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Makoto Ito - One of the best experts on this subject based on the ideXlab platform.
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Molecular mechanism for sphingosine-induced Pseudomonas Ceramidase expression through the transcriptional regulator SphR.
Scientific reports, 2016Co-Authors: Nozomu Okino, Makoto ItoAbstract:Pseudomonas aeruginosa, an opportunistic, but serious multidrug-resistant pathogen, secretes a Ceramidase capable of cleaving the N-acyl linkage of ceramide to generate fatty acids and sphingosine. We previously reported that the secretion of P. aeruginosa Ceramidase was induced by host-derived sphingolipids, through which phospholipase C-induced hemolysis was significantly enhanced. We herein investigated the gene(s) regulating sphingolipid-induced Ceramidase expression and identified SphR, which encodes a putative AraC family transcriptional regulator. Disruption of the sphR gene in P. aeruginosa markedly decreased the sphingomyelin-induced secretion of Ceramidase, reduced hemolytic activity, and resulted in the loss of sphingomyelin-induced Ceramidase expression. A microarray analysis confirmed that sphingomyelin significantly induced Ceramidase expression in P. aeruginosa. Furthermore, an electrophoretic mobility shift assay revealed that SphR specifically bound free sphingoid bases such as sphingosine, dihydrosphingosine, and phytosphingosine, but not sphingomyelin or ceramide. A β-galactosidase-assisted promoter assay showed that sphingosine activated Ceramidase expression through SphR at a concentration of 100 nM. Collectively, these results demonstrated that sphingosine induces the secretion of Ceramidase by promoting the mRNA expression of Ceramidase through SphR, thereby enhancing hemolytic phospholipase C-induced cytotoxicity. These results facilitate understanding of the physiological role of bacterial Ceramidase in host cells.
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Ceramidase enhances phospholipase C-induced hemolysis by Pseudomonas aeruginosa.
The Journal of biological chemistry, 2007Co-Authors: Nozomu Okino, Makoto ItoAbstract:Abstract We previously reported the purification, molecular cloning, and characterization of a neutral Ceramidase from Pseudomonas aeruginosa strain AN17 (Okino, N., Tani, M., Imayama, S., and Ito, M. (1998) J. Biol. Chem. 273, 14368–14373; Okino, N., Ichinose, S., Omori, A., Imayama, S., Nakamura, T., and Ito, M. (1999) J. Biol. Chem. 274, 36616–36622). Interestingly, the gene encoding the enzyme is adjacent to that encoding hemolytic phospholipase C (plcH) in the genome of Pseudomonas aeruginosa, which is a well known pathogen for opportunistic infections. We report here that simultaneous production of PlcH and Ceramidase was induced by several lipids and PlcH-induced hemolysis was significantly enhanced by the action of the Ceramidase. When the strain was cultured with sphingomyelin or phosphatidylcholine, production of both enzymes drastically increased, causing the increase of hemolytic activity in the cell-free culture supernatant. Ceramide and sphingosine were also effective in promoting the production of Ceramidase but not that of PlcH. Furthermore, we found that the hemolytic activity of a Bacillus cereus sphingomyelinase was significantly enhanced by addition of a recombinant Pseudomonas Ceramidase. TLC analysis of the erythrocytes showed that ceramide produced from sphingomyelin by the sphingomyelinase was partly converted to sphingosine by the Ceramidase. A Ceramidase-null mutant strain caused much less hemolysis of sheep erythrocytes than did the wild-type strain. Sphingosine was detected in the erythrocytes co-cultured with the wild-type strain but not the mutant strain. Finally, we found that the enhancement of PlcH-induced hemolysis by the Ceramidase occurred in not only sheep but also human erythrocytes. These results may indicate that the Ceramidase enhances the PlcH-induced cytotoxicity and provide new insights into the role of sphingolipid-degrading enzymes in the pathogenicity of P. aeruginosa.
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Subcellular localization of human neutral Ceramidase expressed in HEK293 cells.
Biochemical and biophysical research communications, 2005Co-Authors: Young Ha Hwang, Nozomu Okino, Motohiro Tani, Tetsuto Nakagawa, Makoto ItoAbstract:Abstract We previously reported that rat and mouse neutral Ceramidases were mainly localized to plasma membranes as a type II integral membrane protein and partly detached from the cells via processing of the N-terminal/anchor sequence when expressed in HEK293 cells [M. Tani, H. Iida, M. Ito, O-glycosylation of mucin-like domain retains the neutral Ceramidase on the plasma membranes as a type II integral membrane protein, J. Biol. Chem. 278 (2003) 10523–10530]. In contrast, the human homologue was exclusively detected in mitochondria when expressed in HEK293 and MCF7 cells as a fusion protein with green fluorescent protein at the N-terminal of the enzyme [S.E. Bawab, P. Roddy, T. Quian, A. Bielawska, J.J. Lemasters, Y.A. Hannun, Molecular cloning and characterization of a human mitochondrial Ceramidase, J. Biol. Chem. 275 (2000) 21508–21513]. Given this discrepancy, we decided to clone the neutral Ceramidase from human kidney cDNA and re-examine the intracellular localization of the enzyme when expressed in HEK293 cells. The putative amino acid sequence of the newly cloned enzyme was identical to that reported for human neutral Ceramidase except at the N-terminal; the new protein was 19 amino acids longer at the N-terminal. We found that the putative full-length human neutral Ceramidase was transported to plasma membranes, but not to mitochondria, possibly via a classical ER/Golgi pathway and localized mainly in plasma membranes when expressed in HEK293 cells. The N-terminal-truncated mutant, previously reported as a human mitochondrial Ceramidase, was also weakly expressed in HEK293 cells but mainly released into the medium possibly due to the insufficient signal/anchor sequence.
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o glycosylation of mucin like domain retains the neutral Ceramidase on the plasma membranes as a type ii integral membrane protein
Journal of Biological Chemistry, 2003Co-Authors: Motohiro Tani, Hiroshi Iida, Makoto ItoAbstract:Abstract Ceramidase is a key enzyme involved in regulating cellular levels of ceramide, sphingosine, and possibly sphigosine 1-phosphate and thus could modulate sphingolipid signaling. Here we report that O-glycosylation of the mucin-like domain of neutral Ceramidases was required for localization to the surface of plasma membranes. The deduced amino acid sequences of the mammalian enzymes contain a serine-threonine-rich domain (mucin box), which follows the signal/anchor sequence, whereas those of bacterial and invertebrate enzymes completely lack a mucin box, suggesting that the specific domain has been acquired during evolution. In HEK293 cells overexpressing Ceramidase, the enzyme was not only secreted into the medium after cleavage of the NH2-terminal signal/anchor sequence but also localized at the plasma membrane as a type II integral membrane protein. Lectin blot analysis using peanut agglutinin revealed that the mucin box of the enzyme is highly glycosylated withO-glycans. Interestingly, a mutant lacking the mucin box or possible O-glycosylation sites in the mucin box was secreted into the medium but not localized at the surface of the cells. Furthermore, a mucin box-fused chimera green fluorescent protein (GFP), but not GFP itself, with the signal/anchor sequence was distributed on the surface of the cells. These results suggest thatO-glycosylation of the mucin box retains proteins on the plasma membranes. We also found that the 112-kDa membrane-bound enzyme from mouse kidney is O-glycosylated, whereas the 94-kDa soluble enzyme from liver is not. These results clearly indicate that post-translational modification of the enzyme withO-glycans is tissue-specific and helps the enzyme to localize at the surface of plasma membranes as a type II membrane protein.
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Purification, Characterization, Molecular Cloning, and Subcellular Distribution of Neutral Ceramidase of Rat Kidney
The Journal of biological chemistry, 2001Co-Authors: Susumu Mitsutake, Motohiro Tani, Nozomu Okino, Kaoru Mori, Sachiyo Ichinose, Akira Omori, Hiroshi Iida, T Nakamura, Makoto ItoAbstract:Abstract Previously, we reported two types of neutral Ceramidase in mice, one solubilized by freeze-thawing and one not. The former was purified as a 94-kDa protein from mouse liver, and cloned (Tani, M., Okino, N., Mori, K., Tanigawa, T., Izu, H., and Ito, M. (2000) J. Biol. Chem. 275, 11229–11234). In this paper, we describe the purification, molecular cloning, and subcellular distribution of a 112-kDa membrane-bound neutral Ceramidase of rat kidney, which was completely insoluble by freeze-thawing. The open reading frame of the enzyme encoded a polypeptide of 761 amino acids having nine putative N-glycosylation sites and one possible transmembrane domain. In the Ceramidase overexpressing HEK293 cells, 133-kDa (Golgi-form) and 113-kDa (endoplasmic reticulum-form) Myc-tagged Ceramidases were detected, whereas these two proteins were converted to a 87-kDa protein concomitantly with loss of activity when expressed in the presence of tunicamycin, indicating that theN-glycosylation process is indispensable for the expression of the enzyme activity. Immunohistochemical analysis clearly showed that the Ceramidase was mainly localized at the apical membrane of proximal tubules, distal tubules, and collecting ducts in rat kidney, while in liver the enzyme was distributed with endosome-like organelles in hepatocytes. Interestingly, the kidney Ceramidase was found to be enriched in the raft microdomains with cholesterol and GM1 ganglioside.
Yusuf A Hannun - One of the best experts on this subject based on the ideXlab platform.
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Neutral Ceramidase: Advances in mechanisms, cell regulation, and roles in cancer
Advances in biological regulation, 2018Co-Authors: Nicolas Coant, Yusuf A HannunAbstract:Abstract Extensive research conducted in the last three decades has identified the roles for the main bioactive sphingolipids, namely ceramide, sphingosine, and sphingosine 1-phosphate (S1P) as key regulators of cellular homeostasis, growth and death. One of the major groups of enzymes in the ceramide pathway, Ceramidases, converts ceramide into sphingosine and fatty acids, with sphingosine being further metabolized to S1P. Thus, these enzymes play important roles in the network controlling the functions associated with these bioactive sphingolipids. Among the family of Ceramidases, neutral Ceramidase (nCDase), which is named according to its optimal pH for catalytic activity, has received increased attention in the last decade. The goal of this review is to provide a brief background on bioactive sphingolipids and the Ceramidases. We then describe more recent advances on nCDase, specifically the resolution of its crystal structure and understanding its roles in cell biology and physiology.
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Functions of neutral Ceramidase in the Golgi apparatus.
Journal of lipid research, 2018Co-Authors: Wataru Sakamoto, Nicolas Coant, Lina M Obeid, Daniel Canals, Yusuf A HannunAbstract:Ceramidases hydrolyze ceramides into sphingosine and fatty acids, with sphingosine being further metabolized into sphingosine-1-phosphate (S1P); thus, Ceramidases control the levels of these bioactive sphingolipids in cells and tissues. Neutral Ceramidase (nCDase) is highly expressed in colorectal tissues, and a recent report showed that nCDase activity is involved in Wnt/β-catenin signaling. In addition, the inhibition of nCDase decreases the development and progression of colorectal tumor growth. Here, to determine the action of nCDase in colorectal cancer cells, we focused on the subcellular localization and metabolic functions of this enzyme in HCT116 cells. nCDase was found to be located in both the plasma membrane and in the Golgi apparatus, but it had minimal effects on basal levels of ceramide, sphingosine, or S1P. Cells overexpressing nCDase were protected from the cell death and Golgi fragmentation induced by C6-ceramide, and they showed reduced levels of C6-ceramide and higher levels of S1P and sphingosine. Furthermore, compartment-specific metabolic functions of the enzyme were probed using C6-ceramide and Golgi-targeted bacterial SMase (bSMase) and bacterial Ceramidase (bCDase). The results showed that Golgi-specific bCDase also demonstrated resistance against the cell death stimulated by C6-ceramide, and it catalyzed the metabolism of ceramides and produced sphingosine in the Golgi. Targeting bSMase to the Golgi resulted in increased levels of ceramide that were attenuated by the expression of nCDase, also supporting its ability to metabolize Golgi-generated ceramide. These results are critical in understanding the functions of nCDase actions in colorectal cancer cells as well as the compartmentalized pathways of sphingolipid metabolism.
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alkaline Ceramidase 2 is essential for the homeostasis of plasma sphingoid bases and their phosphates
The FASEB Journal, 2018Co-Authors: Fang Li, Monica Garciabarros, Jennifer Schrandt, Ashley Snider, Xiancheng Jiang, Mingsong Li, Ruijuan Xu, Izolda Mileva, Yusuf A HannunAbstract:Sphingosine-1-phosphate (S1P) plays important roles in cardiovascular development and immunity. S1P is abundant in plasma because erythrocytes—the major source of S1P—lack any S1P-degrading activity; however, much remains unclear about the source of the plasma S1P precursor, sphingosine (SPH), derived mainly from the hydrolysis of ceramides by the action of Ceramidases that are encoded by 5 distinct genes, acid Ceramidase 1 (ASAH1)/Asah1, ASAH2/Asah2, alkaline Ceramidase 1 (ACER1)/Acer1, ACER2/Acer2, and ACER3/Acer3, in humans/mice. Previous studies have reported that knocking out Asah1 or Asah2 failed to reduce plasma SPH and S1P levels in mice. In this study, we show that knocking out Acer1 or Acer3 also failed to reduce the blood levels of SPH or S1P in mice. In contrast, knocking out Acer2 from either whole-body or the hematopoietic lineage markedly decreased the blood levels of SPH and S1P in mice. Of interest, knocking out Acer2 from whole-body or the hematopoietic lineage also markedly decreased th...
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Anticancer actions of lysosomally targeted inhibitor, LCL521, of acid Ceramidase
PloS one, 2017Co-Authors: Aiping Bai, Cungui Mao, Zdzislaw M. Szulc, Alicja Bielawska, Russell W. Jenkins, Yusuf A HannunAbstract:Acid Ceramidase, which catalyzes ceramide hydrolysis to sphingosine and free fatty acid mainly in the lysosome, is being recognized as a potential therapeutic target for cancer. B13 is an effective and selective acid Ceramidase inhibitor in vitro, but not as effective in cells due to poor access to the lysosomal compartment. In order to achieve targeting of B13 to the lysosome, we designed lysosomotropic N, N-dimethyl glycine (DMG)-conjugated B13 prodrug LCL521 (1,3-di-DMG-B13). Our previous results indicated the efficient delivery of B13 to the lysosome resulted in augmented effects of LCL521 on cellular acid Ceramidase as evaluated by effects on substrate/product levels. Our current studies indicate that functionally, this translated into enhanced inhibition of cell proliferation. Moreover, there were greater synergistic effects of LCL521 with either ionizing radiation or Tamoxifen. Taken together, these results clearly indicate that compartmental targeting for the inhibition of acid Ceramidase is an efficient and valuable therapeutic strategy.
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Ceramidases, roles in sphingolipid metabolism and in health and disease.
Advances in biological regulation, 2016Co-Authors: Nicolas Coant, Cungui Mao, Wataru Sakamoto, Yusuf A HannunAbstract:Over the past three decades, extensive research has been able to determine the biologic functions for the main bioactive sphingolipids, namely ceramide, sphingosine, and sphingosine 1-phosphate (S1P) (Hannun, 1996; Hannun et al., 1986; Okazaki et al., 1989). These studies have managed to define the metabolism, regulation, and function of these bioactive sphingolipids. This emerging body of literature has also implicated bioactive sphingolipids, particularly S1P and ceramide, as key regulators of cellular homeostasis. Ceramidases have the important role of cleaving fatty acid from ceramide and producing sphingosine, thereby controlling the interconversion of these two lipids. Thus far, five human Ceramidases encoded by five different genes have been identified: acid Ceramidase (AC), neutral Ceramidase (NC), alkaline Ceramidase 1 (ACER1), alkaline Ceramidase 2 (ACER2), and alkaline Ceramidase 3 (ACER3). These Ceramidases are classified according to their optimal pH for catalytic activity. AC, which is localized to the lysosomal compartment, has been associated with Farber's disease and is involved in the regulation of cell viability. Neutral Ceramidase, which is localized to the plasma membrane and primarily expressed in the small intestine and colon, is involved in digestion, and has been implicated in colon carcinogenesis. ACER1 which can be found in the endoplasmic reticulum and is highly expressed in the skin, plays an important role in keratinocyte differentiation. ACER2, localized to the Golgi complex and highly expressed in the placenta, is involved in programed cell death in response to DNA damage. ACER3, also localized to the endoplasmic reticulum and the Golgi complex, is ubiquitously expressed, and is involved in motor coordination-associated Purkinje cell degeneration. This review seeks to consolidate the current knowledge regarding these key cellular players.
Nozomu Okino - One of the best experts on this subject based on the ideXlab platform.
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Molecular mechanism for sphingosine-induced Pseudomonas Ceramidase expression through the transcriptional regulator SphR.
Scientific reports, 2016Co-Authors: Nozomu Okino, Makoto ItoAbstract:Pseudomonas aeruginosa, an opportunistic, but serious multidrug-resistant pathogen, secretes a Ceramidase capable of cleaving the N-acyl linkage of ceramide to generate fatty acids and sphingosine. We previously reported that the secretion of P. aeruginosa Ceramidase was induced by host-derived sphingolipids, through which phospholipase C-induced hemolysis was significantly enhanced. We herein investigated the gene(s) regulating sphingolipid-induced Ceramidase expression and identified SphR, which encodes a putative AraC family transcriptional regulator. Disruption of the sphR gene in P. aeruginosa markedly decreased the sphingomyelin-induced secretion of Ceramidase, reduced hemolytic activity, and resulted in the loss of sphingomyelin-induced Ceramidase expression. A microarray analysis confirmed that sphingomyelin significantly induced Ceramidase expression in P. aeruginosa. Furthermore, an electrophoretic mobility shift assay revealed that SphR specifically bound free sphingoid bases such as sphingosine, dihydrosphingosine, and phytosphingosine, but not sphingomyelin or ceramide. A β-galactosidase-assisted promoter assay showed that sphingosine activated Ceramidase expression through SphR at a concentration of 100 nM. Collectively, these results demonstrated that sphingosine induces the secretion of Ceramidase by promoting the mRNA expression of Ceramidase through SphR, thereby enhancing hemolytic phospholipase C-induced cytotoxicity. These results facilitate understanding of the physiological role of bacterial Ceramidase in host cells.
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Ceramidase enhances phospholipase C-induced hemolysis by Pseudomonas aeruginosa.
The Journal of biological chemistry, 2007Co-Authors: Nozomu Okino, Makoto ItoAbstract:Abstract We previously reported the purification, molecular cloning, and characterization of a neutral Ceramidase from Pseudomonas aeruginosa strain AN17 (Okino, N., Tani, M., Imayama, S., and Ito, M. (1998) J. Biol. Chem. 273, 14368–14373; Okino, N., Ichinose, S., Omori, A., Imayama, S., Nakamura, T., and Ito, M. (1999) J. Biol. Chem. 274, 36616–36622). Interestingly, the gene encoding the enzyme is adjacent to that encoding hemolytic phospholipase C (plcH) in the genome of Pseudomonas aeruginosa, which is a well known pathogen for opportunistic infections. We report here that simultaneous production of PlcH and Ceramidase was induced by several lipids and PlcH-induced hemolysis was significantly enhanced by the action of the Ceramidase. When the strain was cultured with sphingomyelin or phosphatidylcholine, production of both enzymes drastically increased, causing the increase of hemolytic activity in the cell-free culture supernatant. Ceramide and sphingosine were also effective in promoting the production of Ceramidase but not that of PlcH. Furthermore, we found that the hemolytic activity of a Bacillus cereus sphingomyelinase was significantly enhanced by addition of a recombinant Pseudomonas Ceramidase. TLC analysis of the erythrocytes showed that ceramide produced from sphingomyelin by the sphingomyelinase was partly converted to sphingosine by the Ceramidase. A Ceramidase-null mutant strain caused much less hemolysis of sheep erythrocytes than did the wild-type strain. Sphingosine was detected in the erythrocytes co-cultured with the wild-type strain but not the mutant strain. Finally, we found that the enhancement of PlcH-induced hemolysis by the Ceramidase occurred in not only sheep but also human erythrocytes. These results may indicate that the Ceramidase enhances the PlcH-induced cytotoxicity and provide new insights into the role of sphingolipid-degrading enzymes in the pathogenicity of P. aeruginosa.
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Subcellular localization of human neutral Ceramidase expressed in HEK293 cells.
Biochemical and biophysical research communications, 2005Co-Authors: Young Ha Hwang, Nozomu Okino, Motohiro Tani, Tetsuto Nakagawa, Makoto ItoAbstract:Abstract We previously reported that rat and mouse neutral Ceramidases were mainly localized to plasma membranes as a type II integral membrane protein and partly detached from the cells via processing of the N-terminal/anchor sequence when expressed in HEK293 cells [M. Tani, H. Iida, M. Ito, O-glycosylation of mucin-like domain retains the neutral Ceramidase on the plasma membranes as a type II integral membrane protein, J. Biol. Chem. 278 (2003) 10523–10530]. In contrast, the human homologue was exclusively detected in mitochondria when expressed in HEK293 and MCF7 cells as a fusion protein with green fluorescent protein at the N-terminal of the enzyme [S.E. Bawab, P. Roddy, T. Quian, A. Bielawska, J.J. Lemasters, Y.A. Hannun, Molecular cloning and characterization of a human mitochondrial Ceramidase, J. Biol. Chem. 275 (2000) 21508–21513]. Given this discrepancy, we decided to clone the neutral Ceramidase from human kidney cDNA and re-examine the intracellular localization of the enzyme when expressed in HEK293 cells. The putative amino acid sequence of the newly cloned enzyme was identical to that reported for human neutral Ceramidase except at the N-terminal; the new protein was 19 amino acids longer at the N-terminal. We found that the putative full-length human neutral Ceramidase was transported to plasma membranes, but not to mitochondria, possibly via a classical ER/Golgi pathway and localized mainly in plasma membranes when expressed in HEK293 cells. The N-terminal-truncated mutant, previously reported as a human mitochondrial Ceramidase, was also weakly expressed in HEK293 cells but mainly released into the medium possibly due to the insufficient signal/anchor sequence.
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Purification and Characterization of Recombinant, Human Acid Ceramidase CATALYTIC REACTIONS AND INTERACTIONS WITH ACID SPHINGOMYELINASE
The Journal of biological chemistry, 2003Co-Authors: Nozomu Okino, Konrad Sandhoff, Arie Dagan, Shimon Gatt, Heike Schulze, Rajwinder Dhami, Edward H. SchuchmanAbstract:Abstract Human acid Ceramidase was overexpressed in Chinese hamster ovary cells by amplification of the transfected, full-length cDNA. The majority of the overexpressed enzyme was secreted into the culture media and purified to apparent homogeneity. The purified protein contained the same 13-(α) and 40 (β)–kDa subunits as human acid Ceramidase from natural sources, had an acidic pH optimum (4.5), and followed normal Michaelis-Menten kinetics using 14C- and BODIPY-labeled C12-ceramide as substrates. Deglycosylation studies showed that the recombinant enzyme contained mostly “high mannose” type oligosaccharides and that two distinct β-subunits were present. Amino acid sequencing of these subunit polypeptides revealed a single N terminus, suggesting that the ∼2–4-kDa molecular mass difference was likely due to C-terminal processing. The purified enzyme also catalyzed ceramide synthesis in vitro using 14C-labeled C12 fatty acid and sphingosine as substrates. Surprisingly, we found that media from the overexpressing hamster cells had increased acid sphingomyelinase activity and that this activity could be co-precipitated with acid Ceramidase using anti-Ceramidase antibodies. Overexpression of acid Ceramidase in normal human skin fibroblasts also led to enhanced acid sphingomyelinase secretion, but this was not observed in Niemann-Pick disease cells. RNA studies showed that this increased activity was not due to overexpression of the endogenous acid sphingomyelinase gene. Uptake studies using mouse macrophages revealed rapid internalization of the acid Ceramidase activity from the hamster cell media but not acid sphingomyelinase. These studies provide new insights into acid Ceramidase and the related lipid hydrolase, acid sphingomyelinase.
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Purification, Characterization, Molecular Cloning, and Subcellular Distribution of Neutral Ceramidase of Rat Kidney
The Journal of biological chemistry, 2001Co-Authors: Susumu Mitsutake, Motohiro Tani, Nozomu Okino, Kaoru Mori, Sachiyo Ichinose, Akira Omori, Hiroshi Iida, T Nakamura, Makoto ItoAbstract:Abstract Previously, we reported two types of neutral Ceramidase in mice, one solubilized by freeze-thawing and one not. The former was purified as a 94-kDa protein from mouse liver, and cloned (Tani, M., Okino, N., Mori, K., Tanigawa, T., Izu, H., and Ito, M. (2000) J. Biol. Chem. 275, 11229–11234). In this paper, we describe the purification, molecular cloning, and subcellular distribution of a 112-kDa membrane-bound neutral Ceramidase of rat kidney, which was completely insoluble by freeze-thawing. The open reading frame of the enzyme encoded a polypeptide of 761 amino acids having nine putative N-glycosylation sites and one possible transmembrane domain. In the Ceramidase overexpressing HEK293 cells, 133-kDa (Golgi-form) and 113-kDa (endoplasmic reticulum-form) Myc-tagged Ceramidases were detected, whereas these two proteins were converted to a 87-kDa protein concomitantly with loss of activity when expressed in the presence of tunicamycin, indicating that theN-glycosylation process is indispensable for the expression of the enzyme activity. Immunohistochemical analysis clearly showed that the Ceramidase was mainly localized at the apical membrane of proximal tubules, distal tubules, and collecting ducts in rat kidney, while in liver the enzyme was distributed with endosome-like organelles in hepatocytes. Interestingly, the kidney Ceramidase was found to be enriched in the raft microdomains with cholesterol and GM1 ganglioside.
Cungui Mao - One of the best experts on this subject based on the ideXlab platform.
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Elusive Roles of the Different Ceramidases in Human Health, Pathophysiology, and Tissue Regeneration.
Cells, 2020Co-Authors: Carolina Duarte, Cungui Mao, Juliet Akkaoui, Chiaki Yamada, Alexandru MovilaAbstract:Ceramide and sphingosine are important interconvertible sphingolipid metabolites which govern various signaling pathways related to different aspects of cell survival and senescence. The conversion of ceramide into sphingosine is mediated by Ceramidases. Altogether, five human Ceramidases—named acid Ceramidase, neutral Ceramidase, alkaline Ceramidase 1, alkaline Ceramidase 2, and alkaline Ceramidase 3—have been identified as having maximal activities in acidic, neutral, and alkaline environments, respectively. All five Ceramidases have received increased attention for their implications in various diseases, including cancer, Alzheimer’s disease, and Farber disease. Furthermore, the potential anti-inflammatory and anti-apoptotic effects of Ceramidases in host cells exposed to pathogenic bacteria and viruses have also been demonstrated. While Ceramidases have been a subject of study in recent decades, our knowledge of their pathophysiology remains limited. Thus, this review provides a critical evaluation and interpretive analysis of existing literature on the role of acid, neutral, and alkaline Ceramidases in relation to human health and various diseases, including cancer, neurodegenerative diseases, and infectious diseases. In addition, the essential impact of Ceramidases on tissue regeneration, as well as their usefulness in enzyme replacement therapy, is also discussed.
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New fluorogenic probes for neutral and alkaline Ceramidases
Journal of lipid research, 2019Co-Authors: Mireia Casasampere, Cungui Mao, Gemma Fabriàs, José Luis Abad, Antonio Delgado, Núria Bielsa, Daniel Riba, Laura Bassas, Josefina CasasAbstract:New fluorogenic Ceramidase substrates derived from the N-acyl modification of our previously reported probes (RBM14) are reported. While none of the new probes were superior to the known RBM14C12 as acid Ceramidase substrates, the corresponding nervonic acid amide (RBM14C24:1) is an efficient and selective substrate for the recombinant human neutral Ceramidase, both in cell lysates and in intact cells. A second generation of substrates, incorporating the natural 2-(N-acylamino)-1,3-diol-4-ene framework (compounds RBM15) is also reported. Among them, the corresponding fatty acyl amides with an unsaturated N-acyl chain can be used as substrates to determine alkaline Ceramidase (ACER)1 and ACER2 activities. In particular, compound RBM15C18:1 has emerged as the best fluorogenic probe reported so far to measure ACER1 and ACER2 activities in a 96-well plate format.
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New fluorogenic probes for neutral and alkaline Ceramidases
'American Society for Biochemistry & Molecular Biology (ASBMB)', 2019Co-Authors: Casasampere Mireia, Cungui Mao, Xu Ruijuan, Bielsa Núria, Riba Daniel, Bassas Laura, Fabriàs Gemma, Abad, José Luis, Delgado Cirilo Antonio, Casas JosefinaAbstract:New fluorogenic Ceramidase substrates derived from the N acyl modification of our previously reported probes (RBM14) are reported. While none of the new probes were superior to the known RBM14C12 as acid Ceramidase substrates, the corresponding nervonic acid amide (RBM14C24:1) is an efficient and selective substrate for the recombinant human neutral Ceramidase, both in cell lysates and in intact cells. A second generation of substrates, incorporating the natural 2 (N acylamino) 1,3 diol 4 ene framework (compounds RBM15) is also reported. Among them, the corresponding fatty acyl amides with an unsaturated N acyl chain can be used as substrates to determine ACER1 and ACER2 activities. In particular, compound RBM15C18:1 has emerged as the best fluorogenic probe reported so far to measure ACER1 and ACER2 activities in a 96 well plate format.Peer reviewe
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A neutral Ceramidase, NlnCDase, is involved in the stress responses of brown planthopper, Nilaparvata lugens (Stål)
Nature Publishing Group, 2018Co-Authors: Xiao-xiao Shi, Cungui Mao, Yuan-jie Huang, Mahfuj-ara Begum, Mu-fei Zhu, Min-jing Zhang, Wen-wu Zhou, Zeng-rong ZhuAbstract:Abstract Ceramidases (CDases) are vital enzymes involved in the biosynthesis of sphingolipids, which are essential components of eukaryotic membranes. The function of these enzymes in insects, however, is poorly understood. We identified a neutral Ceramidase (NlnCDase) from the brown planthopper, Nilaparvata lugens, one of the most destructive hemipteran pests of rice. The C12-ceramide was the most preferred substrate for the NlnCDase enzyme. The activity of the NlnCDase enzyme was highest in the neutral-pH range (pH 6.0). It was inhibited by EGTA, Cs+ and Fe2+, while stimulated by EDTA and Ca2+. Moreover, the NlnCDase has higher transcript level and activity in adults than in eggs and nymphs, and in the reproductive organs (ovaries and spermaries) than in other tissues (i.e. heads, thorax, legs, midguts), which suggested that the NlnCDase might be elevated to mediate developmental process. In addition, transcripts and activity of the NlnCDase were up-regulated under abiotic stresses including starvation, abnormal temperature, and insecticides, and biotic stress of resistant rice varieties. Knocking down NlnCDase by RNA interference increased female survival under starvation and temperature stresses, suggesting that NlnCDase might be involved in the stress response in N. lugens
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Anticancer actions of lysosomally targeted inhibitor, LCL521, of acid Ceramidase
PloS one, 2017Co-Authors: Aiping Bai, Cungui Mao, Zdzislaw M. Szulc, Alicja Bielawska, Russell W. Jenkins, Yusuf A HannunAbstract:Acid Ceramidase, which catalyzes ceramide hydrolysis to sphingosine and free fatty acid mainly in the lysosome, is being recognized as a potential therapeutic target for cancer. B13 is an effective and selective acid Ceramidase inhibitor in vitro, but not as effective in cells due to poor access to the lysosomal compartment. In order to achieve targeting of B13 to the lysosome, we designed lysosomotropic N, N-dimethyl glycine (DMG)-conjugated B13 prodrug LCL521 (1,3-di-DMG-B13). Our previous results indicated the efficient delivery of B13 to the lysosome resulted in augmented effects of LCL521 on cellular acid Ceramidase as evaluated by effects on substrate/product levels. Our current studies indicate that functionally, this translated into enhanced inhibition of cell proliferation. Moreover, there were greater synergistic effects of LCL521 with either ionizing radiation or Tamoxifen. Taken together, these results clearly indicate that compartmental targeting for the inhibition of acid Ceramidase is an efficient and valuable therapeutic strategy.
Andrea Huwiler - One of the best experts on this subject based on the ideXlab platform.
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Effective inhibition of acid and neutral Ceramidases by novel B-13 and LCL-464 analogues.
Bioorganic & medicinal chemistry, 2012Co-Authors: Krishna P. Bhabak, Burkhard Kleuser, Andrea Huwiler, Christoph ArenzAbstract:Induction of apoptosis mediated by the inhibition of Ceramidases has been shown to enhance the efficacy of conventional chemotherapy in several cancer models. Among the inhibitors of Ceramidases reported in the literature, B-13 is considered as a lead compound having good in vitro potency towards acid Ceramidase. Furthermore, owing to the poor activity of B-13 on lysosoamal acid Ceramidase in living cells, LCL-464 a modified derivative of B-13 containing a basic ω-amino group at the fatty acid was reported to have higher potency towards lysosomal acid Ceramidase in living cells. In a search for more potent inhibitors of Ceramidases, we have designed a series of compounds with structural modifications of B-13 and LCL-464. In this study, we show that the efficacy of B-13 in vitro as well as in intact cells can be enhanced by suitable modification of functional groups. Furthermore, a detailed SAR investigation on LCL-464 analogues revealed novel promising inhibitors of aCDase and nCDase. In cell culture studies using the breast cancer cell line MDA-MB-231, some of the newly developed compounds elevated endogenous ceramide levels and in parallel, also induced apoptotic cell death. In summary, this study shows that structural modification of the known Ceramidase inhibitors B-13 and LCL-464 generates more potent Ceramidase inhibitors that are active in intact cells and not only elevates the cellular ceramide levels, but also enhances cell death.
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Nitric oxide induces neutral Ceramidase degradation by the ubiquitin/proteasome complex in renal mesangial cell cultures
FEBS letters, 2002Co-Authors: Rochus Franzen, Josef Pfeilschifter, Andrea HuwilerAbstract:The neutral Ceramidase is a key enzyme in the regulation of cellular ceramide levels. Previously we have reported that stimulation of rat renal mesangial cells with nitric oxide (NO) donors leads to an inhibition of neutral Ceramidase activity which is due to increased degradation of the enzyme. This and the concomitant activation of the sphingomyelinase results in an amplification of ceramide levels. Here, we show that the NO-triggered degradation of neutral Ceramidase involves activation of the ubiquitin/proteasome complex. The specific proteasome inhibitor lactacystin completely reverses the NO-induced degradation of Ceramidase protein and neutral Ceramidase activity. As a consequence, the cellular amount of ceramide, which drastically increases by NO stimulation, is reduced in the presence of lactacystin. Furthermore, ubiquitinated neutral Ceramidase accumulates after NO stimulation. In summary, our data clearly show that the ubiquitin/proteasome complex is an important determinant of neutral Ceramidase activity and thereby regulates the availability of ceramide.
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Nitric Oxide Induces Degradation of the Neutral Ceramidase in Rat Renal Mesangial Cells and Is Counterregulated by Protein Kinase C
The Journal of biological chemistry, 2002Co-Authors: Rochus Franzen, Josef Pfeilschifter, Doriano Fabbro, Armaz Aschrafi, Andrea HuwilerAbstract:Abstract Ceramide levels are strongly increased by stimulation of renal mesangial cells with nitric oxide (NO). This effect was shown previously to be due to a dual action of NO, comprising an activation of sphingomyelinases and an inhibition of Ceramidase activity. In this study we show that the NO-triggered inhibition of neutral Ceramidase activity is paralleled by a down-regulation at the protein level. A complete loss of neutral Ceramidase protein is obtained after 24 h of stimulation. Whereas the selective proteasome inhibitor lactacystin blocked NO-evoked Ceramidase degradation, several caspase inhibitors were ineffective. Moreover, the NO-induced degradation is reversed by the protein kinase C (PKC) activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), and also by the physiological PKC activators platelet-derived growth factor-BB (PDGF), angiotensin II and ATP, resulting in a normalization of neutral Ceramidase protein as well as activity. In vivophosphorylation studies using 32Pi-labeled mesangial cells revealed that TPA, PDGF, angiotensin II, and ATP trigger an increased phosphorylation of the neutral Ceramidase, which is blocked by the broad spectrum PKC inhibitor Ro-31 8220 but not by CGP 41251, which has a preferential action on Ca2+-dependent isoforms, thus suggesting the involvement of a Ca2+-independent PKC isoform. In vitro phosphorylation assays using recombinant PKC isoenzymes and neutral Ceramidase immunoprecipitated from unstimulated mesangial cells show that particularly the PKC-δ isoform and to a lesser extent the PKC-α isoform are efficient in directly phosphorylating neutral Ceramidase. In summary, our data show that NO is able to induce degradation of neutral Ceramidase, thereby promoting accumulation of ceramide in the cell. This effect is reversed by PKC activation, most probably by the PKC-δ isoenzyme, which can directly phosphorylate and thereby prevent neutral Ceramidase degradation. These novel regulatory interactions will provide therapeutically valuable information to target neutral Ceramidase stability and subsequent ceramide accumulation.
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Interleukin-1β Induces Chronic Activation and de Novo Synthesis of Neutral Ceramidase in Renal Mesangial Cells
The Journal of biological chemistry, 2001Co-Authors: Rochus Franzen, Andrea Pautz, Lutz Bräutigam, Gerd Geisslinger, Josef Pfeilschifter, Andrea HuwilerAbstract:The lipid signaling molecule ceramide is formed by the action of acid and neutral sphingomyelinases and degraded by acid and neutral Ceramidases. Short-term stimulation of mesangial cells with the pro-inflammatory cytokine interleukin-1beta (IL-1beta) leads to a rapid and transient increase in neutral sphingomyelinase activity (Kaszkin, M., Huwiler, A., Scholz, K., van den Bosch, H., and Pfeilschifter, J. (1998) FEBS Lett. 440, 163-166). In this study, we report on a second delayed peak of activation occurring after hours of IL-1beta treatment. This second phase of activation was first detectable after 2 h of treatment and steadily increased over the next 2 h, reaching maximal values after 4 h. In parallel, a pronounced increase in neutral Ceramidase activity was observed, accounting for a constant or even decreased level of ceramide after long-term IL-1beta treatment, despite continuous sphingomyelinase activation. The increase in neutral Ceramidase activity was due to expressional up-regulation, as detected by an increase in mRNA levels and enhanced de novo protein synthesis. The increase in neutral Ceramidase protein levels and activity could be blocked dose- dependently by the p38 MAPK inhibitor SB 202190, whereas the classical MAPK pathway inhibitor U0126 and the protein kinase C inhibitor Ro 318220 were ineffective. Moreover, cotreatment of cells for 24 h with IL-1beta and SB 202190 led to an increase in ceramide formation. Interestingly, IL-1beta-stimulated neutral Ceramidase activation was not reduced in mesangial cells isolated from mice deficient in MAPK-activated protein kinase-2, which is a downstream substrate of p38 MAPK, thus suggesting that the p38 MAPK-mediated induction of neutral Ceramidase occurs independently of the MAPK-activated protein kinase-2 pathway. In summary, our results suggest a biphasic regulation of sphingomyelin hydrolysis in cytokine-treated mesangial cells with delayed de novo synthesis of neutral Ceramidase counteracting sphingomyelinase activity and apoptosis. Neutral Ceramidase may thus represent a novel cytoprotective enzyme for mesangial cells exposed to inflammatory stress conditions.