RESUMO
Sphingosine-1-phosphate (S1P) is an important signaling sphingolipid that regulates the immune system, angiogenesis, auditory function, and epithelial and endothelial barrier integrity. Spinster homolog 2 (Spns2) is an S1P transporter that exports S1P to initiate lipid signaling cascades. Modulating Spns2 activity can be beneficial in treatments of cancer, inflammation, and immune diseases. However, the transport mechanism of Spns2 and its inhibition remain unclear. Here, we present six cryo-EM structures of human Spns2 in lipid nanodiscs, including two functionally relevant intermediate conformations that link the inward- and outward-facing states, to reveal the structural basis of the S1P transport cycle. Functional analyses suggest that Spns2 exports S1P via facilitated diffusion, a mechanism distinct from other MFS lipid transporters. Finally, we show that the Spns2 inhibitor 16d attenuates the transport activity by locking Spns2 in the inward-facing state. Our work sheds light on Spns2-mediated S1P transport and aids the development of advanced Spns2 inhibitors.
Assuntos
Inflamação , Lisofosfolipídeos , Humanos , Esfingosina , Proteínas de Transporte de Ânions/fisiologiaRESUMO
Lipids are produced site-specifically in cells and then distributed nonrandomly among membranes via vesicular and nonvesicular trafficking mechanisms. The latter involves soluble amphitropic proteins extracting specific lipids from source membranes to function as molecular solubilizers that envelope their insoluble cargo before transporting it to destination sites. Lipid-binding and lipid transfer structural motifs range from multi-ß-strand barrels, to ß-sheet cups and baskets covered by α-helical lids, to multi-α-helical bundles and layers. Here, we focus on how α-helical proteins use amphipathic helical layering and bundling to form modular lipid-binding compartments and discuss the functional consequences. Preformed compartments generally rely on intramolecular disulfide bridging to maintain conformation (e.g., albumins, nonspecific lipid transfer proteins, saposins, nematode polyprotein allergens/antigens). Insights into nonpreformed hydrophobic compartments that expand and adapt to accommodate a lipid occupant are few and provided mostly by the three-layer, α-helical ligand-binding domain of nuclear receptors. The simple but elegant and nearly ubiquitous two-layer, α-helical glycolipid transfer protein (GLTP)-fold now further advances understanding.
Assuntos
Albuminas/química , Alérgenos/química , Antígenos/química , Proteínas de Transporte/química , Lipídeos/química , Albuminas/genética , Albuminas/metabolismo , Alérgenos/genética , Alérgenos/metabolismo , Animais , Antígenos/genética , Antígenos/metabolismo , Sítios de Ligação , Transporte Biológico , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Expressão Gênica , Humanos , Metabolismo dos Lipídeos , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios ProteicosRESUMO
Stem cells are fundamental units of tissue remodeling whose functions are dictated by lineage-specific transcription factors. Home to epidermal stem cells and their upward-stratifying progenies, skin relies on its secretory functions to form the outermost protective barrier, of which a transcriptional orchestrator has been elusive. KLF5 is a Krüppel-like transcription factor broadly involved in development and regeneration whose lineage specificity, if any, remains unclear. Here we report KLF5 specifically marks the epidermis, and its deletion leads to skin barrier dysfunction in vivo. Lipid envelopes and secretory lamellar bodies are defective in KLF5-deficient skin, accompanied by preferential loss of complex sphingolipids. KLF5 binds to and transcriptionally regulates genes encoding rate-limiting sphingolipid metabolism enzymes. Remarkably, skin barrier defects elicited by KLF5 ablation can be rescued by dietary interventions. Finally, we found that KLF5 is widely suppressed in human diseases with disrupted epidermal secretion, and its regulation of sphingolipid metabolism is conserved in human skin. Altogether, we established KLF5 as a disease-relevant transcription factor governing sphingolipid metabolism and barrier function in the skin, likely representing a long-sought secretory lineage-defining factor across tissue types.
RESUMO
Nearly one-third of nascent proteins are initially targeted to the endoplasmic reticulum (ER), where they are correctly folded and assembled before being delivered to their final cellular destinations. To prevent the accumulation of misfolded membrane proteins, ER-associated degradation (ERAD) removes these client proteins from the ER membrane to the cytosol in a process known as retrotranslocation. Our previous work demonstrated that rhomboid pseudoprotease Dfm1 is involved in the retrotranslocation of ubiquitinated membrane integral ERAD substrates. Herein, we found that Dfm1 associates with the SPOTS complex, which is composed of serine palmitoyltransferase (SPT) enzymes and accessory components that are critical for catalyzing the first rate-limiting step of the sphingolipid biosynthesis pathway. Furthermore, Dfm1 employs an ERAD-independent role for facilitating the ER export and endosome- and Golgi-associated degradation (EGAD) of Orm2, which is a major antagonist of SPT activity. Given that the accumulation of human Orm2 homologs, ORMDLs, is associated with various pathologies, our study serves as a molecular foothold for understanding how dysregulation of sphingolipid metabolism leads to various diseases.
Assuntos
Degradação Associada com o Retículo Endoplasmático , Esfingolipídeos , Humanos , Esfingolipídeos/metabolismo , Ubiquitina/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , HomeostaseRESUMO
Prenatal exposure to environmental agents can influence the fitness of not only the fetus, but also subsequent generations. In a recent study, Wang et al. demonstrated that feeding ursolic acid (UA), a plant-derived compound, to Caenorhabditis elegans mothers during their reproductive period prevented neurodegeneration in not only their offspring, but also the F2 progeny.
Assuntos
Caenorhabditis elegans , Reprodução , Animais , Caenorhabditis elegans/genética , AxôniosRESUMO
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.
Assuntos
Proteínas de Bactérias , Serina C-Palmitoiltransferase , Esfingolipídeos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Serina C-Palmitoiltransferase/metabolismo , Serina C-Palmitoiltransferase/genética , Esfingolipídeos/biossíntese , Oxirredutases/metabolismo , Transporte Proteico , Citoplasma/enzimologia , Caulobacter crescentus/enzimologia , Escherichia coli/enzimologiaRESUMO
Polyploid giant cancer cells (PGCC) are frequently detected in tumors and are increasingly recognized for their roles in chromosomal instability and associated genome evolution that leads to cancer recurrence. We previously reported that therapy stress promotes polyploidy, and that acid ceramidase plays a role in depolyploidization. In this study, we used an RNA-seq approach to gain a better understanding of the underlying transcriptomic changes that occur as cancer cells progress through polyploidization and depolyploidization. Our results revealed gene signatures that are associated with disease-free and/or overall survival in several cancers and identified the cell cycle inhibitor CDKN1A/p21 as the major hub in PGCC and early progeny. Increased expression of p21 in PGCC was limited to the cytoplasm. We previously demonstrated that the sphingolipid enzyme acid ceramidase is dispensable for polyploidization upon therapy stress but plays a crucial role in depolyploidization. The current study demonstrates that treatment of cells with ceramide is not sufficient for p53-independent induction of p21 and that knockdown of acid ceramidase, which hydrolyzes ceramide, does not interfere with upregulation of p21. In contrast, blocking the expression of p21 with UC2288 prevented the induction of acid ceramidase and inhibited both the formation of PGCC from parental cells as well as the generation of progeny from PGCC. Taken together, our data suggest that p21 functions upstream of acid ceramidase and plays an important role in polyploidization and depolyploidization.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21 , Células Gigantes , Neoplasias , Poliploidia , Humanos , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células Gigantes/metabolismo , Células Gigantes/patologia , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , TranscriptomaRESUMO
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.
Assuntos
Serina C-Palmitoiltransferase , Serina , Sphingobacterium , Domínio Catalítico , Cristalização , Medição da Troca de Deutério , Elétrons , Hidrogênio/metabolismo , Palmitoil Coenzima A/metabolismo , Serina/análogos & derivados , Serina/metabolismo , Serina C-Palmitoiltransferase/química , Serina C-Palmitoiltransferase/metabolismo , Sphingobacterium/enzimologia , Sphingobacterium/metabolismo , Esfingosina/análogos & derivados , Esfingosina/biossíntese , Esfingosina/metabolismo , Estereoisomerismo , Especificidade por SubstratoRESUMO
The fatty acid (FA) elongation cycle produces very-long-chain FAs with ≥C21, which have unique physiological functions. Trans-2-enoyl-CoA reductases (yeast, Tsc13; mammals, TECR) catalyze the reduction reactions in the fourth step of the FA elongation cycle and in the sphingosine degradation pathway. However, their catalytic residues and coordinated action in the FA elongation cycle complex are unknown. To reveal these, we generated and analyzed Ala-substituted mutants of 15 residues of Tsc13. An in vitro FA elongation assay showed that nine of these mutants were less active than WT protein, with E91A and Y256A being the least active. Growth complementation analysis, measurement of ceramide levels, and deuterium-sphingosine labeling revealed that the function of the E91A mutant was substantially impaired in vivo. In addition, we found that the activity of FA elongases, which catalyze the first step of the FA elongation cycle, were reduced in the absence of Tsc13. Similar results were observed in Tsc13 E91A-expressing cells, which is attributable to reduced interaction between the Tsc13 E91A mutant and the FA elongases Elo2/Elo3. Finally, we found that E94A and Y248A mutants of human TECR, which correspond to E91A and Y256A mutants of Tsc13, showed reduced and almost no activity, respectively. Based on these results and the predicted three-dimensional structure of Tsc13, we speculate that Tyr256/Tyr248 of Tsc13/TECR is the catalytic residue that supplies a proton to trans-2-enoyl-CoAs. Our findings provide a clue concerning the catalytic mechanism of Tsc13/TECR and the coordinated action in the FA elongation cycle complex.
Assuntos
Ácidos Graxos Dessaturases , Esfingosina , Humanos , Ácidos Graxos Dessaturases/metabolismo , Elongases de Ácidos Graxos/metabolismo , Ácidos Graxos/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Esfingosina/metabolismoRESUMO
Cellular stress has been associated with inflammation, yet precise underlying mechanisms remain elusive. In this study, various unrelated stress inducers were employed to screen for sensors linking altered cellular homeostasis and inflammation. We identified the intracellular pattern recognition receptors NOD1/2, which sense bacterial peptidoglycans, as general stress sensors detecting perturbations of cellular homeostasis. NOD1/2 activation upon such perturbations required generation of the endogenous metabolite sphingosine-1-phosphate (S1P). Unlike peptidoglycan sensing via the leucine-rich repeats domain, cytosolic S1P directly bound to the nucleotide binding domains of NOD1/2, triggering NF-κB activation and inflammatory responses. In sum, we unveiled a hitherto unknown role of NOD1/2 in surveillance of cellular homeostasis through sensing of the cytosolic metabolite S1P. We propose S1P, an endogenous metabolite, as a novel NOD1/2 activator and NOD1/2 as molecular hubs integrating bacterial and metabolic cues.
Assuntos
Inflamação/metabolismo , Lisofosfolipídeos/metabolismo , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Adaptadora de Sinalização NOD2/metabolismo , Esfingosina/análogos & derivados , Animais , Linhagem Celular , Linhagem Celular Tumoral , Feminino , Células HEK293 , Células HeLa , Humanos , Camundongos , NF-kappa B/metabolismo , Peptidoglicano/metabolismo , Transdução de Sinais/fisiologia , Esfingosina/metabolismo , Células THP-1RESUMO
Eisosomes are large hemitubular structures that underlie the invaginated microdomains in the plasma membrane of various ascomycetous fungi, lichens and unicellular algae. In fungi, they are organized by BAR-domain containing proteins of the Pil1 family. Two such proteins, Pil1 and Lsp1, participate in eisosome formation in the yeast Saccharomyces cerevisiae. Under normal laboratory conditions, deletion of the PIL1 gene results in the inability of cells to assemble wild-type-like eisosomes. We found that under certain stress conditions, Lsp1 partially substitutes for the Pil1 function and mediates assembly of eisosomes, specifically following a decrease in the activity of serine palmitoyltransferase, for example, in response to hyperosmotic stress. Besides Lsp1, the assembly of eisosomes lacking Pil1 also requires Seg1 and Nce102 proteins. Using next-generation sequencing, we found that the seg1Δnce102Δpil1Δ strain, which is unable to form eisosomes, overexpresses genes coding for proteins of oxidative phosphorylation and tricarboxylic acid cycle. By contrast, genes involved in DNA repair, ribosome biogenesis and cell cycle are downregulated. Our results identify Lsp1 as a stress-responsive eisosome organizer and indicate several novel functional connections between the eisosome and essential cellular processes.
Assuntos
Proteínas de Saccharomyces cerevisiae , Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Accumulating evidence in several model organisms indicates that reduced sphingolipid biosynthesis promotes longevity, although underlying mechanisms remain unclear. In yeast, sphingolipid depletion induces a state resembling amino acid restriction, which we hypothesized might be due to altered stability of amino acid transporters at the plasma membrane. To test this, we measured surface abundance for a diverse panel of membrane proteins in the presence of myriocin, a sphingolipid biosynthesis inhibitor, in Saccharomyces cerevisiae. Unexpectedly, we found that surface levels of most proteins examined were either unaffected or increased during myriocin treatment, consistent with an observed decrease in bulk endocytosis. In contrast, sphingolipid depletion triggered selective endocytosis of the methionine transporter Mup1. Unlike methionine-induced Mup1 endocytosis, myriocin triggered Mup1 endocytosis that required the Rsp5 adaptor Art2, C-terminal lysine residues of Mup1 and the formation of K63-linked ubiquitin polymers. These findings reveal cellular adaptation to sphingolipid depletion by ubiquitin-mediated remodeling of nutrient transporter composition at the cell surface.
Assuntos
Proteínas de Saccharomyces cerevisiae , Endocitose , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Metionina/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Esfingolipídeos/metabolismo , Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
The COVID-19 pandemic, caused by SARS-CoV-2, has had a significant worldwide impact, affecting millions of people. COVID-19 is characterized by a heterogenous clinical phenotype, potentially involving hyperinflammation and prolonged tissue damage, although the exact underlying mechanisms are yet to be fully understood. Sphingolipid metabolites, which govern cell survival and proliferation, have emerged as key players in inflammatory signaling and cytokine responses. Given the complex metabolic pathway of sphingolipids, this study aimed to understand their potential role in the pathogenesis of COVID-19. We conducted a comprehensive examination of sphingolipid modulations across groups classified based on disease severity, incorporating a time-course in serum and urine samples. Several sphingolipids, including sphingosine, lactosylceramide, and hexosylceramide, emerged as promising indicators of COVID-19 severity, as validated by correlation analyses conducted on both serum and urine samples. Other sphingolipids, such as sphingosine 1-phosphate, ceramides, and deoxy-dihydroceramides, decreased in both COVID-19 patients and individuals with non-COVID infectious diseases. This suggests that these sphingolipids are not specifically associated with COVID-19 but rather with pathological conditions caused by infectious diseases. Our analysis of urine samples revealed elevated levels of various sphingolipids, with changes dependent on disease severity, potentially highlighting the acute kidney injury associated with COVID-19. This study illuminates the intricate relationship between disturbed sphingolipid metabolism, COVID-19 severity, and clinical factors. These findings provide valuable insights into the broader landscape of inflammatory diseases.
Assuntos
COVID-19 , SARS-CoV-2 , Índice de Gravidade de Doença , Esfingolipídeos , COVID-19/metabolismo , COVID-19/sangue , COVID-19/virologia , Humanos , Esfingolipídeos/metabolismo , Esfingolipídeos/sangue , Masculino , Feminino , Pessoa de Meia-Idade , Adulto , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Idoso , Biomarcadores/sangue , Biomarcadores/metabolismoRESUMO
Disruption of sphingolipid homeostasis and signaling has been implicated in diabetes, cancer, cardiometabolic, and neurodegenerative disorders. Yet, mechanisms governing cellular sensing and regulation of sphingolipid homeostasis remain largely unknown. In yeast, serine palmitoyltransferase, catalyzing the first and rate-limiting step of sphingolipid de novo biosynthesis, is negatively regulated by Orm1 and 2. Lowering sphingolipids triggers Orms phosphorylation, upregulation of serine palmitoyltransferase activity and sphingolipid de novo biosynthesis. However, mammalian orthologs ORMDLs lack the N-terminus hosting the phosphosites. Thus, which sphingolipid(s) are sensed by the cells, and mechanisms of homeostasis remain largely unknown. Here, we identify sphingosine-1-phosphate (S1P) as key sphingolipid sensed by cells via S1PRs to maintain homeostasis. The increase in S1P-S1PR signaling stabilizes ORMDLs, restraining SPT activity. Mechanistically, the hydroxylation of ORMDLs at Pro137 allows a constitutive degradation of ORMDLs via ubiquitin-proteasome pathway, preserving SPT activity. Disrupting S1PR/ORMDL axis results in ceramide accrual, mitochondrial dysfunction, impaired signal transduction, all underlying endothelial dysfunction, early event in the onset of cardio- and cerebrovascular diseases. Our discovery may provide the molecular basis for therapeutic intervention restoring sphingolipid homeostasis.
Assuntos
Proteínas de Saccharomyces cerevisiae , Esfingolipídeos , Animais , Humanos , Esfingolipídeos/metabolismo , Serina C-Palmitoiltransferase/genética , Serina C-Palmitoiltransferase/metabolismo , Proteínas de Membrana/metabolismo , Homeostase , Saccharomyces cerevisiae/metabolismo , Mamíferos/metabolismoRESUMO
Cilium formation and regeneration requires new protein synthesis, but the underlying cytosolic translational reprogramming remains largely unknown. Using ribosome footprinting, we performed global translatome profiling during cilia regeneration in Chlamydomonas and uncovered that flagellar genes undergo an early transcriptional activation but late translational repression. This pattern guided our identification of sphingolipid metabolism enzymes, including serine palmitoyltransferase (SPT), as essential regulators for ciliogenesis. Cryo-electron tomography showed that ceramide loss abnormally increased the membrane-axoneme distance and generated bulged cilia. We found that ceramides interact with intraflagellar transport (IFT) particle proteins that IFT motors transport along axoneme microtubules (MTs), suggesting that ceramide-IFT particle-IFT motor-MT interactions connect the ciliary membrane with the axoneme to form rod-shaped cilia. SPT-deficient vertebrate cells were defective in ciliogenesis, and SPT mutations from patients with hereditary sensory neuropathy disrupted cilia, which could be restored by sphingolipid supplementation. These results reveal a conserved role of sphingolipid in cilium formation and link compromised sphingolipid production with ciliopathies.
Assuntos
Axonema , Chlamydomonas , Cílios , Flagelos , Regeneração , Esfingolipídeos , Axonema/química , Axonema/metabolismo , Ceramidas/metabolismo , Chlamydomonas/fisiologia , Cílios/fisiologia , Flagelos/fisiologia , Transporte Proteico , Esfingolipídeos/metabolismoRESUMO
Hemolytic phospholipase C, PlcH, is an important virulence factor for Pseudomonas aeruginosa. PlcH preferentially hydrolyzes sphingomyelin and phosphatidylcholine, and this hydrolysis activity drives tissue damage and inflammation and interferes with the oxidative burst of immune cells. Among other contributors, transcription of plcH was previously shown to be induced by phosphate starvation via PhoB and the choline metabolite, glycine betaine, via GbdR. Here, we show that sphingosine can induce plcH transcription and result in secreted PlcH enzyme activity. This induction is dependent on the sphingosine-sensing transcriptional regulator SphR. The SphR induction of plcH occurs from the promoter for the gene upstream of plcH that encodes the neutral ceramidase, CerN, and transcriptional readthrough of the cerN transcription terminator. Evidence for these conclusions came from mutation of the SphR binding site in the cerN promoter, mutation of the cerN terminator, enhancement of cerN termination by adding the rrnB terminator, and reverse transcriptase PCR (RT-PCR) showing that the intergenic region between cerN and plcH is made as RNA during sphingosine, but not choline, induction. We also observed that, like glycine betaine induction, sphingosine induction of plcH is under catabolite repression control, which likely explains why such induction was not seen in other studies using sphingosine in rich media. The addition of sphingosine as a novel inducer for PlcH points to the regulation of plcH transcription as a site for the integration of multiple host-derived signals. IMPORTANCE: PlcH is a secreted phospholipase C/sphingomyelinase that is important for the virulence of Pseudomonas aeruginosa. Here, we show that sphingosine, which presents itself or as a product of P. aeruginosa sphingomyelinase and ceramidase activity, leads to the induction of plcH transcription. This transcriptional induction occurs from the promoter of the upstream ceramidase gene generating a conditional operon. The transcript on which plcH resides, therefore, is different depending on which host molecule or condition leads to induction, and this may have implications for PlcH post-transcriptional regulation. This work also adds to our understanding of P. aeruginosa with host-derived sphingolipids.
Assuntos
Betaína , Pseudomonas aeruginosa , Betaína/metabolismo , Pseudomonas aeruginosa/metabolismo , Esfingosina/metabolismo , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/metabolismo , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismo , Ceramidases/metabolismoRESUMO
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.
Assuntos
Autofagia , Doença de Niemann-Pick Tipo C , Esfingolipídeos , Esfingolipídeos/metabolismo , Esfingolipídeos/biossíntese , Doença de Niemann-Pick Tipo C/metabolismo , Doença de Niemann-Pick Tipo C/patologia , Doença de Niemann-Pick Tipo C/genética , Humanos , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Animais , Proteína C1 de Niemann-Pick , Serina C-Palmitoiltransferase/metabolismo , Serina C-Palmitoiltransferase/genética , Serina C-Palmitoiltransferase/antagonistas & inibidoresRESUMO
Lipid transport is an essential cellular process with importance to human health, disease development, and therapeutic strategies. Type IV P-type ATPases (P4-ATPases) have been identified as membrane lipid flippases by utilizing nitrobenzoxadiazole (NBD)-labeled lipids as substrates. Among the 14 human type IV P-type ATPases, ATP10D was shown to flip NBD-glucosylceramide (GlcCer) across the plasma membrane. Here, we found that conversion of incorporated GlcCer (d18:1/12:0) to other sphingolipids is accelerated in cells exogenously expressing ATP10D but not its ATPase-deficient mutant. These findings suggest that 1) ATP10D flips unmodified GlcCer as well as NBD-GlcCer at the plasma membrane and 2) ATP10D can translocate extracellular GlcCer, which is subsequently converted to other metabolites. Notably, exogenous expression of ATP10D led to the reduction in cellular hexosylceramide levels. Moreover, the expression of GlcCer flippases, including ATP10D, also reduced cellular hexosylceramide levels in fibroblasts derived from patients with Gaucher disease, which is a lysosomal storage disorder with excess GlcCer accumulation. Our study highlights the contribution of ATP10D to the regulation of cellular GlcCer levels and maintaining lipid homeostasis.
Assuntos
Glucosilceramidas , ATPases do Tipo-P , Humanos , Glucosilceramidas/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Adenosina Trifosfatases/metabolismo , Homeostase , ATPases do Tipo-P/metabolismoRESUMO
Adipogenesis is one of the major mechanisms for adipose tissue expansion, during which spindle-shaped mesenchymal stem cells commit to the fate of adipocyte precursors and differentiate into round-shaped fat-laden adipocytes. Here, we investigated the lipidomic profile dynamics of ex vivo-differentiated brown and white adipocytes derived from the stromal vascular fractions of interscapular brown (iBAT) and inguinal white adipose tissues. We showed that sphingomyelin was specifically enriched in terminally differentiated brown adipocytes, but not white adipocytes. In line with this, freshly isolated adipocytes of iBAT showed higher sphingomyelin content than those of inguinal white adipose tissue. Upon cold exposure, sphingomyelin abundance in iBAT gradually decreased in parallel with reduced sphingomyelin synthase 1 protein levels. Cold-exposed animals treated with an inhibitor of sphingomyelin hydrolases failed to maintain core body temperature and showed reduced oxygen consumption and iBAT UCP1 levels. Conversely, blockade of sphingomyelin synthetic enzymes resulted in enhanced nonshivering thermogenesis, reflected by elevated body temperature and UCP1 levels. Taken together, our results uncovered a relation between sphingomyelin abundance and fine-tuning of UCP1-mediated nonshivering thermogenesis.
Assuntos
Esfingomielinas , Termogênese , Proteína Desacopladora 1 , Animais , Proteína Desacopladora 1/metabolismo , Proteína Desacopladora 1/genética , Esfingomielinas/metabolismo , Camundongos , Masculino , Tecido Adiposo Branco/metabolismo , Tecido Adiposo Marrom/metabolismo , Camundongos Endogâmicos C57BLRESUMO
Multiple isozymes are encoded in the Caenorhabditis elegans genome for the various sphingolipid biosynthesis reactions, but the contributions of individual isozymes are characterized only in part. We developed a simple but effective reversed-phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS) method that enables simultaneous identification and quantification of ceramides (Cer), glucosylceramides (GlcCer), and sphingomyelins (SM) from the same MS run. Validating this sphingolipid profiling method, we show that nearly all 47 quantifiable sphingolipid species found in young adult worms were reduced upon RNA interference (RNAi) of sptl-1 or elo-5, which are both required for synthesis of the id17:1 sphingoid base. We also confirm that HYL-1 and HYL-2, but not LAGR-1, constitute the major ceramide synthase activity with different preference for fatty acid substrates, and that CGT-3, but not CGT-1 and CGT-2, plays a major role in producing GlcCers. Deletion of sms-5 hardly affected SM levels. RNAi of sms-1, sms-2, and sms-3 all lowered the abundance of certain SMs with an odd-numbered N-acyl chains (mostly C21 and C23, with or without hydroxylation). Unexpectedly, sms-2 RNAi and sms-3 RNAi elevated a subset of SM species containing even-numbered N-acyls. This suggests that sphingolipids containing even-numbered N-acyls could be regulated separately, sometimes in opposite directions, from those containing odd-numbered N-acyls, which are presumably monomethyl branched chain fatty acyls. We also find that ceramide levels are kept in balance with those of GlcCers and SMs. These findings underscore the effectiveness of this RPLC-MS/MS method in studies of C. elegans sphingolipid biology.