Serine Palmitoyltransferase Complex Research Articles

Targeting ceramides in 5XFAD mice reduces amyloid plaque deposition and gliosis

AbstractBackgroundAlzheimer’s Disease and other neurodegenerative disorders have been linked to metabolic disorders associated with aberrant lipid deposition. Sphingolipids, such as ceramides, are among the most deleterious lipids that accumulate in the AD brain. Increased ceramides have been measured in post‐mortem AD brain samples and are abundant in Aβ‐plaques. As biomarkers, ceramides in the plasma and cerebrospinal fluid correlate with AD risk. The potential of ceramide‐lowering strategies to treat or prevent AD has been underexplored. We hypothesize that ceramides are causal contributors to AD pathologies and amenable to therapeutic targeting to improve AD outcomes.MethodTo address ceramides in AD, we have targeted serine palmitoyl‐transferase (SPT), the first enzyme and rate‐limiting step in ceramide de novo synthesis, with the drug myriocin in 5XFAD mice on a regular chow diet and an obesogenic high‐fat diet. We have also generated inducible genetic models to study loss‐of‐ceramides (overexpression of the ceramide‐degrading enzyme Asah1) or gain‐of‐ceramides (overexpression of the SPT complex), specifically in neurons in the 5XFAD model. We used immunocytochemistry to assess Aβ plaque numbers and size and both astrocyte and microglial activation and numbers. Cognitive behavior was measured using a Barnes maze.Result5XFAD mice treated with myriocin for 17 weeks had a reduction of hippocampal ceramides. As expected, control vehicle‐treated 5XFAD mice showed a significant memory deficit during behavioral testing. Myriocin‐treated mice had significantly improved memory of the Barnes maze. Treated mice had fewer and smaller Aβ plaques and had a reduction in both astrocyte numbers and arborization in the cortex and hippocampus. Microglia were reduced in the hippocampus of treated mice. In 5XFAD mice with neuron‐specific Asah1 overexpression, Aβ plaque size and number remained unchanged compared to a 5XFAD control. However, Gfap+ astrocytes and Iba1+ microglia/monocyte activation and migration were eliminated, resulting in plaques with no associated glia and glial morphology like non‐5XFAD mice.ConclusionDecreasing ceramide production by myriocin treatment in 5XFAD mice reduced Aβ plaque number and size and reduced gliosis in the cortex and hippocampus. Reducing ceramides in neurons results in the abrogation of gliosis throughout the brain of a 5XFAD mouse.

Murine endothelial serine palmitoyltransferase 1 (SPTLC1) is required for vascular development and systemic sphingolipid homeostasis.

Serine palmitoyl transferase (SPT), the rate-limiting enzyme in the de novo synthesis of sphingolipids (SL), is needed for embryonic development, physiological homeostasis, and response to stress. The functions of de novo SL synthesis in vascular endothelial cells (EC), which line the entire circulatory system, are not well understood. Here, we show that the de novo SL synthesis in EC not only regulates vascular development but also maintains circulatory and peripheral organ SL levels. Mice with an endothelial-specific gene knockout of SPTLC1 (Sptlc1 ECKO), an essential subunit of the SPT complex, exhibited reduced EC proliferation and tip/stalk cell differentiation, resulting in delayed retinal vascular development. In addition, Sptlc1 ECKO mice had reduced retinal neovascularization in the oxygen-induced retinopathy model. Mechanistic studies suggest that EC SL produced from the de novo pathway are needed for lipid raft formation and efficient VEGF signaling. Post-natal deletion of the EC Sptlc1 also showed rapid reduction of several SL metabolites in plasma, red blood cells, and peripheral organs (lung and liver) but not in the retina, part of the central nervous system (CNS). In the liver, EC de novo SL synthesis was important for acetaminophen-induced rapid ceramide elevation and hepatotoxicity. These results suggest that EC-derived SL metabolites are in constant flux between the vasculature, circulatory elements, and parenchymal cells of non-CNS organs. Taken together, our data point to the central role of the endothelial SL biosynthesis in maintaining vascular development, neovascular proliferation, non-CNS tissue metabolic homeostasis, and hepatocyte response to stress.

De novo sphingolipid biosynthesis necessitates detoxification in cancer cells.

SUMMARYSphingolipids 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.

Sphingolipids and Asthma.

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 stabilizesthe 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.

Structural insights into the regulation of human serine palmitoyltransferase complexes

Sphingolipids are essential lipids in eukaryotic membranes. In humans, the first and rate-limiting step of sphingolipid synthesis is catalyzed by the serine palmitoyltransferase (SPT) complex. The SPT complex consists of SPTLC1 and SPTLC2 as catalytic components, and ssSPTa and ORMDL3 as regulatory components. To understand the assembly, substrate processing and regulation of the complex, we determined cryo-electron microscopy structures of the human SPT complex in various functional states at resolutions of 2.6–3.4 Å. The structures elucidate how catalytic components recognize the substrate, as well as how regulatory components modulate the substrate-binding tunnel to control enzyme activity. These findings reveal the molecular mechanism of sphingolipid biogenesis governed by the serine palmitoyltransferase complex.

Structural insights into the regulation of human serine palmitoyltransferase complexes.

Sphingolipids are essential lipids in eukaryotic membranes. In humans, the first and rate-limiting step of sphingolipid synthesis is catalyzed by the serine palmitoyltransferase holocomplex, which consists of catalytic components (SPTLC1 and SPTLC2) and regulatory components (ssSPTa and ORMDL3). However, the assembly, substrate processing and regulation of the complex are unclear. Here, we present 8 cryo-electron microscopy structures of the human serine palmitoyltransferase holocomplex in various functional states at resolutions of 2.6-3.4 Å. The structures reveal not only how catalytic components recognize the substrate, but also how regulatory components modulate the substrate-binding tunnel to control enzyme activity: ssSPTa engages SPTLC2 and shapes the tunnel to determine substrate specificity. ORMDL3 blocks the tunnel and competes with substrate binding through its amino terminus. These findings provide mechanistic insights into sphingolipid biogenesis governed by the serine palmitoyltransferase complex.

Kicking off sphingolipid biosynthesis: structures of the serine palmitoyltransferase complex.

Structures of the human serine palmitoyltransferase complex reveal the overall assembly, regulatory mechanisms and substrate selectivity of this key enzyme in sphingolipid synthesis.

Structural insights into the assembly and substrate selectivity of human SPT-ORMDL3 complex.

Human serine palmitoyltransferase (SPT) complex catalyzes the initial and rate-limiting step in the de novo biosynthesis of all sphingolipids. ORMDLs regulate SPT function, with human ORMDL3 being related to asthma. Here we report three high-resolution cryo-EM structures: the human SPT complex, composed of SPTLC1, SPTLC2 and SPTssa; the SPT-ORMDL3 complex; and the SPT-ORMDL3 complex bound to two substrates, PLP-L-serine (PLS) and a non-reactive palmitoyl-CoA analogue. SPTLC1 and SPTLC2 form a dimer of heterodimers as the catalytic core. SPTssa participates in acyl-CoA coordination, thereby stimulating the SPT activity and regulating the substrate selectivity. ORMDL3 is located in the center of the complex, serving to stabilize the SPT assembly. Our structural and biochemical analyses provide a molecular basis for the assembly and substrate selectivity of the SPT and SPT-ORMDL3 complexes, and lay a foundation for mechanistic understanding of sphingolipid homeostasis and for related therapeutic drug development.

SSSPTA is essential for serine palmitoyltransferase function during development and hematopoiesis

Serine palmitoyltransferase complex (SPT) mediates the first and rate-limiting step in the de novo sphingolipid biosynthetic pathway. The larger subunits SPTLC1 and SPTLC2/SPTLC3 together form the catalytic core while a smaller third subunit either SSSPTA or SSSPTB has been shown to increase the catalytic efficiency and provide substrate specificity for the fatty acyl-CoA substrates. The in vivo biological significance of these smaller subunits in mammals is still unknown. Here, using two null mutants, a conditional null for ssSPTa and a null mutant for ssSPTb, we show that SSSPTA is essential for embryogenesis and mediates much of the known functions of the SPT complex in mammalian hematopoiesis. The ssSPTa null mutants are embryonic lethal at E6.5 much like the Sptlc1 and Sptlc2 null alleles. Mx1-Cre induced deletion of ssSPTa leads to lethality and myelopoietic defect. Chimeric and competitive bone marrow transplantation experiments show that the defect in myelopoiesis is accompanied by an expansion of the Lin−Sca1+c-Kit+ stem and progenitor compartment. Progenitor cells that fail to differentiate along the myeloid lineage display evidence of endoplasmic reticulum stress. On the other hand, ssSPTb null mice are homozygous viable, and analyses of the bone marrow cells show no significant difference in the proliferation and differentiation of the adult hematopoietic compartment. SPTLC1 is an obligatory subunit for the SPT function, and because Sptlc1−/− and ssSPTa−/− mice display similar defects during development and hematopoiesis, we conclude that an SPT complex that includes SSSPTA mediates much of its developmental and hematopoietic functions in a mammalian model.

Combined Omics Approach Identifies Gambogic Acid and Related Xanthones as Covalent Inhibitors of the Serine Palmitoyltransferase Complex

In this study, we identify the natural product gambogic acid as well as structurally related synthetic xanthones as first-in-class covalent inhibitors of the de novo sphingolipid biosynthesis. We apply chemoproteomics to determine that gambogic acid binds to the regulatory small subunit B of the serine palmitoyltransferase complex (SPTSSB). We then test structurally related synthetic xanthones to identify 18 as an equally potent but more selective binder of SPTSSB and show that 18 reduces sphingolipid levels in situ and invivo. Finally, using various biological methods, we demonstrate that 18 induces cellular responses characteristic for diminished sphingosine-1-phosphate (S1P) signaling. This study demonstrates that SPTSSB may become a viable therapeutic target in various diseases with pathological S1P signaling. Furthermore, we believe that our compound will become a valuable tool for studying the sphingolipid metabolism and serve as a blueprint for the development of a new generation of sphingolipid biosynthesis inhibitors.

Dynamics of sphingolipids and the serine palmitoyltransferase complex in rat oligodendrocytes during myelination

Myelin is a unique lipid-rich membrane structure that accelerates neurotransmission and supports neuronal function. Sphingolipids are critical myelin components. Yet sphingolipid content and synthesis have not been well characterized in oligodendrocytes, the myelin-producing cells of the CNS. Here, using quantitative real-time PCR, LC-MS/MS-based lipid analysis, and biochemical assays, we examined sphingolipid synthesis during the peak period of myelination in the postnatal rat brain. Importantly, we characterized sphingolipid production in isolated oligodendrocytes. We analyzed sphingolipid distribution and levels of critical enzymes and regulators in the sphingolipid biosynthetic pathway, with focus on the serine palmitoyltransferase (SPT) complex, the rate-limiting step in this pathway. During myelination, levels of the major SPT subunits increased and oligodendrocyte maturation was accompanied by extensive alterations in the composition of the SPT complex. These included changes in the relative levels of two alternative catalytic subunits, SPTLC2 and -3, in the relative levels of isoforms of the small subunits, ssSPTa and -b, and in the isoform distribution of the SPT regulators, the ORMDLs. Myelination progression was accompanied by distinct changes in both the nature of the sphingoid backbone and the N-acyl chains incorporated into sphingolipids. We conclude that the distribution of these changes among sphingolipid family members is indicative of a selective channeling of the ceramide backbone toward specific downstream metabolic pathways during myelination. Our findings provide insights into myelin production in oligodendrocytes and suggest how dysregulation of the biosynthesis of this highly specialized membrane could contribute to demyelinating diseases.

Abstract 735: A Novel Class of Sphingolipids Mediate Autophagy and Apoptosis in Models of Ischemia

Rationale: Heart failure caused by ischemic cardiomyopathy is the leading cause of death and disability in the USA and world, accounting for 10 million deaths in 2016. Sphingolipids including ceramides and sphingosine-1-phosphate have been demonstrated to play roles in myocardial injury. In general, these lipids are synthesized from serine palmitoyltransferase (SPT) derived using serine and palmitoyl-CoA. While the standard SPT enzyme exists as a heterodimer of two subunits, Sptlc1 and Sptlc2, a new subunit was recently discovered, Sptlc3, which enables the SPT complex to use myristoyl-CoA, thereby synthesizing a previously underappreciated group of atypical sphingolipids. We previously demonstrated that these atypical lipids are a major component of myocardial sphingolipid pools. Furthermore, in contrast to canonical sphingolipids, the atypical lipids promote cardiomyocyte apoptosis. Therefore, the goal of this research is to determine the contribution of these novel lipids to cardiomyocyte apoptosis and therefore whether they may contribute to ischemic injury. Objectives: To determine the contribution of Sptlc3 and atypical sphingolipids to cardiomyocyte apoptosis and ischemic injury and to assess the potential relevance of Sptlc3 to human HF. Methods: Murine primary cardiac fibroblasts and cardiomyocytes were isolated and subject to ischemic induction. Following this, cells were analyzed with TUNEL staining, lipidomic analysis or treatment with a Sptlc3 mimetic. Results: Ischemic cells show significantly increased Sptlc3 expression, non-canonical sphingolipids in the sphingolipid pool and apoptotic cells as compared to their controls. The cells treated with the Sptlc3 mimetic show apoptotic and a non-canonical autophagic pathway being induced in cells. Conclusions: We have identified a novel class of sphingolipids enriched in myocardium of mice with cardiomyopathies. Furthermore, the enzyme that produces these, Sptlc3, is robustly increased in human heart failure. Because the Sptlc3-derived lipids promoted cardiomyocyte apoptosis, we propose that Sptlc3 may mediate myocardial injury in ischemia.

Sphingolipid biosynthesis induces a conformational change in the murine norovirus receptor and facilitates viral infection.

Cellular susceptibility to viral infections is in part determined by the presence of a host cellular receptor. Here we use murine norovirus as a model to uncover an unappreciated connection between an intracellular lipid biosynthetic enzyme and a receptor conformation permissive for viral infection. The serine palmitoyltransferase (SPT) complex is required for de novo sphingolipid biosynthesis and we find that its absence impairs the ability of murine norovirus to bind and enter cells. While, the SPT complex is dispensable for the surface expression of the norovirus receptor, CD300lf, SPT activity is required for CD300lf to adopt a conformation permissive for viral binding. Addition of extracellular ceramide to SPT deficient cells chemically complements both the conformational changes of CD300lf and the cellular susceptibility to murine norovirus infection. Taken together, these data indicate that intracellular sphingolipid biosynthesis regulates the conformation of the murine norovirus receptor, and therefore the tropism of murine norovirus. This indicates that intracellular biosynthetic pathways can regulate viral tropism even when the receptor for a virus is expressed on the target cell surface.

Pharmacological characterization of synthetic serine palmitoyltransferase inhibitors by biochemical and cellular analyses

Human serine palmitoyltransferase (SPT) is a PLP-dependent enzyme residing in the endoplasmic reticulum. It catalyzes the synthesis of 3-ketodihydrosphingosine (3-KDS) from the substrates palmitoyl-CoA and l-serine. It is a rate-limiting enzyme for sphingolipid synthesis in cells. In the present study, we characterized and pharmacologically profiled a series of tetrahydropyrazolopyridine derivatives that potently inhibit human SPT enzymatic activity, including two cell-active derivatives and one fluorescent-labelled derivative. These SPT inhibitors exhibited dual inhibitory activities against SPT2 and SPT3. We used a fluorescent-labelled probe to molecularly assess the inhibitory mechanism and revealed its binding to the SPT2 or SPT3 subunit in the small subunit (ss) SPTa/SPT1/SPT2/or ssSPTa/SPT1/SPT3 functional complexes. One of the SPT inhibitors exhibited a significantly slow dissociation from the SPT complex. We confirmed that our SPT inhibitors suppressed ceramide content in non-small-cell lung cancer cell line, HCC4006, by performing a target engagement analysis. The potency of ceramide reduction correlated to that observed in a recombinant SPT2 enzyme assay. We thus elucidated and provided a fundamental understanding of the molecular mode of action of SPT inhibitors and developed potent, cell-active SPT inhibitors that can be used to clarify the biological function of SPT.

A systematic comparison of all mutations in hereditary sensory neuropathy type I (HSAN I) reveals that the G387A mutation is not disease associated

Hereditary sensory neuropathy type 1 (HSAN I) is an autosomal dominant inherited neurodegenerative disorder of the peripheral nervous system associated with mutations in the SPTLC1 subunit of the serine palmitoyltransferase (SPT). Four missense mutations (C133W, C133Y, V144D and G387A) in SPTLC1 were reported to cause HSAN I. SPT catalyses the condensation of Serine and Palmitoyl-CoA, which is the first and rate-limiting step in the de novo synthesis of ceramides. Earlier studies showed that C133W and C133Y mutants have a reduced activity, whereas the impact of the V144D and G387A mutations on the human enzyme was not tested yet. In this paper, we show that none of the HSAN I mutations interferes with SPT complex formation. We demonstrate that also V144D has a reduced SPT activity, however to a lower extent than C133W and C133Y. In contrast, the G387A mutation showed no influence on SPT activity. Furthermore, the growth phenotype of LY-B cells--a SPTLC1 deficient CHO cell line--could be reversed by expressing either the wild-type SPTLC1 or the G387A mutant, but not the C133W mutant. This indicates that the G387A mutation is most likely not directly associated with HSAN I. These findings were genetically confirmed by the identification of a nuclear HSAN family which showed segregation of the G387A variant as a non-synonymous SNP.

Is the mammalian serine palmitoyltransferase a high-molecular-mass complex?

SPT (serine palmitoyltransferase) catalyses the rate-limiting step for the de novo synthesis of sphingolipids. Mammalian SPT is believed to be a heterodimer composed of two subunits, SPTLC1 and SPTLC2. We reported previously the identification of a new third SPT subunit, SPTLC3. In the present study, we have investigated the structure of the SPT complex in more detail. Pull-down assays with antibodies against SPTLC3 concomitantly co-precipitated SPTLC1 and SPTLC2 in human placenta extracts and SPTLC3 overexpressing human embryonic kidney-293 cells. By size exclusion chromatography, we determined the molecular mass of the functional SPT complex to be approx. 480 kDa. By Blue-native-PAGE experiments we demonstrated that all three SPT subunits (SPTLC1-3) are co-localized within a single SPT complex. On the basis of these results we conclude that the functional SPT is not a dimer, but a higher organized complex, composed of three distinct subunits (SPTLC1, SPTLC2 and SPTLC3) with a molecular mass of 480 kDa. The stoichiometry of SPTLC2 and SPTLC3 in this complex seems not to be fixed and is probably changed dynamically in dependence of the tissue specific SPTLC2 and SPTLC3 expression levels. Based on our own and earlier published data we propose a model of an octameric SPT structure. The observed dynamic composition of the SPT complex could provide a cellular mechanism to adjust SPT activity to tissue specific requirements in sphingolipid synthesis.

Selective Substrate Supply in the Regulation of Yeast de Novo Sphingolipid Synthesis

The heat stress response of Saccharomyces cerevisiae is characterized by transient cell cycle arrest, altered gene expression, degradation of nutrient permeases, trehalose accumulation, and translation initiation of heat shock proteins. Importantly heat stress also induces de novo sphingolipid synthesis upon which many of these subprograms of the heat stress response depend. Despite extensive data addressing the roles for sphingolipids in heat stress, the mechanism(s) by which heat induces sphingolipid synthesis remains unknown. This study was undertaken to determine the events and/or factors required for heat stress-induced sphingolipid synthesis. Data presented indicate that heat does not directly alter the in vitro activity of serine palmitoyltransferase (SPT), the enzyme responsible for initiating de novo sphingolipid synthesis. Moreover deletion of the small peptide Tsc3p, which is thought to maximize SPT activity, specifically reduced production of C(20) sphingolipid species by over 70% but did not significantly decrease overall sphingoid base production. In contrast, the fatty-acid synthase inhibitor cerulenin nearly completely blocked sphingoid base production after heat, indicating a requirement for endogenous fatty acids for heat-mediated sphingoid base synthesis. Consistent with this, genetic studies show that fatty acid import does not contribute to heat-induced de novo synthesis under normal conditions. Interestingly the absence of medium serine also ameliorated heat-induced sphingoid base production, indicating a requirement for exogenous serine for the response, and consistent with this finding, disruption of synthesis of endogenous serine did not affect heat-induced sphingolipid synthesis. Serine uptake assays indicated that heat increased serine uptake from medium by 100% during the first 10 min of heat stress. Moreover treatments that increase serine uptake in the absence of heat including acute medium acidification and glucose treatment also enhanced de novo sphingoid base synthesis equivalent to that induced by heat stress. These data agree with findings from mammalian systems that availability of substrates is a key determinant of flux through sphingolipid synthesis. Moreover data presented here indicate that SPT activity can be driven by several factors that increase serine uptake in the absence of heat. These findings may provide insights into the many systems in which de novo synthesis is increased in the absence of elevated in vitro SPT activity.

Purification of the Serine Palmitoyltransferase Complex Responsible for Sphingoid Base Synthesis by Using Affinity Peptide Chromatography Techniques

Serine palmitoyltransferase (SPT), a membrane-bound enzyme of the endoplasmic reticulum, catalyzes the condensation of palmitoyl coenzyme A (CoA) and L-serine to produce 3-ketodihydrosphingosine. This enzyme contains at least two different subunits, named the LCB1 and LCB2 proteins. In the present study, we expressed a FLAG- and His(6) peptide-tagged version of the hamster LCB1 protein in a Chinese hamster ovary cell mutant strain lacking the endogenous LCB1 subunit and purified SPT from the cells near to homogeneity by affinity peptide chromatography. The endogenous LCB2 protein was co-purified with the tagged LCB1 protein in purification of SPT. In various aspects, including optimum pH, acyl-CoA specificity, and sphingofungin sensitivity, the activity of purified SPT was consistent with the activity detected in lysates of wild-type Chinese hamster ovary cells. The optimum concentration of palmitoyl-CoA for 3-ketodihydrosphingosine formation by purified SPT was approximately 25 microM, and the apparent K(m) of L-serine was 0.28 mM. Competition analysis of the SPT reaction with various serine analogs showed that all of the amino, carboxyl, and hydroxyl groups of L-serine were responsible for the substrate recognition of the enzyme. SDS-polyacrylamide gel electrophoretic analysis of purified SPT, together with immunoprecipitation analysis of metabolically labeled LCB proteins, strongly suggested that the SPT enzyme consisted of the LCB1 and LCB2 proteins with a stoichiometry of 1:1.