ABSTRACT
Sphingolipids are ubiquitous in membranes of eukaryotes and are associated with important cellular functions. Although sphingolipids occur scarcely in bacteria, for some of them they are essential and, in other bacteria, they contribute to fitness and stability of the outer membrane, such as in the well-studied α-proteobacterium Caulobacter crescentus. We previously defined five structural genes for ceramide synthesis in C. crescentus, among them the gene for serine palmitoyltransferase, the enzyme that catalyzes the committed step of sphingolipid biosynthesis. Other mutants affected in genes of this same genomic region show cofitness with a mutant deficient in serine palmitoyltransferase. Here we show that at least two phosphosphingolipids are produced in C. crescentus and that at least another six gene products are needed for the decoration of ceramide upon phosphosphingolipid formation. All eleven genes participating in phosphosphingolipid formation are also required in C. crescentus for membrane stability and for displaying sensitivity towards the antibiotic polymyxin B. The genes for the formation of complex phosphosphingolipids are also required for C. crescentus virulence on Galleria mellonella insect larvae.
Subject(s)
Caulobacter crescentus , Sphingolipids , Caulobacter crescentus/metabolism , Caulobacter crescentus/genetics , Virulence , Animals , Sphingolipids/metabolism , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Serine C-Palmitoyltransferase/metabolism , Serine C-Palmitoyltransferase/genetics , Moths/microbiologyABSTRACT
After recognizing its ligand lipopolysaccharide, Toll-like receptor 4 (TLR4) recruits adaptor proteins to the cell membrane, thereby initiating downstream signaling and triggering inflammation. Whether this recruitment of adaptor proteins is dependent solely on protein-protein interactions is unknown. Here, we report that the sphingolipid sphinganine physically interacts with the adaptor proteins MyD88 and TIRAP and promotes MyD88 recruitment in macrophages. Myeloid cell-specific deficiency in serine palmitoyltransferase long chain base subunit 2, which encodes the key enzyme catalyzing sphingolipid biosynthesis, decreases the membrane recruitment of MyD88 and inhibits inflammatory responses in in vitro bone marrow-derived macrophage and in vivo sepsis models. In a melanoma mouse model, serine palmitoyltransferase long chain base subunit 2 deficiency decreases anti-tumor myeloid cell responses and increases tumor growth. Therefore, sphinganine biosynthesis is required for the initiation of TLR4 signal transduction and serves as a checkpoint for macrophage pattern recognition in sepsis and melanoma mouse models.
Subject(s)
Macrophages , Melanoma , Myeloid Differentiation Factor 88 , Sepsis , Serine C-Palmitoyltransferase , Sphingosine , Toll-Like Receptor 4 , Animals , Toll-Like Receptor 4/metabolism , Sepsis/metabolism , Macrophages/metabolism , Myeloid Differentiation Factor 88/metabolism , Mice , Sphingosine/analogs & derivatives , Sphingosine/metabolism , Melanoma/metabolism , Melanoma/pathology , Melanoma/genetics , Serine C-Palmitoyltransferase/metabolism , Serine C-Palmitoyltransferase/genetics , Humans , Signal Transduction , Disease Models, Animal , Inflammation/metabolism , Receptors, Interleukin-1/metabolism , Receptors, Interleukin-1/genetics , Membrane Glycoproteins/metabolism , Membrane Glycoproteins/genetics , Mice, Inbred C57BL , Mice, Knockout , HEK293 Cells , LipopolysaccharidesABSTRACT
Hereditary sensory and autonomic neuropathy type 1 is an autosomal dominant neuropathy caused by the SPTLC1 or SPTLC2 variants. These variants modify the preferred substrate of serine palmitoyl transferase, responsible for the first step of de novo sphingolipids synthesis, leading to accumulation of cytotoxic deoxysphingolipids. Diagnosis of HSAN1 is based on clinical symptoms, mainly progressive loss of distal sensory keep, and genetic analysis. Aim: Identifying new SPTLC1 or SPTLC2 "gain-of-function" variants raises the question as to their pathogenicity. This work focused on characterizing six new SPTLC1 variants using in silico prediction tools, new meta-scores, 3D modeling, and functional testing to establish their pathogenicity. Methods: Variants from six patients with HSAN1 were studied. In silico, CADD and REVEL scores and the 3D modeling software MITZLI were used to characterize the pathogenic effect of the variants. Functional tests based on plasma sphingolipids quantification (total deoxysphinganine, ceramides, and dihydroceramides) were performed by tandem mass spectrometry. Results: In silico predictors did not provide very contrasting results when functional tests discriminated the different variants according to their impact on deoxysphinganine level or canonical sphingolipids synthesis. Two SPTLC1 variants were newly described as pathogenic: SPTLC1 NM_006415.4:c.998A>G and NM_006415.4:c.1015G>A. Discussion: The combination of the different tools provides arguments to establish the pathogenicity of these new variants. When available, functional testing remains the best option to establish the in vivo impact of a variant. Moreover, the comprehension of metabolic dysregulation offers opportunities to develop new therapeutic strategies for these genetic disorders.
Subject(s)
Hereditary Sensory and Autonomic Neuropathies , Mutation, Missense , Serine C-Palmitoyltransferase , Sphingolipids , Humans , Serine C-Palmitoyltransferase/genetics , Serine C-Palmitoyltransferase/metabolism , Hereditary Sensory and Autonomic Neuropathies/genetics , Hereditary Sensory and Autonomic Neuropathies/diagnosis , Male , Female , Sphingolipids/metabolism , Adult , Middle AgedABSTRACT
Niemann-Pick type C1 (NPC1) disease is a rare neurodegenerative cholesterol and sphingolipid storage disorder primarily due to mutations in the cholesterol-trafficking protein NPC1. In addition to catabolic-derived sphingolipids, NPC1 dysfunction also leads to an increase in de novo sphingolipid biosynthesis, yet little is known about the cellular mechanism involved. Although deletion of NPC1 or inhibition of the NPC1 sterol binding domain enhanced de novo sphingolipid biosynthesis, surprisingly levels of the ORMDLs, the regulatory subunits of serine palmitoyltransferase (SPT), the rate-limiting step in sphingolipid biosynthesis, were also greatly increased. Nevertheless, less ORMDL was bound in the SPT-ORMDL complex despite elevated ceramide levels. Instead, ORMDL colocalized with p62, the selective autophagy receptor, and accumulated in stalled autophagosomes due to defective autophagy in NPC1 disease cells. Restoration of autophagic flux with N-acetyl-L-leucine in NPC1 deleted cells decreased ORMDL accumulation in autophagosomes and reduced de novo sphingolipid biosynthesis and their accumulation. This study revealed a previously unknown link between de novo sphingolipid biosynthesis, ORMDL, and autophagic defects present in NCP1 disease. In addition, we provide further evidence and mechanistic insight for the beneficial role of N-acetyl-L-leucine treatment for NPC1 disease which is presently awaiting approval from the Food and Drug Administration and the European Medicines Agency.
Subject(s)
Autophagy , Niemann-Pick Disease, Type C , Sphingolipids , Sphingolipids/metabolism , Sphingolipids/biosynthesis , Niemann-Pick Disease, Type C/metabolism , Niemann-Pick Disease, Type C/pathology , Niemann-Pick Disease, Type C/genetics , Humans , Membrane Proteins/metabolism , Membrane Proteins/genetics , Animals , Niemann-Pick C1 Protein , Serine C-Palmitoyltransferase/metabolism , Serine C-Palmitoyltransferase/genetics , Serine C-Palmitoyltransferase/antagonists & inhibitorsABSTRACT
Motor neuron diseases and peripheral neuropathies are heterogeneous groups of neurodegenerative disorders that manifest with distinct symptoms due to progressive dysfunction or loss of specific neuronal subpopulations during different stages of development. A few monogenic, neurodegenerative diseases associated with primary metabolic disruptions of sphingolipid biosynthesis have been recently discovered. Sphingolipids are a subclass of lipids that form critical building blocks of all cellular and subcellular organelle membranes including the membrane components of the nervous system cells. They are especially abundant within the lipid portion of myelin. In this review, we will focus on our current understanding of disease phenotypes in three monogenic, neuromuscular diseases associated with pathogenic variants in components of serine palmitoyltransferase, the first step in sphingolipid biosynthesis. These include hereditary sensory and autonomic neuropathy type 1 (HSAN1), a sensory predominant peripheral neuropathy, and two neurodegenerative disorders: juvenile amyotrophic lateral sclerosis affecting the upper and lower motor neurons with sparing of sensory neurons, and a complicated form of hereditary spastic paraplegia with selective involvement of the upper motor neurons and more broad CNS neurodegeneration. We will also review our current understanding of disease pathomechanisms, therapeutic approaches, and the unanswered questions to explore in future studies.
Subject(s)
Neurodegenerative Diseases , Neurodevelopmental Disorders , Serine C-Palmitoyltransferase , Humans , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/metabolism , Hereditary Sensory and Autonomic Neuropathies/genetics , Hereditary Sensory and Autonomic Neuropathies/metabolism , Hereditary Sensory and Autonomic Neuropathies/physiopathology , Neurodegenerative Diseases/metabolism , Serine C-Palmitoyltransferase/metabolism , Serine C-Palmitoyltransferase/genetics , Spastic Paraplegia, Hereditary/genetics , Spastic Paraplegia, Hereditary/metabolism , Sphingolipids/metabolismABSTRACT
T helper 17 (TH17) cells are implicated in autoimmune diseases, and several metabolic processes are shown to be important for their development and function. In this study, we report an essential role for sphingolipids synthesized through the de novo pathway in TH17 cell development. Deficiency of SPTLC1, a major subunit of serine palmitoyl transferase enzyme complex that catalyzes the first and rate-limiting step of de novo sphingolipid synthesis, impaired glycolysis in differentiating TH17 cells by increasing intracellular reactive oxygen species (ROS) through enhancement of nicotinamide adenine dinucleotide phosphate oxidase 2 activity. Increased ROS leads to impaired activation of mammalian target of rapamycin C1 and reduced expression of hypoxia-inducible factor 1-alpha and c-Myc-induced glycolytic genes. SPTLCI deficiency protected mice from developing experimental autoimmune encephalomyelitis and experimental T cell transfer colitis. Our results thus show a critical role for de novo sphingolipid biosynthetic pathway in shaping adaptive immune responses with implications in autoimmune diseases.
Subject(s)
Cell Differentiation , Encephalomyelitis, Autoimmune, Experimental , Serine C-Palmitoyltransferase , Sphingolipids , Th17 Cells , Animals , Sphingolipids/metabolism , Sphingolipids/biosynthesis , Th17 Cells/immunology , Th17 Cells/metabolism , Th17 Cells/cytology , Mice , Encephalomyelitis, Autoimmune, Experimental/metabolism , Encephalomyelitis, Autoimmune, Experimental/pathology , Encephalomyelitis, Autoimmune, Experimental/immunology , Serine C-Palmitoyltransferase/metabolism , Serine C-Palmitoyltransferase/genetics , Reactive Oxygen Species/metabolism , Glycolysis , Mice, Knockout , Colitis/metabolism , Colitis/pathology , Mice, Inbred C57BLABSTRACT
Sphingolipids are produced by nearly all eukaryotes where they play significant roles in cellular processes such as cell growth, division, programmed cell death, angiogenesis, and inflammation. While it was previously believed that sphingolipids were quite rare among bacteria, bioinformatic analysis of the recently identified bacterial sphingolipid synthesis genes suggests that these lipids are likely to be produced by a wide range of microbial species. The sphingolipid synthesis pathway consists of three critical enzymes. Serine palmitoyltransferase catalyzes the condensation of serine with palmitoyl-CoA (or palmitoyl-acyl carrier protein), ceramide synthase adds the second acyl chain, and a reductase reduces the ketone present on the long-chain base. While there is general agreement regarding the identity of these bacterial enzymes, the precise mechanism and order of chemical reactions for microbial sphingolipid synthesis is more ambiguous. Two mechanisms have been proposed. First, the synthesis pathway may follow the well characterized eukaryotic pathway in which the long-chain base is reduced prior to the addition of the second acyl chain. Alternatively, our previous work suggests that addition of the second acyl chain precedes the reduction of the long-chain base. To distinguish between these two models, we investigated the subcellular localization of these three key enzymes. We found that serine palmitoyltransferase and ceramide synthase are localized to the cytoplasm, whereas the ceramide reductase is in the periplasmic space. This is consistent with our previously proposed model wherein the second acyl chain is added in the cytoplasm prior to export to the periplasm where the lipid molecule is reduced.
Subject(s)
Bacterial Proteins , Serine C-Palmitoyltransferase , Sphingolipids , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Serine C-Palmitoyltransferase/metabolism , Serine C-Palmitoyltransferase/genetics , Sphingolipids/biosynthesis , Oxidoreductases/metabolism , Protein Transport , Cytoplasm/enzymology , Caulobacter crescentus/enzymology , Escherichia coli/enzymologyABSTRACT
BACKGROUND: Dysregulated metabolism of bioactive sphingolipids, including ceramides and sphingosine-1-phosphate, has been implicated in cardiovascular disease, although the specific species, disease contexts, and cellular roles are not completely understood. Sphingolipids are produced by the serine palmitoyltransferase enzyme, canonically composed of 2 subunits, SPTLC1 (serine palmitoyltransferase long chain base subunit 1) and SPTLC2 (serine palmitoyltransferase long chain base subunit 2). Noncanonical sphingolipids are produced by a more recently described subunit, SPTLC3 (serine palmitoyltransferase long chain base subunit 3). METHODS: The noncanonical (d16) and canonical (d18) sphingolipidome profiles in cardiac tissues of patients with end-stage ischemic cardiomyopathy and in mice with ischemic cardiomyopathy were analyzed by targeted lipidomics. Regulation of SPTLC3 by HIF1α under ischemic conditions was determined with chromatin immunoprecipitation. Transcriptomics, lipidomics, metabolomics, echocardiography, mitochondrial electron transport chain, mitochondrial membrane fluidity, and mitochondrial membrane potential were assessed in the cSPTLC3KO transgenic mice we generated. Furthermore, morphological and functional studies were performed on cSPTLC3KO mice subjected to permanent nonreperfused myocardial infarction. RESULTS: Herein, we report that SPTLC3 is induced in both human and mouse models of ischemic cardiomyopathy and leads to production of atypical sphingolipids bearing 16-carbon sphingoid bases, resulting in broad changes in cell sphingolipid composition. This induction is in part attributable to transcriptional regulation by HIF1α under ischemic conditions. Furthermore, cardiomyocyte-specific depletion of SPTLC3 in mice attenuates oxidative stress, fibrosis, and hypertrophy in chronic ischemia, and mice demonstrate improved cardiac function and increased survival along with increased ketone and glucose substrate metabolism utilization. Depletion of SPTLC3 mechanistically alters the membrane environment and subunit composition of mitochondrial complex I of the electron transport chain, decreasing its activity. CONCLUSIONS: Our findings suggest a novel essential role for SPTLC3 in electron transport chain function and a contribution to ischemic injury by regulating complex I activity.
Subject(s)
Cardiomyopathies , Electron Transport Complex I , Serine C-Palmitoyltransferase , Animals , Serine C-Palmitoyltransferase/metabolism , Serine C-Palmitoyltransferase/genetics , Mice , Humans , Cardiomyopathies/metabolism , Cardiomyopathies/genetics , Electron Transport Complex I/metabolism , Electron Transport Complex I/genetics , Sphingolipids/metabolism , Mice, Knockout , Myocardial Ischemia/metabolism , Myocardial Ischemia/genetics , Myocardial Ischemia/pathology , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Male , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathologyABSTRACT
Oxidative stress plays a pivotal role in the pathogenesis of several neurodegenerative diseases. Retinal degeneration causes irreversible death of photoreceptor cells, ultimately leading to vision loss. Under oxidative stress, the synthesis of bioactive sphingolipid ceramide increases, triggering apoptosis in photoreceptor cells and leading to their death. This study investigates the effect of L-Cycloserine, a small molecule inhibitor of ceramide biosynthesis, on sphingolipid metabolism and the protection of photoreceptor-derived 661W cells from oxidative stress. The results demonstrate that treatment with L-Cycloserine, an inhibitor of Serine palmitoyl transferase (SPT), markedly decreases bioactive ceramide and associated sphingolipids in 661W cells. A nontoxic dose of L-Cycloserine can provide substantial protection of 661W cells against H2O2-induced oxidative stress by reversing the increase in ceramide level observed under oxidative stress conditions. Analysis of various antioxidant, apoptotic and sphingolipid pathway genes and proteins also confirms the ability of L-Cycloserine to modulate these pathways. Our findings elucidate the generation of sphingolipid mediators of cell death in retinal cells under oxidative stress and the potential of L-Cycloserine as a therapeutic candidate for targeting ceramide-induced degenerative diseases by inhibiting SPT. The promising therapeutic prospect identified in our findings lays the groundwork for further validation in in-vivo and preclinical models of retinal degeneration.
Subject(s)
Apoptosis , Ceramides , Cycloserine , Oxidative Stress , Sphingolipids , Oxidative Stress/drug effects , Cycloserine/pharmacology , Animals , Ceramides/metabolism , Ceramides/pharmacology , Mice , Sphingolipids/metabolism , Apoptosis/drug effects , Photoreceptor Cells, Vertebrate/drug effects , Photoreceptor Cells, Vertebrate/metabolism , Photoreceptor Cells, Vertebrate/pathology , Serine C-Palmitoyltransferase/metabolism , Serine C-Palmitoyltransferase/antagonists & inhibitors , Hydrogen Peroxide/toxicity , Hydrogen Peroxide/pharmacology , Cell Line , Retinal Degeneration/metabolism , Retinal Degeneration/prevention & control , Retinal Degeneration/pathology , Retinal Degeneration/drug therapy , Blotting, Western , Enzyme Inhibitors/pharmacology , Cell Survival/drug effectsABSTRACT
Serine palmitoyltransferase (SPT) catalyzes the pyridoxal-5'-phosphate (PLP)-dependent decarboxylative condensation of l-serine and palmitoyl-CoA to form 3-ketodihydrosphingosine (KDS). Although SPT was shown to synthesize corresponding products from amino acids other than l-serine, it is still arguable whether SPT catalyzes the reaction with d-serine, which is a question of biological importance. Using high substrate and enzyme concentrations, KDS was detected after the incubation of SPT from Sphingobacterium multivorum with d-serine and palmitoyl-CoA. Furthermore, the KDS comprised equal amounts of 2S and 2R isomers. 1H-NMR study showed a slow hydrogen-deuterium exchange at Cα of serine mediated by SPT. We further confirmed that SPT catalyzed the racemization of serine. The rate of the KDS formation from d-serine was comparable to those for the α-hydrogen exchange and the racemization reaction. The structure of the d-serine-soaked crystal (1.65 Å resolution) showed a distinct electron density of the PLP-l-serine aldimine, interpreted as the racemized product trapped in the active site. The structure of the α-methyl-d-serine-soaked crystal (1.70 Å resolution) showed the PLP-α-methyl-d-serine aldimine, mimicking the d-serine-SPT complex prior to racemization. Based on these enzymological and structural analyses, the synthesis of KDS from d-serine was explained as the result of the slow racemization to l-serine, followed by the reaction with palmitoyl-CoA, and SPT would not catalyze the direct condensation between d-serine and palmitoyl-CoA. It was also shown that the S. multivorum SPT catalyzed the racemization of the product KDS, which would explain the presence of (2R)-KDS in the reaction products.
Subject(s)
Serine C-Palmitoyltransferase , Serine , Sphingobacterium , Catalytic Domain , Crystallization , Deuterium Exchange Measurement , Electrons , Hydrogen/metabolism , Palmitoyl Coenzyme A/metabolism , Serine/analogs & derivatives , Serine/metabolism , Serine C-Palmitoyltransferase/chemistry , Serine C-Palmitoyltransferase/metabolism , Sphingobacterium/enzymology , Sphingobacterium/metabolism , Sphingosine/analogs & derivatives , Sphingosine/biosynthesis , Sphingosine/metabolism , Stereoisomerism , Substrate SpecificityABSTRACT
The homeostatic regulation of serine palmitoyltransferase (SPT) activity in yeast involves N-terminal phosphorylation of Orm proteins, while higher eukaryotes lack these phosphorylation sites. Although recent studies have indicated a conserved ceramide-mediated feedback inhibition of the SPT-ORM/ORMDL complex in higher eukaryotes, its conservation and relationship with phosphorylation regulation in yeast remain unclear. Here, we determine the structure of the yeast SPT-Orm2 complex in a dephosphomimetic state and identify an evolutionarily conserved ceramide-sensing site. Ceramide stabilizes the dephosphomimetic Orm2 in an inhibitory conformation, facilitated by an intramolecular ß-sheet between the N- and C-terminal segments of Orm2. Moreover, we find that a phosphomimetic mutant of Orm2, positioned adjacent to its intramolecular ß-sheet, destabilizes the inhibitory conformation of Orm2. Taken together, our findings suggest that both Orm dephosphorylation and ceramide binding are crucial for suppressing SPT activity in yeast. This highlights a distinctive regulatory mechanism in yeast involving the collaborative actions of phosphorylation and ceramide.
Subject(s)
Ceramides , Saccharomyces cerevisiae Proteins , Ceramides/metabolism , Saccharomyces cerevisiae/metabolism , Sphingolipids/metabolism , Phosphorylation , Proteins/metabolism , Serine C-Palmitoyltransferase/genetics , Serine C-Palmitoyltransferase/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
Sphingolipids are a diverse class of lipids with essential functions as determinants of membrane physical properties and as intra- and intercellular signaling agents. Disruption of the normal biochemical processes that establish the levels of individual sphingolipids is associated with a variety of human diseases including cancer, cardiovascular disease, metabolic disease, skin diseases, and lysosomal storage diseases. A unique aspect of this metabolic network is that there is a single enzymatic step that initiates the biosynthetic pathway for all sphingolipids. This step is catalyzed by the enzyme serine palmitoyltranserase (SPT). Under most circumstances SPT condenses serine and the 16-carbon acyl-CoA, palmitoyl-CoA to produce the precursor of all sphingolipids. SPT, a four-subunit protein complex, is subject to classic feedback regulation: when cellular sphingolipids are elevated, SPT activity is inhibited. Ceramide is the sphingolipid sensed by this system and it regulates SPT by directly binding to the complex. The ceramide binding site in the SPT complex, and how ceramide binding results in SPT inhibition, has now been determined in vertebrates, plants, and yeast using molecular modeling and cryo-electron microscopy. Here we discuss the similarities and differences revealed by these resolved structures and the surprising result that ceramide binds at almost identical positions in the SPT complex of these divergent organisms, but accomplishes SPT regulation in very different ways.