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Sphingomyelin synthase related protein is a mammalian phosphatidylethanolamine phospholipase C.

ABSTRACT: Sphingomyelin synthase related protein (SMSr) has no SM synthase activity but has ceramide phosphorylethanolamine (CPE) synthase activity in vitro. Although SMSr is ubiquitously expressed in all tested tissues, the CPE levels in most mammalian tissues or cells are extremely low or undetectable. Therefore, SMSr seems not to be a functional CPE synthase in vivo and its real biological function needs to be elucidated. In this study, we utilized purified recombinant SMSr and adenovirus-mediated SMSr in vivo expression to show that SMSr has phosphatidylethanolamine phospholipases C (PE-PLC) activity, i.e., it can generate DAG through PE hydrolysis in the absence of ceramide. Further, we found that SMSr has no phosphatidylcholine (PC)-PLC, phosphatidylserine (PS)-PLC, phosphatidylglycerol (PG)-PLC, and phosphatidic phosphatase (PAP) activities, indicating that SMSr-mediated PE-PLC activity has specificity. We conclude that SMSr is a mammalian PE-PLC. Importantly, SMSr can regulate steady state levels of PE in vivo, and it should be a new tool for PE-related biological study.

SUBMITTER: Chiang YP

PROVIDER: S-EPMC8387966 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Sphingomyelin synthase–related protein SMSr is a phosphatidylethanolamine phospholipase C protein that promotes nonalcoholic fatty liver disease

Project description:Sphingomyelin synthase (SMS)–related protein (SMSr) is a phosphatidylethanolamine phospholipase C (PE-PLC) molecule that is conserved and ubiquitous in mammals. However, its biological function is still not clear. We previously observed that SMS1 deficiency–mediated glucosylceramide accumulation caused nonalcoholic fatty liver diseases (NAFLD), including nonalcoholic steatohepatitis (NASH) and liver fibrosis. Here, first, we evaluated high-fat diet/fructose-induced NAFLD in Smsr knock out (KO) and WT mice. Second, we evaluated whether SMSr deficiency can reverse SMS1 deficiency-mediated NAFLD, using Sms1/Sms2 double and Sms1/Sms2/Smsr triple KO mice. We found that SMSr/PE-PLC deficiency attenuated high-fat diet/fructoseinduced fatty liver and NASH, and attenuated glucosylceramide accumulation-induced NASH, fibrosis, and tumor formation. Further, we found that SMSr/PE-PLC deficiency reduced the expression of many inflammatory cytokines and fibrosis-related factors, and PE supplementation in vitro or in vivo mimicked the condition of SMSr/PE-PLC deficiency. Furthermore, we demonstrated that SMSr/PEPLC deficiency or PE supplementation effectively prevented membrane-bound βcatenin transfer to the nucleus, thereby preventing tumor-related gene expression. Finally, we observed that patients with NASH had higher SMSr protein levels in the liver, lower plasma PE levels, and lower plasma PE/phosphatidylcholine (PC) ratios, and that human plasma PE levels are negatively associated with TNF-α and TGFβ1 levels. In conclusion, SMSr/PE-PLC deficiency causes PE accumulation, which can attenuate fatty liver, NASH, and fibrosis. These results suggest that SMSr/PE-PLC inhibition therapy may mitigate NAFLD.

2023-08-17 | GSE241036 | GEO

Sphingomyelin synthase–related protein SMSr is a phosphatidylethanolamine phospholipase C protein that promotes nonalcoholic fatty liver disease

Project description:Sphingomyelin synthase–related protein SMSr is a phosphatidylethanolamine phospholipase C protein that promotes nonalcoholic fatty liver disease

| PRJNA1006276 | ENA

Diacylglycerol kinase ? and sphingomyelin synthase-related protein functionally interact via their sterile ? motif domains.

Project description:The ? isozyme of diacylglycerol kinase (DGK?) plays critical roles in lipid signaling by converting diacylglycerol (DG) to phosphatidic acid (PA). We previously demonstrated that DGK? preferably phosphorylates palmitic acid (16:0)- and/or palmitoleic acid (16:1)-containing DG molecular species, but not arachidonic acid (20:4)-containing DG species, which are recognized as DGK substrates derived from phosphatidylinositol turnover, in high glucose-stimulated myoblasts. However, little is known about the origin of these DG molecular species. DGK? and two DG-generating enzymes, sphingomyelin synthase (SMS) 1 and SMS-related protein (SMSr), contain a sterile ? motif domain (SAMD). In this study, we found that SMSr-SAMD, but not SMS1-SAMD, co-immunoprecipitates with DGK?-SAMD. Full-length DGK? co-precipitated with full-length SMSr more strongly than with SMS1. However, SAMD-deleted variants of SMSr and DGK? interacted only weakly with full-length DGK? and SMSr, respectively. These results strongly suggested that DGK? interacts with SMSr through their respective SAMDs. To determine the functional outcomes of the relationship between DGK? and SMSr, we used LC-MS/MS to investigate whether overexpression of DGK? and/or SMSr in COS-7 cells alters the levels of PA species. We found that SMSr overexpression significantly enhances the production of 16:0- or 16:1-containing PA species such as 14:0/16:0-, 16:0/16:0-, 16:0/18:1-, and/or 16:1/18:1-PA in DGK?-overexpressing COS-7 cells. Moreover, SMSr enhanced DGK? activity via their SAMDs in vitro Taken together, these results strongly suggest that SMSr is a candidate DG-providing enzyme upstream of DGK? and that the two enzymes represent a new pathway independent of phosphatidylinositol turnover.

| S-EPMC7062193 | biostudies-literature

Complex formation of sphingomyelin synthase 1 with glucosylceramide synthase increases sphingomyelin and decreases glucosylceramide levels.

Project description:Sphingolipids, including sphingomyelin (SM) and glucosylceramide (GlcCer), are generated by the addition of a polar head group to ceramide (Cer). Sphingomyelin synthase 1 (SMS1) and glucosylceramide synthase (GCS) are key enzymes that catalyze the conversion of Cer to SM and GlcCer, respectively. GlcCer synthesis has been postulated to occur mainly in cis-Golgi, and SM synthesis is thought to occur in medial/trans-Golgi; however, SMS1 and GCS are known to partially co-localize in cisternae, especially in medial/trans-Golgi. Here, we report that SMS1 and GCS can form a heteromeric complex, in which the N terminus of SMS1 and the C terminus of GCS are in close proximity. Deletion of the N-terminal sterile ?-motif of SMS1 reduced the stability of the SMS1-GCS complex, resulting in a significant reduction in SM synthesis in vivo In contrast, chemical-induced heterodimerization augmented SMS1 activity, depending on an increase in the amount and stability of the complex. Fusion of the SMS1 N terminus to the GCS C terminus via linkers of different lengths increased SM synthesis and decreased GlcCer synthesis in vivo These results suggest that formation of the SMS1-GCS heteromeric complex increases SM synthesis and decreases GlcCer synthesis. Importantly, this regulation of relative Cer levels by the SMS1-GCS complex was confirmed by CRISPR/Cas9-mediated knockout of SMS1 or GCS combined with pharmacological inhibition of Cer transport protein in HEK293T cells. Our findings suggest that complex formation between SMS1 and GCS is part of a critical mechanism controlling the metabolic fate of Cer in the Golgi.

| S-EPMC6231140 | biostudies-literature

Sphingomyelin synthase SMS2 displays dual activity as ceramide phosphoethanolamine synthase.

Project description:Sphingolipids are vital components of eukaryotic membranes involved in the regulation of cell growth, death, intracellular trafficking, and the barrier function of the plasma membrane (PM). While sphingomyelin (SM) is the major sphingolipid in mammals, previous studies indicate that mammalian cells also produce the SM analog ceramide phosphoethanolamine (CPE). Little is known about the biological role of CPE or the enzyme(s) responsible for CPE biosynthesis. SM production is mediated by the SM synthases SMS1 in the Golgi and SMS2 at the PM, while a closely related enzyme, SMSr, has an unknown biochemical function. We now demonstrate that SMS family members display striking differences in substrate specificity, with SMS1 and SMSr being monofunctional enzymes with SM and CPE synthase activity, respectively, and SMS2 acting as a bifunctional enzyme with both SM and CPE synthase activity. In agreement with the PM residency of SMS2, we show that both SM and CPE synthase activities are enhanced at the surface of SMS2-overexpressing HeLa cells. Our findings reveal an unexpected diversity in substrate specificity among SMS family members that should enable the design of specific inhibitors to target the biological role of each enzyme individually.

| S-EPMC2759833 | biostudies-literature

Symmetrically substituted dichlorophenes inhibit <i>N</i>-acyl-phosphatidylethanolamine phospholipase D.

Project description:N-Acyl-phosphatidylethanolamine phospholipase D (NAPE-PLD) (EC 3.1.4.4) catalyzes the final step in the biosynthesis of N-acyl-ethanolamides. Reduced NAPE-PLD expression and activity may contribute to obesity and inflammation, but a lack of effective NAPE-PLD inhibitors has been a major obstacle to elucidating the role of NAPE-PLD and N-acyl-ethanolamide biosynthesis in these processes. The endogenous bile acid lithocholic acid (LCA) inhibits NAPE-PLD activity (with an IC50 of 68 μm), but LCA is also a highly potent ligand for TGR5 (EC50 0.52 μm). Recently, the first selective small-molecule inhibitor of NAPE-PLD, ARN19874, has been reported (having an IC50 of 34 μm). To identify more potent inhibitors of NAPE-PLD, here we used a quenched fluorescent NAPE analog, PED-A1, as a substrate for recombinant mouse Nape-pld to screen a panel of bile acids and a library of experimental compounds (the Spectrum Collection). Muricholic acids and several other bile acids inhibited Nape-pld with potency similar to that of LCA. We identified 14 potent Nape-pld inhibitors in the Spectrum Collection, with the two most potent (IC50 = ∼2 μm) being symmetrically substituted dichlorophenes, i.e. hexachlorophene and bithionol. Structure-activity relationship assays using additional substituted dichlorophenes identified key moieties needed for Nape-pld inhibition. Both hexachlorophene and bithionol exhibited significant selectivity for Nape-pld compared with nontarget lipase activities such as Streptomyces chromofuscus PLD or serum lipase. Both also effectively inhibited NAPE-PLD activity in cultured HEK293 cells. We conclude that symmetrically substituted dichlorophenes potently inhibit NAPE-PLD in cultured cells and have significant selectivity for NAPE-PLD versus other tissue-associated lipases.

| S-EPMC7247316 | biostudies-literature

Sphingomyelin synthases 1 and 2 exhibit phosphatidylcholine phospholipase C activity.

Project description:Many studies have confirmed the enzymatic activity of a mammalian phosphatidylcholine (PC) phospholipase C (PLC) (PC-PLC), which produces diacylglycerol (DAG) and phosphocholine through the hydrolysis of PC in the absence of ceramide. However, the protein(s) responsible for this activity have never yet been identified. Based on the fact that tricyclodecan-9-yl-potassium xanthate can inhibit both PC-PLC and sphingomyelin synthase (SMS) activities, and SMS1 and SMS2 have a conserved catalytic domain that could mediate a nucleophilic attack on the phosphodiester bond of PC, we hypothesized that both SMS1 and SMS2 might have PC-PLC activity. In the present study, we found that purified recombinant SMS1 and SMS2 but not SMS-related protein have PC-PLC activity. Moreover, we prepared liver-specific Sms1/global Sms2 double-KO mice. We found that liver PC-PLC activity was significantly reduced and steady-state levels of PC and DAG in the liver were regulated by the deficiency, in comparison with control mice. Using adenovirus, we expressed Sms1 and Sms2 genes in the liver of the double-KO mice, respectively, and found that expressed SMS1 and SMS2 can hydrolyze PC to produce DAG and phosphocholine. Thus, SMS1 and SMS2 exhibit PC-PLC activity in vitro and in vivo.

| S-EPMC8648842 | biostudies-literature

The domain responsible for sphingomyelin synthase (SMS) activity.

Project description:Sphingomyelin synthase (SMS) sits at the crossroads of sphingomyelin (SM), ceramide, diacylglycerol (DAG) metabolism. It utilizes ceramide and phosphatidylcholine as substrates to produce SM and DAG, thereby regulating lipid messengers which play a role in cell survival and apoptosis. There are two isoforms of the enzyme, SMS1 and SMS2. Both SMS1 and SMS2 contain two histidines and one aspartic acid which are evolutionary conserved within the lipid phosphate phosphatase superfamily. In this study, we systematically mutated these amino acids using site-directed mutagenesis and found that each point mutation abolished SMS activity without altering cellular distribution. We also explored the domains which are responsible for cellular distribution of both enzymes. Given their role as a potential regulator of diseases, these findings, coupled with homology modeling of SMS1 and SMS2, will be useful for drug development targeting SMS.

| S-EPMC3426831 | biostudies-literature

Sphingomyelin synthase 1-generated sphingomyelin plays an important role in transferrin trafficking and cell proliferation.

Project description:Transferrin (Tf) endocytosis and recycling are essential for iron uptake and the regulation of cell proliferation. Tf and Tf receptor (TfR) complexes are internalized via clathrin-coated pits composed of a variety of proteins and lipids and pass through early endosomes to recycling endosomes. We investigated the role of sphingomyelin (SM) synthases (SMS1 and SMS2) in clathrin-dependent trafficking of Tf and cell proliferation. We employed SM-deficient lymphoma cells that lacked SMSs and that failed to proliferate in response to Tf. Transfection of SMS1, but not SMS2, enabled these cells to incorporate SM into the plasma membrane, restoring Tf-mediated proliferation. SM-deficient cells showed a significant reduction in clathrin-dependent Tf uptake compared with the parental SM-producing cells. Both SMS1 gene transfection and exogenous short-chain SM treatment increased clathrin-dependent Tf uptake in SM-deficient cells, with the Tf being subsequently sorted to Rab11-positive recycling endosomes. We observed trafficking of the internalized Tf to late/endolysosomal compartments, and this was not dependent on the clathrin pathway in SM-deficient cells. Thus, SMS1-mediated SM synthesis directs Tf-TfR to undergo clathrin-dependent endocytosis and recycling, promoting the proliferation of lymphoma cells.

| S-EPMC3195583 | biostudies-literature

All members in the sphingomyelin synthase gene family have ceramide phosphoethanolamine synthase activity.

Project description:Sphingomyelin synthase-related protein (SMSr) synthesizes the sphingomyelin analog ceramide phosphoethanolamine (CPE) in cells. Previous cell studies indicated that SMSr is involved in ceramide homeostasis and is crucial for cell function. To further examine SMSr function in vivo, we generated Smsr KO mice that were fertile and had no obvious phenotypic alterations. Quantitative MS analyses of plasma, liver, and macrophages from the KO mice revealed only marginal changes in CPE and ceramide as well as other sphingolipid levels. Because SMS2 also has CPE synthase activity, we prepared Smsr/Sms2 double KO mice. We found that CPE levels were not significantly changed in macrophages, suggesting that CPE levels are not exclusively dependent on SMSr and SMS2 activities. We then measured CPE levels in Sms1 KO mice and found that Sms1 deficiency also reduced plasma CPE levels. Importantly, we found that expression of Sms1 or Sms2 in SF9 insect cells significantly increased not only SM but also CPE formation, indicating that SMS1 also has CPE synthase activity. Moreover, we measured CPE synthase Km and Vmax for SMS1, SMS2, and SMSr using different NBD ceramides. Our study reveals that all mouse SMS family members (SMSr, SMS1, and SMS2) have CPE synthase activity. However, neither CPE nor SMSr appears to be a critical regulator of ceramide levels in vivo.

| S-EPMC4340302 | biostudies-literature

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