Ana M. Vacaru

Publications (5)18.38 Total impact

• Article: Ceramide phosphoethanolamine biosynthesis in Drosophila is mediated by a unique ethanolamine phosphotransferase in the Golgi lumen.

  Ana M Vacaru, Joep van den Dikkenberg, Philipp Ternes, Joost C M Holthuis
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  ABSTRACT: Sphingomyelin (SM) is a vital component of mammalian membranes, providing mechanical stability and a structural framework for plasma membrane organization. Its production involves the transfer of phosphocholine from phosphatidylcholine onto ceramide, a reaction catalyzed by SM synthase (SMS) in the Golgi lumen. Drosophila lacks SM and instead synthesizes the SM analogue ceramide phosphoethanolamine (CPE) as the principal membrane sphingolipid. The corresponding CPE synthase shares mechanistic features with enzymes mediating phospholipid biosynthesis via the Kennedy pathway. Using a functional cloning strategy, we here identified a CDP-ethanolamine:ceramide ethanolamine phosphotransferase as the enzyme responsible for CPE production in Drosophila. CPE synthase constitutes a new branch within the CDP-alcohol phosphotransferase superfamily with homologues in Arthropoda (insects, spiders, mites, scorpions), Cnidaria (Hydra, sea anemones) and Mollusca (oysters) but not in most other animal phyla. The enzyme resides in the Golgi complex with its active site facing the lumen, contrary to the membrane topology of other CDP-alcohol phosphotransferases. Our findings open up an important new avenue to address the biological role of CPE, an enigmatic membrane constituent of a wide variety of invertebrate and marine organisms.
  Journal of Biological Chemistry 02/2013; · 4.77 Impact Factor
• Article: Alcohol disrupts endoplasmic reticulum function and protein secretion in hepatocytes.

  Deanna L Howarth, Ana M Vacaru, Orkhontuya Tsedensodnom, Elisabetta Mormone, Natalia Nieto, Lindsey M Costantini, Erik L Snapp, Kirsten C Sadler
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  ABSTRACT: Many alcoholic patients have serum protein deficiency that contributes to their systemic problems. The unfolded protein response (UPR) is induced in response to disequilibrium in the protein folding capability of the endoplasmic reticulum (ER) and is implicated in hepatocyte lipid accumulation and apoptosis, which are associated with alcoholic liver disease (ALD). We investigated whether alcohol affects ER structure, function, and UPR activation in hepatocytes in vitro and in vivo. HepG2 cells expressing human cytochrome P450 2E1 and mouse alcohol dehydrogenase (VL-17A) were treated for up to 48 hours with 50 and 100 mM ethanol. Zebrafish larvae at 4 days postfertilization were exposed to 350 mM ethanol for 32 hours. ER morphology was visualized by fluorescence in cells and transmission electron microscopy in zebrafish. UPR target gene activation was assessed using quantitative PCR, in situ hybridization, and Western blotting. Mobility of the major ER chaperone, BIP, was monitored in cells by fluorescence recovery after photobleaching (FRAP). VL-17A cells metabolized alcohol yet only had slight activation of some UPR target genes following ethanol treatment. However, ER fragmentation, crowding, and accumulation of unfolded proteins as detected by immunofluorescence and FRAP demonstrate that alcohol induced some ER dysfunction despite the lack of UPR activation. Zebrafish treated with alcohol, however, showed modest ER dilation, and several UPR targets were significantly induced. Ethanol metabolism directly impairs ER structure and function in hepatocytes. Zebrafish are a novel in vivo system for studying ALD.
  Alcoholism Clinical and Experimental Research 07/2011; 36(1):14-23. · 3.34 Impact Factor
• Source

  Available from: Joost C M Holthuis

  Article: Sphingomyelin synthase-related protein SMSr controls ceramide homeostasis in the ER.

  Ana M Vacaru, Fikadu G Tafesse, Philipp Ternes, Vangelis Kondylis, Martin Hermansson, Jos F H M Brouwers, Pentti Somerharju, Catherine Rabouille, Joost C M Holthuis
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  ABSTRACT: Ceramides are central intermediates of sphingolipid metabolism with critical functions in cell organization and survival. They are synthesized on the cytosolic surface of the endoplasmic reticulum (ER) and transported by ceramide transfer protein to the Golgi for conversion to sphingomyelin (SM) by SM synthase SMS1. In this study, we report the identification of an SMS1-related (SMSr) enzyme, which catalyses the synthesis of the SM analogue ceramide phosphoethanolamine (CPE) in the ER lumen. Strikingly, SMSr produces only trace amounts of CPE, i.e., 300-fold less than SMS1-derived SM. Nevertheless, blocking its catalytic activity causes a substantial rise in ER ceramide levels and a structural collapse of the early secretory pathway. We find that the latter phenotype is not caused by depletion of CPE but rather a consequence of ceramide accumulation in the ER. Our results establish SMSr as a key regulator of ceramide homeostasis that seems to operate as a sensor rather than a converter of ceramides in the ER.
  The Journal of Cell Biology 07/2009; 185(6):1013-27. · 10.26 Impact Factor
• Article: Mechanisms and impact of ceramide phosphoethanolamine biosynthesis

  Ana M. Vacaru
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  ABSTRACT: Sphingolipids represent an essential class of membrane molecules in eukaryotic cells. They are primarily found in the outer leaflet of the plasma membrane where they help create a rigid and impermeable barrier to the extracellular environment. While sphingomyelin (SM) is the most abundant sphingolipid in mammals and nematodes, insects produce the SM analogue ceramide phosphoethanolamine (CPE) as a major plasma membrane constituent. Little is known about the biological role of CPE or about the enzymes responsible for CPE biosynthesis. SM production is mediated by a SM synthase (SMS) in the lumen of the Golgi. We identified an SMS-related enzyme, SMSr, that catalyses CPE production in the endoplasmic reticulum (ER) of both insect and mammalian cells. Curiously, while SMSr synthesizes only trace amounts of CPE, we discovered that the enzyme is a critical mediator of ceramide homeostasis in the ER with a key role in protecting cells against ceramide-induced suicide. We also found that bulk production of CPE in insects is independent of SMSr but requires a different enzyme (CPES) that shares mechanistic features with the enzymes responsible for phosphatidylethanolamine production via the Kennedy pathway. Using a bioinformatics-based cloning strategy, we identified CPES and found that this enzyme is unique for insects, sea anemones and Hydra. We also show that CPES utilizes CDP-ethanolamine as the donor molecule of the CPE head group. Contrary to SM production in mammals and nematodes, CPE production by CPES seems to occur on the cytosolic surface of the Golgi. Besides uncovering two unique enzymes of sphingolipid biosynthesis, our findings challenge prevailing concepts of membrane lipid organization in animal cells and reveal a novel mechanism of sphingolipid homeostasis.
• Article: Sphingomyelin synthase-related protein SMSr controls ceramide homeostasis in the ER

  Ana M. Vacaru, F.G. Tafesse, P.G. Ternes, V. Kondylis, M. Hermansson, J.F.H.M. Brouwers, P. Somerharju, C. Rabouille, J.C.M. Holthuis
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  ABSTRACT: Ceramides are central intermediates of sphingolipid metabolism with critical functions in cell organization and survival. They are synthesized on the cytosolic surface of the endoplasmic reticulum (ER) and transported by ceramide transfer protein to the Golgi for conversion to sphingomyelin (SM) by SM synthase SMS1. In this study, we report the identification of an SMS1-related (SMSr) enzyme, which catalyses the synthesis of the SM analogue ceramide phosphoethanolamine (CPE) in the ER lumen. Strikingly, SMSr produces only trace amounts of CPE, i.e., 300-fold less than SMS1-derived SM. Nevertheless, blocking its catalytic activity causes a substantial rise in ER ceramide levels and a structural collapse of the early secretory pathway. We find that the latter phenotype is not caused by depletion of CPE but rather a consequence of ceramide accumulation in the ER. Our results establish SMSr as a key regulator of ceramide homeostasis that seems to operate as a sensor rather than a converter of ceramides in the ER.