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1.
Am J Hum Genet ; 108(4): 722-738, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33798445

RESUMO

Progressive myoclonus epilepsies (PMEs) comprise a group of clinically and genetically heterogeneous rare diseases. Over 70% of PME cases can now be molecularly solved. Known PME genes encode a variety of proteins, many involved in lysosomal and endosomal function. We performed whole-exome sequencing (WES) in 84 (78 unrelated) unsolved PME-affected individuals, with or without additional family members, to discover novel causes. We identified likely disease-causing variants in 24 out of 78 (31%) unrelated individuals, despite previous genetic analyses. The diagnostic yield was significantly higher for individuals studied as trios or families (14/28) versus singletons (10/50) (OR = 3.9, p value = 0.01, Fisher's exact test). The 24 likely solved cases of PME involved 18 genes. First, we found and functionally validated five heterozygous variants in NUS1 and DHDDS and a homozygous variant in ALG10, with no previous disease associations. All three genes are involved in dolichol-dependent protein glycosylation, a pathway not previously implicated in PME. Second, we independently validate SEMA6B as a dominant PME gene in two unrelated individuals. Third, in five families, we identified variants in established PME genes; three with intronic or copy-number changes (CLN6, GBA, NEU1) and two very rare causes (ASAH1, CERS1). Fourth, we found a group of genes usually associated with developmental and epileptic encephalopathies, but here, remarkably, presenting as PME, with or without prior developmental delay. Our systematic analysis of these cases suggests that the small residuum of unsolved cases will most likely be a collection of very rare, genetically heterogeneous etiologies.


Assuntos
Dolicóis/metabolismo , Mutação/genética , Epilepsias Mioclônicas Progressivas/genética , Adolescente , Adulto , Idade de Início , Criança , Pré-Escolar , Estudos de Coortes , Variações do Número de Cópias de DNA/genética , Feminino , Glicosilação , Humanos , Íntrons/genética , Masculino , Pessoa de Meia-Idade , Epilepsias Mioclônicas Progressivas/classificação , Sequenciamento Completo do Exoma , Adulto Jovem
2.
Sci Rep ; 11(1): 1411, 2021 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-33446867

RESUMO

The oligosaccharide required for asparagine (N)-linked glycosylation of proteins in the endoplasmic reticulum (ER) is donated by the glycolipid Glc3Man9GlcNAc2-PP-dolichol. Remarkably, whereas glycosylation occurs in the ER lumen, the initial steps of Glc3Man9GlcNAc2-PP-dolichol synthesis generate the lipid intermediate Man5GlcNAc2-PP-dolichol (M5-DLO) on the cytoplasmic side of the ER. Glycolipid assembly is completed only after M5-DLO is translocated to the luminal side. The membrane protein (M5-DLO scramblase) that mediates M5-DLO translocation across the ER membrane has not been identified, despite its importance for N-glycosylation. Building on our ability to recapitulate scramblase activity in proteoliposomes reconstituted with a crude mixture of ER membrane proteins, we developed a mass spectrometry-based 'activity correlation profiling' approach to identify scramblase candidates in the yeast Saccharomyces cerevisiae. Data curation prioritized six polytopic ER membrane proteins as scramblase candidates, but reconstitution-based assays and gene disruption in the protist Trypanosoma brucei revealed, unexpectedly, that none of these proteins is necessary for M5-DLO scramblase activity. Our results instead strongly suggest that M5-DLO scramblase activity is due to a protein, or protein complex, whose activity is regulated at the level of quaternary structure.


Assuntos
Retículo Endoplasmático/enzimologia , Hexosiltransferases/química , Espectrometria de Massas , Proteínas de Membrana/química , Proteínas de Protozoários/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Trypanosoma brucei brucei/enzimologia , Dolicóis/química , Dolicóis/metabolismo , Hexosiltransferases/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Protozoários/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
3.
Plant Sci ; 303: 110773, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33487357

RESUMO

Dolichol is an essential polyisoprenoid within the endoplasmic reticulum of all eukaryotes. It serves as a membrane bound anchor onto which N-glycans are assembled prior to being transferred to nascent polypeptides, many of which enter the secretory pathway. Historically, it has been posited that the accumulation of dolichol represents the 'rate-limiting' step in the evolutionary conserved process of N-glycosylation, which ultimately affects the efficacy of approximately one fifth of the entire eukaryotic proteome. Therefore, this study aimed to enhance dolichol accumulation by manipulating the enzymes involved in its biosynthesis using an established Nicotiana benthamiana platform. Co-expression of a Solanum lycopersicum (tomato) cis-prenyltransferase (CPT) and its cognate partner protein, CPT binding protein (CPTBP), that catalyze the antepenultimate step in dolichol biosynthesis led to a 400-fold increase in the levels of long-chain polyprenols but resulted in only modest increases in dolichol accumulation. However, when combined with a newly characterized tomato polyprenol reductase, dolichol biosynthesis was enhanced by approximately 20-fold. We provide further evidence that in the aquatic macrophyte, Lemna gibba, dolichol is derived exclusively from the mevalonic acid (MVA) pathway with little participation from the evolutionary co-adopted non-MVA pathway. Taken together these results indicate that to effectively enhance the in planta accumulation of dolichol, coordinated synthesis and reduction of polyprenol to dolichol, is strictly required.


Assuntos
Dolicóis/biossíntese , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Tabaco/metabolismo , Lycopersicon esculentum/enzimologia , Lycopersicon esculentum/genética , Lycopersicon esculentum/metabolismo , Redes e Vias Metabólicas , Oxirredutases/genética , Filogenia , Proteínas de Plantas/genética , Tabaco/enzimologia , Tabaco/genética , Transferases/metabolismo
4.
Cell Rep ; 33(2): 108261, 2020 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-33053347

RESUMO

The biogenesis of small extracellular vesicles (sEVs) is regulated by multiple molecular machineries generating considerably heterogeneous vesicle populations, including exosomes and non-exosomal vesicles, with distinct cargo molecules. However, the role of carbohydrate metabolism in generating such vesicle heterogeneity remains largely elusive. Here, we discover that 2-deoxyglucose (2-DG), a well-known glycolysis inhibitor, suppresses the secretion of non-exosomal vesicles by impairing asparagine-linked glycosylation (N-glycosylation) in mouse melanoma cells. Mechanistically, 2-DG is metabolically incorporated into N-glycan precursors, causing precursor degradation and partial hypoglycosylation. N-glycosylation blockade by Stt3a silencing is sufficient to inhibit non-exosomal vesicle secretion. In contrast, N-glycosylation blockade barely influences exosomal secretion of tetraspanin proteins. Functionally, N-glycosylation at specific sites of the hepatocyte growth factor receptor, a cargo protein of non-exosomal vesicles, facilitates its sorting into vesicles. These results uncover a link between N-glycosylation and unconventional vesicle secretion and suggest that N-glycosylation facilitates sEV biogenesis through cargo protein sorting.


Assuntos
Vesículas Extracelulares/metabolismo , Animais , Linhagem Celular Tumoral , Desoxiglucose/metabolismo , Dolicóis/metabolismo , Exossomos/metabolismo , Vesículas Extracelulares/ultraestrutura , Glicosilação , Lipídeos/química , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Proteínas de Membrana/metabolismo , Camundongos , Metástase Neoplásica , Proteínas Proto-Oncogênicas c-met/metabolismo
5.
Sci Rep ; 10(1): 13264, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32764679

RESUMO

The cis-polyisoprenoid lipids namely polyprenols, dolichols and their derivatives are linear polymers of several isoprene units. In eukaryotes, polyprenols and dolichols are synthesized as a mixture of four or more homologues of different length with one or two predominant species with sizes varying among organisms. Interestingly, co-occurrence of polyprenols and dolichols, i.e. detection of a dolichol along with significant levels of its precursor polyprenol, are unusual in eukaryotic cells. Our metabolomics studies revealed that cis-polyisoprenoids are more diverse in the malaria parasite Plasmodium falciparum than previously postulated as we uncovered active de novo biosynthesis and substantial levels of accumulation of polyprenols and dolichols of 15 to 19 isoprene units. A distinctive polyprenol and dolichol profile both within the intraerythrocytic asexual cycle and between asexual and gametocyte stages was observed suggesting that cis-polyisoprenoid biosynthesis changes throughout parasite's development. Moreover, we confirmed the presence of an active cis-prenyltransferase (PfCPT) and that dolichol biosynthesis occurs via reduction of the polyprenol to dolichol by an active polyprenol reductase (PfPPRD) in the malaria parasite.


Assuntos
Dolicóis/isolamento & purificação , Metabolômica/métodos , Plasmodium falciparum/crescimento & desenvolvimento , Vias Biossintéticas , Dolicóis/biossíntese , Regulação da Expressão Gênica no Desenvolvimento , Plasmodium falciparum/metabolismo , Poliprenois/isolamento & purificação , Poliprenois/metabolismo , Proteínas de Protozoários/genética
6.
J Cell Mol Med ; 24(14): 7697-7705, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32542927

RESUMO

Neurite outgrowth inhibitor-B (Nogo-B) is a membrane protein which is extensively expressed in multiple organs, especially in endothelial cells and vascular smooth muscle cells of blood vessels and belongs to the reticulon protein family. Notably, its specific receptor, Nogo-B receptor (NgBR), encoded by NUS1, has been implicated in many crucial cellular processes, such as cholesterol trafficking, lipid metabolism, dolichol synthesis, protein N-glycosylation, vascular remodelling, angiogenesis, tumorigenesis and neurodevelopment. In recent years, accumulating studies have demonstrated the statistically significant changes of NgBR expression levels in human diseases, including Niemann-Pick type C disease, fatty liver, congenital disorders of glycosylation, persistent pulmonary hypertension of the newborn, invasive ductal breast carcinoma, malignant melanoma, non-small cell lung carcinoma, paediatric epilepsy and Parkinson's disease. Besides, both the in vitro and in vivo studies have shown that NgBR overexpression or knockdown contribute to the alteration of various pathophysiological processes. Thus, there is a broad development potential in therapeutic strategies by modifying the expression levels of NgBR.


Assuntos
Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Animais , Transporte Biológico , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Colesterol/metabolismo , Suscetibilidade a Doenças , Dolicóis/biossíntese , Regulação da Expressão Gênica , Glicosilação , Humanos , Metabolismo dos Lipídeos , Sistema Nervoso/metabolismo , Proteínas Nogo/genética , Proteínas Nogo/metabolismo , Ligação Proteica , Receptores de Superfície Celular/química , Pesquisa/tendências , Transdução de Sinais
7.
Clin Chim Acta ; 507: 88-93, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32289257

RESUMO

Dolichol is a membrane lipid which carries monosaccharides and glycans for N-linked protein glycosylation occurring in the endoplasmic reticulum. Recently, some types of congenital disorders of glycosylation (CDG) have been described as consequences of defects in dolichol biosynthesis and metabolism, yet these types of CDG are not detectable by standard screening methods. The aim of this project was to evaluate the potential of dolichol as a biomarker of CDG. Biological material for this study consisted of urine samples from 75 controls, 6 patients with CDG and 43 patients with suspicion of CDG; samples of the frontal cortex, liver, muscle and heart tissues from 2 patients with mutation in the NUS1 gene and controls. Molecular species profiles of dolichol were analyzed by liquid chromatography combined with tandem mass spectrometry. In the control group, a significant correlation between the ratio of dolichol 18 to dolichol 19 (Dol18/Dol19) and age was found in urine. We established a reference range for Dol18/Dol19 from urine samples. The ratio of Dol18/Dol19 was significantly higher in both urine and tissue samples from patients with mutation in NUS1 in comparison to controls. Our results show a novel diagnostic option for patients with rare congenital disorders of glycosylation.


Assuntos
Defeitos Congênitos da Glicosilação/diagnóstico , Defeitos Congênitos da Glicosilação/metabolismo , Dolicóis/química , Dolicóis/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/química , Biomarcadores/metabolismo , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Adulto Jovem
8.
Int J Mol Sci ; 20(20)2019 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-31614738

RESUMO

The essential role of dolichyl phosphate (DolP) as a carbohydrate carrier during protein N-glycosylation is well established. The cellular pool of DolP is derived from de novo synthesis in the dolichol branch of the mevalonate pathway and from recycling of DolPP after each cycle of N-glycosylation, when the oligosaccharide is transferred from the lipid carrier to the protein and DolPP is released and then dephosphorylated. In Saccharomyces cerevisiae, the dephosphorylation of DolPP is known to be catalyzed by the Cwh8p protein. To establish the role of the Cwh8p orthologue in another distantly related yeast species, Candida albicans, we studied its mutant devoid of the CaCWH8 gene. A double Cacwh8∆/Cacwh8∆ strain was constructed by the URA-blaster method. As in S. cerevisiae, the mutant was impaired in DolPP recycling. This defect, however, was accompanied by an elevation of cis-prenyltransferase activity and higher de novo production of dolichols. Despite these compensatory changes, protein glycosylation, cell wall integrity, filamentous growth, and biofilm formation were impaired in the mutant. These results suggest that the defects are not due to the lack of DolP for the protein N-glycosylation but rather that the activity of oligosacharyltransferase could be inhibited by the excess DolPP accumulating in the mutant.


Assuntos
Candida albicans/metabolismo , Dolicóis/biossíntese , Proteínas Fúngicas/genética , Oligossacarídeos de Poli-Isoprenil Fosfato/metabolismo , Processamento de Proteína Pós-Traducional , Pirofosfatases/genética , Candida albicans/crescimento & desenvolvimento , Parede Celular/metabolismo , Dolicóis/genética , Proteínas Fúngicas/metabolismo , Glicosilação , Morfogênese , Pirofosfatases/metabolismo
9.
Molecules ; 24(15)2019 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-31370240

RESUMO

Arabidopsis roots accumulate a complex mixture of dolichols composed of three families, (i.e., short-, medium- and long-chain dolichols), but until now none of the cis-prenyltransferases (CPTs) predicted in the Arabidopsis genome has been considered responsible for their synthesis. In this report, using homo- and heterologous (yeast and tobacco) models, we have characterized the AtCPT1 gene (At2g23410) which encodes a CPT responsible for the formation of long-chain dolichols, Dol-18 to -23, with Dol-21 dominating, in Arabidopsis. The content of these dolichols was significantly reduced in AtCPT1 T-DNA insertion mutant lines and highly increased in AtCPT1-overexpressing plants. Similar to the majority of eukaryotic CPTs, AtCPT1 is localized to the endoplasmic reticulum (ER). Functional complementation tests using yeast rer2Δ or srt1Δ mutants devoid of medium- or long-chain dolichols, respectively, confirmed that this enzyme synthesizes long-chain dolichols, although the dolichol chains thus formed are somewhat shorter than those synthesized in planta. Moreover, AtCPT1 acts as a homomeric CPT and does not need LEW1 for its activity. AtCPT1 is the first plant CPT producing long-chain polyisoprenoids that does not form a complex with the NgBR/NUS1 homologue.


Assuntos
Arabidopsis/enzimologia , Raízes de Plantas/enzimologia , Terpenos/química , Transferases/química , Arabidopsis/genética , Dolicóis/química , Dolicóis/genética , Retículo Endoplasmático , Genoma de Planta/genética , Raízes de Plantas/genética , Transferases/genética
10.
Int J Mol Sci ; 20(12)2019 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-31234450

RESUMO

 Mono-saturated polyprenols (dolichols) have been found in almost all Eukaryotic cells, however, dolichols containing additional saturated bonds at the ω-end, have been identified in A. fumigatus and A. niger. Here we confirm using an LC-ESI-QTOF-MS analysis, that poly-saturated dolichols are abundant in other filamentous fungi, Trichoderma reesei, A. nidulans and Neurospora crassa, while the yeast Saccharomyces cerevisiae only contains the typical mono-saturated dolichols. We also show, using differential scanning calorimetry (DSC) and fluorescence anisotropy of 1,6-diphenyl-l,3,5-hexatriene (DPH) that the structure of dolichols modulates the properties of membranes and affects the functioning of dolichyl diphosphate mannose synthase (DPMS). The activity of this enzyme from T. reesei and S. cerevisiae was strongly affected by the structure of dolichols. Additionally, the structure of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) model membranes was more strongly disturbed by the poly-saturated dolichols from Trichoderma than by the mono-saturated dolichols from yeast. By comparing the lipidome of filamentous fungi with that from S. cerevisiae, we revealed significant differences in the PC/PE ratio and fatty acids composition. Filamentous fungi differ from S. cerevisiae in the lipid composition of their membranes and the structure of dolichols. The structure of dolichols profoundly affects the functioning of dolichol-dependent enzyme, DPMS.


Assuntos
Dolicóis/metabolismo , Proteínas Fúngicas/metabolismo , Fungos/metabolismo , Glicosiltransferases/metabolismo , Aspergillus niger/química , Aspergillus niger/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Dolicóis/análise , Fungos/química , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Modelos Moleculares , Neurospora crassa/química , Neurospora crassa/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Trichoderma/química , Trichoderma/metabolismo
11.
Prensa méd. argent ; 105(4): 214-227, jun 2019. tab, fig
Artigo em Inglês | LILACS, BINACIS | ID: biblio-1045983

RESUMO

The prescription composition of the biologically active dietary supplement (BADS) Oleopren Neuro has been scientifically substantiated through the pharmacological evaluation of the effective agents, polyprenols being the main of them. The technology of an innovative product has been developed, with the establishment of adjustable production parameters securing high organoleptic advantages and stability of the BADS. Regulated indicators of quality and nutritional value have been determined. Sanitary toxicological and hygienic safety indicators of the product under development had been studied, which allowed to establish a shelf life of two years taking the safety margin of three months into account. The possible mechanisms of the dolichols effect on the metabolic status of the organism were considered. Clinical trials of the efficiency and functional focus of a specialized product on a representative group of patients with the dyscirculatory encephalopathy (DE) of vascular genesis stages I ­ II were conducted. The BADS were included in the diet of patients, along with the basic therapy, one capsule twice a day for 10 days. The materials obtained in this article allow to recommend the developed product as an efficient means of increasing the body's resistance to adverse effects of the environment, stressful situations, as well as psychoemotional and physical stress.


Assuntos
Suplementos Nutricionais , Dolicóis/uso terapêutico , Produção de Produtos , Vigilância Sanitária de Produtos , Doenças do Sistema Nervoso/terapia , Valor Nutritivo
12.
Mol Metab ; 24: 1-17, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31003944

RESUMO

OBJECTIVE: Aging is accompanied by loss of brown adipocytes and a decline in their thermogenic potential, which may exacerbate the development of adiposity and other metabolic disorders. Presently, only limited evidence exists describing the molecular alterations leading to impaired brown adipogenesis with aging and the contribution of these processes to changes of systemic energy metabolism. METHODS: Samples of young and aged murine brown and white adipose tissue were used to compare age-related changes of brown adipogenic gene expression and thermogenesis-related lipid mobilization. To identify potential markers of brown adipose tissue aging, non-targeted proteomic and metabolomic as well as targeted lipid analyses were conducted on young and aged tissue samples. Subsequently, the effects of several candidate lipid classes on brown adipocyte function were examined. RESULTS: Corroborating previous reports of reduced expression of uncoupling protein-1, we observe impaired signaling required for lipid mobilization in aged brown fat after adrenergic stimulation. Omics analyses additionally confirm the age-related impairment of lipid homeostasis and reveal the accumulation of specific lipid classes, including certain sphingolipids, ceramides, and dolichols in aged brown fat. While ceramides as well as enzymes of dolichol metabolism inhibit brown adipogenesis, inhibition of sphingosine 1-phosphate receptor 2 induces brown adipocyte differentiation. CONCLUSIONS: Our functional analyses show that changes in specific lipid species, as observed during aging, may contribute to reduced thermogenic potential. They thus uncover potential biomarkers of aging as well as molecular mechanisms that could contribute to the degradation of brown adipocytes, thereby providing potential treatment strategies of age-related metabolic conditions.


Assuntos
Adipócitos Marrons/metabolismo , Envelhecimento/metabolismo , Metabolismo dos Lipídeos , Adipócitos Marrons/citologia , Animais , Biomarcadores/metabolismo , Células Cultivadas , Ceramidas/metabolismo , Dolicóis/metabolismo , Masculino , Metaboloma , Camundongos , Camundongos Endogâmicos C57BL , Proteoma/genética , Proteoma/metabolismo , Esfingolipídeos/metabolismo
13.
Glycobiology ; 28(10): 741-753, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-29939232

RESUMO

In eukaryotes, the biosynthesis of a highly conserved dolichol-linked oligosaccharide (DLO) precursor Glc3Man9GlcNAc2-pyrophosphate-dolichol (PP-Dol) begins on the cytoplasmic face of the endoplasmic reticulum (ER) and ends within the lumen. Two functionally distinguished heteromeric glycosyltransferase (GTase) complexes are responsible for the cytosolic DLO assembly. Alg1, a ß-1, 4 mannosyltransferase (MTase) physically interacts with Alg2 and Alg11 proteins to form the multienzyme complex which catalyzes the addition of all five mannose to generate the Man5GlcNAc2-PP-Dol intermediate. Despite the fact that Alg1 plays a central role in the formation of the multi-MTase has been confirmed, the topological information of Alg1 including the molecular mechanism of membrane association are still poorly understood. Using a combination of bioinformatics and biological approaches, we have undertaken a structural and functional study on Alg1 protein, in which the enzymatic activities of Alg1 and its variants were monitored by a complementation assay using the GALpr-ALG1 yeast strain, and further confirmed by a liquid chromatography-mass spectrometry-based in vitro quantitative assay. Computational and experimental evidence confirmed Alg1 shares structure similarity with Alg13/14 complex, which has been defined as a membrane-associated GT-B GTase. Particularly, we provide clear evidence that the N-terminal transmembrane domain including the following positively charged amino acids and an N-terminal amphiphilic-like α helix domain exposed on the protein surface strictly coordinate the Alg1 orientation on the ER membrane. This work provides detailed membrane topology of Alg1 and further reveals its biological importance at the spatial aspect in coordination of cytosolic DLO biosynthesis.


Assuntos
Membrana Celular/metabolismo , Dolicóis/biossíntese , Manosiltransferases/metabolismo , Oligossacarídeos/biossíntese , Saccharomyces cerevisiae/metabolismo , Membrana Celular/química , Dolicóis/química , Manosiltransferases/química , Manosiltransferases/genética , Oligossacarídeos/química , Conformação Proteica , Saccharomyces cerevisiae/citologia
14.
Biosci Biotechnol Biochem ; 82(6): 947-955, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29297247

RESUMO

The reduction pathway leading to the formation of dolichol was clarified in 2010 with the identification of SRD5A3, which is the polyprenol reductase. The finding inspired us to reanalyze the length of the major chain of polyprenol and dolichol from several plant leaves, including mangrove plants, as well as from animal and fish livers by 2D-TLC. Polyprenol- and dolichol-derived metabolites such as polyprenylacetone and epoxydolichol were found together with rubber-like prenol. This review focuses on analyses of polyprenol and its derivatives, including recently found epoxypolyprenol and polyprenylacetone. Attention has also been paid to the chromatographic behavior of rubber-like prenol on TLC.


Assuntos
Pentanóis/metabolismo , Animais , Cromatografia em Camada Delgada , Dolicóis/metabolismo , Hemiterpenos , Fígado/metabolismo , Folhas de Planta/metabolismo
15.
Biochim Biophys Acta Bioenerg ; 1859(1): 1-7, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29031613

RESUMO

Missense mutations of the human mitochondrial citrate carrier, encoded by the SLC25A1 gene, lead to an autosomal recessive neurometabolic disorder characterised by neonatal-onset encephalopathy with severe muscular weakness, intractable seizures, respiratory distress, and lack of psychomotor development, often resulting in early death. Here, we have measured the effect of all twelve known pathogenic mutations on the transport activity. The results show that nine mutations abolish transport of citrate completely, whereas the other three reduce the transport rate by >70%, indicating that impaired citrate transport is the most likely primary cause of the disease. Some mutations may be detrimental to the structure of the carrier, whereas others may impair key functional elements, such as the substrate binding site and the salt bridge network on the matrix side of the carrier. To understand the consequences of impaired citrate transport on metabolism, the substrate specificity was also determined, showing that the human citrate carrier predominantly transports citrate, isocitrate, cis-aconitate, phosphoenolpyruvate and malate. Although D-2- and L-2 hydroxyglutaric aciduria is a metabolic hallmark of the disease, it is unlikely that the citrate carrier plays a significant role in the removal of hydroxyglutarate from the cytosol for oxidation to oxoglutarate in the mitochondrial matrix. In contrast, computer simulations of central metabolism predict that the export of citrate from the mitochondrion cannot be fully compensated by other pathways, restricting the cytosolic production of acetyl-CoA that is required for the synthesis of lipids, sterols, dolichols and ubiquinone, which in turn explains the severe disease phenotypes.


Assuntos
Proteínas de Transporte de Ânions , Ácido Cítrico/metabolismo , Simulação por Computador , Dolicóis , Proteínas Mitocondriais , Modelos Biológicos , Mutação de Sentido Incorreto , Esteróis , Ubiquinona , Proteínas de Transporte de Ânions/química , Proteínas de Transporte de Ânions/genética , Proteínas de Transporte de Ânions/metabolismo , Transporte Biológico Ativo/genética , Encefalopatias Metabólicas Congênitas/enzimologia , Encefalopatias Metabólicas Congênitas/genética , Domínio Catalítico , Dolicóis/biossíntese , Dolicóis/química , Dolicóis/genética , Humanos , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Transportadores de Ânions Orgânicos , Esteróis/biossíntese , Esteróis/química , Esteróis/metabolismo , Ubiquinona/biossíntese , Ubiquinona/química , Ubiquinona/genética
16.
Genetics ; 207(4): 1371-1386, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28978675

RESUMO

Dolichols are isoprenoid lipids of varying length that act as sugar carriers in glycosylation reactions in the endoplasmic reticulum. In Saccharomyces cerevisiae, there are two cis-prenyltransferases that synthesize polyprenol-an essential precursor to dolichol. These enzymes are heterodimers composed of Nus1 and either Rer2 or Srt1. Rer2-Nus1 and Srt1-Nus1 can both generate dolichol in vegetative cells, but srt1∆ cells grow normally while rer2∆ grows very slowly, indicating that Rer2-Nus1 is the primary enzyme used in mitotically dividing cells. In contrast, SRT1 performs an important function in sporulating cells, where the haploid genomes created by meiosis are packaged into spores. The spore wall is a multilaminar structure and SRT1 is required for the generation of the outer chitosan and dityrosine layers of the spore wall. Srt1 specifically localizes to lipid droplets associated with spore walls, and, during sporulation there is an SRT1-dependent increase in long-chain polyprenols and dolichols in these lipid droplets. Synthesis of chitin by Chs3, the chitin synthase responsible for chitosan layer formation, is dependent on the cis-prenyltransferase activity of Srt1, indicating that polyprenols are necessary to coordinate assembly of the spore wall layers. This work shows that a developmentally regulated cis-prenyltransferase can produce polyprenols that function in cellular processes besides protein glycosylation.


Assuntos
Alquil e Aril Transferases/genética , Quitina Sintase/genética , Dolicóis/genética , Proteínas de Saccharomyces cerevisiae/genética , Esporos Fúngicos/genética , Parede Celular/genética , Quitina/biossíntese , Quitina/genética , Quitosana/química , Quitosana/metabolismo , Dimetilaliltranstransferase/genética , Dolicóis/biossíntese , Retículo Endoplasmático/genética , Haploidia , Meiose/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Esporos Fúngicos/crescimento & desenvolvimento , Tretinoína/análogos & derivados , Tretinoína/metabolismo
17.
Plant Physiol ; 174(2): 857-874, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28385729

RESUMO

The cooperation of the mevalonate (MVA) and methylerythritol phosphate (MEP) pathways, operating in parallel in plants to generate isoprenoid precursors, has been studied extensively. Elucidation of the isoprenoid metabolic pathways is indispensable for the rational design of plant and microbial systems for the production of industrially valuable terpenoids. Here, we describe a new method, based on numerical modeling of mass spectra of metabolically labeled dolichols (Dols), designed to quantitatively follow the cooperation of MVA and MEP reprogrammed upon osmotic stress (sorbitol treatment) in Arabidopsis (Arabidopsis thaliana). The contribution of the MEP pathway increased significantly (reaching 100%) exclusively for the dominating Dols, while for long-chain Dols, the relative input of the MEP and MVA pathways remained unchanged, suggesting divergent sites of synthesis for dominating and long-chain Dols. The analysis of numerically modeled Dol mass spectra is a novel method to follow modulation of the concomitant activity of isoprenoid-generating pathways in plant cells; additionally, it suggests an exchange of isoprenoid intermediates between plastids and peroxisomes.


Assuntos
Arabidopsis/metabolismo , Dolicóis/química , Modelos Teóricos , Espectrometria de Massas por Ionização por Electrospray/métodos , Terpenos/metabolismo , Isótopos de Carbono , Cromatografia Gasosa/métodos , Dolicóis/metabolismo , Eritritol/análogos & derivados , Eritritol/metabolismo , Marcação por Isótopo/métodos , Redes e Vias Metabólicas , Ácido Mevalônico/análogos & derivados , Ácido Mevalônico/química , Ácido Mevalônico/metabolismo , Pressão Osmótica , Fitosteróis/biossíntese , Sorbitol/metabolismo , Fosfatos Açúcares/metabolismo , Xilulose/análogos & derivados , Xilulose/química
18.
Eur J Med Chem ; 125: 952-964, 2017 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-27769035

RESUMO

Citronellyl- and solanesyl-based dolichol linked oligosaccharide (DLO) analogs were synthesized and tested along with undecaprenyl compounds for their ability to inhibit the release of [3H]OSP from [3H]DLO by mammalian liver DLO diphosphatase activity. Solanesyl (C45) and undecaprenyl (C55) compounds were 50-500 fold more potent than their citronellyl (C10)-based counterparts, indicating that the alkyl chain length is important for activity. The relative potency of the compounds within the citronellyl series was different to that of the solanesyl series with citronellyl diphosphate being 2 and 3 fold more potent than citronellyl-PP-GlcNAc2 and citronellyl-PP-GlcNAc, respectively; whereas solanesyl-PP-GlcNAc and solanesyl-PP-GlcNAc2 were 4 and 8 fold more potent, respectively, than solanesyl diphosphate. Undecaprenyl-PP-GlcNAc and bacterial Lipid II were 8 fold more potent than undecaprenyl diphosphate at inhibiting the DLODP assay. Therefore, at least for the more hydrophobic compounds, diphosphodiesters are more potent inhibitors of the DLODP assay than diphosphomonoesters. These results suggest that DLO rather than dolichyl diphosphate might be a preferred substrate for the DLODP activity.


Assuntos
Dolicóis/química , Oligossacarídeos/química , Animais , Fosfatos de Dolicol , Humanos , Fígado/enzimologia , Monoterpenos , Diester Fosfórico Hidrolases/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Açúcares de Poli-Isoprenil Fosfato , Fosfatos de Poli-Isoprenil , Especificidade por Substrato
19.
Am J Med Genet A ; 170(12): 3165-3171, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27480077

RESUMO

Increasing numbers of congenital disorders of glycosylation (CDG) have been reported recently resulting in an expansion of the phenotypes associated with this group of disorders. SRD5A3 codes for polyprenol reductase which converts polyprenol to dolichol. This is a major pathway for dolichol biosynthesis for N-glycosylation, O-mannosylation, C-mannosylation, and GPI anchor synthesis. We present the features of five individuals (three children and two adults) with mutations in SRD5A3 focusing on the variable eye and skin involvement. We compare that to 13 affected individuals from the literature including five adults allowing us to delineate the features that may develop over time with this disorder including kyphosis, retinitis pigmentosa, and cataracts. © 2016 Wiley Periodicals, Inc.


Assuntos
3-Oxo-5-alfa-Esteroide 4-Desidrogenase/genética , Defeitos Congênitos da Glicosilação/genética , Olho/fisiopatologia , Proteínas de Membrana/genética , Pele/fisiopatologia , Adulto , Criança , Defeitos Congênitos da Glicosilação/fisiopatologia , Dolicóis/metabolismo , Feminino , Glicosilação , Homozigoto , Humanos , Masculino , Mutação , Fenótipo , Tretinoína/análogos & derivados , Tretinoína/metabolismo
20.
Methods Enzymol ; 576: 121-45, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27480685

RESUMO

Dolichol and natural rubber are representative cis-polyisoprenoids in primary and secondary metabolism, respectively. Their biosynthesis is catalyzed by cis-prenyltransferase (CPT) by sequential condensations of isopentenyl diphosphates (IPPs) to a priming molecule. Although prokaryotic CPTs have been well characterized, the mechanism of eukaryotic CPTs in cis-polyisoprene biosynthesis was only recently revealed. It was shown that eukaryotes have evolved a unique protein complex, comprised of CPT and CPT-binding protein (CBP), to synthesize cis-polyisoprenoids. In the context of this new discovery, we found discrepancies in literature for CPT or CBP biochemical assays and in vivo CPT complementation using rer2 (yeast CPT) yeast mutant. Our study here shows that rer2 revertants occur at a frequency that cannot be disregarded and are likely accountable for the results that cannot be explained by the CPT/CBP heteroprotein complex model. To make a stable mutant, SRT1 gene (secondary CPT expressed at a basal level in yeast) was additionally deleted in the rer2Δ mutant background. This stable rer2Δ srt1Δ strain was then used to individually or simultaneously express Arabidopsis CPT1 (AtCPT1, At2g17570) and CBP (AtLEW1, At1G11755). We found that the simultaneous expression of Arabidopsis CPT1 and AtLEW1 effectively complements the rer2Δ srt1Δ strain, whereas the individual expression of AtCPT1 alone or AtLEW1 alone failed to rescue the yeast mutant. Microsomes from the dual expresser showed an efficient incorporation of IPPs into cis-polyisoprenoid (30% in 2h). These results showed that the CPT/CBP heteroprotein complex model is valid in Arabidopsis thaliana. Experimental details of these results are described in this methodology paper.


Assuntos
Alquil e Aril Transferases/genética , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Dimetilaliltranstransferase/genética , Dolicóis/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Transferases/genética , Alquil e Aril Transferases/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Vias Biossintéticas , Dimetilaliltranstransferase/metabolismo , Dolicóis/genética , Técnicas de Silenciamento de Genes , Hemiterpenos/genética , Hemiterpenos/metabolismo , Mutação , Compostos Organofosforados/metabolismo , Borracha/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Metabolismo Secundário , Transferases/metabolismo
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