The neutral sphingomyelinase family: identifying biochemical connections

Neutral sphingomyelinases (N-SMases) are considered to be key mediators of stress-induced ceramide production. The extended family of N-SMases is a subset of the DNaseI superfamily and comprises members from bacteria, yeast and mammals. In recent years, the identification and cloning of mammalian N-SMase family members has led to significant advances in understanding their physiological roles and regulation. However, there is still limited information on their regulation at the biochemical and molecular level. In this review, we summarize current knowledge about the biochemical regulation of the eukaryotic N-SMases and identify the major areas where knowledge is lacking. In recent years, research into the roles and regulation of N-SMases has moved in great strides with the cloning and characterization of multiple N-SMase isoforms and the development of knockout mice. However, as researchers continue to move forward in understanding the physiological functions of these various N-SMase isoforms, it has become exceedingly important to define howthese isoforms are regulated at the biochemical and molecular level. This is crucial for the development of future tools to study N-SMase signaling such as, for example, phospho-specific antibodies designating activation states. This is also an important part of identifying novel roles of N-SMases in physiological and pathological states. Finally, only by obtaining a more complete understanding of the workings of these enzymes at the molecular level, will investigators be able to design appropriate compounds that can target and inhibit their activity both efficiently and specifically. Certainly, the last of these is crucial when considering the potential of N-SMases as therapeutic targets. With this in mind, we sincerely hope that the next decade of research will even surpass the last ten years in advancing our understanding of the eukaryotic N-SMase family.     

The Roles of Neutral Sphingomyelinases in Neurological Pathologies

The neutral sphingomyelinases (N-SMases) are a group of Mg²⁺-dependent enzymes with a pH optimum in the neutral range. N-SMases catalyze the conversion of sphingomyelin to ceramide and have been found particularly enriched in brain tissue. N-SMase activity has been implicated in many physiological and pathological processes affecting the brain and nervous system. In this review, we discuss the proposed functions of N-SMase with a particular emphasis on its role in neurological disorders, such as age-related neurodegeneration, Alzheimer’s disease, HIV-associated dementia, atherosclerosis, ischemia–reperfusion injury, and cancer.     

Role for neutral sphingomyelinase-2 in tumor necrosis factor alpha-stimulated expression of vascular cell adhesion molecule-1 (VCAM) and intercellular adhesion molecule-1 (ICAM) in lung epithelial cells: p38 MAPK is an upstream regulator of nSMase2

Neutral sphingomyelinases (N-SMases) are major candidates for stress-induced ceramide production. However, there is little information on the physiological regulation and roles of the cloned N-SMase enzyme, nSMase2. In this study, nSMase2 was found to translocate acutely to the plasma membrane of A549 epithelial cells in response to tumor necrosis factor alpha (TNF-alpha) in a time- and dose-dependent manner. Additionally, TNF-alpha increased N-SMase activity rapidly and transiently both endogenously and in cells overexpressing nSMase2. Furthermore, the translocation of nSMase2 was regulated by p38-alpha MAPK, but not ERK or JNK, and the increase in endogenous N-SMase activity was abrogated by p38 MAPK inhibition. In addition, both p38-alpha MAPK and nSMase2 were implicated in the TNF-alpha-stimulated up-regulation of the adhesion proteins vascular cell adhesion molecule-1 (VCAM) and intercellular adhesion molecule-1 (ICAM), but this was largely independent of NF-kappaB activation. These data reveal p38 MAPK as an upstream regulator of nSMase2 and indicate a role for nSMase2 in pro-inflammatory responses induced by TNF-alpha as a regulator of adhesion proteins.     

Maximizing the expression of mammalian cytochrome P-450 monooxygenase activities in yeast cells

Cytochrome P-450s constitute a superfamily of mono-oxygenases which require the association with specific redox enzymes bound to the endoplasmic reticulum membrane for their activity. Conditions for the functional expression of these mammalian enzymes in yeast cells and the respective merits and limitations of currently used P-450 expression systems, are considered. The dependence of the mouse P-450 IA1 specific activity on the cytochrome expression level in yeast microsomes is studied and results demonstrate that the low amounts of endogenous NADPH-cytochrome P-450 reductase and cytochrome b5 which are naturally present, are limiting for the heterologous monooxygenase activities. The sequences encoding human liver cytochrome b5, the native and a modified form of the yeast NADPH-cytochrome P-450 reductase were cloned by making use of PCR techniques, over-expressed in yeast as functional forms, and characterized. New vectors allowing a high level of mammalian P-450 expression upon induction were also constructed and tested. A strategy for the construction of a co-expression system allowing maximal activity of mammalian cytochrome P-450s is discussed.     

Identification and Characterization of Murine Mitochondria-associated Neutral Sphingomyelinase (MA-nSMase), the Mammalian Sphingomyelin Phosphodiesterase 5

Sphingolipids play important roles in regulating cellular responses. Although mitochondria contain sphingolipids, direct regulation of their levels in mitochondria or mitochondria-associated membranes is mostly unclear. Neutral SMase (N-SMase) isoforms, which catalyze hydrolysis of sphingomyelin (SM) to ceramide and phosphocholine, have been found in the mitochondria of yeast and zebrafish, yet their existence in mammalian mitochondria remains unknown. Here, we have identified and cloned a cDNA based on nSMase homologous sequences. This cDNA encodes a novel protein of 483 amino acids that displays significant homology to nSMase2 and possesses the same catalytic core residues as members of the extended N-SMase family. A transiently expressed V5-tagged protein co-localized with both mitochondria and endoplasmic reticulum markers in MCF-7 and HEK293 cells; accordingly, the enzyme is referred to as mitochondria-associated nSMase (MA-nSMase). MA-nSMase was highly expressed in testis, pancreas, epididymis, and brain. MA-nSMase had an absolute requirement for cations such as Mg²⁺ and Mn²⁺ and activation by the anionic phospholipids, especially phosphatidylserine and the mitochondrial cardiolipin. Importantly, overexpression of MA-nSMase in HEK293 cells significantly increased in vitro N-SMase activity and also modulated the levels of SM and ceramide, indicating that the identified cDNA encodes a functional SMase. Thus, these studies identify and characterize, for the first time, a mammalian MA-nSMase. The characterization of MA-nSMase described here will contribute to our understanding of pathways regulated by sphingolipid metabolites, particularly with reference to the mitochondria and associated organelles.     

Biochemical properties of mammalian neutral sphingomyelinase 2 and its role in sphingolipid metabolism

Neutral sphingomyelinase (N-SMase) is one of the key enzymes involved in the generation of ceramide; however, the gene(s) encoding for the mammalian N-SMase is still not well defined. Previous studies on the cloned nSMase1 had shown that the protein acts primarily as lyso-platelet-activating factor-phospholipase C. Recently the cloning of another putative N-SMase, nSMase2, was reported. In this study, biochemical characterization of the mouse nSMase2 was carried out using the overexpressed protein in yeast cells in which the inositol phosphosphingolipid phospholipase C (Isc1p) was deleted. N-SMase activity was dependent on Mg(2+) and was activated by phosphatidylserine and inhibited by GW4869. The ability of nSMase2 to recognize endogenous sphingomyelin (SM) as substrate was investigated by overexpressing nSMase2 in MCF7 cells. Mass measurements showed a 40% decrease in the SM levels in the overexpressor cells, and labeling studies demonstrated that nSMase2 accelerated SM catabolism. Accordingly, ceramide measurement showed a 60 +/- 15% increase in nSMase2-overexpressing cells compared with the vector-transfected MCF7. The role of nSMase2 in cell growth was next investigated. Stable overexpression of nSMase2 resulted in a 30-40% decrease in the rate of growth at the late exponential phase. Moreover, tumor necrosis factor induced approximately 50% activation of nSMase2 in MCF7 cells overexpressing the enzyme, demonstrating that nSMase2 is a tumor necrosis factor-responsive enzyme. In conclusion, these results 1) show that nSMase2 is a structural gene for nSMase, 2) suggest that nSMase2 acts as a bona fide N-SMase in cells, and 3) implicate nSMase2 in the regulation of cell growth and cell signaling.     

Sphingosine-1-phosphate phosphatases

Sphingosine-1-phosphate is a potent proliferative, survival, and morphogenetic factor, acting as an extracellular ligand for the EDG family of G-protein-coupled receptors and possibly intracellularly through as yet, unidentified targets. It is produced within most, if not all cells by phosphorylation of sphingosine, and is an abundant serum lipid that is released from activated platelets. Sphingosine and sphingosine-1-phosphate are in dynamic equilibrium with each other due to the activities of sphingosine kinase and sphingosine-1-phosphate phosphatase (SPPase). Several SPPase genes have now been cloned, first from yeast and more recently from mammalian cells. By sequence homology, these enzymes can be classified as a subset of membrane bound, Type 2 lipid phosphohydrolases that contain conserved residues within three domains predicted to be at the active site of the enzyme. Outside of the consensus motif, there is very little homology between SPPases and the other type 2 lipid phosphohydrolases in the LPP/PAP family. Type 2 phosphatase activity is Mg+-independent and insensitive to N-ethylmaleimide, and substrate specificity is broad for LPP enzymes, whereas SPPases are highly selective for sphingolipid substrates. SPPase activity in yeast and mammalian cells regulates intracellular sphingosine-1-phosphate levels, and also alters the levels of sphingosine and ceramide, two other signaling molecules that often oppose the actions of sphingosine-1-phosphate. Thus, loss of SPPase in yeast results in high sphingosine-1-phosphate levels and cells are more resistant to stress, and in mammalian cells, overexpression of SPPase elevates ceramide levels and provokes apoptosis.     

Sphingosine-1-Phosphate Phosphatases

Sphingosine-1-phosphate is a potent proliferative, survival, and morphogenetic factor, acting as an extracellular ligand for the EDG family of G-protein-coupled receptors and possibly intracellularly through as yet, unidentified targets. It is produced within most, if not all cells by phosphorylation of sphingosine, and is an abundant serum lipid that is released from activated platelets. Sphingosine and sphingosine-1-phosphate are in dynamic equilibrium with each other due to the activities of sphingosine kinase and sphingosine-1-phosphate phosphatase (SPPase). Several SPPase genes have now been cloned, first from yeast and more recently from mammalian cells. By sequence homology, these enzymes can be classified as a subset of membrane bound, Type 2 lipid phosphohydrolases that contain conserved residues within three domains predicted to be at the active site of the enzyme. Outside of the consensus motif, there is very little homology between SPPases and the other type 2 lipid phosphohydrolases in the LPP/PAP family. Type 2 phosphatase activity is Mg(+)-independent and insensitive to N-ethylmaleimide, and substrate specificity is broad for LPP enzymes, whereas SPPases are highly selective for sphingolipid substrates. SPPase activity in yeast and mammalian cells regulates intracellular sphingosine-1-phosphate levels, and also alters the levels of sphingosine and ceramide, two other signaling molecules that often oppose the actions of sphingosine-1-phosphate. Thus, loss of SPPase in yeast results in high sphingosine-1-phosphate levels and cells are more resistant to stress, and in mammalian cells, overexpression of SPPase elevates ceramide levels and provokes apoptosis.     

Metabolism and functions of phosphatidylserine

Phosphatidylserine (PS) is a quantitatively minor membrane phospholipid that is synthesized by prokaryotic and eukaryotic cells. In this review we focus on genes and enzymes that are involved in PS biosynthesis in bacteria, yeast, plants and mammalian cells and discuss the available information on the regulation of PS biosynthesis in these organisms. The enzymes that synthesize PS are restricted to endoplasmic reticulum membranes in yeast and mammalian cells, yet PS is widely distributed throughout other organelle membranes. Thus, mechanisms of inter-organelle movement of PS, particularly the transport of PS from its site of synthesis to the site of PS decarboxylation in mitochondria, are considered. PS is normally asymmetrically distributed across the membrane bilayer, thus the mechanisms of transbilayer translocation of PS, particularly across the plasma membrane, are also discussed. The exposure of PS on the outside surface of cells is widely believed to play a key role in the removal of apoptotic cells and in initiation of the blood clotting cascade. PS is also the precursor of phosphatidylethanolamine that is made by PS decarboxylase in bacteria, yeast and mammalian cells. Furthermore, PS is required as a cofactor for several important enzymes, such as protein kinase C and Raf-1 kinase, that are involved in signaling pathways.     

Yeast ubiquitin-conjugating enzymes involved in selective protein degradation are essential for cell viability.

Ubiquitin-mediated proteolysis is a major pathway for selective protein degradation in eukaryotic cells. This proteolysis pathway involves the processive covalent attachment of ubiquitin to proteolytic substrates and their subsequent degradation by a specific ATP-dependent protease complex. We have cloned the genes and characterized the function of ubiquitin-conjugating enzymes (UBCs) from the yeast Saccharomyces cerevisiae. UBC1, UBC4 and UBC5 enzymes were found to mediate selective degradation of short-lived and abnormal proteins. These enzymes have overlapping functions and constitute a UBC subfamily essential for growth. UBC1 is specifically required at early stages of growth after germination of spores. UBC4 and UBC5 enzymes generate high molecular weight ubiquitin-protein conjugates and comprise a major ubiquitin-conjugation activity in yeast cells. Moreover, these enzymes are central components of the cellular stress response.     

Identification of a novel alternatively spliced septin.

Septins are a family of cytoskeletal proteins involved in cytokinesis, targeting of proteins to specific sites on the plasma membrane, and cellular morphogenesis. While many aspects of their function in cytokinesis in yeast cells have been investigated, the function of septins in mammalian cells is less well understood. For example, septins are present in post-mitotic neurons, suggesting they have other roles in, for example, establishing cell polarity. The full extent of the septin gene family is not known in mammalian cells. To better understand the septin gene family, we have cloned and characterized a novel mammalian septin.     

Identification of Heat Shock Protein 60 as a Regulator of Neutral Sphingomyelinase 2 and Its Role in Dopamine Uptake

Activation of sphingomyelinase (SMase) by extracellular stimuli is the major pathway for cellular production of ceramide, a bioactive lipid mediator acting through sphingomyelin (SM) hydrolysis. Previously, we reported the existence of six forms of neutral pH–optimum and Mg²⁺-dependent SMase (N-SMase) in the membrane fractions of bovine brain. Here, we focus on N-SMase ε from salt-extracted membranes. After extensive purification by 12,780-fold with a yield of 1.3%, this enzyme was eventually characterized as N-SMase2. The major single band of 60-kDa molecular mass in the active fractions of the final purification step was identified as heat shock protein 60 (Hsp60) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis. Proximity ligation assay and immunoprecipitation study showed that Hsp60 interacted with N-SMase2, prompting us to examine the effect of Hsp60 on N-SMase2 and ceramide production. Interestingly, Hsp60 siRNA treatment significantly increased the protein level of N-SMase2 in N-SMase2-overexpressed HEK293 cells. Furthermore, transfection of Hsp60 siRNA into PC12 cells effectively increased both N-SMase activity and ceramide production and increased dopamine re-uptake with paralleled increase. Taken together, these results show that Hsp60 may serve as a negative regulator in N-SMase2-induced dopamine re-uptake by decreasing the protein level of N-SMase2.     

Enzymatic lysis of microbial cells

Cell wall lytic enzymes are valuable tools for the biotechnologist, with many applications in medicine, the food industry, and agriculture, and for recovering of intracellular products from yeast or bacteria. The diversity of potential applications has conducted to the development of lytic enzyme systems with specific characteristics, suitable for satisfying the requirements of each particular application. Since the first time the lytic enzyme of excellence, lysozyme, was discovered, many investigations have contributed to the understanding of the action mechanisms and other basic aspects of these interesting enzymes. Today, recombinant production and protein engineering have improved and expanded the area of potential applications. In this review, some of the recent advances in specific enzyme systems for bacteria and yeast cells rupture and other applications are examined. Emphasis is focused in biotechnological aspects of these enzymes.     

The human solute carrier gene SLC35B4 encodes a bifunctional nucleotide sugar transporter with specificity for UDP-xylose and UDP-N-acetylglucosamine

The transport of nucleotide sugars from the cytoplasm into the Golgi apparatus is mediated by specialized type III proteins, the nucleotide sugar transporters (NSTs). Transport assays carried out in vitro with Golgi vesicles from mammalian cells showed specific uptake for a total of eight nucleotide sugars. When this study was started, NSTs with transport activities for all but two nucleotide sugars (UDP-Xyl and UDP-Glc) had been cloned. Aiming at identifying these elusive NSTs, bioinformatic methods were used to display putative NST sequences in the human genome. Ten open reading frames were identified, cloned, and heterologously expressed in yeast. Transport capabilities for UDP-Glc and UDP-Xyl were determined with Golgi vesicles isolated from transformed cells. Although a potential UDP-Glc transporter could not be identified due to the high endogenous transport background, the measurement of UDP-Xyl transport was possible on a zero background. Vesicles from yeast cells expressing the human gene SLC35B4 showed specific uptake of UDP-Xyl, and subsequent testing of other nucleotide sugars revealed a second activity for UDP-GlcNAc. Expression of the epitope-tagged SLC35B4 in mammalian cells demonstrated strict Golgi localization. Because decarboxylation of UDP-GlcA is known to produce UDP-Xyl directly in the endoplasmic reticulum and Golgi lumen, our data demonstrate that two ways exist to deliver UDP-Xyl to the Golgi apparatus.     

2-Oxoaldehyde metabolism in microorganisms

The properties of methylglyoxal-metabolizing enzymes in prokaryotic and eukaryotic microorganisms were studied systematically and compared with those of mammalian enzymes. The enzymes constitute a glycolytic bypass and convert methylglyoxal into pyruvate via lactate. The first step in this conversion is catalyzed by glyoxalase I, methylglyoxal reductase, or methylglyoxal dehydrogenase. The regulation of the yeast glyoxalase system was analyzed. The system was closely related to the proliferative states of yeast cells, the activity of the system being high in dividing cells and low in nondividing ones. The gene for the glyoxalase I of Pseudomonas putida and the genes responsible for the activity of glyoxalase I and methylglyoxal reductase in Saccharomyces cerevisiae were cloned and their structural and phenotypic characters studied.     

Ceramide generation in nitric oxide-induced apoptosis. Activation of magnesium-dependent neutral sphingomyelinase via caspase-3

Sodium nitroprusside (SNP), a NO donor, has been recognized as an inducer of apoptosis in various cell lines. Here, we demonstrated the intracellular formation of ceramide, a lipid signal mediator, in SNP-induced apoptosis in human leukemia HL-60 cells and investigated the mechanisms of ceramide generation. The levels of intracellular ceramide increased to, at most, 160% of the control level in a time- and dose-dependent manner when the cells were treated with 1 mM SNP. SNP also decreased the sphingomyelin level to approximately 70% of the control level and increased magnesium-dependent neutral sphingomyelinase (N-SMase) activity to 160% of the control activity 2 h after treatment. Neither acid SMase nor magnesium-independent N-SMase was affected by SNP. Caspases are thought to be key enzymes in apoptotic cell death. Acetyl-Asp-Glu-Val-Asp-aldehyde, a synthetic tetrapeptide inhibitor of caspases, inhibited magnesiumdependent N-SMase, ceramide generation, and apoptosis. Moreover, recombinant purified caspase-3 increased magnesium-dependent N-SMase in a cell-free system. These results suggest that the findings that SNP increased ceramide generation and magnesium-dependent N-SMase activity via caspase-3 are interesting to future study to determine the relation between caspases and sphingolipid metabolites in NO-mediated signaling.     

Function of the cloned putative neutral sphingomyelinase as lyso-platelet activating factor-phospholipase C

Sphingolipids such as ceramide and sphingosine have been regarded as novel signal mediators in cells. However, the mechanisms of generation of these lipids upon various stimulation remain to be elucidated. Neutral sphingomyelinase (N-SMase) is one of the key enzymes in the generation of ceramide, and recently the cloning of a putative N-SMase was reported. Because the function of the protein was unclear in the previous report, we investigated the role it plays in cells. N-SMase activity in cells overexpressing the protein with hexa-histidine tag was immunoprecipitated with anti-hexa-histidine antibody. The metabolism of ceramide and SM was not apparently affected in overexpressing cells. Radiolabeling experiments using [(3)H]palmitic acid or [(3)H]hexadecanol demonstrated an accumulation of 1-O-alkyl-sn-glycerol and a corresponding decrease of 1-alkyl-2-acyl-sn-glycero-3-phosphocholine in overexpressing cells. In vitro studies showed that both 1-acyl-2-lyso-sn-glycero-3-phosphocholine (lyso-PC) and 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine (lyso-platelet activating factor (lyso-PAF)) are good substrates of the protein. In further radiolabeling experiments, 1-acyl-lyso-PC was predominantly and equally metabolized into diacyl-PC in both vector and overexpressing cells. On the other hand, 1-O-alkyl-lyso-PC (lyso-PAF) was metabolized into both diradyl-PC and 1-O-alkyl-glycerol in overexpressing cells but only into diradyl-PC in vector cells. These results suggest that the protein acts as lyso-PAF-PLC rather than lyso-PC-PLC or N-SMase in cells.