The reverse activity of human acid ceramidase

An overexpression system was recently developed to produce and purify recombinant, human acid ceramidase. In addition to ceramide hydrolysis, the purified enzyme was able to catalyze ceramide synthesis using [14C]lauric acid and sphingosine as substrates. Herein we report detailed characterization of this acid ceramidase-associated "reverse activity" and provide evidence that this reaction occurs in situ as well as in vitro. The pH optimum of the reverse reaction was approximately 5.5, as compared with approximately 4.5 for the hydrolysis reaction. Non-ionic detergents and zinc cations inhibited the activity, whereas most other cations were stimulatory. Of note, sphingomyelin also was very inhibitory toward this reaction, whereas the anionic lipids, phosphatidic acid and phosphatidylserine, were stimulatory. Of various sphingosine stereoisomers tested in the reverse reaction, only the natural, D-erythro form could efficiently serve as a substrate. Using D-erythro-sphingosine and lauric acid as substrates, the reaction followed normal Michaelis-Menten kinetics. The Km and Vmax values toward sphingosine were 23.75 microM and 208.3 pmol/microg/h, respectively, whereas for lauric acid they were 73.76 microM and 232.5 pmol/microg/h, respectively. Importantly, the reverse activity was reduced in cell lysates from a Farber disease patient to the same extent as the acid ceramidase activity. Furthermore, when 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)) (NBD)-conjugated lauric acid and sphingosine were added to cultured lymphoblasts from a Farber disease patient in the presence of fumonisin B (1), the conversion to NBD-ceramide was reduced approximately 30% when compared with normal cells. These data provide important new information on human acid ceramidase and further document its central role in sphingolipid metabolism.     

Laminin alpha 3 LG4 module induces matrix metalloproteinase-1 through mitogen-activated protein kinase signaling

The LG4 module of the laminin alpha 3 chain (alpha 3 LG4), a component of epithelial-specific laminin-5, has cell attachment activity and binds syndecan (Utani, A., Nomizu, M., Matsuura, H., Kato, K., Kobayashi, T., Takeda, U., Aota, S., Nielsen, P. K., and Shinkai, H. (2001) J. Biol. Chem. 276, 28779-28788). Here, we show that recombinant alpha 3 LG4 and a 19-mer synthetic peptide (A3G756) within alpha 3 LG4 active for syndecan binding increased the expression of matrix metalloproteinase-1 (MMP-1) in keratinocytes and fibroblasts. This induction was inhibited by heparin and required de novo synthesis of proteins. In keratinocytes, A3G756 up-regulated interleukin (IL)-1 beta and MMP-1 expression and an IL-1 receptor antagonist thoroughly inhibited A3G756-mediated induction of MMP-1. A3G756 also activated p38 mitogen-activated protein kinase (p38 MAPK) and extracellular signal-related kinase (Erk). Studies with specific inhibitors of MAPKs showed that p38 MAPK activation was necessary for both IL-1 beta and MMP-1 induction, but Erk activation was required only for MMP-1 induction. In fibroblasts, IL-1 receptor antagonist did not block A3G756-mediated induction of MMP-1. These results indicated that induction of MMP-1 by alpha 3 LG4 is mediated through the IL-1 beta autocrine loop in keratinocytes but the mechanism of the induction in fibroblasts is different. Our study suggests that the laminin alpha 3 LG4 module may play an important role in tissue remodeling by inducing MMP-1 expression during wound healing.     

Effects of ET-A receptor blockade on eNOS gene expression in chronic hypoxic rat lungs

We tested the hypothesis that pulmonary endothelial nitric oxide synthase (eNOS) gene expression is primarily regulated by hemodynamic factors and is thus increased in rats with chronic hypoxic pulmonary hypertension. Furthermore, we examined the role of endothelin (ET)-1 in this regulatory process, since ET-1 is able to induce eNOS via activation of the ET-B receptor. Therefore, chronic hypoxic rats (10% O(2)) were treated with the selective ET-A receptor antagonist LU-135252 (50 mg x kg(-1) x day(-1)). Right ventricular systolic pressure and cross-sectional medial vascular wall area of pulmonary arteries rose significantly, and eNOS mRNA levels increased 1.8- and 2.6-fold after 2 and 4 wk of hypoxia, respectively (each P < 0.05). Pulmonary ET-1 mRNA and ET-1 plasma levels increased significantly after 4 wk of hypoxia (each P < 0.05). LU-135252 reduced right ventricular systolic pressure, vascular remodeling, and eNOS gene expression in chronic hypoxic rats (each P < 0.05), whereas ET-1 production was not altered. We conclude that eNOS expression in chronic hypoxic rat lungs is modified predominantly by hemodynamic factors, whereas the ET-B receptor-mediated pathway and hypoxia seem to be less important.     

Sphingolipid metabolism during epidermal barrier development in mice

In rodents, a competent skin barrier to water loss is formed within 2 or 3 days prior to birth. Acquisition of barrier function during rat gestation correlates with the formation of a stratum corneum enriched in ceramides, cholesterol, and fatty acids (Aszterbaum, M., G. K. Menon, K. R. Feingold, and M. L. Williams. 1992. Ontogeny of the epidermal barrier to water loss in the rat: correlation of function with stratum corneum structure and lipid content. PEDIATR: Res. 31: 308-317). We analyzed the formation and epidermal localization of glucosylceramides during embryonic skin barrier development in Balb/c mice. Using immunohistochemistry, epidermal glucosylceramides were hardly detectable 3 days prior to birth. After further 24 h of gestation the level of glucosylceramides was maximal and decreased with increasing gestational age. In parallel, glucosylceramides were targeted to the apical side of the outermost granular keratinocyte layer. A spectrum of five distinct epidermal ceramides was present 2 days prior to birth. With ongoing gestation the composition of the ceramide fraction changed markedly. Most importantly, the level of omega-hydroxylated acylceramides decreased paralleled by the formation of the corneocyte lipid envelope. This structure consists of omega-hydroxylated ceramides and fatty acids bound to surface proteins of the corneocytes. The covalent attachment of ceramides or glucosylceramides correlated with the maturation of the stratum corneum and might contribute to its chemical and enzymatic resistance.     

Identification of Ceramide Binding Proteins in Neuronal Cells: A Critical Point of View

Much discussion has centered on the biochemical mechanism by which ceramide is produced and functions as a signalling molecule in cells. To identify proteins involved in ceramide signalling, we synthesized a radioactively labelled ceramide analogue equipped with a photosensitive group: N-(p-trifluoromethyl-diazirinyl)phenyl-ethyl-2-[³⁵S]-2-thioacetyl-d-erythro-C₁₈-sphingosine ([³⁵S]-TDS-ceramide). This compound was then employed in photo-affinity labelling experiments in primary cultured cerebellar neurons. Due to the hydrophobic nature of the compound, most of the cell-associated radioactivity was recovered in the lipid fraction while only about 0.1% of radioactivity was photocoupled to proteins. In order to improve protein labelling the cytosolic fraction of rapidly growing human neuroblastoma cells (SH-SY5Y) was isolated and subjected to ceramide affinity chromatography prior to photo-affinity labelling. Following electrophoresis proteins photocoupled to ceramide were identified by MALDI mass spectrometry in combination with tryptic digestion and turned out to be either cytoskeletal or stress proteins that are highly abundant in cytosol and contain at least one hydrophobic domain.     

L-Malyl-coenzyme A lyase/beta-methylmalyl-coenzyme A lyase from Chloroflexus aurantiacus,a bifunctional enzyme involved in autotrophic CO(2) fixation

The 3-hydroxypropionate cycle is a bicyclic autotrophic CO(2) fixation pathway in the phototrophic Chloroflexus aurantiacus (Bacteria), and a similar pathway is operating in autotrophic members of the Sulfolobaceae (Archaea). The proposed pathway involves in a first cycle the conversion of acetyl-coenzyme A (acetyl-CoA) and two bicarbonates to L-malyl-CoA via 3-hydroxypropionate and propionyl-CoA; L-malyl-CoA is cleaved by L-malyl-CoA lyase into acetyl-CoA and glyoxylate. In a second cycle, glyoxylate and another molecule of propionyl-CoA (derived from acetyl-CoA and bicarbonate) are condensed by a putative beta-methylmalyl-CoA lyase to beta-methylmalyl-CoA, which is converted to acetyl-CoA and pyruvate. The putative L-malyl-CoA lyase gene of C. aurantiacus was cloned and expressed in Escherichia coli, and the recombinant enzyme was purified and studied. Beta-methylmalyl-CoA lyase was purified from cell extracts of C. aurantiacus and characterized. We show that these two enzymes are identical and that both enzymatic reactions are catalyzed by one single bifunctional enzyme, L-malyl-CoA lyase/beta-methylmalyl-CoA lyase. Interestingly, this enzyme works with two different substrates in two different directions: in the first cycle of CO(2) fixation, it cleaves L-malyl-CoA into acetyl-CoA and glyoxylate (lyase reaction), and in the second cycle it condenses glyoxylate with propionyl-CoA to beta-methylmalyl-CoA (condensation reaction). The combination of forward and reverse directions of a reversible enzymatic reaction, using two different substrates, is rather uncommon and reduces the number of enzymes required in the pathway. In summary, L-malyl-CoA lyase/beta-methylmalyl-CoA lyase catalyzes the interconversion of L-malyl-CoA plus propionyl-CoA to beta-methylmalyl-CoA plus acetyl-CoA.     

Formation of complexes between Ca2+.calmodulin and the synapse-associated protein SAP97 requires the SH3 domain-guanylate kinase domain-connecting HOOK region

Mammalian synapse-associated protein SAP97, a structural and functional homolog of Drosophila Dlg, is a membrane-associated guanylate kinase (MAGUK) that is present at pre- and postsynaptic sites as well as in epithelial cell-cell contact sites. It is a multidomain scaffolding protein that shares with other members of the MAGUK protein family a characteristic modular organization composed of three sequential protein interaction motifs known as PDZ domains, followed by an Src homology 3 (SH3) domain, and an enzymatically inactive guanylate kinase (GK)-like domain. Specific binding partners are known for each domain, and different modes of intramolecular interactions have been proposed that particularly involve the SH3 and GK domains and the so-called HOOK region located between these two domains. We identified the HOOK region as a specific site for calmodulin binding and studied the dynamics of complex formation of recombinant calmodulin and SAP97 by surface plasmon resonance spectroscopy. Binding of various SAP97 deletion constructs to immobilized calmodulin was strictly calcium-dependent. From the rate constants of association and dissociation we determined an equilibrium dissociation constant K(d) of 122 nm for the association of calcium-saturated calmodulin and a SAP97 fragment, which encompassed the entire SH3-HOOK-GK module. Comparative structure-based sequence analysis of calmodulin binding regions from various target proteins predicts variable affinities for the interaction of calmodulin with members of the MAGUK protein family. Our findings suggest that calmodulin could regulate the intramolecular interaction between the SH3, HOOK, and GK domains of SAP97.     

Evidence for a copper-binding superfamily of the amyloid precursor protein

The amyloid precursor protein (APP) copper-binding domain (CuBD) has been shown to reduce Cu(II) to Cu(I) and to mediate copper-induced oxidation in vitro. However, little is known about copper binding to the homologous domains of APP and APP family paralogs and orthologs (including amyloid precursor-like proteins from Drosophila melanogaster, Xenopus laevis, and Caenorhabditis elegans) and their effects on Cu-induced oxidation and Cu(I) formation. Here, we show that APP homologues with and without conserved histidine residues at positions 147, 149, and 151 all bind Cu(II). Oxidized peptides were the kinetically favored products of the redox reaction of CuBDs promoting the reduction of Cu(II) to Cu(I). These results reveal a molecular phylogeny-based divergence that has taken place between the ancestral Drosophila APPL and C. elegans APL-1 and the recently evolved APP lineage of CuBDs. Whereas higher species CuBDs have a decreased affinity for Cu(II) and high Cu(II) reducing activities, ancestral CuBDs form very tight binding sites for Cu(II) ions and have low Cu(II) reducing activities. Thus, the APP lineage displays a gain in activity toward promoting Cu(II) reduction and Cu(I) release. The findings suggests that the Cu(II)-binding equilibrium at the phylogenetic stage of Drosophila APPL and C. elegans APL-1 is shifted from the exchangeable Cu(II) pool to the tightly bound, nonexchangeable pool and that ancestral CuBDs may exert antioxidation activities in vivo. The more recently evolved homologues of human APP appear to take advantage of unique redox properties for yet unknown biological functions.