Membrane phospholipids and adrenergic receptor function

We have reviewed the effects on adrenergic receptors by membrane phospholipid alterations secondary to oxidative stress and phospholipases' activity. Experimental evidences indicate that the function of both - and -adrenoceptors is regulated by their phospholipid microdomain; however, the underlying mechanism is still undefined. No information seems to be available on the influence of phospholipids on ₂-adrenoceptors and on all adrenoceptors' subtypes. Thus, further studies are necessary to clarify the role of membrane phospholipids in regulating the function of each member of the adrenergic receptor superfamily.     

Membrane phospholipids of normal and crown gall callus cultures

The relative proportions of different phospholipid species were determined in membrane-enriched fractions from normal and crown-gall callus cultures. In general the pattern of phospholipids was similar, but the tumour cultures contained relatively more phosphatidyl choline and less phosphatidic acid and phosphatidyl inositol than the corresponding normal cultures. These differences are probably not caused by phospholipase-D activity during homogenization.     

The triacylglycerol synthesis enzyme DGAT1 also catalyzes the synthesis of diacylglycerols, waxes, and retinyl esters

The final step of triacylglycerol biosynthesis is catalyzed by acyl CoA:diacylglycerol acyltransferase (DGAT) enzymes. The two known DGATs, DGAT1 and DGAT2, are encoded by unrelated genes. Although both DGAT1 and DGAT2 knockout mice have reduced tissue triacylglycerol contents, they have disparate phenotypes, prompting us to investigate whether the two enzymes have unrecognized functional differences. We now report that DGAT1 exhibits additional acyltransferase activities in vitro, including those of acyl CoA:monoacylglycerol acyltransferase (MGAT), wax monoester and wax diester synthases, and acyl CoA:retinol acyltransferase (ARAT), which catalyze the synthesis of diacylglycerols, wax esters, and retinyl esters, respectively. These activities were demonstrated in in vitro assays with membranes from insect cells or homogenates from COS7 cells overexpressing DGAT1. Wax synthase and ARAT activities were also demonstrated in intact COS7 cells expressing DGAT1. Additionally, cells and tissues from DGAT1-deficient mice exhibited reduced ARAT activity, and the mice had increased levels of unesterified retinol in their livers on a high-retinol diet. Our findings indicate that DGAT1 can utilize a variety of acyl acceptors as substrates in vitro and suggest that these activities may be relevant to the in vivo functions of DGAT1.     

Physiological and microfluorometric studies of reduction and clearance of retinal in bleached rod photoreceptors

The visual cycle comprises a sequence of reactions that regenerate the visual pigment in photoreceptors during dark adaptation, starting with the reduction of all-trans retinal to all-trans retinol and its clearance from photoreceptors. We have followed the reduction of retinal and clearance of retinol within bleached outer segments of red rods isolated from salamander retina by measuring its intrinsic fluorescence. Following exposure to a bright light (bleach), increasing fluorescence intensity was observed to propagate along the outer segments in a direction from the proximal region adjacent to the inner segment toward the distal tip. Peak retinol fluorescence was achieved after approximately 30 min, after which it declined very slowly. Clearance of retinol fluorescence is considerably accelerated by the presence of the exogenous lipophilic substances IRBP (interphotoreceptor retinoid binding protein) and serum albumin. We have used simultaneous fluorometric and electrophysiological measurements to compare the rate of reduction of all-trans retinal to all-trans retinol to the rate of recovery of flash response amplitude in these cells in the presence and absence of IRBP. We find that flash response recovery in rods is modestly accelerated in the presence of extracellular IRBP. These results suggest such substances may participate in the clearance of retinoids from rod photoreceptors, and that this clearance, at least in rods, may facilitate dark adaptation by accelerating the clearance of photoproducts of bleaching.     

Vitamin A receptors of the retina differential binding in light and dark

Vitamin A receptors of the retina appear to be differentially extractable in light and in dark suggesting that they could function as inter- or intra-cellular transport vehicles in the visual cycle.     

Effect of cigarette smoking on high density lipoprotein phospholipids

The effect of acute inhalation of cigarette smoke on high density lipoprotein (HDL) phospholipid composition in White Carneau pigeons was examined. Four treatments included: 1) Shelf Control birds fed a chow diet and retained in their cages; 2) Sham pigeons fed a cholesterol-saturated fat diet and exposed to fresh air by a smoking machine; 3) Low nicotine-low carbon monoxide (LoLo) animals also fed the cholesterol diet and exposed to low concentrations of these cigarette smoke products; and 4) High nicotine-high carbon monoxide (HiHi) birds fed the cholesterol diet and subjected to high concentrations of these inhalants. The cholesterol-fat diet caused an increase in the concentration of most HDL phospholipid classes. Exposure to the HiHi regimen resulted in an increase in the HDL cholesterol/phospholipid ratio and a reduction in the concentration of HDL phosphatidyl ethanolamine, phosphatidyl serine/inositol, sphingomyelin and lysophosphatidyl choline. Cigarette smoking may thus attenuate HDL's anti-atherogenic properties by altering surface phospholipid components.     

A proposed architecture for lecithin cholesterol acyl transferase (LCAT): identification of the catalytic triad and molecular modeling.

The enzyme cholesterol lecithin acyl transferase (LCAT) shares the Ser/Asp-Glu/His triad with lipases, esterases and proteases, but the low level of sequence homology between LCAT and these enzymes did not allow for the LCAT fold to be identified yet. We, therefore, relied upon structural homology calculations using threading methods based on alignment of the sequence against a library of solved three-dimensional protein structures, for prediction of the LCAT fold. We propose that LCAT, like lipases, belongs to the alpha/beta hydrolase fold family, and that the central domain of LCAT consists of seven conserved parallel beta-strands connected by four alpha-helices and separated by loops. We used the conserved features of this protein fold for the prediction of functional domains in LCAT, and carried out site-directed mutagenesis for the localization of the active site residues. The wild-type enzyme and mutants were expressed in Cos-1 cells. LCAT mass was measured by ELISA, and enzymatic activity was measured on recombinant HDL, on LDL and on a monomeric substrate. We identified D345 and H377 as the catalytic residues of LCAT, together with F103 and L182 as the oxyanion hole residues. In analogy with lipases, we further propose that a potential "lid" domain at residues 50-74 of LCAT might be involved in the enzyme-substrate interaction. Molecular modeling of human LCAT was carried out using human pancreatic and Candida antarctica lipases as templates. The three-dimensional model proposed here is compatible with the position of natural mutants for either LCAT deficiency or Fish-eye disease. It enables moreover prediction of the LCAT domains involved in the interaction with the phospholipid and cholesterol substrates.     

Autophagy and phagocytosis converge for better vision

The retinal pigment epithelium (RPE) is a single layer of nonregenerating cells essential to homeostasis in the retina and the preservation of vision. While the RPE perform a number of important functions, 2 essential processes are phagocytosis, which removes the most distal tips of the photoreceptors to support disk renewal, and the visual cycle, which maintains the supply of chromophore for regeneration of photo-bleached visual pigments. We recently reported that these processes are linked by a noncanonical form of autophagy termed LC3-associated phagocytosis (LAP) in which components of the autophagy pathway are co-opted by phagocytosis to recover vitamin A in support of optimal vision. Here we summarize these findings.     

Acidic retinoids synergize with vitamin A to enhance retinol uptake and STRA6, LRAT, and CYP26B1 expression in neonatal lung

Vitamin A (VA) is essential for fetal lung development and postnatal lung maturation. VA is stored mainly as retinyl esters (REs), which may be mobilized for production of retinoic acid (RA). This study was designed 1) to evaluate several acidic retinoids for their potential to increase RE in the lungs of VA-supplemented neonatal rats, and 2) to determine the expression of retinoid homeostatic genes related to retinol uptake, esterification, and catabolism as possible mechanisms. When neonatal rats were treated with VA combined with any one of several acidic retinoids (RA, 9-cis-RA, or Am580, a stable analog of RA), lung RE increased ∼5–7 times more than after an equal amount of VA alone. Retinol uptake and esterification during the period of absorption correlated with increased expression of both STRA6 (retinol-binding protein receptor) and LRAT (retinol esterification), while a reduction in RE after 12 h in Am580-treated, VA-supplemented rats correlated with a strong and persistent increase in CYP26B1 (RA hydroxylase). We conclude that neonatal lung RE can be increased synergistically by VA combined with both natural and synthetic acidic retinoids, concomitant with induction of the dyad of STRA6 and LRAT. However, the pronounced and prolonged induction of CYP26B1 by Am580 may counteract lung RE accumulation after the absorption process is completed.     

Identification of conserved histidines and glutamic acid as key residues for isomerohydrolase activity of RPE65, an enzyme of the visual cycle in the retinal pigment epithelium

We have recently reported that RPE65 from the retinal pigment epithelium is the isomerohydrolase, a critical enzyme in the visual cycle for regeneration of 11-cis retinal, the chromophore for visual pigments. Here, we demonstrated that mutation of any one of the absolutely conserved four histidine and one glutamic acid residues to alanine in RPE65 abolished its isomerohydrolase activity. Substitution of the conserved glutamic acid with glutamine also resulted in loss of the activity. Moreover, these mutations significantly reduced protein stability of RPE65. These results indicate that these conserved residues are essential for the isomerohydrolase activity of RPE65 and its stability.     

Enzymology of retinoic acid biosynthesis and degradation: Thematic Review Series: Fat-Soluble Vitamins: Vitamin A

All-trans-retinoic acid is a biologically active derivative of vitamin A that regulates numerous physiological processes. The concentration of retinoic acid in the cells is tightly regulated, but the exact mechanisms responsible for this regulation are not completely understood, largely because the enzymes involved in the biosynthesis of retinoic acid have not been fully defined. Recent studies using in vitro and in vivo models suggest that several members of the short-chain dehydrogenase/reductase superfamily of proteins are essential for retinoic acid biosynthesis and the maintenance of retinoic acid homeostasis. However, the exact roles of some of these recently identified enzymes are yet to be characterized. The properties of the known contributors to retinoid metabolism have now been better defined and allow for more detailed understanding of their interactions with retinoid-binding proteins and other retinoid enzymes. At the same time, further studies are needed to clarify the interactions between the cytoplasmic and membrane-bound proteins involved in the processing of hydrophobic retinoid metabolites. This review summarizes current knowledge about the roles of various biosynthetic and catabolic enzymes in the regulation of retinoic acid homeostasis and outlines the remaining questions in the field.     

Enzymological analysis of the tumor suppressor A-C1 reveals a novel group of phospholipid-metabolizing enzymes

A-C1 protein is the product of a tumor suppressor gene negatively regulating the oncogene Ras and belongs to the HRASLS (HRAS-like suppressor) subfamily. We recently found that four members of this subfamily expressed in human tissues function as phospholipid-metabolizing enzymes. Here we examined a possible enzyme activity of A-C1. The homogenates of COS-7 cells overexpressing recombinant A-C1s from human, mouse, and rat showed a phospholipase A_1/2 (PLA_1/2) activity toward phosphatidylcholine (PC). This finding was confirmed with the purified A-C1. The activity was Ca²⁺ independent, and dithiothreitol and Nonidet P-40 were indispensable for full activity. Phosphatidylethanolamine (PE) was also a substrate and the phospholipase A₁ (PLA₁) activity was dominant over the PLA₂ activity. Furthermore, the protein exhibited acyltransferase activities transferring an acyl group of PCs to the amino group of PEs and the hydroxyl group of lyso PCs. As for tissue distribution in human, mouse, and rat, A-C1 mRNA was abundantly expressed in testis, skeletal muscle, brain, and heart. These results demonstrate that A-C1 is a novel phospholipid-metabolizing enzyme. Moreover, the fact that all five members of the HRASLS subfamily, including A-C1, show similar catalytic properties strongly suggests that these proteins constitute a new class of enzymes showing PLA_1/2 and acyltransferase activities.     

Membrane proteins and phospholipids as effectors of reverse cholesterol transport

The review highlights the membrane aspect of cholesterol efflux from cell membranes to high density lipoproteins (HDL), an initial stage of reverse cholesterol transport to liver. In addition to traditional viewpoints considering cholesterol transport as the step of sequential lipoprotein transformation, which involves blood plasma apoproteins and proteins transporters, employment of proteomic approaches has shown the active role of cell plasma membranes as cholesterol donors and plasma membrane bound proteins in cholesterol transport. These include ATP-binding ABC-A1 transporter and membrane receptor SR-B1. There is experimental and clinical evidence that impairment of genes encoding these proteins cause impairments of reverse cholesterol transport (e.g. Tangier disease and genetic manipulations with experimental animals.) Although precise mechanism involving these membrane proteins remains unknown it is suggested that ABC-AI with free plasma apoA1 facilitates the efflux of membrane phospholipids and formation of their complex with apoAI. This complex accepts membrane cholesterol, with simultaneous formation of a full HDL particle. In certain cells there is correlation between cholesterol efflux into HDL and expression of SR-BI, which reversibly binds to HDL. This receptor protein may influence molecular organization of membrane phospholipids and cholesterol, facilitating cholesterol efflux. The review also deals with properties of ABC-A1 and SR-B1, putative mechanisms of their effects, the role of these proteins in reverse cholesterol transport and their functional coupling to the phospholipid matrix of biomembranes.     

Site-specific detection and structural characterization of the glycosylation of human plasma proteins lecithin:cholesterol acyltransferase and apolipoprotein D using HPLC/electrospray mass spectrometry and sequential glycosidase digestion.

Site-specific structural characterization of the glycosylation of human lecithin:cholesterol acyltransferase (LCAT) was carried out using microbore reversed-phase high performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC/ESIMS). A recently described mass spectrometric technique involving monitoring of carbohydrate-specific fragment ions during HPLC/ESIMS was employed to locate eight different groups of glycopeptides in a digest of a human LCAT protein preparation. In addition to the four expected N-linked glycopeptides of LCAT, a di-O-linked glycopeptide was detected, as well as three additional glycopeptides. Structural information on the oligosaccharides from all eight glycopeptides was obtained by sequential glycosidase digestion of the glycopeptides followed by HPLC/ESIMS. All four potential N-linked glycosylation sites (Asn20, Asn84, Asn272, and Asn384) of LCAT were determined to contain sialylated triantennary and/or biantennary complex structures. Two unanticipated O-linked glycosylation sites were identified at Thr407 and Ser409 of the LCAT O-linked glycopeptide, each of which contain sialylated galactose beta 1-->3N-acetylgalactosamine structures. The three additional glycopeptides were determined to be from a copurifying protein, apolipoprotein D, which contains potential N-linked glycosylation sites at Asn45 and Asn78. These glycopeptides were determined to bear sialylated triantennary oligosaccharides or fucosylated sialylated biantennary oligosaccharides. Previous studies of LCAT indicated that removal of the glycosylation site at Asn272 converts this protein to a phospholipase (Francone OL, Evangelista L, Fielding CJ, 1993, Biochim Biophys Acta 1166:301-304). Our results indicate that the carbohydrate structures themselves are not the source of this functional discrimination; rather, it must be mediated by the structural environment around Asn272.     

Autophagy and phagocytosis converge for better vision

The retinal pigment epithelium (RPE) is a single layer of nonregenerating cells essential to homeostasis in the retina and the preservation of vision. While the RPE perform a number of important functions, 2 essential processes are phagocytosis, which removes the most distal tips of the photoreceptors to support disk renewal, and the visual cycle, which maintains the supply of chromophore for regeneration of photo-bleached visual pigments. We recently reported that these processes are linked by a noncanonical form of autophagy termed LC3-associated phagocytosis (LAP) in which components of the autophagy pathway are co-opted by phagocytosis to recover vitamin A in support of optimal vision. Here we summarize these findings.     

Colorimetric assay of gramicidin A in the presence of surfactants and phospholipids

Gramicidin A, a hydrophobic polypeptide containing 4 tryptophan residues/molecule, may be determined quantitatively after reaction with 4-(dimethylamino)benzaldehyde, a method previously proposed for tryptophan analysis. The assay may be carried out even in the presence of various surfactants and phospholipids at high concentrations.     

Dietary Modifications of Phospholipid Composition and Biophysical Properties of the Brush Border Membrane Along the Trout Intestine

Brush border membranes (BBM) are isolated from middle and posterior intestine of trout fed either an essential fatty acid-rich diet or a saturated one. The different phospholipid classes are separated, and their fatty acid composition is determined. Fluorescence anisotropy studies are performed using two lipid fluorophores, namely diphenylhexatriene (DPH) and trimethyl-aminodiphenylhexatriene (TMA-DPH). The results indicate that the usual parameters affecting the lipid fluidity such as the phospholipid:protein (PL:PROT), cholesterol:phospholipid (CHOL:PL), and sphingomyelin:phosphatidylcholine (SP:PC) ratios and the unsaturation of the acyl chains are sufficient to explain the fluidity values determined using DPH, but not those obtained with TMA-DPH as a probe. This fluorophore is assessed to be localized only in the external leaflet of the membrane. Hence, it will be affected by the composition of the major phospholipids of this leaflet, sphingomyelin and phosphatidylcholine.     

Changes in the composition of phospholipids in nuclear subfractions of wheat seedlings treated with gibberellin

Macromolecule transport between the cytoplasm and nucleus occurs through the nuclear pore complexes with the help of soluble transport factors. The receptors of GA are not yet identified, and the molecular mechanism of plant response to GA is not known so far. We compared the content of phospholipids in the soluble nuclear fraction and nuclear membrane of wheat seedlings on the third and fourth days of germination after treatment with GA. It was shown that GA induced differently directed changes in the composition of phospholipids in nuclear subfractions tested. Changes in the composition of the nuclear membrane are supposed to be involved in the control of gene expression by GA.     

Cis-retinoids and the chemistry of vision

We discuss here principal biochemical transformations of retinoid molecules in the visual cycle. We focus our analysis on the accumulating evidence of alternate pathways and functional redundancies in the cycle. The efficiency of the visual cycle depends, on one hand, on fast regeneration of the photo-bleached chromophores. On the other hand, it is crucial that the cyclic process should be highly selective to avoid accumulation of byproducts. The state-of-the-art knowledge indicates that single enzymatically active components of the cycle are not strictly selective and may require chaperones to enhance their rates. It appears that protein–protein interactions significantly improve the biological stability of the visual cycle. In particular, synthesis of thermodynamically less stable 11-cis-retinoid conformers is favored by physical interactions of the isomerases present in the retina with cellular retinaldehyde binding protein.