Lipid transfer in plants

Summary Plant cells contain cytosolic proteins, called lipid transfer proteins (LTP), which are able to facilitate in vitro intermembrane transfer of phospholipids. Proteins of this kind from three plants, purified to homogeneity, have several properties in common: molecular mass around 9 kDa, high isoelectric point, lack of specificity for phospholipids, and binding ability for fatty acids. The comparison of their amino acid sequences revealed striking homologies and conserved domains which are probably involved in their function as LTPs. These proteins could play a major role in membrane biogenesis by conveying phospholipids from their site of biosynthesis to membranes unable to form these lipids. Immunochemical methods were used to establish an in vivo correlation between membrane biogenesis and the level of LTP or the amount of LTP synthesized in vitro from mRNAs. The recent isolation of a full-length cDNA allows novel approaches to studying the participation of LTPs in the biogenesis of plant cell membranes.     

Lipid transport pathways in mammalian cells

Summary A major deficit in our understanding of membrane biogenesis in eukaryotes is the definition of mechanisms by which the lipid constituents of cell membranes are transported from their sites of intracellular synthesis to the multiplicity of membranes that constitute a typical cell. A variety of approaches have been used to examine the transport of lipids to different organelles. In many cases the development of new methods has been necessary to study the problem. These methods include cytological examination of cells labeled with fluorescent lipid analogs, improved methods of subcellular fractionation, in situ enzymology that demonstrates lipid translocation by changes in lipid structure, and cell-free reconstitution with isolated organelles. Several general patterns of lipid transport have emerged but there does not appear to be a unifying mechanism by which lipids move among different organelles. Significant evidence now exists for vesicular and metabolic energy-dependent mechanisms as well as mechanisms that are clearly independent of cellular ATP content.     

Lipid characterization and14C-acetate metabolism in catfish taste epithelium

Summary The catfish,Ictalurus punctatus is an important model system for the study of the biochemical mechanisms of taste reception. A detailed lipid analysis of epithelial tissue from the taste organ (barbel) of the catfish has been performed. Polar lipids account for 62±1% of the total, neutrals for 38±1%. Phosphatidyl-cholines, serines and ethanolamines are the major constitutents of the polar fraction. Plasmalogen concentration is high relative to that of non-neural tissues. [¹⁴C]-Acetate is incorporated into cell lipid fractions after incubation of barbel tissue at 37°C for 60 min. Percentage amounts of most lipids change with time during this in vitro incubation. The phospholipids are the most metabolically active fractions. This work yields information for continuing reconstitution experiments and indicates that the taste epithelium of this important model system is a metabolically active tissue capable of supporting lipid turnover/synthesis.This work was supported in part by NIH Research Grant No. NS-23622, NS-22620, and by the Veterans Administration.     

Emerging roles of the oxysterol-binding protein family in metabolism, transport, and signaling

OSBP (oxysterol-binding protein) and ORPs (OSBP-related proteins) constitute an enigmatic eukaryotic protein family that is united by a signature domain that binds oxysterols, sterols, and possibly other hydrophobic ligands. The human genome contains 12 OSBP/ORP family members genes, while that of the budding yeast Saccharomyces cerevisiae encodes seven OSBP homologues (Osh). Of these, Osh4 (also referred to as Kes1) has been the most widely studied to date. Recently, three-dimensional crystal structures of Osh4 with and without sterols bound within the core of the protein were determined. The core consists of 19 anti-parallel β-sheets that form a near-complete β-barrel. Recent work has suggested that Osh proteins facilitate the non-vesicular transport of sterols in vivo and in vitro, while other evidence supports a role for Osh proteins in the regulation of vesicular transport and lipid metabolism.This article will review recent advances in the study of ORP/Osh proteins and will discuss future research issues regarding the ORP/Osh family. Received 17 July 2007; received after revision 14 August 2007; accepted 12 September 2007     

Microtubule transport defects in neurological and ciliary disease

Microtubules are primarily responsible for facilitating long-distance transport of both proteins and organelles. Given the critical role of this process in cellular function, it is not surprising that perturbation of microtubule-based transport can lead to diverse phenotypes in humans, including cancer and neurodegenerative disorders such as Alzheimer or Huntington disease. Recent investigations have also indicated that defects in specialized microtubule-based transport systems, such as mutations affecting the transport of protein particles along the length of cilia (intraflagellar transport) can cause retinal dystrophy, polycystic kidney disease or more complex syndromic phenotypes, such as Bardet-Biedl syndrome. In this review, we discuss recent findings implicating defects in microtubule-associated transport and motor proteins in a variety of diseases, particularly the role of defective microtubular transport in neurological and ciliary disease. These defects frequently display phenotypic consequences that manifest as human disease yet do not cause organismal lethality.Received 7 Janury 2005; received after revision 23 February 2005; accepted 21 March 2005     

Lipid transport in the migrating Monarch butterfly,Danaus p. plexippus

Summary The transport of lipid in the haemolymph of the Monarch butterfly during its fall migration was examined. Diglyceride was the major lipid class of 2 electrophoretically distinct lipoprotein fractions in both males and females. Triglycerides, hydrocarbons, free fatty acids, phosphatidyl cholines and phosphatidyl ethanolamines were minor components of these lipoproteins. Differences in lipid transport attributable to sex were not detected.This study is a contribution of the Missouri Agricultural Experiment Station, Journal series No. 8520.     

Drosophila unconventional myosin VI is involved in intra- and intercellular transport during oogenesis

During mid-oogenesis of Drosophila, cyto plasmic particles are transported within the nurse cells and through ring canals (cytoplasmic bridges) into the oocyte by means of a microfilament-dependent mecha nism. Video-intensified fluorescence timelapse mi croscopy, in combination with microinjections of antibodies directed against Drosophila 95F myosin, have revealed that this unconventional myosin of class VI is involved in the transport processes. The results indicate that certain cytoplasmic particles in the nurse cells move along microfilaments due to their direct association with myosin VI motors. Additional myosin- VI molecules located at the rim of the ring canals seem to be involved in particle transport into the oocyte. Microinjected mitochondria-specific dyes have revealed that some of these particles are mitochondria. Received 3 April 1997; received after revision 5 May 1997; accepted 27 May 1997     

Regulatory mechanisms involved in modulating RGS function

Regulator of G-Protein Signaling (RGS) refers to a conserved 120–125 amino acid motif that was first identified by its ability to negatively regulate G-Protein-Coupled Receptor (GPCR) signalling. Mechanistically, RGSs were found to regulate GPCR responses by binding to and stimulating the GTPase activity of the receptor-activated GTP-bound G α subunits. There are now over 25 mammalian RGSs containing proteins that are reported to carry out a variety of functions, many of which are unrelated to GPCR signalling. RGS proteins range in size from small proteins that contain little more than an RGS box to very large proteins that contain a variety of domains. The selectivity of function of the RGS proteins is attributable to the divergence of the RGS sequences as well as the presence of a variety of functional motifs, which allow them to interact with other proteins. Here we focus on the RGSs that are involved in modulating GPCR signalling by reviewing the diversity of the mechanisms involved in regulating these RGSs. Received 9 February 2006; received after revision 4 May 2006; accepted 22 May 2006     

Lytic transglycosylases in macromolecular transport systems of Gram-negative bacteria

The cell wall of Gram-negative bacteria is essential for the integrity of the bacterial cell but also imposes a physical barrier to trans-envelope transport processes in which DNA and/or proteins are taken up or secreted by complex protein assemblies. The presence of genes encoding lytic transglycosylases in macromolecular transport systems (bacteriophage entry, type II secretion and type IV pilus synthesis, type III secretion, type IV secretion) suggests an important role for these specialised cell-wall-degrading enzymes. Such enzymes are capable of locally enlarging gaps in the peptidoglycan meshwork to allow the efficient assembly and anchoring of supramolecular transport complexes in the cell envelope. In this review, current knowledge on the role and distribution of these specialised murein-degrading enzymes in diverse macromolecular transport systems is summarised and discussed.Received 13 February 2003; received after revision 25 April 2003; accepted 12 May 2003     

Etiology of rheumatoid arthritis

Summary Definite genetic associations with immunological cooperative HLA-D(R) antigens have been demonstrated for rheumatoid arthritis (RA). Microbial etiology has not been proven, but some hope for the supporters of this view is still given by small viruses, plasmids of enteric bacteria or perhaps oncogen-like DNA-sequences. Yet, electrophoretical analysis of membrane proteins or surface glycoproteins of RA synovial cells does not show any differences compared to reference cells. Autoimmunity to several tissue elements has been demonstrated, but most of it is of secondary nature. Antigenicities of type II and III collagens are probably only contributory factors for HLA-DR4 positive individuals. Proteoglycans or minor cartilage collagens have not been extensively studied, so far. Endocrine, dietary or psychological influences might be triggering events for otherwise preloaded individuals.     

Evidence of undiscovered cell regulatory mechanisms: phosphoproteins and protein kinases in mitochondria

The finding that mitochondria contain substrates for protein kinases lead to the discovery that protein kinases are located in the mitochondria of certain tissues and species. These include pyruvate dyhydrogenase kinase, branched-chain α-ketoacid dehydrogenase kinase, protein kinase A, protein kinase Cδ, stress-activated kinase and A-Raf as well as unidentified kinases. Recent evidence suggests that mitochondrial protein kinases may be involved in physiological processes such as apoptosis and steroidogenesis. Additionally, the novel finding of low-molecular-weight GTP-binding proteins in mitochondria suggests the possibility that these may interact with mitochondrial protein kinases to regulate the activity of mitochondrial effector proteins. The fact that there are components of cellular regulatory systems in mitochondria indicates the exciting possibility of undiscovered systems regulating mitochondrial physiology. Received 19 June 2001; received after revision 7 August 2001; accepted 8 August 2001     

Expression of the Stp1 LMW-PTP and inhibition of protein CK2 display a cooperative effect on immunophilin Fpr3 tyrosine phosphorylation and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> growth

Although the yeast genome does not encode bona fide protein tyrosine kinases, tyrosine-phosphorylated proteins are numerous, suggesting that besides dual-specificity kinases, some Ser/Thr kinases are also committed to tyrosine phosphorylation in Saccharomyces cerevisiae. Here we show that blockage of the highly pleiotropic Ser/Thr kinase CK2 with a specific inhibitor synergizes with the overexpression of Stp1 low-molecular-weight protein tyrosine phosphatase (PTP) in inducing a severe growth-defective phenotype, consistent with a prominent role for CK2 in tyrosine phosphorylation in yeast. We also present in vivo evidence that immunophilin Fpr3, the only tyrosine-phosphorylated CK2 substrate recognized so far, interacts with and is dephosphorylated by Spt1. These data disclose a functional correlation between CK2 and LMW-PTPs, and suggest that reversible phosphorylation of Fpr3 plays a role in the regulation of growth rate and budding in S. cerevisiae.Received 15 January 2004; received after revision 20 February 2004; accepted 4 March 2004     

Hydroxylation of demethoxy-Q6 constitutes a control point in yeast coenzyme Q6 biosynthesis

Coenzyme Q is a lipid molecule required for respiration and antioxidant protection. Q biosynthesis in Saccharomyces cerevisiae requires nine proteins (Coq1p–Coq9p). We demonstrate in this study that Q levels are modulated during growth by its conversion from demethoxy-Q (DMQ), a late intermediate. Similar conversion was produced when cells were subjected to oxidative stress conditions. Changes in Q₆/DMQ₆ ratio were accompanied by changes in COQ7 gene mRNA levels encoding the protein responsible for the DMQ hydroxylation, the penultimate step in Q biosynthesis pathway. Yeast coq null mutant failed to accumulate any Q late biosynthetic intermediate. However, in coq7 mutants the addition of exogenous Q produces the DMQ synthesis. Similar effect was produced by over-expressing ABC1/COQ8. These results support the existence of a biosynthetic complex that allows the DMQ₆ accumulation and suggest that Coq7p is a control point for the Q biosynthesis regulation in yeast. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 04 September 2008; received after revision 22 October 2008; accepted 23 October 2008     

Phospholipid metabolism in pancreatic islets

Conclusions The secretion of insulin can be elicited by a wide spectrum of stimuli including nutrients, hormones and neurotransmitters as well as a large number of pharmacological agents such as tumor-promoters and sulphonylureas. The diversity of these secretagogues suggests that islets may be activated through a number of distinct biochemical mechanisms. The work discussed in this review suggests that certain of the above-mentioned secretagogues, especially nutrient and neurotransmitter stimuli, may induce insulin secretion by a mechanism involving enhanced metabolism of inositol-containing lipids. The way in which this process is coupled to secretion is not known, although several possibilities exist. The hydrolysis of phosphoinositides and release of inositol phosphates may result, respectively in altered calcium permeability of the plasma membrane and mobilization of calcium from intracellular sources. The accompanying production of diacylglycerol might also influence membrane permeability and fluidity and also lead to activation of protein kinase C. Diacylglycerol can be phosphorylated to form phosphatidic acid which may play a role as an endogenous ionophore. Finally, inositol lipid breakdown could lead, through diacylglycerol and/or phosphatidic intermediates, to the liberation of arachidonic acid and subsequent conversion to active metabolites of the cyclo-oxygenase and lipoxygenase pathways. Thus, enhanced phospholipid metabolism in islets could, theoretically, result in the generation of a range of intracellular signals which mediate or modulate insulin secretion during stimulation by certain types of secretagogues. Continued investigation is clearly neccessary in order to elucidate the mechanisms by which such secretagogues provoke increased phospholipid metabolism and to understand the role(s) of this process in the regulation of islet function.     

The effect of endotoxin on membrane fatty acid composition in BCG-sensitized mice

Summary The effects of endotoxin on mouse liver phospholipid fatty acid composition have been investigated. Administration of endotoxin fromSalmonella abortus equi led to a decrease in the polyunsaturated fatty acid content of livers from mice sensitized with Bacille Calmette Guérin (GCG). The content of arachidonic acid fell significantly in both the phosphatidylcholine and phosphatidylinositol fractions whereas in the phosphatidylethanolamine fraction the linoleic acid content was significantly reduced. The polyunsaturated fatty acids were replaced by increased amounts of oleic acid and palmitic acid, leading to a reduction in the polyunsaturated to saturated fatty acid ratio.     

Properties and modes of action of specific and non-specific phospholipid transfer proteins

Summary We have described the mode of action of the phosphatidylcholine transfer protein (PC-TP), the phosphatidylinositol transfer protein (PI-TP) and the non-specific lipid transfer protein (nsL-TP) isolated from bovine and rat tissues. PC-TP and PI-TP specifically bind one phospholipid molecule to be carried between membranes. PC-TP, and most likely PI-TP as well, have independent binding sites for thesn-1- andsn-2-fatty acyl chains. These sites have different properties, which may explain the ability of PC-TP and PI-TP to discriminate between positional phospholipid isomers. nsL-TP, which is identical to sterol carrier protein 2, transfers all common phospholipids, cholesterol and oxysterol derivatives between membranes. This protein is very efficient in mediating a net mass transfer of lipids to lipid-deficient membranes. Models for its mode of action, which is clearly different from that of PC-TP and PI-TP, are presented.     

Functional significance of the lipid-protein interface in photosynthetic membranes

The functional significance of the lipid-protein interface in photosynthetic membranes, mainly in thylakoids, is reviewed with emphasis on membrane structure and dynamics. The lipid-protein interface is identified primarily by the restricted molecular dynamics of its lipids as compared with the dynamics in the bulk lipid phase of the membrane. In a broad sense, lipid-protein interfaces comprise solvation shell lipids that are weakly associated with the hydrophobic surface of transmembrane proteins but also include lipids that are strongly and specifically bound to membrane proteins or protein assemblies. The relation between protein-associated lipids and the overall fluidity of the thylakoid membrane is discussed. Spin label electron paramagnetic resonance spectroscopy has been identified as the technique of choice to characterize the protein solvation shell in its highly dynamic nature; biochemical and direct structural methods have revealed an increasing number of protein-bound lipids. The structural and functional roles of these protein-bound lipids are mustered, but in most cases they remain to be determined. As suggested by recent data, the interaction of the non-bilayer-forming lipid, monogalactosyldyacilglycerol (MGDG), with the main light-harvesting chlorophyll a/b-binding protein complexes of photosystem-II (LHCII), the most abundant lipid and membrane protein components on earth, play multiple structural and functional roles in developing and mature thylakoid membranes. A brief outlook to future directions concludes this review.     

Production of pharmaceutical proteins in milk

There is every reason to expect that it will be possible within the next few years to begin to use farm animals to produce large quantities of some of the human proteins that are needed for the treatment of disease. Revolutionary new opportunities for the production of novel proteins in milk have been created by the development of methods for gene transfer. Exploitation of these opportunities depends upon selection and cloning of milk protein genes and identification of the sequences that govern tissue specific hormonally induced expression in the mammary gland. Studies with three genes, ovine -lactoglobulin, rat -casein and whey acidic protein of rat and mouse, suggest that they may all meet this requirement. Fragments of the ovine -lactoglobulin, murine whey acidic protein and rabbit -casein genes have directed production of novel proteins in the milk of transgenic mice, sheep, rabbits and pigs. The proteins were biologically active and usually co-migrated with authentic proteins. In early experiments, protein concentration was low, but our recent observations suggest that fusion genes containing genomic clones direct production of concentrations of protein that are suitable for commercial exploitation. In the longer term, two approaches may offer the potential of more reliable expression. Control elements capable of directing expression that is independent of site of insertion of the gene, but dependent on the number of copies of the gene, have been identified for a small number of genes. The availability of such elements for the milk protein genes would increase the reliability of gene expression considerably. Alternatively, targeted mutation of genes may allow the insertion of coding sequences within an existing gene so avoiding position effects.