Nobiletin: Targeting the Circadian Network to Promote Bioenergetics and Healthy Aging.

The circadian clock is the biological mastermind governing orderly execution of bodily processes throughout the day. In recent years, an emerging topic of broad interest is clock-modulatory agents, including small molecules both of synthetic and natural origins, and their potential applications in disease models. Nobiletin is a naturally occurring flavonoid with the greatest abundance found in citrus peels. Extensive research has shown that Nobiletin is endowed with a wide range of biological activities, yet its mechanism of action remains unclear. We recently found through unbiased chemical screening that Nobiletin impinges on the clock machinery to activate temporal control of downstream processes within the cell and throughout the body. Using animal models of diseases and aging, we and others illustrate potent beneficial effects of Nobiletin on cellular energetics in both periphery and brain to promote healthy aging. Given its excellent safety profile, Nobiletin may represent a promising candidate molecule for development of nutraceutical and chronotherapeutic agents against chronic and age-related neurodegenerative diseases.

Genetic analysis of lipid-protein interactions in Escherichia coli membranes.

Phospholipids play essential roles in defining the membrane permeability barrier, in regulating cellular processes, in providing a support for organization of many membrane-associated processes, and in providing precursors for the synthesis of macromolecules. Although in vitro experiments have provided important information on the role of protein-lipid interactions in cell function, such approaches are limited by the lack of a direct measure for phospholipid function. Genetic approaches can provide direct evidence for a specific role for phospholipids in cell function provided cell viability or membrane structure is not compromised. This review will summarize recent genetic approaches that when coupled with biochemical studies have led to a better understanding of specific functions for phospholipids at the molecular level.

CDP-diacylglycerol synthase of microorganisms.

The synthesis and utilization of CDP-diacylglycerol in mammalian cells was demonstrated over 35 years ago when initial studies were carried out. However, CDP-diacylglycerol synthases and the genes encoding these enzymes have been studied in the greatest detail in Escherichia coli and Saccharomyces cerevisiae. The involvement of CDP-diacylglycerol in regulation of phospholipid metabolism has recently been demonstrated in Saccharomyces cerevisiae, and evidence now exists from studies in Drosophila that this liponucleotide may be important in regulation of lipid-dependent signal transduction processes. The vast amount of biochemical and genetic information on the synthases from microorganisms has led to the cloning of genes that encode CDP-diacylglycerol synthases from somatic cells. The combination of information on these synthases from all organisms will lead to a clearer understanding of the role CDP-diacylglycerol plays in cellular processes.

Reconstituted phosphatidylserine synthase from Escherichia coli is activated by anionic phospholipids and micelle-forming amphiphiles.

The activity of phosphatidylserine (PS) synthase (CDP-1, 2-diacyl-sn-glycerol: l-serine O-phosphatidyltransferase, EC 2.7.8. 8) from Escherichia coli was studied after reconstitution with lipid vesicles of various compositions. PS synthase exhibited practically no activity in the absence of a detergent and with the substrate CDP-diacylglycerol (CDP-DAG) present only in the lipid vesicles. Inclusion of octylglucoside (OG) in the assay mixture increased the activity 20- to 1000-fold, the degree of activation depending on the lipid composition of the vesicles. Inclusion of additional CDP-DAG in the assay mixture increased the activity 5- to 25-fold. When the fraction of phosphatidylglycerol (PG) was increased from 15 to 100 mol% in the vesicles the activity increased 10-fold using the assay mixture containing OG. The highest activities were exhibited with the anionic lipids synthesized by E. coli, namely PG, diphosphatidylglycerol (DPG), and phosphatidic acid, while phosphatidylinositol gave a lower activity. Cryotransmission electron microscopy showed that transformation of the vesicles to micelles brings about an activation of the enzyme that is proportional to the degree of micellization. Thus, the activity of PS synthase is modulated by the lipid aggregate structure and by the fraction and type of anionic phospholipid in the aggregates. The increase in the activity caused by PG and DPG is physiologically relevant; it may be part of a regulatory mechanism that keeps the balance between phosphatidylethanolamine, and the sum of PG and DPG, nearly constant in wild-type E. coli cells.

Effect of divalent cations on lipid organization of cardiolipin isolated from Escherichia coli strain AH930.

Escherichia coli strain AH930 is a lipid biosynthetic mutant, which is unable to synthesize phosphatidylethanolamine. Instead it produces large amounts of phosphatidylglycerol and cardiolipin and has an absolute requirement for certain divalent cations. Cardiolipin was isolated from this mutant strain and its interaction with divalent cations was studied by various biophysical techniques. Monolayer measurements showed that the cations decrease the molecular surface area of cardiolipin in the order Ca2+ approximately Mg2+ > Sr2+ > Ba2+. 31P-NMR and X-ray diffraction measurements demonstrated a comparable sequence for the ability of the cations to promote HII phase formation in dispersions of the E. coli cardiolipin: Ca2+ and Mg2+ induced HII phase formation at 50 degrees C, Sr2+ at 75 degrees C, while Ba2+ was found to be unable to promote HII phase formation in the temperature range measured. Furthermore, all divalent cations were found to increase the temperature at which the transition to the liquid-crystalline phase takes place, which was below 5 degrees C for the lipid in the absence of divalent cations. In the presence of Sr2+, Mg2+ and Ba2+ and at 25 degrees C two lamellar phases were observed, one corresponding to a liquid-crystalline phase, the other to either a gel or a crystalline phase. In the presence of Ca2+ at 25 degrees C and even at 45 degrees C no evidence for a liquid-crystalline phase was obtained and only a crystalline phase could be observed. The ability of the different cations to promote HII phase formation in the isolated E. coli cardiolipin was found to correlate with their ability to support growth of the mutant strain (De Chavigny, A., Heacock, P.N., Dowhan, W. (1991) J. Biol. Chem. 266, 5323-5332), suggesting that cardiolipin with divalent cations can replace the role of phosphatidylethanolamine in the mutant strain, and that this role involves the preference of these lipids for organization in non-bilayer lipid structures.

Cardiolipin binds nonyl acridine orange by aggregating the dye at exposed hydrophobic domains on bilayer surfaces.

10-N-Nonyl acridine orange (NAO) has been used at low concentrations as a fluorescent indicator for cardiolipin (CL) in membranes and bilayers. The mechanism of its selective fluorescence in the presence of CL, and not any other phospholipids, is not understood. The dye might recognize CL by its high pK (pK(2)>8.5). To investigate that, we established that NAO does not exhibit a pK in a pH range between 2.3 and 10.0. A second explanation is that the dye aggregates at hydrophobic domains on bilayers exposed by the CL. We found that a similar spectral shift occurs in the absence of CL in a concentrated solution of the dye in methanol and in the solid state. A model is proposed in which the nonyl group inserts in the bilayer at the hydrophobic surface generated by the presence of four chains on the phospholipid.

Depletion of phosphatidylethanolamine affects secretion of Escherichia coli alkaline phosphatase and its transcriptional expression.

In this report we demonstrate that depletion of the major phospholipid phosphatidylethanolamine, a single non-bilayer forming phospholipid of Escherichia coli, significantly reduces the secretion efficiency of alkaline phosphatase in vivo. Secretion, however, is correlated with the content in membranes of cardiolipin, which in combination with selected divalent cations has a strong tendency to adopt a non-bilayer state indicating the possible involvement of lipid polymorphism in efficient protein secretion. Depletion of this zwitterionic phospholipid also inhibits expression of the protein controlled by the endogenous P(PHO) promoter but not the P(BAD) promoter, which is suggested to be due to the effect of unbalanced phospholipid composition on the orthophosphate signal transduction system (Pho regulon) through an effect on its membrane bound sensor.

Isolation and expression of an isoform of human CDP-diacylglycerol synthase cDNA.

Phosphatidic acid (PA) is a phospholipid involved in signal transduction and in glycerolipid biosynthesis. CDP-diacylglycerol synthase (CDS) or CTP:phosphatidate cytidylyltransferase (EC 2.7.7.41) catalyzes the conversion of PA to CDP-diacylglycerol (CDP-DAG), an important precursor for the synthesis of phosphatidylinositol, phosphatidylglycerol, and cardiolipin. We describe in this study the isolation and characterization of a human cDNA clone that encodes amino acid sequences homologous to Escherichia coli, yeast, and Drosophila CDS sequences. Expression of this human cDNA under the control of a GAL1 promoter in a null cds1 mutant yeast strain complements its growth defect and produces CDS activity when induced with galactose. Transfection of this cDNA into mammalian cells leads to increased CDS activity in cell-free extracts using an in vitro assay that measures the conversion of [alpha-32P]CTP to [32P]CDP-DAG. This increase in CDS activity also leads to increased secretion of tumor necrosis factor-alpha and interleukin-6 from endothelial ECV304 cells upon stimulation with interleukin-1beta, suggesting that CDS overexpression may amplify cellular signaling responses from cytokines.

Cardiolipin in energy transducing membranes.

Cardiolipin is a phospholipid located exclusively in energy transducing membranes such as the bacterial cytoplasmic membrane and the inner membrane of mitochondria. It plays both a structural and a functional role in many multimeric complexes associated with these membranes. The role of cardiolipin in higher order organization of components of the mitochondrial respiratory chain revealed by a combined molecular genetic and biochemical approach is described.

Molecular basis for membrane phospholipid diversity: why are there so many lipids?

Phospholipids play multiple roles in cells by establishing the permeability barrier for cells and cell organelles, by providing the matrix for the assembly and function of a wide variety of catalytic processes, by acting as donors in the synthesis of macromolecules, and by actively influencing the functional properties of membrane-associated processes. The function, at the molecular level, of phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin in specific cellular processes is reviewed, with a focus on the results of combined molecular genetic and biochemical studies in Escherichia coli. These results are compared with primarily biochemical data supporting similar functions for these phospholipids in eukaryotic organisms. The wide range of processes in which specific involvement of phospholipids has been documented explains the need for diversity in phospholipid structure and why there are so many membrane lipids.