Ceramidases: regulators of cellular responses mediated by ceramide, sphingosine, and sphingosine-1-phosphate

Ceramidases catalyze hydrolysis of ceramides to generate sphingosine (SPH), which is phosphorylated to form sphingosine-1-phosphate (S1P). Ceramide, SPH, and S1P are bioactive lipids that mediate cell proliferation, differentiation, apoptosis, adhesion, and migration. Presently, 5 human ceramidases encoded by 5 distinct genes have been cloned: acid ceramidase (AC), neutral ceramidase (NC), alkaline ceramidase 1 (ACER1), alkaline ceramidase 2 (ACER2), and alkaline ceramidase 3 (ACER3). Each human ceramidase has a mouse counterpart. AC, NC, and ACER1-3 have maximal activities in acidic, neutral, and alkaline environments, respectively. ACER1-3 have similar protein sequences but no homology to AC and NC. AC and NC also have distinct protein sequences. The human AC (hAC) was implicated in Farber disease, and hAC may be important for cell survival. The mouse AC (mAC) is needed for early embryo survival. NC is protective against inflammatory cytokines, and the mouse NC (mNC) is required for the catabolism of ceramides in the digestive tract. ACER1 is critical in mediating cell differentiation by controlling the generation of SPH and S1P and that ACER2's role in cell proliferation and survival depends on its expression or the cell type in which it is found. Here, we discuss the role of each ceramidase in regulating cellular responses mediated by ceramides, SPH, and S1P.     

Activity of neutral and alkaline ceramidases on fluorogenic N-acylated coumarin-containing aminodiols

Ceramidases catalyze the cleavage of ceramides into sphingosine and fatty acids. Previously, we reported on the use of the RBM14 fluorogenic ceramide analogs to determine acidic ceramidase activity. In this work, we investigated the activity of other amidohydrolases on RBM14 compounds. Both bacterial and human purified neutral ceramidases (NCs), as well as ectopically expressed mouse neutral ceramidase hydrolyzed RBM14 with different selectivity, depending on the N-acyl chain length. On the other hand, microsomes from alkaline ceramidase (ACER)3 knockdown cells were less competent at hydrolyzing RBM14C12, RBM12C14, and RBM14C16 than controls, while microsomes from ACER2 and ACER3 overexpressing cells showed no activity toward the RBM14 substrates. Conversely, N-acylethanolamine-hydrolyzing acid amidase (NAAA) overexpressing cells hydrolyzed RBM14C14 and RBM14C16 at acidic pH. Overall, NC, ACER3, and, to a lesser extent, NAAA hydrolyze fluorogenic RBM14 compounds. Although the selectivity of the substrates toward ceramidases can be modulated by the length of the N-acyl chain, none of them was specific for a particular enzyme. Despite the lack of specificity, these substrates should prove useful in library screening programs aimed at identifying potent and selective inhibitors for NC and ACER3.     

Alkaline ceramidase family: The first two decades

Ceramidases are a group of enzymes that catalyze the hydrolysis of ceramide, dihydroceramide, and phytoceramide into sphingosine (SPH), dihydrosphingosine (DHS), and phytosphingosine (PHS), respectively, along with a free fatty acid. Ceramidases are classified into the acid, neutral, and alkaline ceramidase subtypes according to the pH optima for their catalytic activity. YPC1 and YDC1 were the first alkaline ceramidase genes to be identified and cloned from the yeast Saccharomyces cerevisiae two decades ago. Subsequently, alkaline ceramidase genes were identified from other species, including one Drosophila melanogaster ACER gene (Dacer), one Arabidopsis thaliana ACER gene (AtACER), three Mus musculus ACER genes (Acer1, Acer2, and Acer3), and three Homo sapiens ACER genes (ACER1, ACER2, and ACER3). The protein products of these genes constitute a large protein family, termed the alkaline ceramidase (ACER) family. All the biochemically characterized members of the ACER family are integral membrane proteins with seven transmembrane segments in the Golgi complex or endoplasmic reticulum, and they each have unique substrate specificity. An increasing number of studies suggest that the ACER family has diverse roles in regulating sphingolipid metabolism and biological processes. Here we discuss the discovery of the ACER family, the biochemical properties, structures, and catalytic mechanisms of its members, and its role in regulating sphingolipid metabolism and biological processes in yeast, insects, plants, and mammals.     

Alkaline ceramidase 3 deficiency aggravates colitis and colitis-associated tumorigenesis in mice by hyperactivating the innate immune system

Increasing studies suggest that ceramides differing in acyl chain length and/or degree of unsaturation have distinct roles in mediating biological responses. However, still much remains unclear about regulation and role of distinct ceramide species in the immune response. Here, we demonstrate that alkaline ceramidase 3 (Acer3) mediates the immune response by regulating the levels of C_18:1-ceramide in cells of the innate immune system and that Acer3 deficiency aggravates colitis in a murine model by augmenting the expression of pro-inflammatory cytokines in myeloid and colonic epithelial cells (CECs). According to the NCBI Gene Expression Omnibus (GEO) database, ACER3 is downregulated in immune cells in response to lipopolysaccharides (LPS), a potent inducer of the innate immune response. Consistent with these data, we demonstrated that LPS downregulated both Acer3 mRNA levels and its enzymatic activity while elevating C_18:1-ceramide, a substrate of Acer3, in murine immune cells or CECs. Knocking out Acer3 enhanced the elevation of C_18:1-ceramide and the expression of pro-inflammatory cytokines in immune cells and CECs in response to LPS challenge. Similar to Acer3 knockout, treatment with C_18:1-ceramide, but not C_18:0-ceramide, potentiated LPS-induced expression of pro-inflammatory cytokines in immune cells. In the mouse model of dextran sulfate sodium-induced colitis, Acer3 deficiency augmented colitis-associated elevation of colonic C_18:1-ceramide and pro-inflammatory cytokines. Acer3 deficiency aggravated diarrhea, rectal bleeding, weight loss and mortality. Pathological analyses revealed that Acer3 deficiency augmented colonic shortening, immune cell infiltration, colonic epithelial damage and systemic inflammation. Acer3 deficiency also aggravated colonic dysplasia in a mouse model of colitis-associated colorectal cancer. Taken together, these results suggest that Acer3 has an important anti-inflammatory role by suppressing cellular or tissue C_18:1-ceramide, a potent pro-inflammatory bioactive lipid and that dysregulation of ACER3 and C_18:1-ceramide may contribute to the pathogenesis of inflammatory diseases including cancer.Ceramides are the central lipid in the metabolic network of sphingolipids, and are generated through the de novo, catabolic and salvage pathways.¹ In the de novo pathway, ceramides are synthesized through multiple steps catalyzed sequentially by serine palmitoyltransferase (SPT), keto-dihydrosphingosine reductase, (dihydro)ceramide synthases (CerSs) and dihydroceramide desaturases. In the catabolic pathways, ceramides are derived from the hydrolysis of sphingomyelins by sphingomyelinases (SMases) or the hydrolysis of glycosphingolipids. In the salvage pathway, ceramides are synthesized from sphingosine (SPH) and fatty acyl-CoA by CerSs. As CerSs (CerS1-6) have distinct specificity toward acyl-CoA chain length and degree of unsaturation, ceramides with various acyl-chains are found in mammalian cells. Upon generation, ceramides can be hydrolyzed by five ceramidases encoded by five distinct genes (ASAH1, ASAH2, ACER1, ACER2 and ACER3). These ceramidases vary in pH optimum for catalytic activity, tissue distribution, cellular localization and substrate specificity,² allowing for regulation of specific ceramides in a cell- or tissue-specific manner.Recent studies have implicated ceramides in regulating the innate immune response. Sakata et al.³ demonstrated that lipopolysaccharides (LPS), a potent inducer of the innate immune response, increases C₁₆-ceramide by activating acid SMase and that inhibition of SMase attenuates LPS-induced production of pro-inflammatory cytokines in THP-1 macrophages. Andreyev et al.⁴ found that ceramides are increased by Toll-like receptor 4 (TLR4)-specific LPS in RAW 264.7 macrophages. Schilling et al.⁵ revealed that LPS and palmitic acid synergistically increase C₁₆-ceramide in primary mouse peritoneal macrophages (PMs) by activating de novo biosynthesis of ceramides and that inhibiting the C₁₆-ceramide increase attenuates LPS-induced production of TNF-α and IL-1β in PMs. A recent study found that LPS increases ceramides in Raw 264.7 macrophages through nuclear factor kappa B (NF-κB)-dependent upregulation of SPT long chain base subunit 2 Sptlc2, a regulator of SPT.⁶ These results suggest that ceramides mediate the immune response in part by enhancing the production of pro-inflammatory cytokines in innate immune cells.Emerging evidence suggests that dysregulation in the innate immune response in inflammatory bowel disease (IBD) contributes to the pathogenesis of the disease.⁷ Consistent with the role of ceramides in potentiating the innate immune response, several studies found that ceramides may have a role in the pathogenesis of IBD. Sakata et al.³ demonstrated that blocking the generation of ceramides with the SMase inhibitor hinders mouse colitis. Fischbeck et al.⁸ showed that increasing ceramides in the gut by supplying mice with dietary sphingomyelins, a precursor of ceramides, aggravates mouse colitis. These results suggest that increased levels of ceramides may contribute to the pathogenesis of IBD.Although the role of ceramides and their generating enzymes in the innate immune response have been well studied, much remains unclear about the role of ceramidases involved in the catabolism of ceramides in this biological response. In this study, we investigated the role of alkaline ceramidase 3 (ACER3)/Acer3 and its substrates in immune response. We demonstrated that Acer3 is downregulated, whereas its substrate, C_18:1-ceramide, is upregulated in murine immune cells and colonic epithelial cells (CECs) during the innate immune response to LPS. Using Acer3 null mice (Acer3^−/−) and their wild-type (Acer3^+/+) littermates, we further discovered that the inverse regulation of Acer3 and C_18:1-ceramide potentiates LPS-induced production of pro-inflammatory cytokines in innate immune cells. More importantly, we found that Acer3 deficiency aggravates dextran sulfate sodium (DSS)-induced colitis and colitis-associated colorectal cancer (CAC) in a murine model. These findings indicate that Acer3/ACER3 and C_18:1-ceramide are novel modulators in the innate immune response and that their dysregulation may contribute to the pathogenesis of inflammatory diseases.     

Molecular cloning and characterization of neutral ceramidase homologue from the red flour beetle, Tribolium castaneum

Ceramidase plays an important role in regulating the metabolism of sphingolipids, such as ceramide, sphingosine (SPH), and sphingosine-1-phosphate (S1P), by controlling the hydrolysis of ceramide. Here we report the cloning and biochemical characterization of a neutral ceramidase from the red flour beetle Tribolium castaneum which is an important storage pest. The Tribolium castaneum neutral ceramidase (Tncer) is a protein of 696 amino acids. It shares a high degree of similarity in protein sequence to neutral ceramidases from various species. Tncer mRNA levels are higher in the adult stage than in pre-adult stages, and they are higher in the reproductive organs than in head, thorax, and midgut. The mature ovary has higher mRNA levels than the immature ovary. Tncer is localized to the plasma membrane. It uses various ceramides (D-erythro-C₆, C₁₂, C₁₆, C_18:1, and C_24:1-ceramide) as substrates and has an abroad pH optimum for its in vitro activity. Tncer has an optimal temperature of 37 °C for its in vitro activity. Its activity is inhibited by Fe²⁺. These results suggest that Tncer has distinct biochemical properties from neutral ceramidases from other species.     

Acid Ceramidase in Melanoma: EXPRESSION,LOCALIZATION, AND EFFECTS OF PHARMACOLOGICAL INHIBITION*

Acid ceramidase (AC) is a lysosomal cysteine amidase that controls sphingolipid signaling by lowering the levels of ceramides and concomitantly increasing those of sphingosine and its bioactive metabolite, sphingosine 1-phosphate. In the present study, we evaluated the role of AC-regulated sphingolipid signaling in melanoma. We found that AC expression is markedly elevated in normal human melanocytes and proliferative melanoma cell lines, compared with other skin cells (keratinocytes and fibroblasts) and non-melanoma cancer cells. High AC expression was also observed in biopsies from human subjects with Stage II melanoma. Immunofluorescence studies revealed that the subcellular localization of AC differs between melanocytes (where it is found in both cytosol and nucleus) and melanoma cells (where it is primarily localized to cytosol). In addition to having high AC levels, melanoma cells generate lower amounts of ceramides than normal melanocytes do. This down-regulation in ceramide production appears to result from suppression of the de novo biosynthesis pathway. To test whether AC might contribute to melanoma cell proliferation, we blocked AC activity using a new potent (IC₅₀ = 12 nm) and stable inhibitor. AC inhibition increased cellular ceramide levels, decreased sphingosine 1-phosphate levels, and acted synergistically with several, albeit not all, antitumoral agents. The results suggest that AC-controlled sphingolipid metabolism may play an important role in the control of melanoma proliferation.     

Effect of Lipid Particle Biogenesis on the Subcellular Distribution of Squalene in the Yeast Saccharomyces cerevisiae

Squalene belongs to the group of isoprenoids and is a precursor for the synthesis of sterols, steroids, and ubiquinons. In the yeast Saccharomyces cerevisiae, the amount of squalene can be increased by variation of growth conditions or by genetic manipulation. In this report, we show that a hem1Δ mutant accumulated a large amount of squalene, which was stored almost exclusively in cytoplasmic lipid particles/droplets. Interestingly, a strain bearing a hem1Δ deletion in a dga1Δlro1Δare1Δare2Δ quadruple mutant background (QMhem1Δ), which is devoid of the classical storage lipids, triacylglycerols and steryl esters, and lacks lipid particles, accumulated squalene at similar amounts as the hem1Δ mutant in a wild type background. In QMhem1Δ, however, increased amounts of squalene were found in cellular membranes, especially in microsomes. The fact that QMhem1Δ did not form lipid particles indicated that accumulation of squalene solely was not sufficient to initiate proliferation of lipid particles. Most importantly, these results also demonstrated that (i) squalene was not lipotoxic under the conditions tested, and (ii) organelle membranes in yeast can accommodate relatively large quantities of this non-polar lipid without compromising cellular functions. In summary, localization of squalene as described here can be regarded as an unconventional example of non-polar lipid storage in cellular membranes.     

The pmr Gene,Encoding a Ca2+-ATPase,Is Required for Calcium and Manganese Homeostasis and Normal Development of Hyphae and Conidia in Neurospora crassa

The pmr gene is predicted to encode a Ca²⁺-ATPase in the secretory pathway. We examined two strains of Neurospora crassa that lacked PMR: the Δpmr strain, in which pmr was completely deleted, and pmr^RIP, in which the gene was extensively mutated. Both strains had identical, complex phenotypes. Compared to the wild type, these strains required high concentrations of calcium or manganese for optimal growth and had highly branched, slow-growing hyphae. They conidiated poorly, and the shape and size of the conidia were abnormal. Calcium accumulated in the Δpmr strains to only 20% of the wild-type level. High concentrations of MnCl₂ (1 to 5 mM) in growth medium partially suppressed the morphological defects but did not alter the defect in calcium accumulation. The Δpmr Δnca-2 double mutant (nca-2 encodes a Ca²⁺-ATPase in the plasma membrane) accumulated 8-fold more calcium than the wild type, and the morphology of the hyphae was more similar to that of wild-type hyphae. Previous experiments failed to show a function for nca-1, which encodes a SERCA-type Ca²⁺-ATPase in the endoplasmic reticulum (B. J. Bowman, S. Abreu, E. Margolles-Clark, M. Draskovic, and E. J. Bowman, Eukaryot. Cell 10:654-661, 2011). The pmr^RIP Δnca-1 double mutant accumulated small amounts of calcium, like the Δpmr strain, but exhibited even more extreme morphological defects. Thus, PMR can apparently replace NCA-1 in the endoplasmic reticulum, but NCA-1 cannot replace PMR. The morphological defects in the Δpmr strain are likely caused, in part, by insufficient concentrations of calcium and manganese in the Golgi compartment; however, PMR is also needed to accumulate normal levels of calcium in the whole cell.     

Substrate-specificities of acid and alkaline ceramidases in fibroblasts from patients with Farber disease and controls

The specific activity of acid ceramidase (N-acylsphingosine deacylase, EC 3.5.1.23) was measured at pH4.5 in normal fibroblasts and in fibroblasts from patients with Farber disease and obligate heterozygotes. Greater activity was found when the synthetically made ceramide substrates contained shorter-chain fatty acids or higher content of double bonds. Acid ceramidase activities towards N-lauroyl- (C_12:0), N-myristoyl- (C_14:0) and N-palmitoyl- (C_16:0) sphingosine (C_18:1) were respectively about 38, 26 and 6 times higher than the activity towards the N-stearoyl (C_18:0) substrate. The activity towards N-linolenoylsphingosine (C_18:3/C_18:1), N-linoleoylsphingosine (C_18:2/C_18:1) and N-oleoylsphingosine (C_18:1/C_18:1) were respectively about 5, 4 and 3 times higher than the activity towards N-stearoylsphingosine (C_18:0/C_18:1). The activity towards N-stearoyldihydrosphingosine (C_18:0/C_18:0) was about 40% of that towards N-stearoylsphingosine. Fibroblast alkaline ceramidase possessed significant activity only towards ceramides of unsaturated fatty acids, with a pH optimum of about 9.0. Deficiency of acid ceramidase activity in fibroblasts from patients with Farber disease and intermediate activities in obligate heterozygotes were demonstrated with all ceramides examined except for N-hexanoylsphingosine (C_6:0/C_18:1), whereas alkaline ceramidase activity was unaffected. Comparative kinetic studies of acid ceramidase activity with N-lauroylsphingosine and N-oleoylsphingosine demonstrated about 5 (2–12)-fold and 7 (4–17)-fold higher K_m values in fibroblasts from patients with Farber disease as compared with normal controls. N-Lauroylsphingosine, towards which acid ceramidase activity in control fibroblasts was about 10 times higher than that towards N-oleoylsphingosine, may serve as a better substrate for enzymic diagnosis of Farber disease as well as for further characterization of the catalytically defective acid ceramidase.     

Functional and structural characterization of α-(1->2) branching sucrase derived from DSR-E glucansucrase

ΔN₁₂₃-glucan-binding domain-catalytic domain 2 (ΔN₁₂₃-GBD-CD2) is a truncated form of the bifunctional glucansucrase DSR-E from Leuconostoc mesenteroides NRRL B-1299. It was constructed by rational truncation of GBD-CD2, which harbors the second catalytic domain of DSR-E. Like GBD-CD2, this variant displays α-(1→2) branching activity when incubated with sucrose as glucosyl donor and (oligo-)dextran as acceptor, transferring glucosyl residues to the acceptor via a ping-pong bi-bi mechanism. This allows the formation of prebiotic molecules containing controlled amounts of α-(1→2) linkages. The crystal structure of the apo α-(1→2) branching sucrase ΔN₁₂₃-GBD-CD2 was solved at 1.90 Å resolution. The protein adopts the unusual U-shape fold organized in five distinct domains, also found in GTF180-ΔN and GTF-SI glucansucrases of glycoside hydrolase family 70. Residues forming subsite −1, involved in binding the glucosyl residue of sucrose and catalysis, are strictly conserved in both GTF180-ΔN and ΔN₁₂₃-GBD-CD2. Subsite +1 analysis revealed three residues (Ala-2249, Gly-2250, and Phe-2214) that are specific to ΔN₁₂₃-GBD-CD2. Mutation of these residues to the corresponding residues found in GTF180-ΔN showed that Ala-2249 and Gly-2250 are not directly involved in substrate binding and regiospecificity. In contrast, mutant F2214N had lost its ability to branch dextran, although it was still active on sucrose alone. Furthermore, three loops belonging to domains A and B at the upper part of the catalytic gorge are also specific to ΔN₁₂₃-GBD-CD2. These distinguishing features are also proposed to be involved in the correct positioning of dextran acceptor molecules allowing the formation of α-(1→2) branches.     

Degradation of glycosphingolipids in oyster: ceramide glycanase and ceramidase in the hepatopancreas of oyster, <Emphasis Type="Italic">Crassostrea virginica</Emphasis>

The hepatopancreas of oyster, Crassostrea virginica, was found to contain two unique glycosphingolipid (GSL) cleaving enzymes, ceramide glycanase (CGase) and ceramidase. These two enzymes were found to be tightly associated together through the consecutive purification steps including gel filtration, hydrophobic interaction and cation-exchange chromatographies. They were separated only by preparatory SDS-PAGE. The purified CGase was found to have a molecular mass of 52 kDa and pH optimum of 3.2–3.3. This enzyme prefers to hydrolyze the acidic GSLs, II³SO₃LacCer and gangliosides over the neutral GSLs. Oyster ceramidase was found to have a molecular mass of 88 kDa and pH optimum of 4–4.5. Since oyster ceramidase greatly prefers ceramides with C₆ to C₈ fatty acids, C₆-ceramide (N-hexanoyl-D-sphingosine) was used as the substrate for its purification and characterization. The oyster acid ceramidase also catalyzed the synthesis of ceramide from a sphingosine and a fatty acid. For the synthesis, C₁₆ and C₁₈ fatty acids were the best precursors. The amino acid sequences of the two cyanogenbromide peptides derived from the purified ceramidase were found to have similarities to those of several neutral and alkaline ceramidases reported. The tight association of CGase and ceramidase may indicate that CGase in oyster hepatopancreas acts as a vehicle to release ceramide from GSLs for subsequent generation of sphingosines and fatty acids by ceramidase to serve as signaling factors and energy source.     

The ATPase activity of Fml1 is essential for its roles in homologous recombination and DNA repair

In fission yeast, the DNA helicase Fml1, which is an orthologue of human FANCM, is a key component of the machinery that drives and governs homologous recombination (HR). During the repair of DNA double-strand breaks by HR, it limits the occurrence of potentially deleterious crossover recombinants, whereas at stalled replication forks, it promotes HR to aid their recovery. Here, we have mutated conserved residues in Fml1’s Walker A (K99R) and Walker B (D196N) motifs to determine whether its activities are dependent on its ability to hydrolyse ATP. Both Fml1^K99R and Fml1^D196N are proficient for DNA binding but totally deficient in DNA unwinding and ATP hydrolysis. In vivo both mutants exhibit a similar reduction in recombination at blocked replication forks as a fml1Δ mutant indicating that Fml1’s motor activity, fuelled by ATP hydrolysis, is essential for its pro-recombinogenic role. Intriguingly, both fml1^K99R and fml1^D196N mutants exhibit greater sensitivity to genotoxins and higher levels of crossing over during DSB repair than a fml1Δ strain. These data suggest that without its motor activity, the binding of Fml1 to its DNA substrate can impede alternative mechanisms of repair and crossover avoidance.     

Autophosphorylation-independent and -dependent Functions of Focal Adhesion Kinase during Development

Focal adhesion kinase (FAK) regulates numerous cellular functions and is critical for processes ranging from embryo development to cancer progression. Although autophosphorylation on Tyr-397 appears required for FAK functions in vitro, its role in vivo has not been established. We addressed this question using a mutant mouse (fakΔ) deleted of exon 15, which encodes Tyr-397. The resulting mutant protein FAKΔ is an active kinase expressed at normal levels. Our results demonstrate that the requirement for FAK autophosphorylation varies during development. FAK^Δ/Δ embryos developed normally up to embryonic day (E) 12.5, contrasting with the lethality at E8.5 of FAK-null embryos. Thus, autophosphorylation on Tyr-397 is not required for FAK to achieve its functions until late mid-gestation. However, FAK^Δ/Δ embryos displayed hemorrhages, edema, delayed artery formation, vascular remodeling defects, multiple organ abnormalities, and overall developmental retardation at E13.5–14.5, and died thereafter demonstrating that FAK autophosphorylation is also necessary for normal development. Fibroblasts derived from mutant embryos had a normal stellate morphology and expression of focal adhesion proteins, Src family members, p53, and Pyk2. In contrast, in FAK^Δ/Δ fibroblasts and endothelial cells, spreading and lamellipodia formation were altered with an increased size and number of focal adhesions, enriched in FAKΔ. FAK mutation also decreased fibroblast proliferation. These results show that the physiological functions of FAK in vivo are achieved through both autophosphorylation-independent and autophosphorylation-dependent mechanisms.     

Contribution of Residue B5 to the Folding and Function of Insulin and IGF-I: CONSTRAINTS AND FINE-TUNING IN THE EVOLUTION OF A PROTEIN FAMILY*

Proinsulin exhibits a single structure, whereas insulin-like growth factors refold as two disulfide isomers in equilibrium. Native insulin-related growth factor (IGF)-I has canonical cystines (A6—A11, A7–B7, and A20—B19) maintained by IGF-binding proteins; IGF-swap has alternative pairing (A7–A11, A6—B7, and A20—B19) and impaired activity. Studies of mini-domain models suggest that residue B5 (His in insulin and Thr in IGFs) governs the ambiguity or uniqueness of disulfide pairing. Residue B5, a site of mutation in proinsulin causing neonatal diabetes, is thus of broad biophysical interest. Here, we characterize reciprocal B5 substitutions in the two proteins. In insulin, His^B5 → Thr markedly destabilizes the hormone (ΔΔG_u 2.0 ± 0.2 kcal/mol), impairs chain combination, and blocks cellular secretion of proinsulin. The reciprocal IGF-I substitution Thr^B5 → His (residue 4) specifies a unique structure with native ¹H NMR signature. Chemical shifts and nuclear Overhauser effects are similar to those of native IGF-I. Whereas wild-type IGF-I undergoes thiol-catalyzed disulfide exchange to yield IGF-swap, His^B5-IGF-I retains canonical pairing. Chemical denaturation studies indicate that His^B5 does not significantly enhance thermodynamic stability (ΔΔG_u 0.2 ± 0.2 kcal/mol), implying that the substitution favors canonical pairing by destabilizing competing folds. Whereas the activity of Thr^B5-insulin is decreased 5-fold, His^B5-IGF-I exhibits 2-fold increased affinity for the IGF receptor and augmented post-receptor signaling. We propose that conservation of Thr^B5 in IGF-I, rescued from structural ambiguity by IGF-binding proteins, reflects fine-tuning of signal transduction. In contrast, the conservation of His^B5 in insulin highlights its critical role in insulin biosynthesis.     

Alkaline Ceramidase 2 (ACER2) and Its Product Dihydrosphingosine Mediate the Cytotoxicity of N-(4-Hydroxyphenyl)retinamide in Tumor Cells

Increased generation of dihydrosphingosine (DHS), a bioactive sphingolipid, has been implicated in the cytotoxicity of the synthetic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) in tumor cells. However, how 4-HPR increases DHS remains unclear. Here we demonstrate that 4-HPR increases the expression of ACER2, which catalyzes the hydrolysis of dihydroceramides to generate DHS, and that ACER2 up-regulation plays a key role in mediating the 4-HPR-induced generation of DHS as well as the cytotoxicity of 4-HPR in tumor cells. Treatment with 4-HPR induced the accumulation of dihydroceramides (DHCs) in tumor cells by inhibiting dihydroceramide desaturase (DES) activity, which catalyzes the conversion of DHCs to ceramides. Treatment with 4-HPR also increased ACER2 expression through a retinoic acid receptor-independent and caspase-dependent manner. Overexpression of ACER2 augmented the 4-HPR-induced generation of DHS as well as 4-HPR cytotoxicity, and 4-HPR-induced death in tumor cells, whereas knocking down ACER2 had the opposite effects. ACER2 overexpression, along with treatment with GT11, another DES inhibitor, markedly increased cellular DHS, leading to tumor cell death, whereas ACER2 overexpression or GT11 treatment alone failed to do so, suggesting that both ACER2 up-regulation and DES inhibition are necessary and sufficient to mediate 4-HPR-induced DHS accumulation, cytotoxicity, and death in tumor cells. Taken together, these results suggest that up-regulation of the ACER2/DHS pathway mediates the cytotoxicity of 4-HPR in tumor cells and that up-regulating or activating ACER2 may improve the anti-cancer activity of 4-HRR and other DHC-inducing agents.     

Effects of ceramide,ceramidase inhibition and expression of ceramide kinase on cytosolic phospholipase A2α; additional role of ceramide-1-phosphate in phosphorylation and Ca2+ signaling

Ceramide and the metabolites including ceramide-1-phosphate (C1P) and sphingosine are reported to regulate the release of arachidonic acid (AA) and/or phospholipase A₂ (PLA₂) activity in many cell types including lymphocytes. Recent studies established that C1P, a product of ceramide kinase, interacts directly with Ca²⁺ binding regions in the C2 domain of α type cytosolic PLA₂ (cPLA₂α), leading to translocation of the enzyme from the cytosol to the perinuclear region in cells. However, a precise mechanism for C1P-induced activation of cPLA₂α has not been well elucidated; such as the phosphorylation signal caused by the extracellular signal-regulated kinases (ERK1/2) pathway, a downstream of the protein kinase C activation with 4β-phorbol myristate acetate (PMA), is required or not. In the present study, we showed that the increase in intracellular ceramide levels (exogenously added cell permeable ceramides and an inhibition of ceramidase by (1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol and the increase in C1P formation by transfection with the vector for human ceramide kinase significantly enhanced the Ca²⁺ ionophore (A23187) -induced release of AA via cPLA₂α's activation in CHO cells. Ceramides did not show additional effects on the release from the cells treated with the inhibitor of ceramidase. Ceramides and C2-C1P neither had effect on the intracellular mobilization of Ca²⁺ nor the phosphorylation of cPLA₂α in cells. A23187/PMA-induced release of AA was enhanced by ceramides and C2-C1P and by expression of ceramide kinase. Our findings suggest that C1P is a stimulatory factor on cPLA₂α that is independent of the Ca²⁺ signal and the PKC-ERK-mediated phosphorylation signal.