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Plant, Cell & Environment Feb 2022Dolichols (Dols), ubiquitous components of living organisms, are indispensable for cell survival. In plants, as well as other eukaryotes, Dols are crucial for...
Dolichols (Dols), ubiquitous components of living organisms, are indispensable for cell survival. In plants, as well as other eukaryotes, Dols are crucial for post-translational protein glycosylation, aberration of which leads to fatal metabolic disorders in humans and male sterility in plants. Until now, the mechanisms underlying Dol accumulation remain elusive. In this study, we have analysed the natural variation of the accumulation of Dols and six other isoprenoids among more than 120 Arabidopsis thaliana accessions. Subsequently, by combining QTL and GWAS approaches, we have identified several candidate genes involved in the accumulation of Dols, polyprenols, plastoquinone and phytosterols. The role of two genes implicated in the accumulation of major Dols in Arabidopsis-the AT2G17570 gene encoding a long searched for cis-prenyltransferase (CPT3) and the AT1G52460 gene encoding an α/β-hydrolase-is experimentally confirmed. These data will help to generate Dol-enriched plants which might serve as a remedy for Dol-deficiency in humans.
Topics: Arabidopsis; Arabidopsis Proteins; Dolichols; Hydrolases; Transferases
PubMed: 34778961
DOI: 10.1111/pce.14223 -
Journal of Lipid Research Dec 2020HMG-CoA reductase (Hmgcr) is the rate-limiting enzyme in the mevalonate pathway and is inhibited by statins. In addition to cholesterol, Hmgcr activity is also required...
HMG-CoA reductase (Hmgcr) is the rate-limiting enzyme in the mevalonate pathway and is inhibited by statins. In addition to cholesterol, Hmgcr activity is also required for synthesizing nonsterol isoprenoids, such as dolichol, ubiquinone, and farnesylated and geranylgeranylated proteins. Here, we investigated the effects of Hmgcr inhibition on nonsterol isoprenoids in the liver. We have generated new genetic models to acutely delete genes in the mevalonate pathway in the liver using AAV-mediated delivery of Cre-recombinase (AAV-) or CRISPR/Cas9 (AAV-CRISPR). The genetic deletion of by AAV- resulted in extensive hepatocyte apoptosis and compensatory liver regeneration. At the biochemical level, we observed decreased levels of sterols and depletion of the nonsterol isoprenoids, dolichol and ubiquinone. At the cellular level, -null hepatocytes showed ER stress and impaired N-glycosylation. We further hypothesized that the depletion of dolichol, essential for N-glycosylation, could be responsible for ER stress. Using AAV-CRISPR, we somatically disrupted dehydrodolichyl diphosphate synthase subunit (), encoding a branch point enzyme required for dolichol biosynthesis. -null livers showed ER stress and impaired N-glycosylation, along with apoptosis and regeneration. Finally, the combined deletion of and synergistically exacerbated hepatocyte ER stress. Our data show a critical role for mevalonate-derived dolichol in the liver and suggest that dolichol depletion is at least partially responsible for ER stress and apoptosis upon potent Hmgcr inhibition.
Topics: Endoplasmic Reticulum Stress; Gene Deletion; Hydroxymethylglutaryl CoA Reductases; Liver; Terpenes
PubMed: 33109681
DOI: 10.1194/jlr.RA120001006 -
Glycobiology Dec 2021Although Halobacterium salinarum provided the first example of N-glycosylation outside the Eukarya, much regarding such post-translational modification in this...
Although Halobacterium salinarum provided the first example of N-glycosylation outside the Eukarya, much regarding such post-translational modification in this halophilic archaea remains either unclear or unknown. The composition of an N-linked glycan decorating both the S-layer glycoprotein and archaellins offers one such example. Originally described some 40 years ago, reports from that time on have presented conflicted findings regarding the composition of this glycan, as well as differences between the protein-bound glycan and that version of the glycan attached to the lipid upon which it is assembled. To clarify these points, liquid chromatography-electrospray ionization mass spectrometry was employed here to revisit the composition of this glycan both when attached to selected asparagine residues of target proteins and when bound to the lipid dolichol phosphate upon which the glycan is assembled. Such efforts revealed the N-linked glycan as corresponding to a tetrasaccharide comprising a hexose, a sulfated hexuronic acid, a hexuronic acid and a second sulfated hexuronic acid. When attached to dolichol phosphate but not to proteins, the same tetrasaccharide is methylated on the final sugar. Moreover, in the absence of the oligosaccharyltransferase AglB, there is an accumulation of the dolichol phosphate-linked methylated and disulfated tetrasaccharide. Knowing the composition of this glycan at both the lipid- and protein-bound stages, together with the availability of gene deletion approaches for manipulating Hbt. salinarum, will allow delineation of the N-glycosylation pathway in this organism.
Topics: Dolichol Phosphates; Dolichols; Glycoproteins; Glycosylation; Halobacterium salinarum; Haloferax volcanii; Phosphates; Spectrometry, Mass, Electrospray Ionization
PubMed: 34314490
DOI: 10.1093/glycob/cwab080 -
Memorias Do Instituto Oswaldo Cruz Aug 2011The development of new drugs is one strategy for malaria control. Biochemical pathways localised in the apicoplast of the parasite, such as the synthesis of isoprenic... (Review)
Review
The development of new drugs is one strategy for malaria control. Biochemical pathways localised in the apicoplast of the parasite, such as the synthesis of isoprenic precursors, are excellent targets because they are different or absent in the human host. Isoprenoids are a large and highly diverse group of natural products with many functions and their synthesis is essential for the parasite's survival. During the last few years, the genes, enzymes, intermediates and mechanisms of this biosynthetic route have been elucidated. In this review, we comment on some aspects of the methylerythritol phosphate pathway and discuss the presence of diverse isoprenic products such as dolichol, ubiquinone, carotenoids, menaquinone and isoprenylated proteins, which are biosynthesised during the intraerythrocytic stages of Plasmodium falciparum.
Topics: Carotenoids; Dolichols; Erythrocytes; Humans; Plasmodium falciparum; Protein Prenylation; Terpenes; Ubiquinone; Vitamin K 2
PubMed: 21881768
DOI: 10.1590/s0074-02762011000900018 -
The Biochemical Journal Sep 1980A derivative of dolichol was formed and then chemically degraded to a small fragment containing the sole centre of asymmetry of the original molecule. Polarimetric...
A derivative of dolichol was formed and then chemically degraded to a small fragment containing the sole centre of asymmetry of the original molecule. Polarimetric comparison of this derivative with a standard prepared from (R)-citronellol showed dolichol to have an S-configuration at C-3. To determine the optical purity of dolichol a diastereoisomeric derivative was prepared and compared with standard diastereoisomers, which could be resolved by high-pressure liquid chromatography. Dolichols from pig liver, human liver and hen oviduct were analysed by this procedure and were all found to be greater than 95% S-configuration.
Topics: Aldehydes; Chromatography, High Pressure Liquid; Diterpenes; Dolichols; Molecular Conformation; Optical Rotatory Dispersion; Stereoisomerism
PubMed: 7213338
DOI: 10.1042/bj1890441 -
Biological & Pharmaceutical Bulletin Mar 2008We examined the correlations between serum dolichol levels and laboratory test parameters in patients affected by disease, as well as the distribution of dolichol in...
We examined the correlations between serum dolichol levels and laboratory test parameters in patients affected by disease, as well as the distribution of dolichol in sera from patients with hyperbetalipoproteinemia and hyperalphalipoproteinemia. Serum dolichol was evaluated by a reverse-phase HPLC method. After centrifugation, the serum dolichol found in healthy controls was mainly associated with medium-sized particles of the high-density lipoprotein (HDL) fraction. For patients with hyperbetalipoproteinemia, serum dolichol was also associated with the medium HDL fractions. However, for hyperalphalipoproteinemia patients the levels of large HDL and serum dolichol were increased, and serum dolichol was mainly associated with the large HDL fraction. On laboratory tests of components, the dolichol level was not correlated with the values for markers of the liver and biliary system, with the values of renal function markers, with creatine kinase activity, amylase activity or uric acid concentration, but was correlated with total cholesterol, HDL-cholesterol and apoA-I concentrations, and with lactate dehydrogenase (LDH) activity. These results suggest that serum dolichol exclusively localized in HDL, and in subpopulation, that in normocholesterolemia or hyperbeta-cholesterolemia is associated with HDL(3), which is small sized and high density HDL, however, that in hyperalphacholesterolemia is associated with HDL(2), which is large sized and lower density HDL.
Topics: Apolipoproteins; Chromatography, High Pressure Liquid; Dolichols; Female; Humans; Hyperlipoproteinemias; Lipoproteins, HDL; Lipoproteins, LDL; Male; Middle Aged; Triglycerides
PubMed: 18310889
DOI: 10.1248/bpb.31.340 -
Methods in Enzymology 2011Glycosylation is a complex form of protein modification occurring in the secretory pathway. The addition of N- and O-glycans affects intracellular processes like the...
Glycosylation is a complex form of protein modification occurring in the secretory pathway. The addition of N- and O-glycans affects intracellular processes like the folding and trafficking of most glycoproteins. To better understand the impact of glycosylation in protein folding and maturation, parameters like glycosylation site occupancy and oligosaccharide structure must be measured quantitatively. In this chapter, we describe current methods enabling the determination of N-glycosylation by assessment of cellular dolichol phosphate levels, dolichol-linked oligosaccharides, and the occupancy of N-glycosylation sites. We also provide detailed methods for the analysis of O-glycosylation, whose role in intracellular protein maturation is often overlooked.
Topics: Animals; Chromatography, High Pressure Liquid; Dolichol Phosphates; Dolichols; Glycoproteins; Glycosylation; Humans; Oligosaccharides; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Unfolded Protein Response
PubMed: 21329800
DOI: 10.1016/B978-0-12-385928-0.00010-9 -
The Journal of Biological Chemistry Mar 1985The effect of dolichols, polyprenols, dolichol esterified with fatty acids, and dolichyl phosphate on the structure and fluidity of model membranes was studied using 31P...
The effect of dolichols, polyprenols, dolichol esterified with fatty acids, and dolichyl phosphate on the structure and fluidity of model membranes was studied using 31P NMR, small-angle x-ray scattering, differential scanning calorimetry, and freeze-fracture electron microscopy. These studies suggest that dolichol and dolichol derivatives destabilize unsaturated phosphatidylethanolamine containing bilayer structures and promote hexagonal II phase formation; high concentrations of dolichol induce lipid structures characterized by "isotropic" 31P NMR and particulate fracture faces; dolichol, contrary to cholesterol, has no effect on the thermotropic behavior of membranes consisting of phosphatidylcholine, while dolichyl-P incorporation abolishes the transition from the gel to liquid crystalline phase in 1,2-dimyristoyl-sn-glycero-3-phosphocholine; both dolichol and dolichyl-P increase the fatty acid fluidity in phosphatidylethanolamine mixtures; the effect of dolichol on bilayer structure and fluidity is more pronounced with increasing number of isoprene residues; dolichol esters are only soluble to a limited extent in the bilayer and segregates into domains at low concentrations; the results are consistent with a localization of dolichyl-P in which the phosphate group is oriented to the water interphase. The induction of hexagonal II phase by dolichyl-P may elicit the transmembrane movement of glycosylated lipid intermediate.
Topics: Calorimetry, Differential Scanning; Diterpenes; Dolichol Phosphates; Dolichols; Esters; Fatty Acids; Freeze Fracturing; Humans; Magnetic Resonance Spectroscopy; Membrane Fluidity; Membrane Lipids; Membranes, Artificial; Microscopy, Electron; Models, Molecular; Phospholipids; Polyisoprenyl Phosphates; Scattering, Radiation; Terpenes
PubMed: 3919007
DOI: No ID Found -
Scientific Reports Aug 2020The cis-polyisoprenoid lipids namely polyprenols, dolichols and their derivatives are linear polymers of several isoprene units. In eukaryotes, polyprenols and dolichols...
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.
Topics: Biosynthetic Pathways; Dolichols; Gene Expression Regulation, Developmental; Metabolomics; Plasmodium falciparum; Polyprenols; Protozoan Proteins
PubMed: 32764679
DOI: 10.1038/s41598-020-70246-0 -
The Journal of Biological Chemistry Oct 1985Dolichol phosphate-mannose (dol-P-mannose) has been shown previously to stimulate the reaction: dolichol phosphate + UDP-[3H]GlcNAc----[3H]GlcNAc-P-P-polyprenols (Kean,... (Comparative Study)
Comparative Study
Dolichol phosphate-mannose (dol-P-mannose) has been shown previously to stimulate the reaction: dolichol phosphate + UDP-[3H]GlcNAc----[3H]GlcNAc-P-P-polyprenols (Kean, E. L. (1982) J. Biol. Chem. 257, 7952-7954). Further studies on this phenomenon are described, using microsomes from the retina of the embryonic chick as the major source of enzyme. Neither dolichol-P-glucose nor retinyl-P-mannose showed this stimulatory activity. Phosphatidylglycerol also stimulated this same process and was most active among a variety of phospholipids which were tested, in accord with previous reports. The presence of GDP-2-deoxy-2-fluoro-D-mannose or GTP had no effect on the reaction. The apparent activation constant for dolichol-P-mannose was 2.2 microM, and for phosphatidylglycerol, 401 microM. The major product (90% or greater) obtained under basal and stimulatory conditions was GlcNAc-P-P-dolichol and the site of the stimulatory effect was the glucosaminyltransferase catalyzing the formation of this compound. The effects of stimulation on the kinetic properties were similar for both activators: increases in the Vmax of the reactions of 7-10-fold; increases in apparent Km for UDP-GlcNAc of 5-7-fold; a 3-fold decrease in apparent Km for dolichol-phosphate. When present together, a mutual inhibition of stimulation was observed compared to the additive effect from dol-P-mannose or phosphatidylglycerol alone. Although a substrate for the reaction, dolichol phosphate repressed the stimulation by dolichol-P-mannose but not that by phosphatidylglycerol. Dol-P-glucose, while not an activator of the reaction, acted as a negative modifier of the stimulation by dol-P-mannose by acting as a competitive inhibitor of the stimulation. The stimulatory phenomenon was observed in microsomes prepared from a variety of tissues from the embryonic chick and from postnatal tissue after partial delipidation. The addition of pyrophosphatase inhibitors did not bring about stimulation of GlcNAc-lipid synthesis, but did enhance the effect. These studies extend the previous observations of the participation of dolichol-P-mannose and phosphatidylglycerol as allosteric activators of GlcNAc-lipid synthesis and indicate additional aspects of metabolic regulation of the dolichol pathway.
Topics: Animals; Brain; Chick Embryo; Dolichol Monophosphate Mannose; Dolichol Phosphates; Dose-Response Relationship, Drug; Guanosine Diphosphate Mannose; Kinetics; Microsomes; Phosphatidylglycerols; Phospholipids; Polyisoprenyl Phosphate Monosaccharides; Polyisoprenyl Phosphate Sugars; Rats; Retina; Uridine Diphosphate N-Acetylglucosamine
PubMed: 2413026
DOI: No ID Found