RESUMO
A woman with ichthyosis, contractures, and progressive neuropathy represents the first case of phosphoserine aminotransferase deficiency diagnosed and treated in an adult. She has novel compound heterozygous mutations in the gene PSAT1. Treatment with high dose oral L-serine completely resolved the ichthyosis. Consideration of this diagnosis is important because early treatment with L-serine repletion can halt progression of neurodegeneration and potentially improve neurological disabilities. As exome sequencing becomes more widely implemented in the diagnostic evaluation of progressive neurodegenerative phenotypes, adult neurologists and geneticists will increasingly encounter later onset manifestations of inborn errors of metabolism classically considered in infancy and early childhood.
Assuntos
Anormalidades Congênitas/genética , Ictiose/genética , Serina/biossíntese , Transaminases/genética , Adulto , Pré-Escolar , Anormalidades Congênitas/patologia , Feminino , Retardo do Crescimento Fetal/genética , Retardo do Crescimento Fetal/metabolismo , Retardo do Crescimento Fetal/patologia , Humanos , Ictiose/metabolismo , Ictiose/patologia , Deformidades Congênitas dos Membros/genética , Deformidades Congênitas dos Membros/patologia , Microcefalia/genética , Microcefalia/patologia , Transtornos Psicomotores/genética , Transtornos Psicomotores/patologia , Convulsões/genética , Convulsões/patologia , Serina/deficiência , Serina/genética , Esfingolipídeos/deficiência , Esfingolipídeos/genética , Transaminases/deficiência , Sequenciamento do ExomaRESUMO
Shiga toxin (Stx) is a major virulence factor of enterohemorrhagic Escherichia coli (E. coli). Stx consists of one enzymatic A subunit and five B subunits (StxB) that are involved in binding. The StxB pentamer specifically recognizes a glycosphingolipid, globotriaosylceramide (Gb3), as a receptor; therefore, it can be used as a probe to detect Gb3. This chapter describes the preparation of recombinant Stx1B proteins using E. coli, their conjugation with fluorescent dyes, and their application for flow cytometry. The prepared fluorescent StxB proteins bound to cells of several lines, including the HeLa human cervix adenocarcinoma cell line and the THP-1 human monocytic leukemia cell line. Furthermore, the probe was useful for confirmation of several sphingolipid-deficient HeLa cell lines that were constructed using genome editing.
Assuntos
Escherichia coli Êntero-Hemorrágica/metabolismo , Rodaminas/química , Toxinas Shiga/isolamento & purificação , Ácidos Sulfônicos/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/isolamento & purificação , Citometria de Fluxo , Edição de Genes , Células HeLa , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Toxinas Shiga/química , Esfingolipídeos/deficiência , Células THP-1RESUMO
We wish to delineate a novel, and rapidly expanding, group of inborn errors of metabolism with neurological/muscular presentations: the defects in phospholipids, sphingolipids and long chain fatty acids biosynthesis. At least 14 disorders have been described so far. Clinical presentations are diverse but can be divided into (1) diseases of the central nervous system; (2) peripheral neuropathies; and (3) muscular/cardiac presentations. (1) Leukodystrophy and/or iron deposits in basal ganglia is a common feature of phospholipase A2 deficiency, fatty acid hydroxylase deficiency, and pantothenate kinase-associated neurodegeneration. Infantile epilepsy has been reported in GM3 synthetase deficiency. Spastic quadriplegia with ichthyosis and intellectual disability are the presenting signs of the elongase 4 deficiency and the Sjogren-Larsson syndrome caused by fatty aldehyde dehydrogenase deficiency. Spastic paraplegia and muscle wasting are also seen in patients with mutations in the neuropathy target esterase gene. (2) Peripheral neuropathy is a prominent feature in PHARC syndrome due to α/ß-hydrolase 12 deficiency, and in hereditary sensory autonomic neuropathy type I due to serine palmitoyl-CoA transferase deficiency. (3) Muscular/cardiac presentations include recurrent myoglobinuria in phosphatidate phosphatase 1 (Lipin1) deficiency; cardiomyopathy and multivisceral involvement in Barth syndrome secondary to tafazzin mutations; congenital muscular dystrophy due to choline kinase deficiency, Sengers syndrome due to acylglycerol kinase deficiency and Chanarin Dorfman syndrome due to α/ß- hydrolase 5 deficiency. These synthesis defects of complex lipid molecules stand at the frontier between classical inborn errors of metabolism and other genetic diseases involving the metabolism of structural proteins.
Assuntos
Ácidos Graxos/biossíntese , Erros Inatos do Metabolismo Lipídico/classificação , Fosfolipídeos/biossíntese , Esfingolipídeos/biossíntese , Animais , Humanos , Erros Inatos do Metabolismo Lipídico/diagnóstico , Erros Inatos do Metabolismo Lipídico/genética , Erros Inatos do Metabolismo Lipídico/metabolismo , Doenças Metabólicas/classificação , Doenças Metabólicas/diagnóstico , Doenças Metabólicas/genética , Modelos Biológicos , Fosfolipídeos/deficiência , Esfingolipídeos/deficiênciaRESUMO
Sphingolipids are essential components of eukaryotic cell membranes. We recently showed that the function of the serotonin(1A) receptor is impaired upon metabolic depletion of sphingolipids using fumonisin B(1) (FB(1)), a specific inhibitor of ceramide synthase. Serotonin(1A) receptors belong to the family of G-protein coupled receptors and are implicated in the generation and modulation of various cognitive, behavioral and developmental functions. Since function and dynamics of membrane receptors are often coupled, we monitored the lateral dynamics of the serotonin(1A) receptor utilizing fluorescence recovery after photobleaching (FRAP) under these conditions. Our results show an increase in mobile fraction of the receptor upon sphingolipid depletion, while the diffusion coefficient of the receptor did not exhibit any significant change. These novel results constitute the first report on the effect of sphingolipid depletion on the mobility of the serotonin(1A) receptor. Our results assume greater relevance in the broader context of the emerging role of receptor mobility in understanding cellular signaling.
Assuntos
Receptor 5-HT1A de Serotonina/metabolismo , Esfingolipídeos/deficiência , Animais , Células CHO , Cricetinae , Cricetulus , Fumonisinas/farmacologia , Humanos , Oxirredutases/antagonistas & inibidores , FotodegradaçãoRESUMO
Sphingolipids are essential components of eukaryotic cell membranes and are thought to be involved in a variety of cellular functions. Mycotoxins such as fumonisins can disrupt sphingolipid metabolism, and treatment with fumonisins represents an efficient approach to modulate cellular sphingolipid levels. In this work, we modulated sphingolipid levels in CHO cells stably expressing the human serotonin(1A) receptor by metabolically inhibiting the biosynthesis of sphingolipids using fumonisin B(1). Serotonin(1A) receptors belong to the family of seven-transmembrane domain receptors that couple to G-proteins and are implicated in the generation and modulation of various cognitive, behavioral, and developmental functions. We explored the function of serotonin(1A) receptors under sphingolipid-depleted conditions by monitoring ligand binding, G-protein coupling, and downstream signaling of the receptor. Importantly, our results show that the function of the serotonin(1A) receptor is impaired upon metabolic depletion of sphingolipids, although the membrane receptor level does not exhibit any reduction. Interestingly, we find that the replenishment of sphingolipids using sphingosine results in restoration of ligand binding of serotonin(1A) receptors, demonstrating that the loss of ligand binding due to metabolic depletion of sphingolipids is reversible. These novel results demonstrate that sphingolipids are necessary for ligand binding and downstream signaling of the human serotonin(1A) receptor. We discuss possible mechanisms of specific interaction of sphingolipids with the serotonin(1A) receptor that could involve the proposed "sphingolipid-binding domain" (SBD).
Assuntos
Receptor 5-HT1A de Serotonina/metabolismo , Esfingolipídeos/metabolismo , Animais , Células CHO , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Cricetinae , Cricetulus , Inibidores Enzimáticos/farmacologia , Fumonisinas/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Ligantes , Oxirredutases/antagonistas & inibidores , Ligação Proteica , Esfingolipídeos/biossíntese , Esfingolipídeos/deficiênciaRESUMO
Sphingolipids containing 2-hydroxylated fatty acids are among the most abundant lipid components of the myelin sheath and therefore are thought to play an important role in formation and function of myelin. To prove this hypothesis, we generated mice lacking a functional fatty acid 2-hydroxylase (FA2H) gene. FA2H-deficient (FA2H(-/-)) mice lacked 2-hydroxylated sphingolipids in the brain and in peripheral nerves. In contrast, nonhydroxylated galactosylceramide was increased in FA2H(-/-) mice. However, oligodendrocyte differentiation examined by in situ hybridization with cRNA probes for proteolipid protein and PDGFalpha receptor and the time course of myelin formation were not altered in FA2H(-/-) mice compared with wild-type littermates. Nerve conduction velocity measurements of sciatic nerves revealed no significant differences between FA2H(-/-) and wild-type mice. Moreover, myelin of FA2H(-/-) mice up to 5 months of age appeared normal at the ultrastructural level, in the CNS and peripheral nervous system. Myelin thickness and g-ratios were normal in FA2H(-/-) mice. Aged (18-month-old) FA2H(-/-) mice, however, exhibited scattered axonal and myelin sheath degeneration in the spinal cord and an even more pronounced loss of stainability of myelin sheaths in sciatic nerves. These results show that structurally and functionally normal myelin can be formed in the absence of 2-hydroxylated sphingolipids but that its long-term maintenance is strikingly impaired. Because axon degeneration appear to start rather early with respect to myelin degenerations, these lipids might be required for glial support of axon function.
Assuntos
Amidoidrolases/deficiência , Axônios/fisiologia , Bainha de Mielina/patologia , Degeneração Neural/genética , Degeneração Neural/patologia , Esfingolipídeos/deficiência , Fatores Etários , Animais , Animais Recém-Nascidos , Axônios/ultraestrutura , Comportamento Animal , Diferenciação Celular/genética , Sistema Nervoso Central/crescimento & desenvolvimento , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/patologia , Comportamento Exploratório , Regulação da Expressão Gênica no Desenvolvimento/genética , Metabolismo dos Lipídeos/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Proteína Básica da Mielina/metabolismo , Bainha de Mielina/ultraestrutura , Condução Nervosa/fisiologia , Oligodendroglia/fisiologia , Nervo Óptico/metabolismo , Nervo Óptico/fisiopatologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
Chronic and persistent lung infections cause the majority of morbidity and mortality in patients with cystic fibrosis (CF). Galactosyl ceramide has been previously shown to be involved in Pseudomonas internalization. Therefore, we assessed ceramide levels in the plasma of patients with CF and compared them to healthy volunteers using high-performance liquid chromatography followed by mass spectrometry. Our results demonstrate that patients with CF display significantly lower levels of several ceramide sphingolipid species, specifically C14:0, C20:1, C22:0, C22:1, and C24:0 ceramides, and dihydroxy ceramide (DHC16:0). We report that Cftr-knockout mice display diminished ceramide levels in CF-related organs (lung, pancreas, ileum, and plasma) compared with their littermate controls. Since it has been previously reported that in vitro treatment with fenretinide induced ceramide in neuroblastoma cell lines, we decided to test this drug in vivo using our Cftr-knockout mice in an attempt to correct this newly identified defect in ceramide levels. We demonstrate that treatment with fenretinide is able to increase ceramide concentrations in CF-related organs. We further assessed the biological effect of fenretinide on the ability of Cftr-knockout mice to combat lung infection with P. aeruginosa. Our data show dramatic improvement in the ability of Cftr-knockout mice to control P. aeruginosa infection. Overall, these findings not only document a novel deficiency in several ceramide species in patients with CF, but also demonstrate a pharmacologic means to correct this defect in Cftr-knockout mice. Our data provide a strong rationale for clinical intervention that may benefit patients with CF suffering from CF lung disease.
Assuntos
Anticarcinógenos/farmacologia , Ceramidas/deficiência , Fibrose Cística/sangue , Fenretinida/farmacologia , Infecções por Pseudomonas/sangue , Pseudomonas aeruginosa , Esfingolipídeos/deficiência , Adulto , Animais , Anticarcinógenos/uso terapêutico , Linhagem Celular Tumoral , Ceramidas/sangue , Cromatografia Líquida de Alta Pressão , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Fibrose Cística/microbiologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Feminino , Fenretinida/uso terapêutico , Humanos , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos CFTR , Camundongos Knockout , Pessoa de Meia-Idade , Especificidade de Órgãos/genética , Infecções por Pseudomonas/genética , Esfingolipídeos/sangueRESUMO
The nicotinic acetylcholine receptor (AChR) is the prototype ligand-gated ion channel, and its function is dependent on its lipid environment. In order to study the involvement of sphingolipids (SL) in AChR trafficking, we used pharmacological approaches to dissect the SL biosynthetic pathway in CHO-K1/A5 cells heterologously expressing the muscle-type AChR. When SL biosynthesis was impaired, the cell surface targeting of AChR diminished with a concomitant increase in the intracellular receptor pool. The SL-inhibiting drugs increased unassembled AChR forms, which were retained at the endoplasmic reticulum (ER). These effects on AChR biogenesis and trafficking could be reversed by the addition of exogenous SL, such as sphingomyelin. On the basis of these effects we propose a 'chaperone-like' SL intervention at early stages of the AChR biosynthetic pathway, affecting both the efficiency of the assembly process and subsequent receptor trafficking to the cell surface.
Assuntos
Receptores Nicotínicos/metabolismo , Esfingolipídeos/fisiologia , Rede trans-Golgi/metabolismo , Animais , Vias Biossintéticas/fisiologia , Bungarotoxinas/farmacocinética , Linhagem Celular , Cricetinae , Cricetulus , Interações Medicamentosas , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Ácidos Graxos Monoinsaturados/farmacologia , Fumonisinas/farmacologia , Imunossupressores/farmacologia , Microscopia de Fluorescência/métodos , Transporte Proteico/genética , Esfingolipídeos/antagonistas & inibidores , Esfingolipídeos/deficiência , Esfingomielinas/farmacologia , Temperatura , Rede trans-Golgi/efeitos dos fármacosRESUMO
It has been previously demonstrated that depletion of cholesterol inhibits endosome to Golgi transport. Whether this inhibition is due to disruption of sphingolipid- and cholesterol-containing lipid rafts that are selected for Golgi transport or whether there is a physical requirement of cholesterol for either membrane deformations, facilitating formation of transport vesicles, or for recruitment of cytosolic constituents is not obvious. To investigate this in more detail, we have studied endosome to Golgi transport of ricin in sphingolipid-deficient cells using either a mutant cell line that does not express serine palmitoyltransferase, the first enzyme in sphingolipid biosynthesis, or a specific inhibitor, myriocin, of the same enzyme. Depletion of sphingolipids gave an increased sensitivity to ricin, and this increased sensitivity was inhibited by addition of sphingolipids. Importantly, endosome to Golgi transport of ricin, measured as sulfation of a modified ricin molecule, was increased in sphingolipid-deficient cells. No effect was seen on other pathways taken by ricin. Interestingly, cholesterol depletion inhibited endosome to Golgi transport even in cells with reduced levels of sphingolipids, suggesting that cholesterol as such is required for formation of transport vesicles. Our results indicate that the presence of sphingolipids actually limits and may function to control endosome to Golgi transport of ricin.
Assuntos
Endossomos/fisiologia , Complexo de Golgi/fisiologia , Ricina/metabolismo , Esfingolipídeos/deficiência , Animais , Células CHO , Cricetinae , Cricetulus , Transporte Proteico/fisiologia , Ricina/toxicidadeRESUMO
In filamentous fungi, the stabilization of a polarity axis is likely to be a pivotal event underlying the emergence of a germ tube from a germinating spore. Recent results implicate the polarisome in this process and also suggest that it requires localized membrane organization. Here, we employ a chemical genetic approach to demonstrate that ceramide synthesis is necessary for the formation of a stable polarity axis in the model fungus Aspergillus nidulans. We demonstrate that a novel compound (HSAF) produced by a bacterial biocontrol agent disrupts polarized growth and leads to loss of membrane organization and formin localization at hyphal tips. We show that BarA, a putative acyl-CoA-dependent ceramide synthase that is unique to filamentous fungi mediates the effects of HSAF. Moreover, A. nidulans possesses a second likely ceramide synthase that is essential and also regulates hyphal morphogenesis. Our results suggest that filamentous fungi possess distinct pools of ceramide that make independent contributions to polarized hyphal growth, perhaps through the formation of specialized lipid microdomains that regulate organization of the cytoskeleton.
Assuntos
Aspergillus nidulans/citologia , Aspergillus nidulans/enzimologia , Polaridade Celular , Oxirredutases/metabolismo , Acil Coenzima A/metabolismo , Antifúngicos/metabolismo , Aspergillus nidulans/crescimento & desenvolvimento , Farmacorresistência Fúngica , Proteínas Fúngicas/metabolismo , Hifas/citologia , Oxigenases de Função Mista/metabolismo , Modelos Biológicos , Mutação/genética , Fenótipo , Filogenia , Proteínas de Saccharomyces cerevisiae/metabolismo , Esfingolipídeos/deficiência , Esfingolipídeos/metabolismoRESUMO
In addition to their crucial role in membrane structure in Saccharomyces cerevisiae, sphingolipids serve vital roles in various aspects of yeast biology including endocytosis, intracellular protein transport and stress responses. Although previous studies have unequivocally demonstrated the sphingolipid requirements for these processes, few studies have contributed mechanistic information. We have used a systems approach including microarray, lipidomics and metabolic modelling to better understand (i) biochemical relationships between various branches of sphingolipid metabolism and pathways and contributing pathways such as fatty acid metabolism and phospholipid synthesis, (ii) the changes in cellular sphingolipid composition under various conditions and (iii) the effects of these changes on the transcriptional profiles and subsequently, cell phenotypes. Thus far, these approaches have indicated roles for sphingolipids in major transcriptional changes in response to heat stress, carbon source utilization, sporulation, cell wall integrity and other basic cellular functions. Although the yeast genome is fully sequenced, nearly 50% of all transcribed open reading frames remain uncharacterized with regard to cellular function; therefore, a major advantage of this approach is the ability to identify both biochemical and biological roles for enzymes and their products within broad cellular contexts.