Background: In contrast to their yeast orthologues, the mechanism by which mammalian ORMDLs regul... more Background: In contrast to their yeast orthologues, the mechanism by which mammalian ORMDLs regulate serine palmitoyltransferase is not understood. Results: Overexpression of serine palmitoyltransferase in HEK293 cells results in increased long-chain base synthesis and an activity-dependent increase in ORMDL expression. Conclusion: A product of ceramide synthase mediates regulation of ORMDL expression and inhibition of serine palmitoyltransferase. Significance: Serine palmitoyltransferase activity indirectly regulates ORMDL expression.
The YBR159w gene encodes the major 3-ketoreductase activity of the elongase system of enzymes req... more The YBR159w gene encodes the major 3-ketoreductase activity of the elongase system of enzymes required for very long-chain fatty acid (VLCFA) synthesis. Mutants lacking the YBR159w gene display many of the phenotypes that have previously been described for mutants with defects in fatty acid elongation. These phenotypes include reduced VLCFA synthesis, accumulation of high levels of dihydrosphingosine and phytosphingosine, and accumulation of medium-chain ceramides. In vitro elongation assays confirm that the ybr159Δ mutant is deficient in the reduction of the 3-ketoacyl intermediates of fatty acid elongation. The ybr159Δ mutant also displays reduced dehydration of the 3-OH acyl intermediates of fatty acid elongation, suggesting that Ybr159p is required for the stability or function of the dehydratase activity of the elongase system. Green fluorescent protein- tagged Ybr159p co-localizes and co-immunoprecipitates with other elongating enzymes, Elo3p and Tsc13p. Whereas VLCFA synthesi...
The Saccharomyces cerevisiae SCS7 and SUR2 genes are members of a gene family that encodes enzyme... more The Saccharomyces cerevisiae SCS7 and SUR2 genes are members of a gene family that encodes enzymes that desaturate or hydroxylate lipids. Sur2p is required for the hydroxylation of C-4 of the sphingoid moiety of ceramide, and Scs7p is required for the hydroxylation of the very long chain fatty acid. Neither SCS7 nor SUR2 are essential for growth, and lack of the Scs7p- or Sur2p- dependent hydroxylation does not prevent the synthesis of mannosyldiinositolphosphorylceramide, the mature sphingolipid found in yeast. Deletion of either gene suppresses the Ca2+-sensitive phenotype of csg2Δ mutants, which arises from overaccumulation of inositolphosphorylceramide due to a defect in sphingolipid mannosylation. Characterization of scs7 and sur2 mutants is expected to provide insight into the function of ceramide hydroxylation
Maintenance of sphingolipid homeostasis is critical for cell growth and programmed cell death (PC... more Maintenance of sphingolipid homeostasis is critical for cell growth and programmed cell death (PCD). Serine palmitoyltransferase (SPT), composed of LCB1 and LCB2 subunits, catalyzes the primary regulatory point for sphingolipid synthesis. Small subunits of SPT (ssSPT) that strongly stimulate SPT activity have been identified in mammals, but the role of ssSPT in eukaryotic cells is unclear. Candidate Arabidopsis thaliana ssSPTs, ssSPTa and ssSPTb, were identified and characterized. Expression of these 56–amino acid polypeptides in a Saccharomyces cerevisiae SPT null mutant stimulated SPT activity from the Arabidopsis LCB1/LCB2 heterodimer by \u3e100-fold through physical interaction with LCB1/LCB2. ssSPTa transcripts were more enriched in all organs and \u3e400-fold more abundant in pollen than ssSPTb transcripts. Accordingly, homozygous ssSPTa T-DNA mutants were not recoverable, and 50% nonviable pollen was detected in heterozygous ssspta mutants. Pollen viability was recovered by e...
Saccharomyces cerevisiae csg2Δ mutants accumulate the sphingolipid inositolphosphorylceramide, wh... more Saccharomyces cerevisiae csg2Δ mutants accumulate the sphingolipid inositolphosphorylceramide, which renders the cells Ca2+-sensitive. Temperature-sensitive mutations that suppress the Ca2+ sensitivity of csg2Δ mutants were isolated and characterized to identify genes that encode sphingolipid synthesis enzymes. These temperature-sensitive csg2Δ suppressors (tsc) fall into 15 complementation groups. The TSC10/YBR265w gene was found to encode 3-ketosphinganine reductase, the enzyme that catalyzes the second step in the synthesis of phytosphingosine, the long chain base found in yeast sphingolipids. 3-Ketosphinganine reductase (Tsc10p) is essential for growth in the absence of exogenous dihydrosphingosine or phytosphingosine. Tsc10p is a member of the short chain dehydrogenase/reductase protein family. The tsc10 mutants accumulate 3-ketosphinganine and microsomal membranes isolated from tsc10 mutants have low 3-ketosphinganine reductase activity. His6-tagged Tsc10p was expressed in Esc...
A number of Saccharomyces cerevisiae membranebound oxidoreductases were examined for potential ro... more A number of Saccharomyces cerevisiae membranebound oxidoreductases were examined for potential roles in microsomal fatty acid elongation, by assaying heterologous elongating activities in individual deletion mutants. One yeast gene, YBR159w, was identified as being required for activity of both the Caenorhabditis elegans elongase PEA1 (F56H11.4) and the Arabidopsis thaliana elongase FAE1. Ybr159p shows some limited homology to human steroid dehydrogenases and is a member of the short-chain alcohol dehydrogenase superfamily. Disruption of YBR159w is not lethal, in contrast to previous reports, although the mutants are slow growing and display high temperature sensitivity. Both Ybr159p and an Arabidopsis homologue were shown to restore heterologous elongase activities when expressed in ybr159Δ mutants. Biochemical characterization of microsomal preparations from ybr159Δ cells revealed a primary perturbation in β-ketoacyl reduction, confirming the assignment of YBR159w as encoding a co...
fantastic team, have been of tremendous assistance to me. They are always there to help me with a... more fantastic team, have been of tremendous assistance to me. They are always there to help me with all the paper work. My sincere thanks to Ms. Janet Anastasi, former graduate office secretary, for her personal and professional support during the initial period of my graduate studies. I would like to acknowledge the Biomedical Instrumental Center for constant and timely service with DNA sequencing and oligonucleotide synthesis. My sincere thanks to all the members of the biochemistry department for creating a cheerful and stimulating environment. My thanks to Ms. Karen Williams for taking the hassle out of ordering supplies. I am grateful to all my friends and colleagues who have made everything look positive and enjoyable. I express my deepest gratitude to Dr. Gouri Chattopadhyay, who was always there for me, and helped me both scientifically and personally and provided me a home away from home. Last but not the least, I would like to thank the Uniformed Services University of the Health Sciences for providing me with the opportunity to enroll and complete my dissertation. vi DEDICATION To my loving parents, and my family. I will always appreciate all the support and encouragement I got from my sister Gouri, brother-in-law, Manas. I would like to thank a very special man Sambuddha who always believed in me. COPYRIGHT STATEMENT The author hereby certifies that the use of any copyrighted material in the dissertation manuscript entitled: "Small Subunits of Serine Palmitoyltransferase (ssSPTs) and their physiological roles" is appropriately acknowledged and, beyond brief excerpts.
Proceedings of the National Academy of Sciences of the United States of America, Jan 24, 2016
Sphingolipids exhibit extreme functional and chemical diversity that is in part determined by the... more Sphingolipids exhibit extreme functional and chemical diversity that is in part determined by their hydrophobic moiety, ceramide. In mammals, the fatty acyl chain length variation of ceramides is determined by six (dihydro)ceramide synthase (CerS) isoforms. Previously, we and others showed that mutations in the major neuron-specific CerS1, which synthesizes 18-carbon fatty acyl (C18) ceramide, cause elevation of long-chain base (LCB) substrates and decrease in C18 ceramide and derivatives in the brain, leading to neurodegeneration in mice and myoclonus epilepsy with dementia in humans. Whether LCB elevation or C18 ceramide reduction leads to neurodegeneration is unclear. Here, we ectopically expressed CerS2, a nonneuronal CerS producing C22-C24 ceramides, in neurons of Cers1-deficient mice. Surprisingly, the Cers1 mutant pathology was almost completely suppressed. Because CerS2 cannot replenish C18 ceramide, the rescue is likely a result of LCB reduction. Consistent with this hypoth...
Sphingolipid synthesis is tightly regulated in eukaryotes. This regulation in plants ensures suff... more Sphingolipid synthesis is tightly regulated in eukaryotes. This regulation in plants ensures sufficient sphingolipids to support growth, while limiting accumulation of sphingolipid metabolites that induce programmed cell death (PCD). Serine palmitoyltransfersase (SPT) catalyzes the first step in sphingolipid biosynthesis and is considered the primary sphingolipid homeostatic regulatory point. In this report, Arabidopsis putative SPT regulatory proteins, orosomucoid-like proteins AtORM1 and AtORM2 were found to physically interact with the Arabidopsis SPT and to suppress SPT activity when co-expressed with Arabidopsis SPT subunits LCB1 and LCB2 and the small subunit of SPT in a yeast SPT-deficient mutant. Consistent with a role in SPT suppression, AtORM1 and AtORM2 overexpression lines displayed increased resistance to the PCD-inducing mycotoxin fumonisin B1 (FB1), with an accompanying reduced accumulation of long-chain bases (LCBs) and C16-fatty acid-containing ceramides relative to...
Sphingolipid (SL) biosynthesis is negatively regulated by the highly conserved endoplasmic reticu... more Sphingolipid (SL) biosynthesis is negatively regulated by the highly conserved endoplasmic reticulum-localized Orm family proteins. Defective SL synthesis in Saccharomyces cerevisiae leads to increased phosphorylation and inhibition of Orm proteins by the kinase Ypk1. Here we present evidence that the yeast morphogenesis checkpoint kinase, Swe1, regulates SL biosynthesis independent of the Ypk1 pathway. Deletion of the Swe1 kinase renders mutant cells sensitive to serine palmitoyltransferase inhibition due to impaired sphingoid long-chain base synthesis. Based on these data and previous results, we suggest that Swe1 kinase perceives alterations in SL homeostasis, activates SL synthesis, and may thus represent the missing regulatory link that controls the SL rheostat during the cell cycle. * This work was supported by Austrian Science Funds (FWF) Projects SFB F 3005 LIPOTOX and Ph.D. program W903 Molecular Enzymology and NAWI Graz (to S. D. K.) and by National Institutes of Health Grant R21HD080181 (to T. D.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors declare that they have no conflicts of interest with the contents of this article.
Fundamental changes in the composition and distribution of lipids within the brain are believed t... more Fundamental changes in the composition and distribution of lipids within the brain are believed to contribute to the cognitive decline associated with Alzheimer’s disease (AD). The mechanisms by which these changes in lipid composition affect cellular function and ultimately cognition are not well understood. Although “candidate gene” approaches can provide insight into the effects of dysregulated lipid metabolism they require a preexisting understanding of the molecular targets of individual lipid species. In this report we combine unbiased gene expression profiling with a genome-wide chemogenomic screen to identify the mitochondria as an important downstream target of PC(O-16:0/2:0), a neurotoxic lipid species elevated in AD. Further examination revealed that PC(O-16:0/2:0) similarly promotes a global increase in ceramide accumulation in human neurons which was associated with mitochondrial-derived reactive oxygen species (ROS) and toxicity. These findings suggest that PC(O-16:0/2...
Proceedings of the National Academy of Sciences of the United States of America, Jan 20, 2015
Sphingolipids typically have an 18-carbon (C18) sphingoid long chain base (LCB) backbone. Althoug... more Sphingolipids typically have an 18-carbon (C18) sphingoid long chain base (LCB) backbone. Although sphingolipids with LCBs of other chain lengths have been identified, the functional significance of these low-abundance sphingolipids is unknown. The LCB chain length is determined by serine palmitoyltransferase (SPT) isoenzymes, which are trimeric proteins composed of two large subunits (SPTLC1 and SPTLC2 or SPTLC3) and a small subunit (SPTssa or SPTssb). Here we report the identification of an Sptssb mutation, Stellar (Stl), which increased the SPT affinity toward the C18 fatty acyl-CoA substrate by twofold and significantly elevated 20-carbon (C20) LCB production in the mutant mouse brain and eye, resulting in surprising neurodegenerative effects including aberrant membrane structures, accumulation of ubiquitinated proteins on membranes, and axon degeneration. Our work demonstrates that SPT small subunits play a major role in controlling SPT activity and substrate affinity, and in s...
To highlight the importance of sphingolipids and their metabolites in plant biology. The completi... more To highlight the importance of sphingolipids and their metabolites in plant biology. The completion of the arabidopsis genome provides a platform for the identification and functional characterization of genes involved in sphingolipid biosynthesis. Using the yeast Saccharomyces cerevisiae as an experimental model, this review annotates arabidopsis open reading frames likely to be involved in sphingolipid metabolism. A number of these open reading frames have already been subject to functional characterization, though the majority still awaits investigation. Plant-specific aspects of sphingolipid biology (such as enhanced long chain base heterogeneity) are considered in the context of the emerging roles for these lipids in plant form and function. Arabidopsis provides an excellent genetic and post-genomic model for the characterization of the roles of sphingolipids in higher plants.
Sphingolipid synthesis is initiated by condensation of Ser with palmitoyl-CoA producing 3-ketodih... more Sphingolipid synthesis is initiated by condensation of Ser with palmitoyl-CoA producing 3-ketodihydrosphinganine (3-KDS), which is reduced by a 3-KDS reductase to dihydrosphinganine. Ser palmitoyltransferase is essential for plant viability. Arabidopsis thaliana contains two genes (At3g06060/TSC10A and At5g19200/TSC10B) encoding proteins with significant similarity to the yeast 3-KDS reductase, Tsc10p. Heterologous expression in yeast of either Arabidopsis gene restored 3-KDS reductase activity to the yeast tsc10Δ mutant, confirming both as bona fide 3-KDS reductase genes. Consistent with sphingolipids having essential functions in plants, double mutant progeny lacking both genes were not recovered from crosses of single tsc10A and tsc10B mutants. Although the 3-KDS reductase genes are functionally redundant and ubiquitously expressed in Arabidopsis, 3-KDS reductase activity was reduced to 10% of wild-type levels in the loss-of-function tsc10a mutant, leading to an altered sphingoli...
Maintenance of sphingolipid homeostasis is critical for cell growth and programmed cell death (PC... more Maintenance of sphingolipid homeostasis is critical for cell growth and programmed cell death (PCD). Serine palmitoyltransferase (SPT), composed of LCB1 and LCB2 subunits, catalyzes the primary regulatory point for sphingolipid synthesis. Small subunits of SPT (ssSPT) that strongly stimulate SPT activity have been identified in mammals, but the role of ssSPT in eukaryotic cells is unclear. Candidate Arabidopsis thaliana ssSPTs, ssSPTa and ssSPTb, were identified and characterized. Expression of these 56–amino acid polypeptides in a Saccharomyces cerevisiae SPT null mutant stimulated SPT activity from the Arabidopsis LCB1/LCB2 heterodimer by >100-fold through physical interaction with LCB1/LCB2. ssSPTa transcripts were more enriched in all organs and >400-fold more abundant in pollen than ssSPTb transcripts. Accordingly, homozygous ssSPTa T-DNA mutants were not recoverable, and 50% nonviable pollen was detected in heterozygous ssspta mutants. Pollen viability was recovered by...
Unbiased lipidomic approaches have identified impairments in glycerophosphocholine second messeng... more Unbiased lipidomic approaches have identified impairments in glycerophosphocholine second messenger metabolism in patients with Alzheimer's disease. Specifically, we have shown that amyloid-b42 signals the intraneuronal accumulation of PC(O-16:0/2:0) which is associated with neurotoxicity. Similar to neuronal cells, intracellular accumulation of PC(O-16:0/2:0) is also toxic to Saccharomyces cerevisiae, making yeast an excellent model to decipher the pathological effects of this lipid. We previously reported that phospholipase D, a phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P 2)-binding protein, was relocalized in response to PC(O-16:0/2:0), suggesting that this neurotoxic lipid may remodel lipid signaling networks. Here we show that PC(O-16:0/2:0) regulates the distribution of the PtdIns(4)P 5-kinase Mss4 and its product PtdIns(4,5)P 2 leading to the formation of invaginations at the plasma membrane (PM). We further demonstrate that the effects of PC(O-16:0/2:0) on the distribution of PM PtdIns(4,5)P 2 pools are in part mediated by changes in the biosynthesis of long chain bases (LCBs) and ceramides. A combination of genetic, biochemical and cell imaging approaches revealed that PC(O-16:0/2:0) is also a potent inhibitor of signaling through the Target of rampamycin complex 2 (TORC2). Together, these data provide mechanistic insight into how specific disruptions in phosphocholine second messenger metabolism associated with Alzheimer's disease may trigger larger network-wide disruptions in ceramide and phosphoinositide second messenger biosynthesis and signaling which have been previously implicated in disease progression.
The autosomal dominant peripheral sensory neuropathy HSAN1 results from mutations in the LCB1 sub... more The autosomal dominant peripheral sensory neuropathy HSAN1 results from mutations in the LCB1 subunit of serine palmitoyltransferase (SPT). Serum from patients and transgenic mice expressing a disease-causing mutation (C133W) contain elevated levels of 1-deoxysphinganine (1-deoxySa), which presumably arise from inappropriate condensation of alanine with palmitoyl-CoA. Mutant heterodimeric SPT is catalytically inactive. However, mutant heterotrimeric SPT has ϳ10-20% of wild-type activity and supports growth of yeast cells lacking endogenous SPT. In addition, long chain base profiling revealed the synthesis of significantly more 1-deoxySa in yeast and mammalian cells expressing the heterotrimeric mutant enzyme than in cells expressing wild-type enzyme. Wild-type and mutant enzymes had similar affinities for serine. Surprisingly, the enzymes also had similar affinities for alanine, indicating that the major affect of the C133W mutation is to enhance activation of alanine for condensation with the acyl-CoA substrate. In vivo synthesis of 1-deoxySa by the mutant enzyme was proportional to the ratio of alanine to serine in the growth media, suggesting that this ratio can be used to modulate the relative synthesis of sphinganine and 1-deoxySa. By expressing SPT as a single-chain fusion protein to ensure stoichiometric expression of all three subunits, we showed that GADD153, a marker for endoplasmic reticulum stress, was significantly elevated in cells expressing mutant heterotrimers. GADD153 was also elevated in cells treated with 1-deoxySa. Taken together, these data indicate that the HSAN1 mutations perturb the active site of SPT resulting in a gain of function that is responsible for the HSAN1 phenotype. Serine palmitoyltransferase (SPT) 3 catalyzes the committed and rate-limiting step in sphingolipid biosynthesis. Until recently, it was believed that there was a single isozyme of this enzyme containing two subunits, LCB1 and LCB2, with the catalytic domain located at the interface and composed of residues from each subunit. However, Hornemann et al. showed that there was a second isoform of LCB2 (1). More recently, we
The YBR159w gene encodes the major 3-ketoreductase activity of the elongase system of enzymes req... more The YBR159w gene encodes the major 3-ketoreductase activity of the elongase system of enzymes required for very long-chain fatty acid (VLCFA) synthesis. Mutants lacking the YBR159w gene display many of the phenotypes that have previously been described for mutants with defects in fatty acid elongation. These phenotypes include reduced VLCFA synthesis, accumulation of high levels of dihydrosphingosine and phytosphingosine, and accumulation of medium-chain ceramides. In vitro elongation assays confirm that the ybr159⌬ mutant is deficient in the reduction of the 3-ketoacyl intermediates of fatty acid elongation. The ybr159⌬ mutant also displays reduced dehydration of the 3-OH acyl intermediates of fatty acid elongation, suggesting that Ybr159p is required for the stability or function of the dehydratase activity of the elongase system. Green fluorescent protein-tagged Ybr159p co-localizes and co-immunoprecipitates with other elongating enzymes, Elo3p and Tsc13p. Whereas VLCFA synthesis is essential for viability, the ybr159⌬ mutant cells are viable (albeit very slowly growing) and do synthesize some VLCFA. This suggested that a functional ortholog of Ybr159p exists that is responsible for the residual 3-ketoreductase activity. By disrupting the orthologs of Ybr159w in the ybr159⌬ mutant we found that the ybr159⌬ayr1⌬ double mutant was inviable, suggesting that Ayr1p is responsible for the residual 3-ketoreductase activity.
1 The abbreviations used are: SPT, serine palmitoyltransferase; LCB, long chain base; AONS, 8-ami... more 1 The abbreviations used are: SPT, serine palmitoyltransferase; LCB, long chain base; AONS, 8-amino-7-oxononanoate synthase; ts, temperature sensitive; bp, base pair(s); PHS, phytosphingosine; ORF, open reading frame; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; HA, hemagglutinin; PCR, polymerase chain reaction; SCS, suppressor of the Ca 2ϩ-sensitive phenotype of the csg2⌬ mutant.
Background: In contrast to their yeast orthologues, the mechanism by which mammalian ORMDLs regul... more Background: In contrast to their yeast orthologues, the mechanism by which mammalian ORMDLs regulate serine palmitoyltransferase is not understood. Results: Overexpression of serine palmitoyltransferase in HEK293 cells results in increased long-chain base synthesis and an activity-dependent increase in ORMDL expression. Conclusion: A product of ceramide synthase mediates regulation of ORMDL expression and inhibition of serine palmitoyltransferase. Significance: Serine palmitoyltransferase activity indirectly regulates ORMDL expression.
The YBR159w gene encodes the major 3-ketoreductase activity of the elongase system of enzymes req... more The YBR159w gene encodes the major 3-ketoreductase activity of the elongase system of enzymes required for very long-chain fatty acid (VLCFA) synthesis. Mutants lacking the YBR159w gene display many of the phenotypes that have previously been described for mutants with defects in fatty acid elongation. These phenotypes include reduced VLCFA synthesis, accumulation of high levels of dihydrosphingosine and phytosphingosine, and accumulation of medium-chain ceramides. In vitro elongation assays confirm that the ybr159Δ mutant is deficient in the reduction of the 3-ketoacyl intermediates of fatty acid elongation. The ybr159Δ mutant also displays reduced dehydration of the 3-OH acyl intermediates of fatty acid elongation, suggesting that Ybr159p is required for the stability or function of the dehydratase activity of the elongase system. Green fluorescent protein- tagged Ybr159p co-localizes and co-immunoprecipitates with other elongating enzymes, Elo3p and Tsc13p. Whereas VLCFA synthesi...
The Saccharomyces cerevisiae SCS7 and SUR2 genes are members of a gene family that encodes enzyme... more The Saccharomyces cerevisiae SCS7 and SUR2 genes are members of a gene family that encodes enzymes that desaturate or hydroxylate lipids. Sur2p is required for the hydroxylation of C-4 of the sphingoid moiety of ceramide, and Scs7p is required for the hydroxylation of the very long chain fatty acid. Neither SCS7 nor SUR2 are essential for growth, and lack of the Scs7p- or Sur2p- dependent hydroxylation does not prevent the synthesis of mannosyldiinositolphosphorylceramide, the mature sphingolipid found in yeast. Deletion of either gene suppresses the Ca2+-sensitive phenotype of csg2Δ mutants, which arises from overaccumulation of inositolphosphorylceramide due to a defect in sphingolipid mannosylation. Characterization of scs7 and sur2 mutants is expected to provide insight into the function of ceramide hydroxylation
Maintenance of sphingolipid homeostasis is critical for cell growth and programmed cell death (PC... more Maintenance of sphingolipid homeostasis is critical for cell growth and programmed cell death (PCD). Serine palmitoyltransferase (SPT), composed of LCB1 and LCB2 subunits, catalyzes the primary regulatory point for sphingolipid synthesis. Small subunits of SPT (ssSPT) that strongly stimulate SPT activity have been identified in mammals, but the role of ssSPT in eukaryotic cells is unclear. Candidate Arabidopsis thaliana ssSPTs, ssSPTa and ssSPTb, were identified and characterized. Expression of these 56–amino acid polypeptides in a Saccharomyces cerevisiae SPT null mutant stimulated SPT activity from the Arabidopsis LCB1/LCB2 heterodimer by \u3e100-fold through physical interaction with LCB1/LCB2. ssSPTa transcripts were more enriched in all organs and \u3e400-fold more abundant in pollen than ssSPTb transcripts. Accordingly, homozygous ssSPTa T-DNA mutants were not recoverable, and 50% nonviable pollen was detected in heterozygous ssspta mutants. Pollen viability was recovered by e...
Saccharomyces cerevisiae csg2Δ mutants accumulate the sphingolipid inositolphosphorylceramide, wh... more Saccharomyces cerevisiae csg2Δ mutants accumulate the sphingolipid inositolphosphorylceramide, which renders the cells Ca2+-sensitive. Temperature-sensitive mutations that suppress the Ca2+ sensitivity of csg2Δ mutants were isolated and characterized to identify genes that encode sphingolipid synthesis enzymes. These temperature-sensitive csg2Δ suppressors (tsc) fall into 15 complementation groups. The TSC10/YBR265w gene was found to encode 3-ketosphinganine reductase, the enzyme that catalyzes the second step in the synthesis of phytosphingosine, the long chain base found in yeast sphingolipids. 3-Ketosphinganine reductase (Tsc10p) is essential for growth in the absence of exogenous dihydrosphingosine or phytosphingosine. Tsc10p is a member of the short chain dehydrogenase/reductase protein family. The tsc10 mutants accumulate 3-ketosphinganine and microsomal membranes isolated from tsc10 mutants have low 3-ketosphinganine reductase activity. His6-tagged Tsc10p was expressed in Esc...
A number of Saccharomyces cerevisiae membranebound oxidoreductases were examined for potential ro... more A number of Saccharomyces cerevisiae membranebound oxidoreductases were examined for potential roles in microsomal fatty acid elongation, by assaying heterologous elongating activities in individual deletion mutants. One yeast gene, YBR159w, was identified as being required for activity of both the Caenorhabditis elegans elongase PEA1 (F56H11.4) and the Arabidopsis thaliana elongase FAE1. Ybr159p shows some limited homology to human steroid dehydrogenases and is a member of the short-chain alcohol dehydrogenase superfamily. Disruption of YBR159w is not lethal, in contrast to previous reports, although the mutants are slow growing and display high temperature sensitivity. Both Ybr159p and an Arabidopsis homologue were shown to restore heterologous elongase activities when expressed in ybr159Δ mutants. Biochemical characterization of microsomal preparations from ybr159Δ cells revealed a primary perturbation in β-ketoacyl reduction, confirming the assignment of YBR159w as encoding a co...
fantastic team, have been of tremendous assistance to me. They are always there to help me with a... more fantastic team, have been of tremendous assistance to me. They are always there to help me with all the paper work. My sincere thanks to Ms. Janet Anastasi, former graduate office secretary, for her personal and professional support during the initial period of my graduate studies. I would like to acknowledge the Biomedical Instrumental Center for constant and timely service with DNA sequencing and oligonucleotide synthesis. My sincere thanks to all the members of the biochemistry department for creating a cheerful and stimulating environment. My thanks to Ms. Karen Williams for taking the hassle out of ordering supplies. I am grateful to all my friends and colleagues who have made everything look positive and enjoyable. I express my deepest gratitude to Dr. Gouri Chattopadhyay, who was always there for me, and helped me both scientifically and personally and provided me a home away from home. Last but not the least, I would like to thank the Uniformed Services University of the Health Sciences for providing me with the opportunity to enroll and complete my dissertation. vi DEDICATION To my loving parents, and my family. I will always appreciate all the support and encouragement I got from my sister Gouri, brother-in-law, Manas. I would like to thank a very special man Sambuddha who always believed in me. COPYRIGHT STATEMENT The author hereby certifies that the use of any copyrighted material in the dissertation manuscript entitled: "Small Subunits of Serine Palmitoyltransferase (ssSPTs) and their physiological roles" is appropriately acknowledged and, beyond brief excerpts.
Proceedings of the National Academy of Sciences of the United States of America, Jan 24, 2016
Sphingolipids exhibit extreme functional and chemical diversity that is in part determined by the... more Sphingolipids exhibit extreme functional and chemical diversity that is in part determined by their hydrophobic moiety, ceramide. In mammals, the fatty acyl chain length variation of ceramides is determined by six (dihydro)ceramide synthase (CerS) isoforms. Previously, we and others showed that mutations in the major neuron-specific CerS1, which synthesizes 18-carbon fatty acyl (C18) ceramide, cause elevation of long-chain base (LCB) substrates and decrease in C18 ceramide and derivatives in the brain, leading to neurodegeneration in mice and myoclonus epilepsy with dementia in humans. Whether LCB elevation or C18 ceramide reduction leads to neurodegeneration is unclear. Here, we ectopically expressed CerS2, a nonneuronal CerS producing C22-C24 ceramides, in neurons of Cers1-deficient mice. Surprisingly, the Cers1 mutant pathology was almost completely suppressed. Because CerS2 cannot replenish C18 ceramide, the rescue is likely a result of LCB reduction. Consistent with this hypoth...
Sphingolipid synthesis is tightly regulated in eukaryotes. This regulation in plants ensures suff... more Sphingolipid synthesis is tightly regulated in eukaryotes. This regulation in plants ensures sufficient sphingolipids to support growth, while limiting accumulation of sphingolipid metabolites that induce programmed cell death (PCD). Serine palmitoyltransfersase (SPT) catalyzes the first step in sphingolipid biosynthesis and is considered the primary sphingolipid homeostatic regulatory point. In this report, Arabidopsis putative SPT regulatory proteins, orosomucoid-like proteins AtORM1 and AtORM2 were found to physically interact with the Arabidopsis SPT and to suppress SPT activity when co-expressed with Arabidopsis SPT subunits LCB1 and LCB2 and the small subunit of SPT in a yeast SPT-deficient mutant. Consistent with a role in SPT suppression, AtORM1 and AtORM2 overexpression lines displayed increased resistance to the PCD-inducing mycotoxin fumonisin B1 (FB1), with an accompanying reduced accumulation of long-chain bases (LCBs) and C16-fatty acid-containing ceramides relative to...
Sphingolipid (SL) biosynthesis is negatively regulated by the highly conserved endoplasmic reticu... more Sphingolipid (SL) biosynthesis is negatively regulated by the highly conserved endoplasmic reticulum-localized Orm family proteins. Defective SL synthesis in Saccharomyces cerevisiae leads to increased phosphorylation and inhibition of Orm proteins by the kinase Ypk1. Here we present evidence that the yeast morphogenesis checkpoint kinase, Swe1, regulates SL biosynthesis independent of the Ypk1 pathway. Deletion of the Swe1 kinase renders mutant cells sensitive to serine palmitoyltransferase inhibition due to impaired sphingoid long-chain base synthesis. Based on these data and previous results, we suggest that Swe1 kinase perceives alterations in SL homeostasis, activates SL synthesis, and may thus represent the missing regulatory link that controls the SL rheostat during the cell cycle. * This work was supported by Austrian Science Funds (FWF) Projects SFB F 3005 LIPOTOX and Ph.D. program W903 Molecular Enzymology and NAWI Graz (to S. D. K.) and by National Institutes of Health Grant R21HD080181 (to T. D.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors declare that they have no conflicts of interest with the contents of this article.
Fundamental changes in the composition and distribution of lipids within the brain are believed t... more Fundamental changes in the composition and distribution of lipids within the brain are believed to contribute to the cognitive decline associated with Alzheimer’s disease (AD). The mechanisms by which these changes in lipid composition affect cellular function and ultimately cognition are not well understood. Although “candidate gene” approaches can provide insight into the effects of dysregulated lipid metabolism they require a preexisting understanding of the molecular targets of individual lipid species. In this report we combine unbiased gene expression profiling with a genome-wide chemogenomic screen to identify the mitochondria as an important downstream target of PC(O-16:0/2:0), a neurotoxic lipid species elevated in AD. Further examination revealed that PC(O-16:0/2:0) similarly promotes a global increase in ceramide accumulation in human neurons which was associated with mitochondrial-derived reactive oxygen species (ROS) and toxicity. These findings suggest that PC(O-16:0/2...
Proceedings of the National Academy of Sciences of the United States of America, Jan 20, 2015
Sphingolipids typically have an 18-carbon (C18) sphingoid long chain base (LCB) backbone. Althoug... more Sphingolipids typically have an 18-carbon (C18) sphingoid long chain base (LCB) backbone. Although sphingolipids with LCBs of other chain lengths have been identified, the functional significance of these low-abundance sphingolipids is unknown. The LCB chain length is determined by serine palmitoyltransferase (SPT) isoenzymes, which are trimeric proteins composed of two large subunits (SPTLC1 and SPTLC2 or SPTLC3) and a small subunit (SPTssa or SPTssb). Here we report the identification of an Sptssb mutation, Stellar (Stl), which increased the SPT affinity toward the C18 fatty acyl-CoA substrate by twofold and significantly elevated 20-carbon (C20) LCB production in the mutant mouse brain and eye, resulting in surprising neurodegenerative effects including aberrant membrane structures, accumulation of ubiquitinated proteins on membranes, and axon degeneration. Our work demonstrates that SPT small subunits play a major role in controlling SPT activity and substrate affinity, and in s...
To highlight the importance of sphingolipids and their metabolites in plant biology. The completi... more To highlight the importance of sphingolipids and their metabolites in plant biology. The completion of the arabidopsis genome provides a platform for the identification and functional characterization of genes involved in sphingolipid biosynthesis. Using the yeast Saccharomyces cerevisiae as an experimental model, this review annotates arabidopsis open reading frames likely to be involved in sphingolipid metabolism. A number of these open reading frames have already been subject to functional characterization, though the majority still awaits investigation. Plant-specific aspects of sphingolipid biology (such as enhanced long chain base heterogeneity) are considered in the context of the emerging roles for these lipids in plant form and function. Arabidopsis provides an excellent genetic and post-genomic model for the characterization of the roles of sphingolipids in higher plants.
Sphingolipid synthesis is initiated by condensation of Ser with palmitoyl-CoA producing 3-ketodih... more Sphingolipid synthesis is initiated by condensation of Ser with palmitoyl-CoA producing 3-ketodihydrosphinganine (3-KDS), which is reduced by a 3-KDS reductase to dihydrosphinganine. Ser palmitoyltransferase is essential for plant viability. Arabidopsis thaliana contains two genes (At3g06060/TSC10A and At5g19200/TSC10B) encoding proteins with significant similarity to the yeast 3-KDS reductase, Tsc10p. Heterologous expression in yeast of either Arabidopsis gene restored 3-KDS reductase activity to the yeast tsc10Δ mutant, confirming both as bona fide 3-KDS reductase genes. Consistent with sphingolipids having essential functions in plants, double mutant progeny lacking both genes were not recovered from crosses of single tsc10A and tsc10B mutants. Although the 3-KDS reductase genes are functionally redundant and ubiquitously expressed in Arabidopsis, 3-KDS reductase activity was reduced to 10% of wild-type levels in the loss-of-function tsc10a mutant, leading to an altered sphingoli...
Maintenance of sphingolipid homeostasis is critical for cell growth and programmed cell death (PC... more Maintenance of sphingolipid homeostasis is critical for cell growth and programmed cell death (PCD). Serine palmitoyltransferase (SPT), composed of LCB1 and LCB2 subunits, catalyzes the primary regulatory point for sphingolipid synthesis. Small subunits of SPT (ssSPT) that strongly stimulate SPT activity have been identified in mammals, but the role of ssSPT in eukaryotic cells is unclear. Candidate Arabidopsis thaliana ssSPTs, ssSPTa and ssSPTb, were identified and characterized. Expression of these 56–amino acid polypeptides in a Saccharomyces cerevisiae SPT null mutant stimulated SPT activity from the Arabidopsis LCB1/LCB2 heterodimer by >100-fold through physical interaction with LCB1/LCB2. ssSPTa transcripts were more enriched in all organs and >400-fold more abundant in pollen than ssSPTb transcripts. Accordingly, homozygous ssSPTa T-DNA mutants were not recoverable, and 50% nonviable pollen was detected in heterozygous ssspta mutants. Pollen viability was recovered by...
Unbiased lipidomic approaches have identified impairments in glycerophosphocholine second messeng... more Unbiased lipidomic approaches have identified impairments in glycerophosphocholine second messenger metabolism in patients with Alzheimer's disease. Specifically, we have shown that amyloid-b42 signals the intraneuronal accumulation of PC(O-16:0/2:0) which is associated with neurotoxicity. Similar to neuronal cells, intracellular accumulation of PC(O-16:0/2:0) is also toxic to Saccharomyces cerevisiae, making yeast an excellent model to decipher the pathological effects of this lipid. We previously reported that phospholipase D, a phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P 2)-binding protein, was relocalized in response to PC(O-16:0/2:0), suggesting that this neurotoxic lipid may remodel lipid signaling networks. Here we show that PC(O-16:0/2:0) regulates the distribution of the PtdIns(4)P 5-kinase Mss4 and its product PtdIns(4,5)P 2 leading to the formation of invaginations at the plasma membrane (PM). We further demonstrate that the effects of PC(O-16:0/2:0) on the distribution of PM PtdIns(4,5)P 2 pools are in part mediated by changes in the biosynthesis of long chain bases (LCBs) and ceramides. A combination of genetic, biochemical and cell imaging approaches revealed that PC(O-16:0/2:0) is also a potent inhibitor of signaling through the Target of rampamycin complex 2 (TORC2). Together, these data provide mechanistic insight into how specific disruptions in phosphocholine second messenger metabolism associated with Alzheimer's disease may trigger larger network-wide disruptions in ceramide and phosphoinositide second messenger biosynthesis and signaling which have been previously implicated in disease progression.
The autosomal dominant peripheral sensory neuropathy HSAN1 results from mutations in the LCB1 sub... more The autosomal dominant peripheral sensory neuropathy HSAN1 results from mutations in the LCB1 subunit of serine palmitoyltransferase (SPT). Serum from patients and transgenic mice expressing a disease-causing mutation (C133W) contain elevated levels of 1-deoxysphinganine (1-deoxySa), which presumably arise from inappropriate condensation of alanine with palmitoyl-CoA. Mutant heterodimeric SPT is catalytically inactive. However, mutant heterotrimeric SPT has ϳ10-20% of wild-type activity and supports growth of yeast cells lacking endogenous SPT. In addition, long chain base profiling revealed the synthesis of significantly more 1-deoxySa in yeast and mammalian cells expressing the heterotrimeric mutant enzyme than in cells expressing wild-type enzyme. Wild-type and mutant enzymes had similar affinities for serine. Surprisingly, the enzymes also had similar affinities for alanine, indicating that the major affect of the C133W mutation is to enhance activation of alanine for condensation with the acyl-CoA substrate. In vivo synthesis of 1-deoxySa by the mutant enzyme was proportional to the ratio of alanine to serine in the growth media, suggesting that this ratio can be used to modulate the relative synthesis of sphinganine and 1-deoxySa. By expressing SPT as a single-chain fusion protein to ensure stoichiometric expression of all three subunits, we showed that GADD153, a marker for endoplasmic reticulum stress, was significantly elevated in cells expressing mutant heterotrimers. GADD153 was also elevated in cells treated with 1-deoxySa. Taken together, these data indicate that the HSAN1 mutations perturb the active site of SPT resulting in a gain of function that is responsible for the HSAN1 phenotype. Serine palmitoyltransferase (SPT) 3 catalyzes the committed and rate-limiting step in sphingolipid biosynthesis. Until recently, it was believed that there was a single isozyme of this enzyme containing two subunits, LCB1 and LCB2, with the catalytic domain located at the interface and composed of residues from each subunit. However, Hornemann et al. showed that there was a second isoform of LCB2 (1). More recently, we
The YBR159w gene encodes the major 3-ketoreductase activity of the elongase system of enzymes req... more The YBR159w gene encodes the major 3-ketoreductase activity of the elongase system of enzymes required for very long-chain fatty acid (VLCFA) synthesis. Mutants lacking the YBR159w gene display many of the phenotypes that have previously been described for mutants with defects in fatty acid elongation. These phenotypes include reduced VLCFA synthesis, accumulation of high levels of dihydrosphingosine and phytosphingosine, and accumulation of medium-chain ceramides. In vitro elongation assays confirm that the ybr159⌬ mutant is deficient in the reduction of the 3-ketoacyl intermediates of fatty acid elongation. The ybr159⌬ mutant also displays reduced dehydration of the 3-OH acyl intermediates of fatty acid elongation, suggesting that Ybr159p is required for the stability or function of the dehydratase activity of the elongase system. Green fluorescent protein-tagged Ybr159p co-localizes and co-immunoprecipitates with other elongating enzymes, Elo3p and Tsc13p. Whereas VLCFA synthesis is essential for viability, the ybr159⌬ mutant cells are viable (albeit very slowly growing) and do synthesize some VLCFA. This suggested that a functional ortholog of Ybr159p exists that is responsible for the residual 3-ketoreductase activity. By disrupting the orthologs of Ybr159w in the ybr159⌬ mutant we found that the ybr159⌬ayr1⌬ double mutant was inviable, suggesting that Ayr1p is responsible for the residual 3-ketoreductase activity.
1 The abbreviations used are: SPT, serine palmitoyltransferase; LCB, long chain base; AONS, 8-ami... more 1 The abbreviations used are: SPT, serine palmitoyltransferase; LCB, long chain base; AONS, 8-amino-7-oxononanoate synthase; ts, temperature sensitive; bp, base pair(s); PHS, phytosphingosine; ORF, open reading frame; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; HA, hemagglutinin; PCR, polymerase chain reaction; SCS, suppressor of the Ca 2ϩ-sensitive phenotype of the csg2⌬ mutant.
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Papers by teresa dunn