Geranylgeranyl pyrophosphate is crucial for neuronal survival but has no special role in Purkinje cell degeneration in Niemann Pick type C1 disease

Niemann Pick type C (NPC1) is a rare fatal hereditary cholesterol storage disease associated with a massive Purkinje cells loss. The mechanisms leading to neurodegeneration are still poorly understood. Different laboratories pointed to hypersensitivity to cytotoxic effects of statins (HMG‐CoA reductase inhibitors) in NPC1 and suggested an underlying lack of geranylgeranyl pyrophosphate (GGPP). GGPP is a non‐sterol isoprenoid essential for cell survival and differentiation. We measured GGPP levels in cerebella of a NPC1 mouse model and of wild‐type littermates and found a physiological increase of GGPP levels between post‐natal days 21 and 49 in wild‐type mice but not in NPC mice. This further supports the hypothesis that Purkinje cell loss may be due to an extremely low level of GGPP. The progressive Purkinje cell loss in NPC starts between p21 and p49. To test the hypothesis, we used long‐term organotypic slice cultures of NPC1 mice that display the natural history of NPC1 disease in vitro and tested if chronic administration of GGPP might prevent Purkinje cell loss. We did not see a beneficial effect. This suggests, in contrast to the expectations, that the relative lack of GGPP may not significantly contribute to mechanisms of Purkinje cell loss in NPC1.

     

Lycopene regulation of cholesterol synthesis and efflux in human macrophages

Hypercholesterolemia is one of the most important risk factors for atherosclerosis, and tomato lycopene has been suggested to have beneficial effects against such a disease, although the exact molecular mechanism is unknown. We tested the hypothesis that lycopene may exert its antiatherogenic role through changes in cholesterol metabolism. Incubation of THP-1 cells with lycopene (0.5–2 μM) dose-dependently reduced intracellular total cholesterol. Such an effect was associated with a decrease in reduction of 3-hydroxy-3-methylglutaryl coenzyme A reductase expression and with an increase in ABCA1 and caveolin-1 (cav-1) expressions. In addition, lycopene enhanced RhoA levels in the cytosolic fraction, activating peroxisome proliferator-activated receptor gamma (PPARγ) and liver X receptor alpha expressions. Concomitant addition of lycopene and the PPARγ inhibitor GW9662 or lycopene and mevalonate blocked the carotenoid-induced increase in ABCA1 and cav-1 expressions. These results imply a potential role of lycopene in attenuating foam cell formation and, therefore, in preventing atherosclerosis by a cascade mechanism involving inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase, RhoA inactivation and subsequent increase in PPARγ and liver X receptor alpha activities and enhancement of ABCA1 and cav-1 expressions.     

Inactivation of the geranylgeranyl reductase (ChlP) gene in the cyanobacterium Synechocystis sp. PCC 6803

Geranylgeranyl reductase catalyses the reduction of geranylgeranyl pyrophosphate to phytyl pyrophosphate required for synthesis of chlorophylls, phylloquinone and tocopherols. The gene chlP (ORF sll1091) encoding the enzyme has been inactivated in the cyanobacterium Synechocystis sp. PCC 6803. The resulting ΔchlP mutant accumulates exclusively geranylgeranylated chlorophyll a instead of its phytylated analogue as well as low amounts of α-tocotrienol instead of α-tocopherol. Whereas the contents of chlorophyll and total carotenoids are decreased, abundance of phycobilisomes is increased in ΔchlP cells. The mutant assembles functional photosystems I and II as judged from 77 K fluorescence and electron transport measurements. However, the mutant is unable to grow photoautotrophically due to instability and rapid degradation of the photosystems in the absence of added glucose. We suggest that instability of the photosystems in ΔchlP is directly related to accumulation of geranylgeranylated chlorophyll a. Increased rigidity of the chlorophyll isoprenoid tail moiety due to three additional CC bonds is the likely cause of photooxidative stress and reduced stability of photosynthetic pigment-protein complexes assembled with geranylgeranylated chlorophyll a in the ΔchlP mutant.     

The first prenylation step in hyperforin biosynthesis

Prenylation reactions contribute considerably to the diversity of natural products. Polyprenylated secondary metabolites include hyperforin which is both quantitatively and pharmacologically a major constituent of the medicinal plant Hypericum perforatum (St. John's wort). Cell cultures of the related species Hypericum calycinum were found to contain a prenyltransferase activity which is likely to catalyze the first prenylation step in hyperforin biosynthesis. The enzyme was soluble and dependent on a divalent cation, with Fe2+ leading to maximum activity (Km=3.8 mM). The preferred prenyl donor was DMAPP (Km=0.46 mM) and the preferred prenyl acceptor was phlorisobutyrophenone (Km=0.52 mM). A broad pH optimum from 6.5 to 8.5 and a temperature optimum from 35 to 40 degrees C were observed. The formation of hyperforins in H. calycinum cell cultures was preceded by an increase in dimethylallyltransferase activity, with the maximum specific activity being 3.6 microkat/kg protein.     

Distribution, metabolism and function of dolichol and polyprenols

Polyisoprenoid alcohols consisting of 9 or more isoprene units are present in all living cells. They can be fully unsaturated (polyprenols) or alpha-saturated (dolichol). Dolichol forms may have additional saturation at or near the omega-end. Some species contain ony dolichol or only polyprenols while others have nearly equal amounts of both types. Some polyisoprenoid alcohols consist entirely of trans isoprene units but most, including dolichol, contain both trans and cis units. Considerable advances in lipid methodology have occurred since the first review of polyisoprenoid alcohols by Hemming in 1974. For example, direct analysis of both dolichol and Dol-P by HPLC has replaced earlier methods which were often both insensitive and inaccurate. The availability of radiolabeled dolichol and polyprenols has facilitated studies concerning the metabolism and distribution of these compounds. Those studies suggest that only a small portion of the dolichol present in cells is likely to be involved in glycosylation. Polyisoprenoid alcohols are usually present at a family of homologues where each differs in size by one isoprene unit. Little or no size related specificity has been observed for any reaction involving dolichol or polyisoprenol intermediates. The overall length of polyisoprenoid alcohols may, however, affect the manner in which these compounds influence the physical and biochemical properties of membranes. Studies on the biosynthetic pathway leading from cis, trans Pol-PP by phosphatase action. The formation of the dolichol backbone from a polyprenol requires the action of an additional enzyme, an alpha-saturase. This enzyme does not always act at the level of a single common substrate, since Pol-PP, Pol-P, and polyprenol all appear to be utilized as substrates. The major product of the de novo pathway differs among different species. Dol-P would appear to be the most energy efficient end-product since it can participate directly in glycoprotein formation. Most often, however, Dol-P is not the major product of metabolic labeling experiments. In some cases, dolichol is formed so that rephosphorylation is required to provide Dol-P for participation in glycoprotein formation. The kinase responsible for this phosphorylation appears to bypass the considerable stores of dolichol present in tissues (i.e. sea urchin eggs) in favor of dolichol derived directly from de novo synthesis. Although HMGR is a major regulatory component of the pathway leading to polyisoprenoid alcohols and cholesterol, control is most often not co-ordinated.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hydrolysis of allylic phosphates by enzymes from the flavedo of Citrus sinensis

Acid phosphatase activities have been partially purified from an aqueous extract of an acetone powder from orange flavedo. The use of a gel filtration step with an ionic gradient allowed a dissociation of proteins from pigments, thus facilitating purification and stabilization of the enzymes. The enzymes do not require metals for full activity, and they hydrolysed a wide spectrum of phosphorylated substrates. C₁₀–C₂₀ allylic pyrophosphates and monophosphates were hydrolysed sequentially by these ‘prenylphosphatases’. The final product was the corresponding unrearranged prenyl alcohol. This demonstrated the absence of E-Z isomerization and suggested an OP bond cleavage. Prenylphosphatases exhibited a certain degree of chain length specificity. Although the E or Z conformation of the C-2 double bond was not important, its presence was required for full activity. Excess prenylpyrophosphate inhibited the rate of formation of alcohols, most likely through the inhibition of phosphomonoesterase activity. These prenylphosphatases generated the alcoholic components of essential oils from the corresponding pyrophosphates and removed them from the chain lengthening process.     

Protein dolichylation in Plasmodium falciparum

We performed reverse-phase thin-layer chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC) analysis of polyisoprenoids released by sulfonium-salt cleavage with methyl iodide from Plasmodium falciparum proteins labeled with [3H]FPP or [3H]GGPP and showed that a dolichol of 11 isoprene units is bound to 21-28-kDa protein clusters from trophozoite and schizont stages. The dolichol structure was confirmed by electrospray-ionization mass spectrometry analysis. Treatment with protein synthesis inhibitors and RP-HPLC analysis of the proteolytic digestion products from parasite proteins labeled with [35S]cysteine and [3H]FPP showed that the attachment of dolichol to protein is a post-translational event and probably occurs via a covalent bond to cysteine residues.     

Uncovering the complex metabolic network underlying diterpenoid phytoalexin biosynthesis in rice and other cereal crop plants

Rice (Oryza sativa) is a staple food crop and serves as a model cereal crop plant for scientific study. Phytochemical investigations of the agronomically devastating rice blast disease have identified a number of rice phytoalexins exhibiting significant direct anti-fungal activity against the causative agent, Magneporthe grisea. Current evidence strongly indicates that these phytoalexins, largely a family of labdane-related diterpenoids, are important as general antibiotics, and that similar phytoalexins are produced more broadly throughout the cereal crop family. From the extensive sequence information available for rice it has been possible to functionally identify the genes for the enzymes catalyzing the two consecutive cyclization reactions that initiate biosynthesis of these labdane-related diterpenoid phytoalexins. This has led to several insights into the underlying evolution of diterpene biosynthesis throughout the cereal crop family. The hydrocarbon olefins resulting from cyclization must be further elaborated to form bioactive natural products and, because not much is currently known, necessarily speculative biosynthetic pathways for these processes are presented. Given the significant antibiotic activity of the labdane-related diterpenoid phytoalexins from rice, and the presence of similar secondary metabolism throughout the cereal crop plant family, study of this type of biosynthesis will continue to be an area of active investigation.     

Rv0989c encodes a novel (E)-geranyl diphosphate synthase facilitating decaprenyl diphosphate biosynthesis in Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) has a highly complex cell wall, which is required for both bacterial survival and infection. Cell wall biosynthesis is dependent on decaprenyl diphosphate as a glyco-carrier, which is hence an essential metabolite in this pathogen. Previous biochemical studies indicated (E)-geranyl diphosphate (GPP) is required for the synthesis of decaprenyl diphosphate. Here we demonstrate that Rv0989c encodes the “missing” GPP synthase, representing the first such enzyme to be characterized from bacteria, and which presumably is involved in decaprenyl diphosphate biosynthesis in Mtb. Our investigation also has revealed previously unrecognized substrate plasticity of the farnesyl diphosphate synthases from Mtb, resolving previous discrepancies between biochemical and genetic studies of cell wall biosynthesis.     

Impact of advanced glycation end products (AGEs) signaling in coronary artery disease

Coronary artery disease remains the leading cause of mortality in adult diabetic population with however, a high predominance also in non-diabetic subjects. In search of common molecular mechanisms and metabolic by-products with potential pathogenic role, increased advanced glycation end products (AGEs) present a critical biomarker for CAD development in both cases. Interaction of AGEs with their transmembrane cell receptor, RAGE in endothelial and smooth muscle cells as well as in platelets, activates intracellular signaling that leads to endothelial injury, modulation of vascular smooth muscle cell function and altered platelet activity. Furthermore, tissue accumulation of AGEs affects current treatment approaches being involved in stent restenosis. The present review provides an update of AGE-induced molecular mechanisms involved in CAD pathophysiology while it discusses emerging therapeutic interventions targeting AGE reduction and AGE-RAGE signaling with beneficial clinical outcome.