RESUMO
1-Deoxysphingolipids (deoxySLs) are atypical sphingolipids that are elevated in the plasma of patients with type 2 diabetes and hereditary sensory and autonomic neuropathy type 1 (HSAN1). Clinically, diabetic neuropathy and HSAN1 are very similar, suggesting the involvement of deoxySLs in the pathology of both diseases. However, very little is known about the biology of these lipids and the underlying pathomechanism. We synthesized an alkyne analog of 1-deoxysphinganine (doxSA), the metabolic precursor of all deoxySLs, to trace the metabolism and localization of deoxySLs. Our results indicate that the metabolism of these lipids is restricted to only some lipid species and that they are not converted to canonical sphingolipids or fatty acids. Furthermore, exogenously added alkyne-doxSA [(2S,3R)-2-aminooctadec-17-yn-3-ol] localized to mitochondria, causing mitochondrial fragmentation and dysfunction. The induced mitochondrial toxicity was also shown for natural doxSA, but not for sphinganine, and was rescued by inhibition of ceramide synthase activity. Our findings therefore indicate that mitochondrial enrichment of an N-acylated doxSA metabolite may contribute to the neurotoxicity seen in diabetic neuropathy and HSAN1. Hence, we provide a potential explanation for the characteristic vulnerability of peripheral nerves to elevated levels of deoxySLs.
Assuntos
Diabetes Mellitus Tipo 2/sangue , Neuropatias Diabéticas/sangue , Neuropatias Hereditárias Sensoriais e Autônomas/sangue , Esfingolipídeos/sangue , Animais , Diabetes Mellitus Tipo 2/patologia , Neuropatias Diabéticas/patologia , Neuropatias Hereditárias Sensoriais e Autônomas/patologia , Humanos , Lipídeos/sangue , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Oxirredutases/metabolismo , Nervos Periféricos/metabolismo , Nervos Periféricos/patologia , Esfingolipídeos/síntese química , Esfingolipídeos/farmacologiaRESUMO
ORMDL proteins are believed to be negative regulators of serine palmitoyltransferase (SPT), which catalyzes the first and rate limiting step in sphingolipid (SL) de novo synthesis. Several single-nucleotide polymorphisms (SNPs) that are close to the ORMDL3 locus have been reported to increase ORMDL3 expression and to be associated with an elevated risk for early childhood asthma; however, the direct effect of ORMDL3 expression on SPT activity and its link to asthma remains elusive. In this study, we investigated whether ORMDL3 expression is associated with changes in SPT activity and total SL levels. Ormdl3-knockout (Ormdl3-/-) and transgenic (Ormdl3Tg/wt) mice were generated to study the effect of ORMDL3 on total SL levels in plasma and tissues. Cellular SPT activity was measured in mouse embryonic fibroblasts from Ormdl3-/- mice, as well as in HEK293 cells in which ORMDL3 was overexpressed and silenced. Furthermore, we analyzed the association of the reported ORMDL3 asthma SNPs with plasma sphingoid bases in a population-based cohort of 971 individuals. Total C18-long chain bases were not significantly altered in the plasma and tissues of Ormdl3-/- mice, whereas C18-sphinganine showed a small and significant increase in plasma, lung, and liver tissues. Mouse embryonic fibroblast cells from Ormdl3-/- mice did not show an altered SPT activity compared with Ormdl3+/- and Ormdl3+/+ mice. Overexpression or knockdown of ORMDL3 in HEK293 cells did not alter SPT activity; however, parallel knockdown of all 3 ORMDL isoforms increased enzyme activity significantly. A significant association of the annotated ORMDL3 asthma SNPs with plasma long-chain sphingoid base levels could not be confirmed. ORMDL3 expression levels seem not to be directly associated with changes in SPT activity. ORMDL3 might influence de novo sphingolipid metabolism downstream of SPT.-Zhakupova, A., Debeuf, N., Krols, M., Toussaint, W., Vanhoutte, L., Alecu, I., Kutalik, Z., Vollenweider, P., Ernst, D., von Eckardstein, A., Lambrecht, B. N., Janssens, S., Hornemann, T. ORMDL3 expression levels have no influence on the activity of serine palmitoyltransferase.
Assuntos
Metabolismo dos Lipídeos/fisiologia , Proteínas de Membrana/metabolismo , Isoformas de Proteínas/metabolismo , Serina C-Palmitoiltransferase/metabolismo , Animais , Asma/metabolismo , Células HEK293 , Humanos , Pulmão/metabolismo , Proteínas de Membrana/genética , Camundongos Knockout , Polimorfismo de Nucleotídeo Único/genética , Esfingolipídeos/sangueRESUMO
Peripheral neuropathy is a major dose-limiting side effect of paclitaxel and cisplatin chemotherapy. In the current study, we tested the involvement of a novel class of neurotoxic sphingolipids, the 1-deoxysphingolipids. 1-Deoxysphingolipids are produced when the enzyme serine palmitoyltransferase uses l-alanine instead of l-serine as its amino acid substrate. We tested whether treatment of cells with paclitaxel (250 nM, 1 µM) and cisplatin (250 nM, 1 µM) would result in elevated cellular levels of 1-deoxysphingolipids. Our results revealed that paclitaxel, but not cisplatin treatment, caused a dose-dependent elevation of 1-deoxysphingolipids levels and an increase in the message and activity of serine palmitoyltransferase (P < 0.05). We also tested whether there is an association between peripheral neuropathy symptoms [evaluated by the European Organization for Research and Treatment of Cancer (EORTC) QLQ-chemotherapy-induced peripheral neuropathy-20 (CIPN20) instrument] and the 1-deoxysphingolipid plasma levels (measured by mass spectrometry) in 27 patients with breast cancer who were treated with paclitaxel chemotherapy. Our results showed that there was an association between the incidence and severity of neuropathy and the levels of very-long-chain 1-deoxyceramides such as C24 (P < 0.05), with the strongest association being with motor neuropathy (P < 0.001). Our data from cells and from patients with breast cancer suggest that 1-deoxysphingolipids, the very-long-chain in particular, play a role as molecular intermediates of paclitaxel-induced peripheral neuropathy.
Assuntos
Neoplasias da Mama , Neurotoxinas/sangue , Paclitaxel/efeitos adversos , Doenças do Sistema Nervoso Periférico , Esfingolipídeos/sangue , Adolescente , Adulto , Neoplasias da Mama/sangue , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Feminino , Células HEK293 , Humanos , Pessoa de Meia-Idade , Paclitaxel/administração & dosagem , Doenças do Sistema Nervoso Periférico/sangue , Doenças do Sistema Nervoso Periférico/induzido quimicamenteRESUMO
1-Deoxysphingolipids (1-deoxySL) are atypical sphingolipids that are formed by the enzyme serine palmitoyltransferase (SPT) due to a promiscuous use of L-alanine over its canonical substrate L-serine. Several mutations in SPT are associated with the hereditary sensory and autonomic neuropathy type I (HSAN1). The current hypothesis is that these mutations induce a permanent shift in the affinity from L-serine toward L-alanine which results in a pathologically increased 1-deoxySL formation in HSAN1 patients. Also, wild-type SPT forms 1-deoxySL under certain conditions, and elevated levels were found in individuals with the metabolic syndrome and diabetes. However, the molecular mechanisms which control the substrate shift of the wild-type enzyme are not understood. Here, we report a novel SPTLC2-S384F variant in two unrelated HSAN1 families. Affected patients showed elevated plasma 1-deoxySL levels and expression of the S384F mutant in HEK293 cells increased 1-deoxySL formation. Previously, S384 has been reported as one of the two (S384 and Y387) putative phosphorylation sites in SPTLC2. The phosphorylation of wild-type SPTLC2 was confirmed by isoelectric focusing. The impact of an S384 phosphorylation on SPT activity was tested by creating mutants mimicking either a constitutively phosphorylated (S384D, S384E) or non-phosphorylated (S384A, Y387F, Y387F+S384A) protein. The S384D but not the S384E variant was associated with increased 1-deoxySL formation. The other mutations had no influence on activity and substrate affinity. In summary, our data show that S384F is a novel mutation in HSAN1 and that the substrate specificity of wild-type SPT might by dynamically regulated by a phosphorylation at this position.
Assuntos
Neuropatias Hereditárias Sensoriais e Autônomas/genética , Serina C-Palmitoiltransferase/genética , Idoso , Sequência de Aminoácidos , Substituição de Aminoácidos , Sequência Conservada , Eletroforese em Gel Bidimensional , Feminino , Células HEK293 , Heterozigoto , Homozigoto , Humanos , Focalização Isoelétrica , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Dados de Sequência Molecular , Condução Nervosa , Linhagem , Fosforilação , Fosfosserina/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Serina C-Palmitoiltransferase/química , Serina C-Palmitoiltransferase/fisiologia , Especificidade da Espécie , Esfingolipídeos/metabolismo , Especificidade por SubstratoRESUMO
OBJECTIVE: To describe the clinical and neurophysiologic phenotype of a family with hereditary sensory and autonomic neuropathy type 1 (HSANI) due to a novel mutation in SPTLC2 and to characterize the biochemical properties of this mutation. METHODS: We screened 107 patients with HSAN who were negative for other genetic causes for mutations in SPTLC2. The biochemical properties of a new mutation were characterized in cell-free and cell-based activity assays. RESULTS: A novel mutation (A182P) was found in 2 subjects of a single family. The phenotype of the 2 subjects was an ulcero-mutilating sensory-predominant neuropathy as described previously for patients with HSANI, but with prominent motor involvement and earlier disease onset in the first decade of life. Affected patients had elevated levels of plasma 1-deoxysphingolipids (1-deoxySLs). Biochemically, the A182P mutation was associated with a reduced canonical activity but an increased alternative activity with alanine, which results in largely increased 1-deoxySL levels, supporting their pathogenicity. CONCLUSION: This study confirms that mutations in SPTLC2 are associated with increased deoxySL formation causing HSANI.
Assuntos
Neuropatias Hereditárias Sensoriais e Autônomas/genética , Mutação/genética , Serina C-Palmitoiltransferase/genética , Esfingolipídeos/sangue , Adulto , Neuropatias Hereditárias Sensoriais e Autônomas/sangue , Neuropatias Hereditárias Sensoriais e Autônomas/classificação , Neuropatias Hereditárias Sensoriais e Autônomas/fisiopatologia , Humanos , Linhagem , Fenótipo , Análise de Sequência de DNARESUMO
The enzyme serine palmitoyltransferase (SPT) catalyzes the rate-limiting step in the de novo synthesis of sphingolipids. Previously the mammalian SPT was described as a heterodimer composed of two subunits, SPTLC1 and SPTLC2. Recently we identified a novel third SPT subunit (SPTLC3). SPTLC3 shows about 68% identity to SPTLC2 and also includes a pyridoxal phosphate consensus motif. Here we report that the overexpression of SPTLC3 in HEK293 cells leads to the formation of two new sphingoid base metabolites, namely C(16)-sphinganine and C(16)-sphingosine. SPTLC3-expressing cells have higher in vitro SPT activities with lauryl- and myristoyl-CoA than SPTLC2-expressing cells, and SPTLC3 mRNA expression levels correlate closely with the C(16)-sphinganine synthesis rates in various human and murine cell lines. Approximately 15% of the total sphingolipids in human plasma contain a C(16) backbone and are found in the high density and low density but not the very low density lipoprotein fraction. In conclusion, we show that the SPTLC3 subunit generates C(16)-sphingoid bases and that sphingolipids with a C(16) backbone constitute a significant proportion of human plasma sphingolipids.