[Show abstract][Hide abstract] ABSTRACT: 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-16:0/2:0)-dependent mitochondrial dysfunction may be an underlying contributing factor to the ROS production associated with AD.
Full-text · Article · Jan 2016 · Scientific Reports
[Show abstract][Hide abstract] ABSTRACT: 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.
Preview · Article · Dec 2015 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: 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 specifying sphingolipid LCB chain length in vivo. Moreover, our studies also suggest that excessive C20 LCBs or C20 LCB-containing sphingolipids impair protein homeostasis and neural functions.
No preview · Article · Oct 2015 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: The relationship between serine palmitoyltransferase (SPT) activity and ORMDL regulation of sphingolipid biosynthesis was
investigated in mammalian HEK293 cells. Each of the three human ORMDLs reduced the increase in long-chain base synthesis seen
after overexpression of wild-type SPT or SPT containing the C133W mutation in hLCB1, which produces the non-catabolizable
sphingoid base, 1-deoxySa. ORMDL-dependent repression of sphingoid base synthesis occurred whether SPT was expressed as individual
subunits or as a heterotrimeric single-chain SPT fusion protein. Overexpression of the single-chain SPT fusion protein under
the control of a tetracycline-inducible promoter in stably transfected cells resulted in increased endogenous ORMDL expression.
This increase was not transcriptional; there was no significant increase in any of the ORMDL mRNAs. Increased ORMDL protein
expression required SPT activity since overexpression of a catalytically inactive SPT with a mutation in hLCB2a had little
effect. Significantly, increased ORMDL expression was also blocked by myriocin inhibition of SPT as well as fumonisin inhibition
of the ceramide synthases, suggesting that increased expression is a response to a metabolic signal. Moreover, blocking ORMDL
induction with fumonisin treatment resulted in significantly greater increases in in vivo SPT activity than was seen when ORMDLs were allowed to increase, demonstrating the physiological significance of this response.
No preview · Article · Nov 2014 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Sphingolipid levels are tightly regulated to maintain cellular homeostasis. During pathologic conditions such as in aging, inflammation, and metabolic and neurodegenerative diseases, levels of some sphingolipids, including the bioactive metabolite ceramide are elevated. Sphingolipid metabolism has been linked to autophagy, a critical catabolic process in both normal cell function and disease; however, the in vivo relevance of the interaction is not well understood. Here we show that blocking autophagy in liver by deletion of the Atg7 gene, which is essential for autophagosome formation, causes an increase in sphingolipid metabolites including ceramide. We also show that overexpression of SPT to elevate de novo sphingolipid biosynthesis induces autophagy in the liver. The results reveal autophagy as a process that limits excessive ceramide levels and that is induced by excessive elevation of de novo sphingolipid synthesis in liver. Dysfunctional autophagy may be an underlying mechanism causing elevations in ceramide that may contribute to pathogenesis in diseases.
Full-text · Article · Oct 2014 · The Journal of Lipid Research
[Show abstract][Hide abstract] ABSTRACT: Unbiased lipidomic approaches have identified impairments in glycerophosphocholine second messenger metabolism in patients with Alzheimer's disease. Specifically, we have shown that amyloid-β42 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)P2)-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)P2 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)P2 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.
[Show abstract][Hide abstract] ABSTRACT: Very long chain fatty acids (VLCFAs) are essential fatty acids with multiple functions, including ceramide synthesis. Although the components of the VLCFA biosynthetic machinery have been elucidated, how their activity is regulated to meet the cell's metabolic demand remains unknown. The goal of this study was to identify mechanisms that regulate the rate of VLCFA synthesis, and we discovered that the fatty acid elongase Elo2 is regulated by phosphorylation. Elo2 phosphorylation is induced upon inhibition of TORC1 and requires GSK3. Expression of nonphosphorylatable Elo2 profoundly alters the ceramide spectrum, reflecting aberrant VLCFA synthesis. Furthermore, VLCFA depletion results in constitutive activation of autophagy, which requires sphingoid base phosphorylation. This constitutive activation of autophagy diminishes cell survival, indicating that VLCFAs serve to dampen the amplitude of autophagy. Together, our data reveal a function for TORC1 and GSK3 in the regulation of VLCFA synthesis that has important implications for autophagy and cell homeostasis.
[Show abstract][Hide abstract] ABSTRACT: 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 expression of wild-type ssSPTa or ssSPTb under control of the ssSPTa promoter, indicating ssSPTa and ssSPTb functional redundancy. SPT activity and sensitivity to the PCD-inducing mycotoxin fumonisin B1 (FB1) were increased by ssSPTa overexpression. Conversely, SPT activity and FB1 sensitivity were reduced in ssSPTa RNA interference lines. These results demonstrate that ssSPTs are essential for male gametophytes, are important for FB1 sensitivity, and limit sphingolipid synthesis in planta.
[Show abstract][Hide abstract] ABSTRACT: The pyridoxal 5'-phosphate (PLP)-dependent enzyme serine palmitoyltransferase (SPT) catalyses the first step of de novo sphingolipid biosynthesis. The core human enzyme is a membrane-bound heterodimer composed of two subunits (hLCB1 and hLCB2a/b), and mutations in both hLCB1 (e.g., C133W and C133Y) and hLCB2a (e.g., V359M, G382V, and I504F) have been identified in patients with hereditary sensory and autonomic neuropathy type I (HSAN1), an inherited disorder that affects sensory and autonomic neurons. These mutations result in substrate promiscuity, leading to formation of neurotoxic deoxysphingolipids found in affected individuals. Here we measure the activities of the hLCB2a mutants in the presence of ssSPTa and ssSPTb and find that all decrease enzyme activity. High resolution structural data of the homodimeric SPT enzyme from the bacterium Sphingomonas paucimobilis (Sp SPT) provides a model to understand the impact of the hLCB2a mutations on the mechanism of SPT. The three human hLCB2a HSAN1 mutations map onto Sp SPT (V246M, G268V, and G385F), and these mutant mimics reveal that the amino acid changes have varying impacts; they perturb the PLP cofactor binding, reduce the affinity for both substrates, decrease the enzyme activity, and, in the most severe case, cause the protein to be expressed in an insoluble form.
[Show abstract][Hide abstract] ABSTRACT: Sphingolipids (SLs) are essential components of cellular membranes formed from the condensation of L-serine and a long-chain acyl thioester. This first step is catalysed by the pyridoxal 5-phosphate (PLP)-dependent enzyme serine palmitoyltransferase (SPT) which is a promising therapeutic target. The fungal natural product myriocin is a potent inhibitor of SPT and is widely-used to block SL biosynthesis despite a lack of a detailed understanding of its molecular mechanism. By combining spectroscopy, mass spectrometry, x-ray crystallography and kinetics we have characterised the molecular details of SPT inhibition by myriocin. Myriocin initially forms an external aldimine with PLP at the active site and a structure of the resulting co-complex explains its nanomolar affinity for the enzyme. This co-complex then catalytically degrades via an unexpected 'retro-aldol like' cleavage mechanism to a C18 aldehyde which in turn acts as a suicide inhibitor of SPT by covalent modification of the essential catalytic lysine. This surprising dual mechanism of inhibition rationalises the extraordinary potency and longevity of myriocin inhibition.
No preview · Article · Aug 2013 · Journal of the American Chemical Society
[Show abstract][Hide abstract] ABSTRACT: Plant sphingolipids are structurally diverse molecules that are important as membrane components and bioactive molecules. An appreciation of the relationship between structural diversity and functional significance of plant sphingolipids is emerging through characterization of Arabidopsis mutants coupled with advanced analytical methods. It is increasingly apparent that modifications such as hydroxylation and desaturation of the sphingolipid nonpolar long-chain bases and fatty acids influence their metabolic routing to particular complex sphingolipid classes and their functions in signaling pathways and other cellular processes, such as membrane protein targeting. Here, we review recent reports investigating some of the more prevalent sphingolipid structural modifications and their functional importance in plants.
Full-text · Article · Mar 2013 · Current opinion in plant biology
[Show abstract][Hide abstract] ABSTRACT: The topological and functional organization of the two isoforms of the small subunits of human serine palmitoyltransferase
(hssSPTs) that activate the catalytic hLCB1/hLCB2 heterodimer was investigated. A variety of experimental approaches placed
the N termini of the ssSPTs in the cytosol, their C termini in the lumen, and showed that they contain a single transmembrane
domain. Deletion analysis revealed that the ability to activate the heterodimer is contained in a conserved 33-amino acid
core domain that has the same membrane topology as the full-length protein. In combination with analysis of isoform chimera
and site-directed mutagenesis, a single amino acid residue in this core (Met25 in ssSPTa and Val25 in ssSPTb) was identified which confers specificity for palmitoyl- or stearoyl-CoA, respectively, in both yeast and mammalian
cells. This same residue also determines which isoform is a better activator of a mutant heterodimer, hLCB1S331F/hLCB2a, which has increased basal SPT activity and decreased amino acid substrate selectivity. This suggests that the role
of the ssSPTs is to increase SPT activity without compromising substrate specificity. In addition, the observation that the
C-terminal domains of ssSPTa and ssSPTb, which are highly conserved within each subfamily but are the most divergent regions
between isoform subfamilies, are not required for activation of the heterodimer or for acyl-CoA selectivity suggests that
the ssSPTs have additional roles that remain to be discovered.
Preview · Article · Feb 2013 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: SLs (sphingolipids) are composed of fatty acids and a polar head group derived from L-serine. SLs are essential components of all eukaryotic and many prokaryotic membranes but S1P (sphingosine 1-phosphate) is also a potent signalling molecule. Recent efforts have sought to inventory the large and chemically complex family of SLs (LIPID MAPS Consortium). Detailed understanding of SL metabolism may lead to therapeutic agents specifically directed at SL targets. We have studied the enzymes involved in SL biosynthesis; later stages are species-specific, but all core SLs are synthesized from the condensation of L-serine and a fatty acid thioester such as palmitoyl-CoA that is catalysed by SPT (serine palmitoyltransferase). SPT is a PLP (pyridoxal 5'-phosphate)-dependent enzyme that forms 3-KDS (3-ketodihydrosphingosine) through a decarboxylative Claisen-like condensation reaction. Eukaryotic SPTs are membrane-bound multi-subunit enzymes, whereas bacterial enzymes are cytoplasmic homodimers. We use bacterial SPTs (e.g. from Sphingomonas) to probe their structure and mechanism. Mutations in human SPT cause a neuropathy [HSAN1 (hereditary sensory and autonomic neuropathy type 1)], a rare SL metabolic disease. How these mutations perturb SPT activity is subtle and bacterial SPT mimics of HSAN1 mutants affect the enzyme activity and structure of the SPT dimer. We have also explored SPT inhibition using various inhibitors (e.g. cycloserine). A number of new subunits and regulatory proteins that have a direct impact on the activity of eukaryotic SPTs have recently been discovered. Knowledge gained from bacterial SPTs sheds some light on the more complex mammalian systems. In the present paper, we review historical aspects of the area and highlight recent key developments.
Full-text · Article · Jun 2012 · Biochemical Society Transactions
[Show abstract][Hide abstract] ABSTRACT: 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 sphingolipid profile. This perturbation of sphingolipid biosynthesis in the Arabidopsis tsc10a mutant leads an altered leaf ionome, including increases in Na, K, and Rb and decreases in Mg, Ca, Fe, and Mo. Reciprocal grafting revealed that these changes in the leaf ionome are driven by the root and are associated with increases in root suberin and alterations in Fe homeostasis.
[Show abstract][Hide abstract] ABSTRACT: Emiliania huxleyi is the host for the coccolithovirus (EhV), which is responsible for the demise of large oceanic blooms formed by this alga. The EhV-86 virus genome sequence has identified several genes apparently involved in sphingolipid metabolism. Recently, an unusual glucosylceramide from E. huxleyi infected with EhV-86 was isolated, implicating sphingolipids in the lysis of this alga. However, the EhV-86-encoded genes contain only a subset of the activities required to generate the novel sphingolipid, implying that its synthesis is the result of coordinated interactions between algal- and viral-encoded biosynthetic enzymes. Here, we discuss the likely role for EhV-86 open reading frames (ORFs) in the synthesis of novel sphingolipids and also consider the concept of the trans-dominant manipulation of lipid metabolism.
No preview · Article · Oct 2010 · Trends in Plant Science
[Show abstract][Hide abstract] ABSTRACT: 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 approximately 10-20% 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.
[Show abstract][Hide abstract] ABSTRACT: Iron is an essential cofactor for enzymes involved in numerous cellular processes, yet little is known about the impact of
iron deficiency on cellular metabolism or iron proteins. Previous studies have focused on changes in transcript and proteins
levels in iron-deficient cells, yet these changes may not reflect changes in transport activity or flux through a metabolic
pathway. We analyzed the metabolomes and transcriptomes of yeast grown in iron-rich and iron-poor media to determine which
biosynthetic processes are altered when iron availability falls. Iron deficiency led to changes in glucose metabolism, amino
acid biosynthesis, and lipid biosynthesis that were due to deficiencies in specific iron-dependent enzymes. Iron-sulfur proteins
exhibited loss of iron cofactors, yet amino acid synthesis was maintained. Ergosterol and sphingolipid biosynthetic pathways
had blocks at points where heme and diiron enzymes function, whereas Ole1, the essential fatty acid desaturase, was resistant
to iron depletion. Iron-deficient cells exhibited depletion of most iron enzyme activities, but loss of activity during iron
deficiency did not consistently disrupt metabolism. Amino acid homeostasis was robust, but iron deficiency impaired lipid
synthesis, altering the properties and functions of cellular membranes.
Full-text · Article · Mar 2010 · Journal of Biological Chemistry