RESUMO
Mutations in the SPTLC1 subunit of serine palmitoyltransferase (SPT) cause an adult-onset, hereditary sensory, and autonomic neuropathy type I (HSAN1). We previously reported that mice bearing a transgene-expressing mutant SPTLC1 (tgSPTLC1(C133W)) show a reduction in SPT activity and hyperpathia at 10 months of age. Now analyzed at a later age, we find these mice develop sensory loss with a distal small fiber neuropathy and peripheral myelinopathy. This phenotype is largely reversed when these mice are crossed with transgenic mice overexpressing wild-type SPTLC1 showing that the mutant SPTLC1 protein is not inherently toxic. Simple loss of SPT activity also cannot account for the HSAN1 phenotype, since heterozygous SPTLC1 knock-out mice have reduced SPT activity but are otherwise normal. Rather, the presence of two newly identified, potentially deleterious deoxysphingoid bases in the tgSPTLC1(C133W), but not in the wild-type, double-transgenic tgSPTLC1(WT + C133W) or SPTLC1(+/-) mice, suggests that the HSAN1 mutations alter amino acid selectivity of the SPT enzyme such that palmitate is condensed with alanine and glycine, in addition to serine. This observation is consistent with the hypothesis that HSAN1 is the result of a gain-of-function mutation in SPTLC1 that leads to accumulation of a toxic metabolite.
Assuntos
Expressão Gênica , Neuropatias Hereditárias Sensoriais e Autônomas/genética , Fenótipo , Subunidades Proteicas/genética , Serina C-Palmitoiltransferase/genética , Esfingolipídeos/metabolismo , Animais , Cricetinae , Neuropatias Hereditárias Sensoriais e Autônomas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Knockout , Camundongos Transgênicos , Subunidades Proteicas/biossíntese , Subunidades Proteicas/fisiologia , Serina C-Palmitoiltransferase/biossíntese , Serina C-Palmitoiltransferase/fisiologia , Esfingolipídeos/toxicidadeRESUMO
Mutations in enzymes involved in sphingolipid metabolism and trafficking cause a variety of neurological disorders, but details of the molecular pathophysiology remain obscure. SPTLC1 encodes one subunit of serine palmitoyltransferase (SPT), the rate-limiting enzyme in sphingolipid synthesis. Mutations in SPTLC1 cause hereditary sensory and autonomic neuropathy (type I) (HSAN1), an adult onset, autosomal dominant neuropathy. HSAN1 patients have reduced SPT activity. Expression of mutant SPTLC1 in yeast and mammalian cell cultures dominantly inhibits SPT activity. We created transgenic mouse lines that ubiquitously overexpress either wild-type (SPTLC1(WT)) or mutant SPTLC1 (SPTLC1(C133W)). We report here that SPTLC1(C133W) mice develop age-dependent weight loss and mild sensory and motor impairments. Aged SPTLC1(C133W) mice lose large myelinated axons in the ventral root of the spinal cord and demonstrate myelin thinning. There is also a loss of large myelinated axons in the dorsal roots, although the unmyelinated fibers are preserved. In the dorsal root ganglia, IB4 staining is diminished, whereas expression of the injury-induced transcription factor ATF3 is increased. These mice represent a novel mouse model of peripheral neuropathy and confirm the link between mutant SPT and neuronal dysfunction.
Assuntos
Envelhecimento/genética , Genes Dominantes , Neuropatias Hereditárias Sensoriais e Autônomas/enzimologia , Neuropatias Hereditárias Sensoriais e Autônomas/genética , Mutação , Serina C-Palmitoiltransferase/genética , Animais , Axônios/patologia , Comportamento Animal/fisiologia , Células CHO , Cricetinae , Cricetulus , Feminino , Neuropatias Hereditárias Sensoriais e Autônomas/patologia , Neuropatias Hereditárias Sensoriais e Autônomas/fisiopatologia , Masculino , Camundongos , Camundongos Transgênicos , Pâncreas Exócrino/patologia , Serina C-Palmitoiltransferase/antagonistas & inibidores , Serina C-Palmitoiltransferase/metabolismo , TransfecçãoRESUMO
The structural organization and topology of the Lcb1p subunit of yeast and mammalian serine palmitoyltransferases (SPT) were investigated. In the yeast protein, three membrane-spanning domains were identified by insertion of glycosylation and factor Xa cleavage sites at various positions. The first domain of the yeast protein, located between residues 50 and 84, was not required for the stability, membrane association, interaction with Lcb2p, or enzymatic activity. Deletion of the comparable domain of the mammalian protein SPTLC1 also had little effect on its function, demonstrating that this region is not required for membrane localization or heterodimerization with SPTLC2. The second and third membrane-spanning domains of yeast Lcb1p, located between residues 342 and 371 and residues 425 and 457, respectively, create a luminal loop of approximately 60 residues. In contrast to the first membrane-spanning domain, the second and third membrane-spanning domains were both required for Lcb1p stability. In addition, mutations in the luminal loop destabilized the SPT heterodimer indicating that this region of the protein is important for SPT structure and function. Mutations in the extreme carboxyl-terminal region of Lcb1p also disrupted heterodimer formation. Taken together, these data suggest that in contrast to other members of the alpha-oxoamine synthases that are soluble homodimers, the Lcb1p and Lcb2p subunits of the SPT heterodimer may interact in the cytosol, as well as within the membrane and/or the lumen of the endoplasmic reticulum.
Assuntos
Aciltransferases/química , Aciltransferases/metabolismo , Alelos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Western Blotting , Células CHO , Membrana Celular/metabolismo , Códon , Cricetinae , Citosol/metabolismo , Dimerização , Retículo Endoplasmático/metabolismo , Fator Xa/química , Deleção de Genes , Genes Reporter , Teste de Complementação Genética , Glicosilação , Proteínas de Fluorescência Verde/metabolismo , Microssomos Hepáticos/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Plasmídeos/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Serina C-PalmitoiltransferaseRESUMO
Methods to systematically test drugs against all possible proteins in a cell are needed to identify the targets underlying their therapeutic action and unwanted effects. Here, we show that a genome-wide drug-induced haploinsufficiency screen by using yeast can reveal drug mode of action in yeast and can be used to predict drug mode of action in human cells. We demonstrate that dihydromotuporamine C, a compound in preclinical development that inhibits angiogenesis and metastasis by an unknown mechanism, targets sphingolipid metabolism. The systematic, unbiased and genome-wide nature of this technique makes it attractive as a general approach to identify cellular pathways affected by drugs.
Assuntos
Antifúngicos/farmacologia , Deleção de Genes , Genoma Fúngico , Saccharomyces cerevisiae/genética , Deleção de Sequência , Animais , Testes de Sensibilidade Microbiana , Ploidias , Saccharomyces cerevisiae/efeitos dos fármacos , Transdução de Sinais , Esfingolipídeos/biossínteseRESUMO
Serine palmitoyltransferase catalyses the committed step in sphingolipid synthesis, the condensation of serine with palmitoyl-CoA to form 3-ketosphinganine. Two proteins, Lcb1p and Lcb2p, are essential for enzyme activity and a third protein, the 80-amino acid Tsc3p, stimulates the activity of serine palmitoyltransferase several-fold. Tsc3p physically associates with a complex of Lcb1p-Lcb2p and stimulates enzyme activity posttranslationally, but its precise function is not known. Tsc3p is essential for cell viability only at elevated temperatures, although serine palmitoyltransferase activity is reduced in the tsc3 delta mutant, even at permissive growth temperatures. Tsc3p is apparently not required for any essential process besides stimulation of serine palmitoyltransferase at 37 degrees C, since providing sphingoid bases to the growth medium reverses the temperature-sensitive growth phenotype of the tsc3 delta mutant. To gain further insight into the function of Tsc3p, suppressor mutants that eliminate the Tsc3p requirement for growth at 37 degrees C were isolated and characterized. These studies show that dominant mutations in the Lcb2p subunit of serine palmitoyltransferase suppress the temperature-sensitive growth phenotype of the tsc3 delta null mutant by increasing the Tsc3p-independent serine palmitoyltransferase activity.
Assuntos
Aciltransferases/genética , Proteínas de Transporte/genética , Proteínas de Membrana/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimologia , Supressão Genética/genética , Aciltransferases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Proteínas de Transporte/metabolismo , Genes Fúngicos/genética , Teste de Complementação Genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Serina C-Palmitoiltransferase , Esfingolipídeos/biossíntese , TemperaturaRESUMO
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 ybr159Delta mutant is deficient in the reduction of the 3-ketoacyl intermediates of fatty acid elongation. The ybr159Delta 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 ybr159Delta 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 ybr159Delta mutant we found that the ybr159Deltaayr1Delta double mutant was inviable, suggesting that Ayr1p is responsible for the residual 3-ketoreductase activity.
Assuntos
3-Hidroxiacil-CoA Desidrogenases/genética , Acetiltransferases/genética , Genes Fúngicos , Microssomos/enzimologia , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , 3-Hidroxiacil-CoA Desidrogenases/metabolismo , Acetiltransferases/metabolismo , Sequência de Bases , Cálcio/metabolismo , Ceramidas/metabolismo , Cromossomos Fúngicos , Primers do DNA , Elongases de Ácidos Graxos , Genes Letais , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Microscopia de Fluorescência , Fenótipo , Proteínas Recombinantes de Fusão/genética , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
It was recently demonstrated that mutations in the human SPTLC1 gene, encoding the Lcb1p subunit of serine palmitoyltransferase (SPT), cause hereditary sensory neuropathy type I . As a member of the subfamily of pyridoxal 5'-phosphate enzymes known as the alpha-oxoamine synthases, serine palmitoyltransferase catalyzes the committed step of sphingolipid synthesis. The residues that are mutated to cause hereditary sensory neuropathy type I reside in a highly conserved region of Lcb1p that is predicted to be a catalytic domain of Lcb1p on the basis of alignments with other members of the alpha-oxoamine synthase family. We found that the corresponding mutations in the LCB1 gene of Saccharomyces cerevisiae reduce serine palmitoyltransferase activity. These mutations are dominant and decrease serine palmitoyltransferase activity by 50% when the wild-type and mutant LCB1 alleles are coexpressed. We also show that serine palmitoyltransferase is an Lcb1p small middle dotLcb2p heterodimer and that the mutated Lcb1p proteins retain their ability to interact with Lcb2p. Modeling studies suggest that serine palmitoyltransferase is likely to have a single active site that lies at the Lcb1p small middle dotLcb2p interface and that the mutations in Lcb1p reside near the lysine in Lcb2p that is expected to form the Schiff's base with the pyridoxal 5'-phosphate cofactor. Furthermore, mutations in this lysine and in a histidine residue that is also predicted to be important for pyridoxal 5'-phosphate binding to Lcb2p also dominantly inactivate SPT similar to the hereditary sensory neuropathy type 1-like mutations in Lcb1p.
Assuntos
Aciltransferases/genética , Aciltransferases/metabolismo , Neuropatias Hereditárias Sensoriais e Autônomas/genética , Mutação , Alelos , Sequência de Aminoácidos , Sítios de Ligação , Western Blotting , Cálcio/metabolismo , Catálise , Cromatografia Líquida , Dimerização , Diploide , Neuropatias Hereditárias Sensoriais e Autônomas/enzimologia , Histidina/química , Lisina/química , Microssomos Hepáticos/metabolismo , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Plasmídeos/metabolismo , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae , Homologia de Sequência de Aminoácidos , Serina C-Palmitoiltransferase , Esfingolipídeos/biossíntese , Esfingolipídeos/metabolismoRESUMO
A number of Saccharomyces cerevisiae membrane-bound 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 ybr159Delta mutants. Biochemical characterization of microsomal preparations from ybr159Delta cells revealed a primary perturbation in beta-ketoacyl reduction, confirming the assignment of YBR159w as encoding a component of the microsomal elongase.