebrafish embryos has a similar role of maternal lipid utilization as in human infants, and how Cel contributes to the utilization of yolk lipids in zebrafish. The cel1 and cel2 genes were expressed ubiquitously in the blastodisc and yolk syncytial layer before 24 hpf, and in the exocrine pancreas after 72 hpf. The cel1 and cel2 morphants exhibited developmental retardation and yolk sac retention. The total cholesterol, cholesterol ester, free cholesterol, and triglyceride were reduced in the morphants' body while accumulated in the yolk (except triglyceride). The FFA content of whole embryos was much lower in morphants than in standard controls. Moreover, the delayed development in cel (cel1/cel2) double morphants was partially rescued by FFA and cholesterol supplementation. Delayed and weakened cholesterol ester transport to the brain and eyes was observed in cel morphants. Correspondingly, shrunken midbrain tectum, microphthalmia, pigmentation-delayed eyes as well as down-regulated Shh target genes were observed in the CNS of double morphants. Interestingly, cholesterol injections reversed these CNS alterations. Our findings suggested that cel genes participate in the lipid releasing from yolk sac to developing body, thereby contributing to the normal growth rate and CNS development in zebrafish.
Assuntos
Carboxilesterase/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Transtornos do Crescimento/genética , Saco Vitelino/enzimologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Animais Geneticamente Modificados , Carboxilesterase/genética , Sistema Nervoso Central/embriologia , Colesterol/metabolismo , Ésteres do Colesterol/metabolismo , Modelos Animais de Doenças , Embrião não Mamífero , Desenvolvimento Embrionário , Técnicas de Silenciamento de Genes , Transtornos do Crescimento/embriologia , Transtornos do Crescimento/enzimologia , Proteínas Hedgehog/metabolismo , Humanos , Metabolismo dos Lipídeos , Morfolinos/administração & dosagem , Morfolinos/genética , Pâncreas Exócrino/embriologia , Pâncreas Exócrino/enzimologia , Triglicerídeos/metabolismo , Saco Vitelino/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
Peters plus syndrome (MIM #261540 PTRPLS), characterized by defects in eye development, prominent forehead, hypertelorism, short stature and brachydactyly, is caused by mutations in the ß3-glucosyltransferase (B3GLCT) gene. Protein O-fucosyltransferase 2 (POFUT2) and B3GLCT work sequentially to add an O-linked glucose ß1-3fucose disaccharide to properly folded thrombospondin type 1 repeats (TSRs). Forty-nine proteins are predicted to be modified by POFUT2, and nearly half are members of the ADAMTS superfamily. Previous studies suggested that O-linked fucose is essential for folding and secretion of POFUT2-modified proteins and that B3GLCT-mediated extension to the disaccharide is essential for only a subset of targets. To test this hypothesis and gain insight into the origin of PTRPLS developmental defects, we developed and characterized two mouse B3glct knockout alleles. Using these models, we tested the role of B3GLCT in enabling function of ADAMTS9 and ADAMTS20, two highly conserved targets whose functions are well characterized in mouse development. The mouse B3glct mutants developed craniofacial and skeletal abnormalities comparable to PTRPLS. In addition, we observed highly penetrant hydrocephalus, white spotting and soft tissue syndactyly. We provide strong genetic and biochemical evidence that hydrocephalus and white spotting in B3glct mutants resulted from loss of ADAMTS20, eye abnormalities from partial reduction of ADAMTS9 and cleft palate from loss of ADAMTS20 and partially reduced ADAMTS9 function. Combined, these results provide compelling evidence that ADAMTS9 and ADAMTS20 were differentially sensitive to B3GLCT inactivation and suggest that the developmental defects in PTRPLS result from disruption of a subset of highly sensitive POFUT2/B3GLCT targets such as ADAMTS20.
Assuntos
Proteínas ADAMTS/metabolismo , Proteína ADAMTS9/metabolismo , Fenda Labial/metabolismo , Córnea/anormalidades , Glicosiltransferases/deficiência , Transtornos do Crescimento/metabolismo , Deformidades Congênitas dos Membros/metabolismo , Alelos , Animais , Fenda Labial/enzimologia , Fenda Labial/genética , Córnea/enzimologia , Córnea/metabolismo , Modelos Animais de Doenças , Feminino , Fucosiltransferases/genética , Fucosiltransferases/metabolismo , Sistema da Enzima Desramificadora do Glicogênio/metabolismo , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Transtornos do Crescimento/enzimologia , Transtornos do Crescimento/genética , Deformidades Congênitas dos Membros/enzimologia , Deformidades Congênitas dos Membros/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Organogênese/genéticaRESUMO
Across eukaryotes, disruption of DNA replication causes an S phase checkpoint response, which regulates multiple processes, including inhibition of replication initiation and fork stabilization. How these events are coordinated remains poorly understood. Here, we show that the replicative helicase component Cdc45 targets the checkpoint kinase Rad53 to distinct replication complexes in the budding yeast Saccharomyces cerevisiae. Rad53 binds to forkhead-associated (FHA) interaction motifs in an unstructured loop region of Cdc45, which is phosphorylated by Rad53 itself, and this interaction is necessary for the inhibition of origin firing through Sld3. Cdc45 also recruits Rad53 to stalled replication forks, which we demonstrate is important for the response to replication stress. Finally, we show that a Cdc45 mutation found in patients with Meier-Gorlin syndrome disrupts the functional interaction with Rad53 in yeast. Together, we present a single mechanism by which a checkpoint kinase targets replication initiation and elongation complexes, which may be relevant to human disease.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2/metabolismo , Dano ao DNA , Reparo do DNA , Replicação do DNA , DNA Fúngico/biossíntese , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Ciclo Celular/genética , Quinase do Ponto de Checagem 2/genética , Microtia Congênita/enzimologia , Microtia Congênita/genética , DNA Fúngico/genética , Proteínas de Ligação a DNA/genética , Transtornos do Crescimento/enzimologia , Transtornos do Crescimento/genética , Humanos , Micrognatismo/enzimologia , Micrognatismo/genética , Mutação , Proteínas Nucleares/genética , Patela/anormalidades , Patela/enzimologia , Fosforilação , Ligação Proteica , Pontos de Checagem da Fase S do Ciclo Celular , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
PURPOSE: Pathogenic variations in genes encoding aminoacyl-tRNA synthetases (ARSs) are increasingly associated with human disease. Clinical features of autosomal recessive ARS deficiencies appear very diverse and without apparent logic. We searched for common clinical patterns to improve disease recognition, insight into pathophysiology, and clinical care. METHODS: Symptoms were analyzed in all patients with recessive ARS deficiencies reported in literature, supplemented with unreported patients evaluated in our hospital. RESULTS: In literature, we identified 107 patients with AARS, DARS, GARS, HARS, IARS, KARS, LARS, MARS, RARS, SARS, VARS, YARS, and QARS deficiencies. Common symptoms (defined as present in ≥4/13 ARS deficiencies) included abnormalities of the central nervous system and/or senses (13/13), failure to thrive, gastrointestinal symptoms, dysmaturity, liver disease, and facial dysmorphisms. Deep phenotyping of 5 additional patients with unreported compound heterozygous pathogenic variations in IARS, LARS, KARS, and QARS extended the common phenotype with lung disease, hypoalbuminemia, anemia, and renal tubulopathy. CONCLUSION: We propose a common clinical phenotype for recessive ARS deficiencies, resulting from insufficient aminoacylation activity to meet translational demand in specific organs or periods of life. Assuming residual ARS activity, adequate protein/amino acid supply seems essential instead of the traditional replacement of protein by glucose in patients with metabolic diseases.
Assuntos
Aminoacil-tRNA Sintetases/deficiência , Doenças Genéticas Inatas/enzimologia , Doenças Genéticas Inatas/genética , Aminoacil-tRNA Sintetases/genética , Doenças do Sistema Nervoso Central/enzimologia , Doenças do Sistema Nervoso Central/genética , Criança , Insuficiência de Crescimento/enzimologia , Insuficiência de Crescimento/genética , Transtornos da Alimentação e da Ingestão de Alimentos/enzimologia , Transtornos da Alimentação e da Ingestão de Alimentos/genética , Feminino , Genes Recessivos , Transtornos do Crescimento/enzimologia , Transtornos do Crescimento/genética , Humanos , Hepatopatias/enzimologia , Hepatopatias/genética , Masculino , FenótipoRESUMO
Background The phosphoglucomutase 1 (PGM1) enzyme plays a central role in glucose homeostasis by catalyzing the inter-conversion of glucose 1-phosphate and glucose 6-phosphate. Recently, PGM1 deficiency has been recognized as a cause of the congenital disorders of glycosylation (CDGs). Methods Two Chinese Han pediatric patients with recurrent hypoglycemia, hepatopathy and growth retardation are described in this study. Targeted gene sequencing (TGS) was performed to screen for causal genetic variants in the genome of the patients and their parents to determine the genetic basis of the phenotype. Results DNA sequencing identified three variations of the PGM1 gene (NM_002633.2). Patient 1 had a novel homozygous mutation (c.119delT, p.Ile40Thrfs*28). In patient 2, we found a compound heterozygous mutation of c.1172G>T(p.Gly391Val) (novel) and c.1507C>T(p.Arg503*) (known pathogenic). Conclusions This report deepens our understanding of the clinical features of PGM1 mutation. The early molecular genetic analysis and multisystem assessment were here found to be essential to the diagnosis of PGM1-CDG and the provision of timely and proper treatment.
Assuntos
Biomarcadores/metabolismo , Doença de Depósito de Glicogênio/genética , Mutação , Fosfoglucomutase/genética , Criança , Pré-Escolar , Feminino , Doença de Depósito de Glicogênio/enzimologia , Doença de Depósito de Glicogênio/patologia , Transtornos do Crescimento/enzimologia , Transtornos do Crescimento/genética , Transtornos do Crescimento/patologia , Humanos , Hipoglicemia/enzimologia , Hipoglicemia/genética , Hipoglicemia/patologia , Hepatopatias/enzimologia , Hepatopatias/genética , Hepatopatias/patologia , Masculino , Fenótipo , PrognósticoRESUMO
Obstructive sleep apnea (OSA) is common in pregnancy and may compromise fetal and even postnatal development. We developed an animal model to determine if maternal OSA could have lasting effects in offspring. Pregnant Sprague-Dawley rats were exposed to reduced ambient O2 from 21 to 4-5%, approximately once per minute [chronic intermittent hypoxia (CIH)] for 8 h/day during gestation days 3-19. Similarly handled animals exposed to ambient air served as controls (HC). Offspring were studied for body growth and cardiovascular function for 8 postnatal weeks. Compared with HC, prenatal CIH led to growth restriction, indicated by smaller body weight and tibial length, and higher arterial blood pressure in both male and female offspring. Compared with same-sex HC, CIH males showed abdominal obesity (greater ratio of abdominal fat weight to body weight or tibial length), left ventricular (LV) hypertrophy (greater heart weight-to-tibial length ratio and LV posterior wall diastolic thickness), elevated LV contractility (increases in LV ejection fraction, end-systolic pressure-volume relations, and preload recruitable stroke work), elevated LV and arterial stiffness (increased end-diastolic pressure-volume relationship and arterial elasticity), and LV oxidative stress (greater lipid peroxide content). Compared with female CIH offspring, male CIH offspring had more profound changes in blood pressure (BP), cardiac function, myocardial lipid peroxidase (LPO) content, and abdominal adiposity. Rodent prenatal CIH exposure, mimicking human maternal OSA, exerts detrimental morphological and cardiovascular effects on developing offspring; the model may provide useful insights of OSA effects in humans. NEW & NOTEWORTHY Obstructive sleep apnea is common in human pregnancy. Following maternal exposure to chronic intermittent hypoxia, a hallmark of sleep apnea, both sexes of rat offspring showed growth retardation, with males being more vulnerable to hypertension and dysfunctional left ventricular changes. This model is useful to study detrimental effects of maternal obstructive sleep apnea on developing offspring in humans.
Assuntos
Transtornos do Crescimento/etiologia , Hipóxia/fisiopatologia , Complicações na Gravidez/fisiopatologia , Efeitos Tardios da Exposição Pré-Natal , Apneia Obstrutiva do Sono/fisiopatologia , Animais , Pressão Arterial , Modelos Animais de Doenças , Ecocardiografia , Feminino , Transtornos do Crescimento/enzimologia , Masculino , Obesidade Abdominal/etiologia , Estresse Oxidativo , Gravidez , Ratos Sprague-DawleyRESUMO
In higher eukaryotes, cell proliferation is regulated by class I phosphatidylinositol 3-kinase (PI3K), which transduces stimuli received from neighboring receptors by local generation of PtdIns(3,4,5)P3 in cellular membranes. PI3K is a heterodimeric protein consisting of a regulatory and a catalytic subunit (p85 and p110 respectively). Heterologous expression of p110α in Saccharomyces cerevisiae leads to toxicity by conversion of essential PtdIns(4,5)P2 into futile PtdIns(3,4,5)P3, providing a humanized yeast model for functional studies on this pathway. Here, we report expression and functional characterization in yeast of all regulatory and catalytic human PI3K isoforms, and exploitation of the most suitable setting to functionally assay panels of tumor- and germ line-associated PI3K mutations, with indications to the limits of the system. The activity of p110α in yeast was not compromised by truncation of its N-terminal adaptor-binding domain (ABD) or inactivation of the Ras-binding domain (RBD). In contrast, a cluster of positively charged residues at the C2 domain was essential. Expression of a membrane-driven p65α oncogenic-truncated version of p85α, but not the full-length protein, led to enhanced activity of α, ß, and δ p110 isoforms. Mutations impairing the inhibitory regulation exerted by the p85α iSH2 domain on the C2 domain of p110α yielded the latter non-responsive to negative regulation, thus reproducing this oncogenic mechanism in yeast. However, p85α germ line mutations associated with short stature, hyperextensibility of joints and/or inguinal hernia, ocular depression, Rieger anomaly, and teething delay (SHORT) syndrome did not increase PI3K activity in this model, supporting the idea that SHORT syndrome-associated p85α mutations operate through mechanisms different from the canonical disruption of inhibitory p85-p110 interactions typical of cancer.
Assuntos
Classe Ia de Fosfatidilinositol 3-Quinase/genética , Mutação , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Transtornos do Crescimento/enzimologia , Transtornos do Crescimento/genética , Transtornos do Crescimento/patologia , Humanos , Hipercalcemia/enzimologia , Hipercalcemia/genética , Hipercalcemia/patologia , Immunoblotting , Doenças Metabólicas/enzimologia , Doenças Metabólicas/genética , Doenças Metabólicas/patologia , Modelos Biológicos , Neoplasias/enzimologia , Neoplasias/genética , Neoplasias/patologia , Nefrocalcinose/enzimologia , Nefrocalcinose/genética , Nefrocalcinose/patologia , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Lon protease is a nuclear-encoded, mitochondrial ATP-dependent protease highly conserved throughout the evolution, crucial for the maintenance of mitochondrial homeostasis. Lon acts as a chaperone of misfolded proteins, and is necessary for maintaining mitochondrial DNA. The impairment of these functions has a deep impact on mitochondrial functionality and morphology. An altered expression of Lon leads to a profound reprogramming of cell metabolism, with a switch from respiration to glycolysis, which is often observed in cancer cells. Mutations of Lon, which likely impair its chaperone properties, are at the basis of a genetic inherited disease named of the cerebral, ocular, dental, auricular, skeletal (CODAS) syndrome. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.
Assuntos
Anormalidades Craniofaciais/genética , DNA Mitocondrial/genética , Anormalidades do Olho/genética , Transtornos do Crescimento/genética , Luxação Congênita de Quadril/genética , Mitocôndrias/enzimologia , Chaperonas Moleculares/química , Mutação , Osteocondrodisplasias/genética , Protease La/química , Anormalidades Dentárias/genética , Reprogramação Celular , Anormalidades Craniofaciais/enzimologia , Anormalidades Craniofaciais/patologia , DNA Mitocondrial/metabolismo , Anormalidades do Olho/enzimologia , Anormalidades do Olho/patologia , Transtornos do Crescimento/enzimologia , Transtornos do Crescimento/patologia , Luxação Congênita de Quadril/enzimologia , Luxação Congênita de Quadril/patologia , Homeostase , Humanos , Mitocôndrias/patologia , Modelos Moleculares , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Osteocondrodisplasias/enzimologia , Osteocondrodisplasias/patologia , Protease La/genética , Protease La/metabolismo , Dobramento de Proteína , Anormalidades Dentárias/enzimologia , Anormalidades Dentárias/patologiaRESUMO
The phosphatidylinositol 3 kinase (PI3K) pathway regulates fundamental cellular processes such as metabolism, proliferation, and survival. A central component in this pathway is the p85α regulatory subunit, encoded by PIK3R1. Using whole-exome sequencing, we identified a heterozygous PIK3R1 mutation (c.1945C>T [p.Arg649Trp]) in two unrelated families affected by partial lipodystrophy, low body mass index, short stature, progeroid face, and Rieger anomaly (SHORT syndrome). This mutation led to impaired interaction between p85α and IRS-1 and reduced AKT-mediated insulin signaling in fibroblasts from affected subjects and in reconstituted Pik3r1-knockout preadipocytes. Normal PI3K activity is critical for adipose differentiation and insulin signaling; the mutated PIK3R1 therefore provides a unique link among lipodystrophy, growth, and insulin signaling.
Assuntos
Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Transtornos do Crescimento/enzimologia , Hipercalcemia/enzimologia , Doenças Metabólicas/enzimologia , Nefrocalcinose/enzimologia , Transdução de Sinais , Adipócitos/metabolismo , Adolescente , Adulto , Idoso , Sequência de Aminoácidos , Índice de Massa Corporal , Diferenciação Celular , Classe Ia de Fosfatidilinositol 3-Quinase/genética , Análise Mutacional de DNA , Ativação Enzimática , Exoma , Feminino , Fibroblastos/metabolismo , Técnicas de Inativação de Genes , Triagem de Portadores Genéticos , Predisposição Genética para Doença , Genética Populacional/métodos , Transtornos do Crescimento/patologia , Humanos , Hipercalcemia/patologia , Proteínas Substratos do Receptor de Insulina/genética , Proteínas Substratos do Receptor de Insulina/metabolismo , Masculino , Doenças Metabólicas/patologia , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mutação , Nefrocalcinose/patologia , Linhagem , Adulto Jovem , Domínios de Homologia de srcRESUMO
AIM: Some cases of idiopathic short stature (ISS) may be caused by defects in the modulation of the negative feedback regulation of the growth hormone receptor (GHR)/ Janus kinase (JAK)2/signal transducers and activators of transcription (STAT)5 signaling pathway. The cytosolic tyrosine phosphatases, protein tyrosine phosphatase 1B (PTP1B) and Src homology 2 (SH2) domain-containing protein-tyrosine phosphatase-1 (SHP-1), the later which translocates to the nucleus after activation, interact with JAK2 in a GH-dependent manner. The possible contribution of PTP1B and SHP-1 to GH signaling in fibroblasts from ISS patients has not been studied. METHODS: We determined the basal protein content of PTP1B and SHP-1 in the presence of recombinant human GH (rhGH) for 24 h in skin fibroblast cultures, obtained from patients with ISS, and were compared with a normal height control children group. JAK2 activation was determined in both groups. RESULTS: JAK2 activation was delayed in fibroblasts from ISS patients compared to controls. Under basal conditions, the protein content of SHP-1 was lower in ISS, and after incubation with rhGH, it decreased in the non-nuclear and nuclear fraction of controls, but not in ISS patients. The protein content of PTP1B, however, increased in a similar fashion in fibroblasts from both ISS and control children. CONCLUSION: The delayed activation of JAK2 and the lack of response of SHP-1 after incubation with GH in fibroblasts from ISS patients, suggests that the growth retardation observed in some of these children may be mediated in part by this phosphotyrosine phosphatase.
Assuntos
Transtornos do Crescimento/enzimologia , Hormônio do Crescimento Humano/metabolismo , Janus Quinase 2/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Transdução de Sinais , Pele/enzimologia , Estatura , Núcleo Celular/enzimologia , Núcleo Celular/metabolismo , Células Cultivadas , Criança , Desenvolvimento Infantil , Ativação Enzimática , Transtornos do Crescimento/metabolismo , Transtornos do Crescimento/patologia , Hormônio do Crescimento Humano/genética , Humanos , Janus Quinase 2/química , Cinética , Masculino , Fosforilação , Processamento de Proteína Pós-Traducional , Receptores da Somatotropina/agonistas , Receptores da Somatotropina/metabolismo , Proteínas Recombinantes/metabolismo , Pele/metabolismo , Pele/patologiaRESUMO
DNA polymerase ε (Polε) is a large, four-subunit polymerase that is conserved throughout the eukaryotes. Its primary function is to synthesize DNA at the leading strand during replication. It is also involved in a wide variety of fundamental cellular processes, including cell cycle progression and DNA repair/recombination. Here, we report that a homozygous single base pair substitution in POLE1 (polymerase ε 1), encoding the catalytic subunit of Polε, caused facial dysmorphism, immunodeficiency, livedo, and short stature ("FILS syndrome") in a large, consanguineous family. The mutation resulted in alternative splicing in the conserved region of intron 34, which strongly decreased protein expression of Polε1 and also to a lesser extent the Polε2 subunit. We observed impairment in proliferation and G1- to S-phase progression in patients' T lymphocytes. Polε1 depletion also impaired G1- to S-phase progression in B lymphocytes, chondrocytes, and osteoblasts. Our results evidence the developmental impact of a Polε catalytic subunit deficiency in humans and its causal relationship with a newly recognized, inherited disorder.
Assuntos
DNA Polimerase II/genética , Ossos Faciais/anormalidades , Transtornos do Crescimento/enzimologia , Transtornos do Crescimento/genética , Síndromes de Imunodeficiência/enzimologia , Síndromes de Imunodeficiência/genética , Livedo Reticular/enzimologia , Livedo Reticular/genética , Adolescente , Adulto , Processamento Alternativo , Sequência de Bases , Estatura/genética , Proliferação de Células , Criança , Pré-Escolar , Cromossomos Humanos Par 12/genética , Consanguinidade , Análise Mutacional de DNA , DNA Polimerase II/deficiência , Feminino , França , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Expressão Gênica , Genes Recessivos , Humanos , Síndromes de Imunodeficiência/imunologia , Íntrons , Livedo Reticular/patologia , Masculino , Linhagem , Mutação Puntual , Proteínas de Ligação a Poli-ADP-Ribose , Síndrome , Adulto JovemRESUMO
Cytosolic aminopeptidase P1 (APP1) is one of the three known mammalian aminopeptidase Ps (APPs) that cleave the N-terminal amino acid residue of peptides in which the penultimate amino acid is proline. In mammals, many biologically active peptides have a highly conserved N-terminal penultimate proline. However, little is known about the physiological role of APP1. In addition, there is no direct evidence to associate a deficiency in APP1 with metabolic diseases. Although two human subjects with reduced APP activity exhibited peptiduria, it is unclear which of the three APP isoforms is responsible for this disorder. In this study, we generated APP1-deficient mice by knocking out Xpnpep1. Mouse APP1 deficiency causes severe growth retardation, microcephaly, and modest lethality. In addition, imino-oligopeptide excretion was observed in urine samples from APP1-deficient mice. These results suggest an essential role for APP1-mediated peptide metabolism in body and brain development, and indicate a strong causal link between APP1 deficiency and peptiduria.
Assuntos
Aminopeptidases/genética , Transtornos do Crescimento/enzimologia , Microcefalia/enzimologia , Peptídeos/urina , Animais , Transtornos do Crescimento/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microcefalia/genéticaRESUMO
Like DNA replication, centrosomes are licensed to duplicate once per cell division cycle to ensure genetic stability. In addition to regulating DNA replication, the Orc1 subunit of the human origin recognition complex controls centriole and centrosome copy number. Here we report that Orc1 harbors a PACT centrosome-targeting domain and a separate domain that differentially inhibits the protein kinase activities of Cyclin E-CDK2 and Cyclin A-CDK2. A cyclin-binding motif (Cy motif) is required for Orc1 to bind Cyclin A and inhibit Cyclin A-CDK2 kinase activity but has no effect on Cyclin E-CDK2 kinase activity. In contrast, Orc1 inhibition of Cyclin E-CDK2 kinase activity occurs by a different mechanism that is affected by Orc1 mutations identified in Meier-Gorlin syndrome patients. The cyclin/CDK2 kinase inhibitory domain of Orc1, when tethered to the PACT domain, localizes to centrosomes and blocks centrosome reduplication. Meier-Gorlin syndrome mutations that disrupt Cyclin E-CDK2 kinase inhibition also allow centrosome reduplication. Thus, Orc1 contains distinct domains that control centrosome copy number and DNA replication. We suggest that the Orc1 mutations present in some Meier-Gorlin syndrome patients contribute to the pronounced microcephaly and dwarfism observed in these individuals by altering centrosome duplication in addition to DNA replication defects.
Assuntos
Centrossomo/patologia , Transtornos do Crescimento/genética , Micrognatismo/genética , Complexo de Reconhecimento de Origem/genética , Sequência de Aminoácidos , Linhagem Celular Tumoral , Centríolos/metabolismo , Microtia Congênita , Quinases Ciclina-Dependentes/metabolismo , Orelha/anormalidades , Transtornos do Crescimento/enzimologia , Células HEK293 , Humanos , Micrognatismo/enzimologia , Dados de Sequência Molecular , Mutação , Patela/anormalidades , Patela/enzimologia , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/metabolismo , Alinhamento de SequênciaRESUMO
We describe a three-generation family with tall stature, scoliosis and macrodactyly of the great toes and a heterozygous p.Val883Met mutation in Npr2, the gene that encodes the CNP receptor NPR2 (natriuretic peptide receptor 2). When expressed in HEK293A cells, the mutant Npr2 cDNA generated intracellular cGMP (cyclic guanosine monophosphate) in the absence of CNP ligand. In the presence of CNP, cGMP production was greater in cells that had been transfected with the mutant Npr2 cDNA compared to wild-type cDNA. Transgenic mice in which the mutant Npr2 was expressed in chondrocytes driven by the promoter and intronic enhancer of the Col11a2 gene exhibited an enhanced production of cGMP in cartilage, leading to a similar phenotype to that observed in the patients. In addition, blood cGMP concentrations were elevated in the patients. These results indicate that p.Val883Met is a constitutive active gain-of-function mutation and elevated levels of cGMP in growth plates lead to the elongation of long bones. Our findings reveal a critical role for NPR2 in skeletal growth in both humans and mice, and may provide a potential target for prevention and treatment of diseases caused by impaired production of cGMP.
Assuntos
Condrócitos , Transtornos do Crescimento , Deformidades Congênitas dos Membros , Mutação de Sentido Incorreto , Receptores do Fator Natriurético Atrial , Adolescente , Substituição de Aminoácidos , Animais , Condrócitos/enzimologia , Condrócitos/patologia , GMP Cíclico , DNA Complementar , Dedos/anormalidades , Dedos/patologia , Expressão Gênica , Transtornos do Crescimento/enzimologia , Transtornos do Crescimento/genética , Transtornos do Crescimento/patologia , Células HEK293 , Humanos , Deformidades Congênitas dos Membros/enzimologia , Deformidades Congênitas dos Membros/genética , Deformidades Congênitas dos Membros/patologia , Masculino , Camundongos , Camundongos Transgênicos , Receptores do Fator Natriurético Atrial/genética , Receptores do Fator Natriurético Atrial/metabolismoRESUMO
Growth hormone (GH) is the most frequently used treatment in children with idiopathic short stature (ISS). Aromatase inhibitor (AI) therapy is still in an experimental state, and both final height (FH) and long-term efficacy data in ISS have not been published. We present a 14.5-year-old boy with ISS and a height of 142.7 cm [standard deviation score (SDS) -2.79]. Based on the baseline bone age (BA) of 13.5-14 years, his predicted adult height (PAH) by Bayley/Pinneau was 154 cm (SDS -3.77)-158.2 (SDS -3.15). After a 5-year letrozole monotherapy, FH was 169 cm (SDS -1.57) showing a height difference between PAH and FH from 10.8 to 15 cm. No permanent side effects of the medication have been observed. Both a transient occurrence and a spontaneous recovery of decreased bone mineral apparent density were seen, verified by dual-energy X-ray absorptiometry. Spinal magnetic resonance imaging revealed no vertebral abnormalities. All therapy might be an effective and low-cost alternative to the use of GH. Further controlled trials should prove efficacy and safety of long-term AI therapy in boys with ISS.
Assuntos
Inibidores da Aromatase/uso terapêutico , Aromatase/metabolismo , Estatura/efeitos dos fármacos , Transtornos do Crescimento/tratamento farmacológico , Transtornos do Crescimento/enzimologia , Nitrilas/uso terapêutico , Triazóis/uso terapêutico , Adolescente , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/efeitos dos fármacos , Humanos , Letrozol , Masculino , RadiografiaRESUMO
Carnitine palmitoyltransferase-1c (CPT1c) is a newly identified and poorly understood brain-specific CPT1 homologue. Here, we have generated a new animal model that allows the conditional expression of CPT1c in a tissue specific and/or temporal manner via Cre-lox mediated recombination. Brain-specific, exogenous expression of CPT1c was achieved by crossing transgenic CPT1c mice to Nestin-Cre mice. The resulting double transgenic mice (CPT1c-TgN) displayed severe growth retardation in the postnatal period with a stunted development at 2 weeks of age. CPT1c-TgN mice had a greater than 2.3-fold reduction in brain weight. Even with this degree of microencephaly, CPT1c-TgN mice were viable and fertile and exhibited normal post-weaning growth. When fed a high fat diet CPT1c-TgN mice were protected from weight gain and the difference in body weight between CPT1c-TgN and control mice was further exaggerated. Conversely, low fat, high carbohydrate feeding partially reversed the body weight defects in CPT1c-TgN mice. Analysis of total brain lipids of low fat fed mice revealed a depletion of total very long chain fatty acids in adult CPT1c-TgN mice which was not evident in high fat fed CPT1c-TgN mice. These data show that CPT1c can elicit profound effects on brain physiology and total fatty acid profiles, which can be modulated by the nutritional composition of the diet.
Assuntos
Encéfalo/enzimologia , Encéfalo/crescimento & desenvolvimento , Carnitina O-Palmitoiltransferase/metabolismo , Carnitina O-Palmitoiltransferase/fisiologia , Microcefalia/enzimologia , Animais , Animais Recém-Nascidos , Glicemia/metabolismo , Western Blotting , Peso Corporal/fisiologia , Encéfalo/patologia , Dieta , Gorduras na Dieta/farmacologia , Metabolismo Energético/genética , Metabolismo Energético/fisiologia , Ácidos Graxos/metabolismo , Ácidos Graxos/farmacologia , Transtornos do Crescimento/enzimologia , Transtornos do Crescimento/genética , Malonil Coenzima A/metabolismo , Camundongos , Camundongos Transgênicos , Microcefalia/patologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Rnd proteins are a subfamily of Rho GTPases involved in the control of actin cytoskeleton dynamics and other cell functions such as motility, proliferation and survival. Unlike other members of the Rho family, Rnd proteins lack GTPase activity and therefore remain constitutively active. We have recently described that RhoE/Rnd3 is expressed in the Central Nervous System and that it has a role in promoting neurite formation. Despite their possible relevance during development, the role of Rnd proteins in vivo is not known. To get insight into the in vivo function of RhoE we have generated mice lacking RhoE expression by an exon trapping cassette. RhoE null mice (RhoE gt/gt) are smaller at birth, display growth retardation and early postnatal death since only half of RhoE gt/gt mice survive beyond postnatal day (PD) 15 and 100% are dead by PD 29. RhoE gt/gt mice show an abnormal body position with profound motor impairment and impaired performance in most neurobehavioral tests. Null mutant mice are hypoactive, show an immature locomotor pattern and display a significant delay in the appearance of the hindlimb mature responses. Moreover, they perform worse than the control littermates in the wire suspension, vertical climbing and clinging, righting reflex and negative geotaxis tests. Also, RhoE ablation results in a delay of neuromuscular maturation and in a reduction in the number of spinal motor neurons. Finally, RhoE gt/gt mice lack the common peroneal nerve and, consequently, show a complete atrophy of the target muscles. This is the first model to study the in vivo functions of a member of the Rnd subfamily of proteins, revealing the important role of Rnd3/RhoE in the normal development and suggesting the possible involvement of this protein in neurological disorders.