RESUMO
Alkaline ceramidases (ACERs) are a class of poorly understood transmembrane enzymes controlling the homeostasis of ceramides. They are implicated in human pathophysiology, including progressive leukodystrophy, colon cancer as well as acute myeloid leukemia. We report here the crystal structure of the human ACER type 3 (ACER3). Together with computational studies, the structure reveals that ACER3 is an intramembrane enzyme with a seven transmembrane domain architecture and a catalytic Zn2+ binding site in its core, similar to adiponectin receptors. Interestingly, we uncover a Ca2+ binding site physically and functionally connected to the Zn2+ providing a structural explanation for the known regulatory role of Ca2+ on ACER3 enzymatic activity and for the loss of function in E33G-ACER3 mutant found in leukodystrophic patients.
Assuntos
Ceramidase Alcalina/metabolismo , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Ceramidase Alcalina/química , Ceramidase Alcalina/genética , Animais , Sítios de Ligação/genética , Cálcio/metabolismo , Cristalografia por Raios X , Células HEK293 , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutação Puntual , Conformação Proteica , Receptores de Adiponectina/química , Células Sf9 , SpodopteraRESUMO
Ypc1p (yeast phyto-ceramidase 1) and Ydc1p (yeast dihydroceramidase 1) are alkaline ceramide hydrolases that reside in the ER (endoplasmic reticulum). Ypc1p can catalyse the reverse reaction, i.e. the condensation of non-esterified fatty acids with phytosphingosine or dihydrosphingosine and overexpression of YPC1 or YDC1 can provide enough ceramide synthesis to rescue the viability of cells lacking the normal acyl-CoA-dependent ceramide synthases. To better understand the coexistence of acyl-CoA-dependent ceramide synthases and ceramidases in the ER we investigated the membrane topology of Ypc1p by probing the cysteine residue accessibility of natural and substituted cysteines with membrane non-permeating mass-tagged probes. The N- and C-terminal ends of Ypc1p are oriented towards the lumen and cytosol respectively. Two of the five natural cysteines, Cys27 and Cys219, are essential for enzymatic activity and form a disulfide bridge. The data allow the inference that all of the amino acids of Ypc1p that are conserved in the Pfam PF05875 ceramidase motif and the CREST {alkaline ceramidase, PAQR [progestin and adipoQ (adiponectin) receptor] receptor, Per1 (protein processing in the ER 1), SID-1 (sister disjunction 1) and TMEM8 (transmembrane protein 8)} superfamily are located in or near the ER lumen. Microsomal assays using a lysine residue-specific reagent show that the reverse ceramidase activity can only be blocked when the reagent has access to Ypc1p from the lumenal side. Overall the data suggest that the active site of Ypc1p resides at the lumenal side of the ER membrane.
Assuntos
Ceramidase Alcalina/química , Membranas Intracelulares/enzimologia , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Ceramidase Alcalina/genética , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Sequência Conservada/genética , Cisteína/química , Cisteína/genética , Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/genética , Membranas Intracelulares/química , Dados de Sequência Molecular , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Human alkaline ceramidase 2 (ACER2) plays an important role in cellular responses by regulating the hydrolysis of ceramides in cells. Here we report its biochemical characterization, membrane topology, and activity regulation. Recombinant ACER2 was expressed in yeast mutant cells (Deltaypc1Deltaydc1) that lack endogenous ceramidase activity, and microsomes from ACER2-expressiong yeast cells were used to biochemically characterize ACER2. ACER2 catalyzed the hydrolysis of various ceramides and followed Michaelis-Menten kinetics. ACER2 required Ca(2+) for both its in vitro and cellular activities. ACER2 has 7 putative transmembrane domains, and its amino (N) and carboxyl (C) termini were found to be oriented in the lumen of the Golgi complex and cytosol, respectively. ACER2 mutant (ACER2DeltaN36) lacking the N-terminal tail (the first 36 amino acid residues) exhibited undetectable activity and was mislocalized to the endoplasmic reticulum, suggesting that the N-terminal tail is necessary for both ACER2 activity and Golgi localization. ACER2 mutant (ACER2DeltaN13) lacking the first 13 residues was also mislocalized to the endoplasmic reticulum although it retained ceramidase activity. Overexpression of ACER2, ACER2DeltaN13, but not ACER2DeltaN36 increased the release of sphingosine 1-phosphate from cells, suggesting that its mislocalization does not affect the ability of ACER2 to regulate sphingosine 1-phosphate secretion. However, overexpression of ACER2 but not ACER2DeltaN13 or ACER2DeltaN36 inhibited the glycosylation of integrin beta1 subunit and Lamp1, suggesting that its mistargeting abolishes the ability of ACER2 to regulation protein glycosylation. These data suggest that ACER2 has broad substrate specificity and requires Ca(2+) for its activity and that ACER2 has the cytosolic C terminus and luminal N terminus, which are essential for its activity, correct cellular localization, and regulation for protein glycosylation.
