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1.
Sci Rep ; 12(1): 16740, 2022 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-36202842

RESUMO

Hyperlipidemia is considered as one of the major systemic factors associated with the development of diabetic retinopathy, and animal models have documented that its presence in a hyperglycemic environment exacerbates cytosolic ROS production (via activation of the Rac1-Nox2 axis) and mitochondrial damage. Hyperglycemia also accelerates Rac1 transcription via dynamic DNA methylation-hydroxymethylation of its promoter. In diabetes, ceramide metabolism in the retina is impaired and its accumulation is increased. Our aim was to investigate the effect of inhibition of the rate limiting enzyme of the de novo ceramide biosynthesis, serine palmitoyl-transferase (SPT), on Rac1 activation in diabetic retinopathy. Using human retinal endothelial cells, transfected with SPT-siRNA, and incubated in 20 mM D-glucose in the presence or absence of 50 µM palmitate (glucolipotoxic and glucotoxic, respectively), activities of Rac1 and Nox2, and ROS levels were quantified. For Rac1 transcriptional activation, 5 hydroxymethyl cytosine (5hmC) levels at its promoter were quantified. Key parameters were confirmed in retinal microvessels from streptozotocin-induced diabetic mice on a normal diet (type 1 diabetic model) or on a high-fat diet (45% kcal, type 2 diabetic model), injected intravitreally with SPT-siRNA. Compared to normal glucose, cells in high glucose, with or without palmitic acid, had increased Rac1-Nox2-ROS signaling, Rac1 transcripts and 5hmC levels at its promoter. Inhibition of SPT by SPT-siRNA or myriocin prevented glucotoxic- and glucolipotoxic-induced increase in Rac1-Nox2-ROS signaling and 5hmC at the Rac1 promoter. Similarly, in both type 1 and type 2 diabetic mouse models, SPT-siRNA attenuated the increase in the Rac1-Nox2-ROS axis and 5hmC at the Rac1 promoter. Thus, inhibition of the rate limiting enzyme of ceramide de novo biosynthesis, SPT, regulates activation of DNA methylation-hydroxymethylation machinery and prevents increased Rac1 transcription. This ameliorates the activation of Rac1-Nox2 signaling and protects the mitochondria from damaging cytosolic ROS, which prevents accelerated capillary cell loss. These results further raise the importance of regulating lipid levels in diabetic patients with dyslipidemia.


Assuntos
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Retinopatia Diabética , Animais , Ceramidas/metabolismo , Citosina/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Retinopatia Diabética/metabolismo , Células Endoteliais/metabolismo , Glucose/metabolismo , Humanos , Camundongos , NADPH Oxidase 2/metabolismo , Palmitatos/farmacologia , Ácido Palmítico/farmacologia , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Serina/metabolismo , Serina C-Palmitoiltransferase/metabolismo , Serina C-Palmitoiltransferase/farmacologia , Estreptozocina/farmacologia , Proteínas rac1 de Ligação ao GTP/metabolismo
2.
J Lipid Res ; 63(10): 100281, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36115594

RESUMO

Serine palmitoyltransferase (SPT) predominantly incorporates serine and fatty acyl-CoAs into diverse sphingolipids (SLs) that serve as structural components of membranes and signaling molecules within or amongst cells. However, SPT also uses alanine as a substrate in the contexts of low serine availability, alanine accumulation, or disease-causing mutations in hereditary sensory neuropathy type I, resulting in the synthesis and accumulation of 1-deoxysphingolipids (deoxySLs). These species promote cytotoxicity in neurons and impact diverse cellular phenotypes, including suppression of anchorage-independent cancer cell growth. While altered serine and alanine levels can promote 1-deoxySL synthesis, they impact numerous other metabolic pathways important for cancer cells. Here, we combined isotope tracing, quantitative metabolomics, and functional studies to better understand the mechanistic drivers of 1-deoxySL toxicity in cancer cells. We determined that both alanine treatment and SPTLC1C133W expression induce 1-deoxy(dihydro)ceramide synthesis and accumulation but fail to broadly impact intermediary metabolism, abundances of other lipids, or growth of adherent cells. However, we found that spheroid culture and soft agar colony formation were compromised when endogenous 1-deoxySL synthesis was induced via SPTLC1C133W expression. Consistent with these impacts on anchorage-independent cell growth, we observed that 1-deoxySL synthesis reduced plasma membrane endocytosis. These results highlight a potential role for SPT promiscuity in linking altered amino acid metabolism to plasma membrane endocytosis.


Assuntos
Neoplasias , Serina C-Palmitoiltransferase , Serina C-Palmitoiltransferase/metabolismo , Ágar/metabolismo , Esfingolipídeos/metabolismo , Serina/química , Ceramidas/metabolismo , Alanina/metabolismo , Membrana Celular/metabolismo , Redes e Vias Metabólicas , Endocitose , Neoplasias/metabolismo
3.
Cell Rep ; 40(13): 111415, 2022 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-36170811

RESUMO

Sphingolipids play important signaling and structural roles in cells. Here, we find that during de novo sphingolipid biosynthesis, a toxic metabolite is formed with critical implications for cancer cell survival. The enzyme catalyzing the first step in this pathway, serine palmitoyltransferase complex (SPT), is upregulated in breast and other cancers. SPT is dispensable for cancer cell proliferation, as sphingolipids can be salvaged from the environment. However, SPT activity introduces a liability as its product, 3-ketodihydrosphingosine (3KDS), is toxic and requires clearance via the downstream enzyme 3-ketodihydrosphingosine reductase (KDSR). In cancer cells, but not normal cells, targeting KDSR induces toxic 3KDS accumulation leading to endoplasmic reticulum (ER) dysfunction and loss of proteostasis. Furthermore, the antitumor effect of KDSR disruption can be enhanced by increasing metabolic input (via high-fat diet) to allow greater 3KDS production. Thus, de novo sphingolipid biosynthesis entails a detoxification requirement in cancer cells that can be therapeutically exploited.


Assuntos
Neoplasias , Serina C-Palmitoiltransferase , Lipogênese , Oxirredutases/metabolismo , Serina/metabolismo , Serina C-Palmitoiltransferase/metabolismo , Esfingolipídeos/metabolismo , Esfingosina/análogos & derivados
4.
Microbiol Spectr ; 10(5): e0133122, 2022 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-36121228

RESUMO

Serine palmitoyltransferase catalyzes the first step of the sphingolipid biosynthesis. Recently, sphingolipid homeostasis has been connected to several human diseases, making serine palmitoyltransferases an interesting therapeutic target. Known and efficient serine palmitoyltransferase-inhibitors are sphingofungins, a group of natural products isolated from fungi. To further characterize newly isolated sphingofungins, we designed an easy to use colorimetric serine palmitoyltransferase activity assay using FadD, which can be performed in 96-well plates. Because sphingofungins exert antifungal activitiy as well, we compared the in vitro assay results with an in vivo growth assay using Saccharomyces cerevisiae. The reported experiments showed differences among the assayed sphingofungins, highlighting an increase of activity based on the saturation levels of the polyketide tail. IMPORTANCE Targeting the cellular sphingolipid metabolism is often discussed as a potential approach to treat associated human diseases such as cancer and Alzheimer's disease. Alternatively, it is also a possible target for the development of antifungal compounds, which are direly needed. A central role is played by the serine palmitoyltransferase, which catalyzes the initial and rate limiting step of sphingolipid de novo synthesis and, as such, the development of inhibitory compounds for this enzyme is of interest. Our work here established an alternative approach for determining the activity of serine palmitoyltransferase adding another tool for the validation of its inhibition. We also determined the effect of different modifications to sphingofungins on their inhibitory activity against serine palmitoyltransferase, revealing important differences on said activity against enzymes of bacterial and fungal origin.


Assuntos
Produtos Biológicos , Policetídeos , Humanos , Serina C-Palmitoiltransferase/metabolismo , Serina C-Palmitoiltransferase/farmacologia , Antifúngicos/farmacologia , Policetídeos/farmacologia , Aciltransferases/metabolismo , Aciltransferases/farmacologia , Saccharomyces cerevisiae , Esfingolipídeos/farmacologia , Serina/farmacologia
5.
FEBS Lett ; 596(18): 2345-2363, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35899376

RESUMO

Modern cell membranes contain a bewildering complexity of lipids, among them sphingolipids (SLs). Advances in mass spectrometry have led to the realization that the number and combinatorial complexity of lipids, including SLs, is much greater than previously appreciated. SLs are generated de novo by four enzymes, namely serine palmitoyltransferase, 3-ketodihydrosphingosine reductase, ceramide synthase and dihydroceramide Δ4-desaturase 1. Some of these enzymes depend on the availability of specific substrates and cofactors, which are themselves supplied by other complex metabolic pathways. The evolution of these four enzymes is poorly understood and likely depends on the co-evolution of the metabolic pathways that supply the other essential reaction components. Here, we introduce the concept of the 'anteome', from the Latin ante ('before') to describe the network of metabolic ('omic') pathways that must have converged in order for these pathways to co-evolve and permit SL synthesis. We also suggest that the current origin of life and evolutionary models lack appropriate experimental support to explain the appearance of this complex metabolic pathway and its anteome.


Assuntos
Serina C-Palmitoiltransferase , Esfingolipídeos , Ceramidas/metabolismo , Ácidos Graxos Dessaturases/metabolismo , Espectrometria de Massas , Redes e Vias Metabólicas , Serina C-Palmitoiltransferase/metabolismo , Esfingolipídeos/metabolismo
6.
J Lipid Res ; 63(7): 100235, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35654151

RESUMO

Ceramides are essential lipids for skin permeability barrier function, and a wide variety of ceramide species exist in the stratum corneum (SC). Although ceramides with long-chain bases (LCBs) of various lengths have been identified in the human SC, a quantitative analysis that distinguishes ceramide species with different LCB chain lengths has not been yet published. Therefore, the whole picture of human SC ceramides remains unclear. Here, we conducted LC/MS/MS analyses to detect individual ceramide species differing in both the LCB and FA chain lengths and quantified 1,327 unbound ceramides and 254 protein-bound ceramides: the largest number of ceramide species reported to date. Ceramides containing an LCB whose chain length was C16-26 were present in the human SC. Of these, C18 (28.6%) was the most abundant, followed by C20 (24.8%) and C22 (12.8%). Each ceramide class had a characteristic distribution of LCB chain lengths and was divided into five groups according to this distribution. There was almost no difference in FA composition between the ceramide species containing LCBs of different chain lengths. Furthermore, we demonstrated that one of the serine palmitoyltransferase (SPT) complexes, SPTLC1/SPTLC3/SPTSSB, was able to produce C16-24 LCBs. The expression levels of all subunits constituting the SPT complexes increased during keratinocyte differentiation, resulting in the observed chain-length diversity of LCBs in the human SC. This study provides a molecular basis for elucidating human SC ceramide diversity and the pathogenesis of skin disorders.


Assuntos
Ceramidas , Espectrometria de Massas em Tandem , Ceramidas/metabolismo , Epiderme/metabolismo , Humanos , Permeabilidade , Serina C-Palmitoiltransferase/genética , Serina C-Palmitoiltransferase/metabolismo
7.
Adv Exp Med Biol ; 1372: 31-46, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35503172

RESUMO

Atherosclerosis is the formation of fibrofatty lesions in the arterial wall, and this inflammatory state of the artery is the main cause of advanced pathological processes, including myocardial infarction and stroke. Dyslipidemic conditions with excess cholesterol accumulate within the arterial vessel wall and initiate atherogenic processes. Following vascular reaction and lipid accumulation, the vascular wall gradually thickens. Together with the occurrence of local inflammation, early atherosclerotic lesions lead to advanced pathophysiological events, plaque rupture, and thrombosis. Ceramide and sphingomyelin have emerged as major risk factors for atherosclerosis and coronary artery disease. Currently, the clinical association between de novo sphingolipid biosynthesis and coronary artery disease has been established. Furthermore, therapeutic strategies to modulate this pathway, especially those involving serine palmitoyltransferase and sphingomyelin synthase, against atherosclerosis, cancer, type 2 diabetes, and non-alcoholic fatty liver disease are actively under development. In this chapter, we focus on the relationship between de novo sphingolipid biosynthesis and coronary artery disease.


Assuntos
Aterosclerose , Doença da Artéria Coronariana , Diabetes Mellitus Tipo 2 , Aterosclerose/metabolismo , Humanos , Serina C-Palmitoiltransferase/genética , Serina C-Palmitoiltransferase/metabolismo , Esfingolipídeos
8.
Adv Exp Med Biol ; 1372: 145-155, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35503179

RESUMO

Asthma is the most prevalent chronic respiratory disease worldwide and the leading serious chronic illness in children. Clinical characteristics are wheezing, reversible airway obstruction, airway inflammation, and airway hyperreactivity. Asthma susceptibility is influenced by genes and environment. 17q12-21 is the most significant genetic asthma susceptibility locus and single nucleotide polymorphisms (SNPs) within that high-risk locus are linked to increased expression of the Ormdl sphingolipid biosynthesis regulator (ORMDL) 3. ORMDL3 is an endoplasmic reticulum protein that stabilizes the serine palmitoyl transferase (SPT) complex that regulates sphingolipid de novo synthesis. Sphingolipids essential for formation and integrity of cellular membranes and bioactive molecules that regulate key cellular processes can be synthesized de novo and through recycling pathways. Their metabolism is tightly regulated through feedback regulation. ORMDL3 inhibits de novo synthesis when it engages subunit 1 of the SPT complex. This chapter focuses on the effect of decreased sphingolipid synthesis on asthma features and summarizes studies in mouse models and in children with and without asthma.


Assuntos
Asma , Esfingolipídeos , Animais , Asma/genética , Asma/metabolismo , Suscetibilidade a Doenças , Proteínas de Membrana/metabolismo , Camundongos , Polimorfismo de Nucleotídeo Único , Serina C-Palmitoiltransferase/genética , Serina C-Palmitoiltransferase/metabolismo , Esfingolipídeos/metabolismo
9.
Adv Exp Med Biol ; 1372: 169-188, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35503181

RESUMO

Sphingolipids are the major lipid components on cellular membranes especially on lipid raft regions, intermediating various important biological functions for eukaryotic cells. Sphingolipid metabolism pathways can utilize sugar, protein, nucleic acid, and other metabolites participating lipid transport in the circulation, play an essential role in maintaining cell homeostasis and are related to a variety of different diseases including lysosomal storage disorders (LSDs), Gaucher disease, etc. The dynamic balance of sphingolipid levels in organisms is regulated by a series of sphingolipid synthases, hydrolases, and metabolic enzymes, such as sphingomyelinase (SMase), sphingomyelin synthase (SMS), serine palmitoyltransferase (SPT), ceramide synthase (CerS), glucosylceramide synthase (GCS), etc. Thus, sphingolipids and its related enzymes are potential targets for drug discoveries and receive great research interests by medicinal chemist. In this chapter, we will discuss the relationship between sphingolipids and the regulating enzymes involved in sphingolipid metabolisms, and systematically summarize the advances in the development of new drugs in the field.


Assuntos
Serina C-Palmitoiltransferase , Esfingolipídeos , Ceramidas/metabolismo , Desenvolvimento de Medicamentos , Homeostase , Metabolismo dos Lipídeos , Serina C-Palmitoiltransferase/metabolismo , Esfingolipídeos/metabolismo
10.
Adv Exp Med Biol ; 1372: 47-56, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35503173

RESUMO

Sphingolipids (SL) are a class of chemically diverse lipids that have important structural and physiological functions in eukaryotic cells. SL entail a long chain base (LCB) as the common structural element, which is typically formed by the condensation of L-serine and long chain acyl-CoA. This condensation is the first and the rate-limiting step in the de novo SL synthesis and catalyzed by the enzyme serine palmitoyltransferase (SPT). Although palmitoyl-CoA is the preferred substrate, SPT can also metabolize other acyl-CoAs, thereby forming a variety of LCBs, which differ in structures and functions. The mammalian SPT enzyme is composed of three core subunits: SPTLC1, SPTLC2, and SPTLC3. Whereas SPTLC1 and SPTLC2 are ubiquitously expressed, SPTLC3 expression is restricted to a few specific tissues. The SPTLC1 subunit is essential and can associate with either SPTLC2 or SPTLC3 to form an active enzyme. Depending on the stoichiometry of the SPTLC2 and SPTLC3 subunits, the spectrum of SPT products varies. While SPTLC1 and SPTLC2 primarily form C18 and C20 LCBs, the combination of SPTLC1 and SPTLC3 produces a broader spectrum of LCBs. Genetic and population based studies have shown that SPTLC3 expression and function are associated with an altered plasma SL profile and an increased risk for cardio-metabolic diseases. Animal and in vitro studies showed that SPTLC3 might be involved in hepatic and cardiac pathology and could be a therapeutic target for these conditions.Here we present an overview of the current data on the role of SPTLC3 in normal and pathological conditions.


Assuntos
Doenças Metabólicas , Serina C-Palmitoiltransferase , Animais , Coenzima A , Humanos , Mamíferos/metabolismo , Doenças Metabólicas/genética , Serina , Serina C-Palmitoiltransferase/química , Serina C-Palmitoiltransferase/genética , Serina C-Palmitoiltransferase/metabolismo , Esfingolipídeos/metabolismo
11.
Exp Mol Med ; 54(5): 573-584, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35513574

RESUMO

Endoplasmic reticulum (ER) stress is induced by various conditions, such as inflammation and the presence of excess nutrients. Abnormal accumulation of unfolded proteins leads to the activation of a collective signaling cascade, termed the unfolded protein response (UPR). ER stress is reported to perturb hepatic insulin response metabolism while promoting insulin resistance. Here, we report that ER stress regulates the de novo biosynthesis of sphingolipids via the activation of serine palmitoyltransferase (SPT), a rate-limiting enzyme involved in the de novo biosynthesis of ceramides. We found that the expression levels of Sptlc1 and Sptlc2, the major SPT subunits, were upregulated and that the cellular concentrations of ceramide and dihydroceramide were elevated by acute ER stress inducers in primary hepatocytes and HepG2 cells. Sptlc2 was upregulated and ceramide levels were elevated by tunicamycin in the livers of C57BL/6J wild-type mice. Analysis of the Sptlc2 promoter demonstrated that the transcriptional activation of Sptlc2 was mediated by the spliced form of X-box binding protein 1 (sXBP1). Liver-specific Sptlc2 transgenic mice exhibited increased ceramide levels in the liver and elevated fasting glucose levels. The insulin response was reduced by the inhibition of the phosphorylation of insulin receptor ß (IRß). Collectively, these results demonstrate that ER stress induces activation of the de novo biosynthesis of ceramide and contributes to the progression of hepatic insulin resistance via the reduced phosphorylation of IRß in hepatocytes.


Assuntos
Resistência à Insulina , Serina C-Palmitoiltransferase , Regulação para Cima , Animais , Ceramidas/metabolismo , Estresse do Retículo Endoplasmático , Insulina/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Serina C-Palmitoiltransferase/genética , Serina C-Palmitoiltransferase/metabolismo , Ativação Transcricional
12.
Cells ; 11(7)2022 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-35406688

RESUMO

Skeletal muscles account for ~80% of insulin-stimulated glucose uptake and play a key role in lipid metabolism. Consumption of a high-fat diet (HFD) contributes to metabolic changes in muscles, including the development of insulin resistance. The studies carried out to date indicate that the accumulation of biologically active lipids, such as long-chain acyl-CoA, diacylglycerols and ceramides, play an important role in the development of insulin resistance in skeletal muscles. Unfortunately, it has not yet been clarified which of these lipid groups plays the dominant role in inducing these disorders. In order to explore this topic further, we locally silenced the gene encoding serine palmitoyltransferase (SPT) in the gastrocnemius muscle of animals with HFD-induced insulin resistance. This enzyme is primarily responsible for the first step of de novo ceramide biosynthesis. The obtained results confirm that the HFD induces the development of whole-body insulin resistance, which results in inhibition of the insulin pathway. This is associated with an increased level of biologically active lipids in the muscles. Our results also demonstrate that silencing the SPT gene with the shRNA plasmid reduces the accumulation of ceramides in gastrocnemius muscle, which, in turn, boosts the activity of the insulin signaling pathway. Furthermore, inhibition of ceramide synthesis does not significantly affect the content of other lipids, which suggests the leading role of ceramide in the lipid-related induction of skeletal muscle insulin resistance.


Assuntos
Ceramidas , Resistência à Insulina , Serina C-Palmitoiltransferase , Animais , Ceramidas/metabolismo , Dieta Hiperlipídica , Inativação Gênica , Insulina/metabolismo , Resistência à Insulina/genética , Camundongos , Músculo Esquelético/metabolismo , Serina C-Palmitoiltransferase/genética , Serina C-Palmitoiltransferase/metabolismo
13.
Int J Mol Sci ; 23(4)2022 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-35216351

RESUMO

It is known that metabolic disturbances, including obesity, predispose to an increased incidence of cardiovascular diseases. Elevated consumption of dietary fat results in intramyocardial accumulation of lipids and their biologically active derivatives, which can disrupt the contractile function of the heart, its metabolism, and intracellular signaling pathways. Therefore, alternative methods, such as phytocannabinoids, are being sought for the treatment of obesity-related effects. In a model of rodent obesity (seven weeks of high-fat-diet (HFD) regime), we used cannabidiol-CBD therapy (intraperitoneal injections for 14 days; 10 mg/kg). High-performance and gas-liquid chromatographies were applied in order to determine sphingolipids in the heart and plasma as well as Western blotting for protein expression. Two-week CBD administration significantly inhibited the de novo ceramide synthesis pathway in the heart of HFD fed rats by lowering sphinganine and sphinganine-1-phosphate contents. The above reductions were accompanied by markedly diminished expressions of myocardial serine palmitoyltransferase 1 and 2 as well as ceramide synthase 5 and 6 in the HFD group with 2-week CBD treatment. To our knowledge, this research is the first that reveals unknown effects of CBD treatment on the heart, i.e., amelioration of de novo ceramide synthesis pathway in obese rats.


Assuntos
Vias Biossintéticas/efeitos dos fármacos , Canabidiol/farmacologia , Ceramidas/metabolismo , Regulação para Baixo/efeitos dos fármacos , Miocárdio/metabolismo , Obesidade/metabolismo , Animais , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Insulina/metabolismo , Resistência à Insulina/fisiologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipogênese/efeitos dos fármacos , Masculino , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Ratos , Ratos Wistar , Serina C-Palmitoiltransferase/metabolismo , Esfingolipídeos/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo
14.
Sci Rep ; 12(1): 955, 2022 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-35046440

RESUMO

Suppression of a specific gene effect can be achieved by genetic as well as chemical methods. Each approach may hide unexpected drawbacks, usually in the form of side effects. In the present study, the specific inhibitor myriocin was used to block serine palmitoyltransferase (SPT), the first enzyme in the sphingolipid synthetic pathway, in CHO cells. The subsequent biophysical changes in plasma membranes were measured and compared with results obtained with a genetically modified CHO cell line containing a defective SPT (the LY-B cell line). Similar effects were observed with both approaches: sphingomyelin values were markedly decreased in myriocin-treated CHO cells and, in consequence, their membrane molecular order (measured as laurdan general polarization) and mechanical resistance (AFM-measured breakthrough force values) became lower than in the native, non-treated cells. Cells treated with myriocin reacted homeostatically to maintain membrane order, synthesizing more fully saturated and less polyunsaturated GPL than the non-treated ones, although they achieved it only partially, their plasma membranes remaining slightly more fluid and more penetrable than those from the control cells. The good agreement between results obtained with very different tools, such as genetically modified and chemically treated cells, reinforces the use of both methods and demonstrates that both are adequate for their intended use, i.e. the complete and specific inhibition of sphingolipid synthesis in CHO cells, without apparent unexpected effects.


Assuntos
Membrana Celular/efeitos dos fármacos , Ácidos Graxos Monoinsaturados/farmacologia , Serina C-Palmitoiltransferase/metabolismo , Esfingolipídeos/biossíntese , Animais , Células CHO , Membrana Celular/metabolismo , Cricetulus , Lipidômica , Serina C-Palmitoiltransferase/antagonistas & inibidores , Serina C-Palmitoiltransferase/genética
15.
Am J Respir Cell Mol Biol ; 66(3): 302-311, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34851798

RESUMO

The 17q21 asthma susceptibility locus includes asthma risk alleles associated with decreased sphingolipid synthesis, likely resulting from increased expression of ORMDL3. ORMDL3 inhibits serine-palmitoyl transferase (SPT), the rate-limiting enzyme of de novo sphingolipid synthesis. There is evidence that decreased sphingolipid synthesis is critical to asthma pathogenesis. Children with asthma and 17q21 asthma risk alleles display decreased sphingolipid synthesis in blood cells. Reduced SPT activity results in airway hyperreactivity, a hallmark feature of asthma. 17q21 asthma risk alleles are also linked to childhood infections with human rhinovirus (RV). This study evaluates the interaction of RV with the de novo sphingolipid synthesis pathway, and the alterative effects of concurrent SPT inhibition in SPT-deficient mice and human airway epithelial cells. In mice, RV infection shifted lung sphingolipid synthesis gene expression to a pattern that resembles genetic SPT deficiency, including decreased expression of Sptssa, a small SPT subunit. This pattern was pronounced in lung epithelial cellular adhesion molecule (EpCAM+) cells and reproduced in human bronchial epithelial cells. RV did not affect Sptssa expression in lung CD45+ immune cells. RV increased sphingolipids unique to the de novo synthesis pathway in mouse lung and human airway epithelial cells. Interestingly, these de novo sphingolipid species were reduced in the blood of RV-infected wild-type mice. RV exacerbated SPT deficiency-associated airway hyperreactivity. Airway inflammation was similar in RV-infected wild-type and SPT-deficient mice. This study reveals the effects of RV infection on the de novo sphingolipid synthesis pathway, elucidating a potential mechanistic link between 17q21 asthma risk alleles and rhinoviral infection.


Assuntos
Proteínas de Membrana , Rhinovirus , Animais , Criança , Humanos , Pulmão/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Serina C-Palmitoiltransferase/genética , Serina C-Palmitoiltransferase/metabolismo , Esfingolipídeos/metabolismo
16.
Chem Biol Drug Des ; 99(3): 373-381, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34862732

RESUMO

Sphingolipids (SLs) are vital for cells as forming membrane and transducing signals. The first step for de novo biosynthesis of SLs is catalyzed by the pyridoxal-5'-phosphate (PLP)-dependent enzyme serine palmitoyltransferase (SPT), which has been proven to be a promising drug target for treating various diseases. However, there are few SPT-specific inhibitors have been identified so far. Myriocin, a natural fungal product, is confirmed as the most potent inhibitor of SPT and has been widely used, but studies of its molecular mechanism are still underway. Besides, there is no intact co-crystal structure of SPT-binding myriocin until now. Aiming to uncover the interaction mechanism between SPT- and PLP-binding myriocin at the molecular level, a systematic computational strategy was performed in this present study. Firstly, covalent docking was implemented to preliminarily predict the binding pose SPT/PLP-myriocin aldimine and its structurally similar intermediate SPT/PLP-ß-ketoacid aldimine. Secondly, two binding complexes were treated as initial structures to perform molecular dynamics simulations and binding free energy calculations. The calculated docking scores and predicted binding energies were consistent with the reported bioactivities. Finally, the binding mechanism of myriocin binding with SPT was meticulously described, and the key residues making favorable contributions were highlighted. Taken together, the current study could provide some important information and valuable guidance for further rational screening, design, and modification of potent specific SPT inhibitors.


Assuntos
Inibidores Enzimáticos/química , Ácidos Graxos Monoinsaturados/química , Simulação de Acoplamento Molecular , Sítios de Ligação , Inibidores Enzimáticos/metabolismo , Ácidos Graxos Monoinsaturados/metabolismo , Simulação de Dinâmica Molecular , Ligação Proteica , Serina C-Palmitoiltransferase/antagonistas & inibidores , Serina C-Palmitoiltransferase/metabolismo , Termodinâmica
17.
Life Sci Alliance ; 5(2)2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34785538

RESUMO

The accumulation of sphingolipid species in the cell contributes to the development of obesity and neurological disease. However, the subcellular localization of sphingolipid-synthesizing enzymes is unclear, limiting the understanding of where and how these lipids accumulate inside the cell and why they are toxic. Here, we show that SPTLC2, a subunit of the serine palmitoyltransferase (SPT) complex, catalyzing the first step in de novo sphingolipid synthesis, localizes dually to the ER and the outer mitochondrial membrane. We demonstrate that mitochondrial SPTLC2 interacts and forms a complex in trans with the ER-localized SPT subunit SPTLC1. Loss of SPTLC2 prevents the synthesis of mitochondrial sphingolipids and protects from palmitate-induced mitochondrial toxicity, a process dependent on mitochondrial ceramides. Our results reveal the in trans assembly of an enzymatic complex at an organellar membrane contact site, providing novel insight into the localization of sphingolipid synthesis and the composition and function of ER-mitochondria contact sites.


Assuntos
Retículo Endoplasmático/metabolismo , Mitocôndrias/metabolismo , Serina C-Palmitoiltransferase/metabolismo , Transporte Biológico , Complexos Multienzimáticos/metabolismo
18.
J Lipid Res ; 62: 100121, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34560079

RESUMO

Leukotrienes (LTs) and sphingolipids are critical lipid mediators participating in numerous cellular signal transduction events and developing various disorders, such as bronchial hyperactivity leading to asthma. Enzymatic reactions initiating production of these lipid mediators involve 5-lipoxygenase (5-LO)-mediated conversion of arachidonic acid to LTs and serine palmitoyltransferase (SPT)-mediated de novo synthesis of sphingolipids. Previous studies have shown that endoplasmic reticulum membrane protein ORM1-like protein 3 (ORMDL3) inhibits the activity of SPT and subsequent sphingolipid synthesis. However, the role of ORMDL3 in the synthesis of LTs is not known. In this study, we used peritoneal-derived mast cells isolated from ORMDL3 KO or control mice and examined their calcium mobilization, degranulation, NF-κB inhibitor-α phosphorylation, and TNF-α production. We found that peritoneal-derived mast cells with ORMDL3 KO exhibited increased responsiveness to antigen. Detailed lipid analysis showed that compared with WT cells, ORMDL3-deficient cells exhibited not only enhanced production of sphingolipids but also of LT signaling mediators LTB4, 6t-LTB4, LTC4, LTB5, and 6t-LTB5. The crosstalk between ORMDL3 and 5-LO metabolic pathways was supported by the finding that endogenous ORMDL3 and 5-LO are localized in similar endoplasmic reticulum domains in human mast cells and that ORMDL3 physically interacts with 5-LO. Further experiments showed that 5-LO also interacts with the long-chain 1 and long-chain 2 subunits of SPT. In agreement with these findings, 5-LO knockdown increased ceramide levels, and silencing of SPTLC1 decreased arachidonic acid metabolism to LTs to levels observed upon 5-LO knockdown. These results demonstrate functional crosstalk between the LT and sphingolipid metabolic pathways, leading to the production of lipid signaling mediators.


Assuntos
Araquidonato 5-Lipoxigenase/metabolismo , Eicosanoides/metabolismo , Proteínas de Membrana/metabolismo , Serina C-Palmitoiltransferase/metabolismo , Esfingolipídeos/metabolismo , Animais , Eicosanoides/análise , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Esfingolipídeos/análise
19.
Toxicology ; 458: 152831, 2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-34097992

RESUMO

Aryl hydrocarbon receptor (AHR) activation via 2,3,7,8-tetrachlorodibenzofuran (TCDF) induces the accumulation of hepatic lipids. Here we report that AHR activation by TCDF (24  µg/kg body weight given orally for five days) induced significant elevation of hepatic lipids including ceramides in mice, was associated with increased expression of key ceramide biosynthetic genes, and increased activity of their respective enzymes. Results from chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assay (EMSA) and cell-based reporter luciferase assays indicated that AHR directly activated the serine palmitoyltransferase long chain base subunit 2 (Sptlc2, encodes serine palmitoyltransferase 2 (SPT2)) gene whose product catalyzes the initial rate-limiting step in de novo sphingolipid biosynthesis. Hepatic ceramide accumulation was further confirmed by mass spectrometry-based lipidomics. Taken together, our results revealed that AHR activation results in the up-regulation of Sptlc2, leading to ceramide accumulation, thus promoting lipogenesis, which can induce hepatic lipid accumulation.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Ceramidas/biossíntese , Lipogênese/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Ativação Metabólica/efeitos dos fármacos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Benzofuranos/farmacologia , Ceramidas/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Lipidômica , Fígado/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Hidrocarboneto Arílico/genética , Serina C-Palmitoiltransferase/genética , Serina C-Palmitoiltransferase/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Triglicerídeos/metabolismo
20.
Nat Med ; 27(7): 1197-1204, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34059824

RESUMO

Amyotrophic lateral sclerosis (ALS) is a progressive, neurodegenerative disease of the lower and upper motor neurons with sporadic or hereditary occurrence. Age of onset, pattern of motor neuron degeneration and disease progression vary widely among individuals with ALS. Various cellular processes may drive ALS pathomechanisms, but a monogenic direct metabolic disturbance has not been causally linked to ALS. Here we show SPTLC1 variants that result in unrestrained sphingoid base synthesis cause a monogenic form of ALS. We identified four specific, dominantly acting SPTLC1 variants in seven families manifesting as childhood-onset ALS. These variants disrupt the normal homeostatic regulation of serine palmitoyltransferase (SPT) by ORMDL proteins, resulting in unregulated SPT activity and elevated levels of canonical SPT products. Notably, this is in contrast with SPTLC1 variants that shift SPT amino acid usage from serine to alanine, result in elevated levels of deoxysphingolipids and manifest with the alternate phenotype of hereditary sensory and autonomic neuropathy. We custom designed small interfering RNAs that selectively target the SPTLC1 ALS allele for degradation, leave the normal allele intact and normalize sphingolipid levels in vitro. The role of primary metabolic disturbances in ALS has been elusive; this study defines excess sphingolipid biosynthesis as a fundamental metabolic mechanism for motor neuron disease.


Assuntos
Esclerose Amiotrófica Lateral/metabolismo , Esfingolipídeos/biossíntese , Adolescente , Adulto , Alelos , Sequência de Aminoácidos , Esclerose Amiotrófica Lateral/enzimologia , Esclerose Amiotrófica Lateral/genética , Sistemas CRISPR-Cas , Criança , Feminino , Genes Dominantes , Células HEK293 , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Serina C-Palmitoiltransferase/genética , Serina C-Palmitoiltransferase/metabolismo , Adulto Jovem
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