Sterol carrier protein-2: structure reveals function

The multiple actions of sterol carrier protein-2 (SCP-2) in intracellular lipid circulation and metabolism originate from its gene and protein structure. The SCP-x/pro-SCP-2 gene is a fusion gene with separate initiation sites coding for 15-kDa pro-SCP-2 (no enzyme activity) and 58-kDa SCP-x (a 3-ketoacyl CoA thiolase). Both proteins share identical cDNA and amino acid sequences for 13-kDa SCP-2 at their C-termini. Cellular 13-kDa SCP-2 derives from complete, posttranslational cleavage of the 15-kDa pro-SCP-2 and from partial posttranslational cleavage of 58-kDa SCP-x. Putative physiological functions of SCP-2 have been proposed on the basis of enhancement of intermembrane lipid transfer (e.g., cholesterol, phospholipid) and activation of enzymes involved in fatty acyl CoA transacylation (cholesterol esters, phosphatidic acid) in vitro, in transfected cells, and in genetically manipulated animals. At least four important SCP-2 structural domains have been identified and related to specific functions. First, the 46-kDa N-terminal presequence present in 58-kDa SCP-x is a 3-ketoacyl-CoA thiolase specific for branched-chain acyl CoAs. Second, the N-terminal 20 amino acid presequence in 15-kDa pro-SCP-2 dramatically modulates the secondary and tertiary structure of SCP-2 as well as potentiating its intracellular targeting coded by the C-terminal peroxisomal targeting sequence. Third, the N-terminal 32 amino acids form an amphipathic a-helical region, one face of which represents a membrane-binding domain. Positively charged amino acid residues in one face of the amphipathic helices allow SCP-2 to bind to membrane surfaces containing anionic phospholipids. Fourth, the hydrophobic faces of the N-terminal amphipathic a helices along with beta strands 4, 5, and helix D form a ligand-binding cavity able to accommodate multiple types of lipids (e. g., fatty acids, fatty acyl CoAs, cholesterol, phospholipids, isoprenoids). Two-dimensional 1H-15N heteronuclear single quantum coherence spectra of both apo-SCP-2 and of the 1:1 oleate-SCP-2 complex, obtained at pH 6.7, demonstrated the homogenous formation of holo-SCP-2. While comparison of the apo- and holoprotein amide fingerprints revealed about 60% of the resonances remaining essentially unchanged, 12 assigned amide residues underwent significant chemical-shift changes upon oleic acid binding. These residues were localized in three regions: the juncture of helices A and B, the mid-section of the beta sheet, and the interface formed by the region of beta strands 4, 5, and helix D. Circular dichroism also showed that these chemical-shift changes, upon oleic acid binding, did not alter the secondary structure of SCP-2. The nuclear magnetic resonance chemical shift difference data, along with mapping of the nearby hydrophobic residues, showed the oleic acid-binding site to be comprised of a pocket created by the face of the beta sheet, helices A and B on one end, and residues associated with beta strands 4, 5, and helix D at the other end of the binding cavity. Furthermore, the hydrophobic nature of the previously ill-defined C-terminus suggested that these 20 amino acids may form a 'hydrophobic cap' which closes around the oleic acid upon binding. Thus, understanding the structural domains of the SCP-x/pro-SCP-2 gene and its respective posttranslationally processed proteins has provided new insights into their functions in intracellular targeting and metabolism of lipids.     

COX-2与CYPlBl在人乳腺癌中的表达及意义

目的探讨人乳腺癌组织中COX-2和CYPlBl的表达规律及与临床病理特征的关系,对认识其在乳腺癌发生发展中的作用奠定初步病理学基础。方法应用免疫组织化学方法检测42例乳腺癌组织中COX-2和CYPlBl的表达情况,结合临床病理指标进行研究分析。结果在乳腺癌中COX-2和CYPIBl表达阳性率分别为78．6％和73．8％。COX-2的阳性表达与乳腺癌淋巴结转移、组织学分级显著相关（P〈0．05）;CYPlBI的阳性表达与组织学分级、ER表达显著相关（P〈0．05）。乳腺癌COX-2与CYPlBI表达呈正相关（r=0．481,P〈0．01）。结论人乳腺癌组织存在COX-2与CYPlBl的共同阳性表达,COX-2可能促进CYPlBl表达,共同作用参与乳腺癌的发生发展。     

Selenoprotein P

Selenoprotein P (SeP) is an extracellular, monomeric glycoprotein containing up to 10 selenocysteine residues in the polypeptide chain. It is ubiquitously expressed in mammalian tissues, and in human plasma it accounts for at least 40% of the total selenium concentration. SeP binds to heparin and cell membranes, and is associated with endothelial cells. SeP in human plasma protects against peroxynitrite-mediated oxidation and reduces phospholipid hydroperoxide in vitro, in accordance with the presumption that it has a function as an extracellular oxidant defense. Immunochemical assays have demonstrated that its concentration in plasma varies much with selenium intake, but other factors also have an influence.     

Molecular modeling of mammalian cytochrome P450s

The cytochrome P450s are a superfamily of hemoprotein enzymes responsible for the metabolism of a wide variety of xenobiotic and endogenous compounds. The individual P450s exhibit unique substrate specificity and stereoselectivity profiles which reflect corresponding differences in primary sequence and tertiary structure. In the absence of an experimental structure, models for mammalian P450s have been generated by their homology with bacterial P450s of known structure. The rather low sequence identity between target and template proteins renders P450 modeling a challenging task. However, the substrate recognition properties of several P450s are consistent with recently developed working models. This review summarizes the major concepts and current approaches of molecular modeling of P450s. Received 28 September 1999; received after revision 25 November 1999; accepted 31 December 1999     

Zur Kenntnis der Blattlipide vonAcer platanoides L. während der herbstlichen Vergilbung

Summary The lipids of green and yellow leaves ofAcer platanoides L. have been investigated. Yellow leaves were found to contain phytyl linolenate, a further example of the formation of esters containing terpenoid alcohols and unsaturated fatty acids in yellow autumn leaves. The determination af squalene in leaves of the same tree showed an increase of squalene content during the whole season and the autumn colouration. Regarding the leaf lipids we conclude autumn colouration to be characterized by 3 facts: (1) Lipid degradation, (2) formation of esters and (3) accumulation of lipid intermediates.     

Functions of fatty acid binding proteins

Summary Cytosolic fatty acid binding proteins (FABP) belong to a gene family of which eight members have been conclusively identified. These 14–15 kDa proteins are abundantly expressed in a highly tissue-specific manner. Although the functions of the cytosolic FABP are not clearly established, they appear to enhance the transfer of long-chain fatty acids between artificial and native lipid membranes, and also to have a stimulatory effect on a number of enzymes of fatty acid metabolism in vitro. These findings, as well as the tissue expression, ligand binding properties, ontogeny and regulation of these proteins provide a considerable body of indirect evidence supporting a broad role for the FABP in the intracellular transport and metabolism of long-chain fatty acids. The available data also support the existence of structure- and tissue-specific specialization of function among different members of the FABP gene family. Moreover, FABP may also have a possible role in the modulation of cell growth and proliferation, possibly by virtue of their affinity for ligands such as prostaglandins, leukotrienes and fatty acids, which are known to influence cell growth activity. FABP structurally unrelated to the cytosolic gene family have also been identified in the plasma membranes of several tissues (FABP_pm). These proteins have not been fully characterized to date, but strong evidence suggests that they function in the transport of long-chain fatty acids across the plasma membrane.     

Thiamine pyrophosphate: An essential cofactor for the α-oxidation in mammals – implications for thiamine deficiencies?

The identification of 2-hydroxyphytanoyl-CoA lyase (2-HPCL), a thiamine pyrophosphate (TPP)-dependent peroxisomal enzyme involved in the α-oxidation of phytanic acid and of 2-hydroxy straight chain fatty acids, pointed towards a role of TPP in these processes. Until then, TPP had not been implicated in mammalian peroxisomal metabolism. The effect of thiamine deficiency on 2-HPCL and α-oxidation has not been studied, nor have possible adverse effects of deficient α-oxidation been considered in the pathogenesis of diseases associated with thiamine shortage, such as thiamine-responsive megaloblastic anemia (TRMA). Experiments with cultured cells and animal models showed that α-oxidation is controlled by the thiamine status of the cell/tissue/organism, and suggested that some pathological consequences of thiamine starvation could be related to impaired α-oxidation. Whereas accumulation of phytanic acid and/or 2-hydroxyfatty acids or their α-oxidation intermediates in TRMA patients given a normal supply of thiamine is unlikely, this may not be true when malnourished. Received 23 December 2005; received after revision 10 April 2006; accepted 28 April 2006     

Cellular uptake of long-chain fatty acids: role of membrane-associated fatty-acid-binding/transport proteins

The critical importance of long-chain fatty acids in cellular homeostasis demands an efficient uptake system for these fatty acids and their metabolism in tissues. Increasing evidence suggests that the plasma-membrane-associated and cytoplasmic fatty-acid-binding proteins are involved in cellular fatty acid uptake, transport and metabolism in tissues. These binding proteins may also function in the fine tuning of cellular events by modulating the metabolism of long-chain fatty acids implicated in the regulation of cell growth and various cellular functions. Several membrane-associated fatty-acid-binding/transport proteins such as plasma membrane fatty-acid-binding protein (FABPpm, 43 kDa), fatty acid translocase (FAT, 88 kDa) and fatty acid transporter protein (FATP, 63 kDa) have been identified. In the feto-placental unit, preferential transport of maternal plasma arachidonic and docosahexaenoic acids across the placenta is of critical importance for fetal growth and development. Our studies have shown that arachidonic and docosahexaenoic acids are preferentially taken up by placental trophoblasts for fetal transport. The existence of a fatty-acid-transport system comprising multiple membrane-binding proteins (FAT, FATP and FABPpm) in human placenta may be essential to facilitate the preferential transport of maternal plasma fatty acids in order to meet the requirements of the growing fetus. The preferential uptake of arachidonic and docosahexaenoic acids by the human placenta has the net effect of shunting these maternal plasma fatty acids towards the fetus. The roles of plasma membrane-associated binding/transport proteins (FABPpm, FAT and FATP) in tissue-specific fatty acid uptake and metabolism are discussed.     

Functions of fatty acid binding proteins

Cytosolic fatty acid binding proteins (FABP) belong to a gene family of which eight members have been conclusively identified. These 14-15 kDa proteins are abundantly expressed in a highly tissue-specific manner. Although the functions of the cytosolic FABP are not clearly established, they appear to enhance the transfer of long-chain fatty acids between artificial and native lipid membranes, and also to have a stimulatory effect on a number of enzymes of fatty acid metabolism in vitro. These findings, as well as the tissue expression, ligand binding properties, ontogeny and regulation of these proteins provide a considerable body of indirect evidence supporting a broad role for the FABP in the intracellular transport and metabolism of long-chain fatty acids. The available data also support the existence of structure- and tissue-specific specialization of function among different members of the FABP gene family. Moreover, FABP may also have a possible role in the modulation of cell growth and proliferation, possibly by virtue of their affinity for ligands such as prostaglandins, leukotrienes and fatty acids, which are known to influence cell growth activity. FABP structurally unrelated to the cytosolic gene family have also been identified in the plasma membranes of several tissues (FABPpm). These proteins have not been fully characterized to date, but strong evidence suggest that they function in the transport of long-chain fatty acids across the plasma membrane.     

Regulation of Plasmodium falciparum Pfnek3 relies on phosphorylation at its activation loop and at threonine 82

A mitogen-activated protein kinase (MAPK), Pfmap2, has been identified in Plasmodium falciparum. However, its bona fide activator remains elusive as no MAPK kinase (MAPKK) homologues have been found so far. Instead, Pfnek3, a NIMA (never in mitosis, Aspergillus)-related kinase, was earlier reported to display a MAPKK-like activity due to its activating effect on Pfmap2. In this study, the regulatory mechanism of Pfnek3 was investigated. Pfnek3 was found to possess a SSEQSS motif within its activation loop that fulfills the consensus SXXXS/T phospho-activating sequence of MAPKKs. Functional analyses of the SSEQSS motif by site-directed mutagenesis revealed that phosphorylation of residues S221 and S226 is essential for mediating Pfnek3 activity. Moreover, via tandem mass-spectrometry, residue T82 was uncovered as an additional phosphorylation site involved in Pfnek3 activation. Collectively, these results provide valuable insights into the potential in vivo regulation of Pfnek3, with residues T82, S221 and S226 functioning as phospho-activating sites.     

Enzymatic reduction of fatty acids and acyl-CoAs to long chain aldehydes and alcohols

Summary The properties of enzymatic systems involved in the synthesis of long chain aldehydes and alcohols have been reviewed. Fatty acid and acyl-CoA reductases are widely distributed and generate fatty alcohols for ether lipid and was ester synthesis as well as fatty aldehydes for bacterial bioluminescence. Fatty alcohol is generally the major product of fatty acid reduction in crude or membrane systems, although reductases which release fatty aldehydes as products have also been purified. The reduction of fatty acid proceeds through the ATP-dependent formation of acyl intermediates such as acyl-CoA and acyl protein, followed by reduction to aldehyde and alcohol with NAD(P)H. In most cases, both the rate of fatty acid conversion and acyl chain specificity of the reaction are determined at the level of reduction of the intermediate. The reduction of fatty acids represents the major pathway for the control of the synthesis of fatty aldehydes and alcohols. Several other enzymatic reactions involved in lipid degradation also release fatty aldehydes but do not apear to play an important role in long chain alcohol synthesis.     

Enzymatic reduction of fatty acids and acyl-CoAs to long chain aldehydes and alcohols

The properties of enzymatic systems involved in the synthesis of long chain aldehydes and alcohols have been reviewed. Fatty acid and acyl-CoA reductases are widely distributed and generate fatty alcohols for ether lipid and wax ester synthesis as well as fatty aldehydes for bacterial bioluminescence. Fatty alcohol is generally the major product of fatty acid reduction in crude or membrane systems, although reductases which release fatty aldehydes as products have also been purified. The reduction of fatty acid proceeds through the ATP-dependent formation of acyl intermediates such as acyl-CoA and acyl protein, followed by reduction to aldehyde and alcohol with NAD(P)H. In most cases, both the rate of fatty acid conversion and acyl chain specificity of the reaction are determined at the level of reduction of the intermediate. The reduction of fatty acids represents the major pathway for the control of the synthesis of fatty aldehydes and alcohols. Several other enzymatic reactions involved in lipid degradation also release fatty aldehydes but do not appear to play an important role in long chain alcohol synthesis.     

Fatty acid modulation of antiestrogen action and antiestrogen-binding protein in cultured lymphoid cells

Nonsteroidal antiestrogens reversibly and specifically inhibited the proliferation of two estrogen receptor-negative lymphoid cell lines (EL4 and Raji) in a dose-dependent manner. [3H]Thymidine incorporation of concanavalin A-stimulated primary splenocytes was also inhibited by 10(-6) M clomiphene (1-[4-(2-diethylaminoethoxy)phenyl]-1,2-diphenyl-2-chloroethylene). The antiproliferative effect could be prevented by the simultaneous presence in the growth medium of 10(-5) M linoleic acid or 10(-5) M arachidonic acid but not by 10(-6) M estradiol. Both lymphoid cell lines had high affinity antiestrogen-binding sites whose affinity could be altered by conditions of growth. Growth of EL4 cells in RPMI 1640 medium supplemented with charcoal-pretreated 5% fetal calf serum (charcoal-stripped medium) resulted in significantly higher affinity (Kd 0.54 nM +/- 0.11 nM; n = 6) than growth in medium supplemented with untreated serum (complete medium) (Kd = 1.68 nM +/- 0.48 nM; n = 6) (p less than 0.001). This change in affinity was partly due to removal of fatty acids from the growth medium by charcoal pretreatment, since addition of 10(-5) M linoleic acid or 10(-5) M gamma-linolenic to charcoal-stripped medium decreased the affinity of the antiestrogen-binding protein. In contrast, growth in 10(-5) M stearic acid or 10(-5) M oleic acid did not significantly alter the affinity of the antiestrogen-binding protein, whereas 10(-5) M palmitic acid significantly increased its affinity. The same fatty acids were also tested for their intrinsic effects on EL4 cell proliferation. Oleic, linoleic and gamma-linolenic acids were growth stimulatory while stearic and palmitic acids were not. Thus linoleic and gamma-linolenic acids whose presence in the growth medium was associated with decreased affinity of [3H]tamoxifen (1-[4-(2-dimethylaminoethoxy)phenyl]-1,2-diphenylbut-1(Z)-ene) binding to the intracellular antiestrogen-binding protein were also growth stimulatory. Unsaturated fatty acids have previously been shown to inhibit binding of [3H]tamoxifen to the antiestrogen-binding protein in a cell-free system. The present observations demonstrate that unsaturated fatty acids also modify the affinity of the antiestrogen-binding protein in intact cells.     

Identification of an oviposition-regulating pheromone in the European grapevine moth,Lobesia botrana (Lepidoptera: Tortricidae)

The oviposition of the European grapevine moth (EGVM)Lobesia botrana can be deterred by an extract of conspecific eggs corresponding to 20 egg equivalents. The reduction of the oviposition behavior is dose-dependent. Nine chemicals have been extracted from the eggs and identified as straight chain fatty acids and esters of fatty acids. A mixture of these rather simple molecules induces the same levels of deterrence as the total extract. It might be possible to use oviposition regulating pheromone in the future for the control of EGVM populations.     

The alpha-kinase family: an exceptional branch on the protein kinase tree

The alpha-kinase family represents a class of atypical protein kinases that display little sequence similarity to conventional protein kinases. Early studies on myosin heavy chain kinases in Dictyostelium discoideum revealed their unusual propensity to phosphorylate serine and threonine residues in the context of an alpha-helix. Although recent studies show that some members of this family can also phosphorylate residues in non-helical regions, the name alpha-kinase has remained. During evolution, the alpha-kinase domains combined with many different functional subdomains such as von Willebrand factor-like motifs (vWKa) and even cation channels (TRPM6 and TRPM7). As a result, these kinases are implicated in a large variety of cellular processes such as protein translation, Mg²⁺ homeostasis, intracellular transport, cell migration, adhesion, and proliferation. Here, we review the current state of knowledge on different members of this kinase family and discuss the potential use of alpha-kinases as drug targets in diseases such as cancer.     

Introduction: lipid-binding proteins: novel aspects

Lipid-binding proteins function to transport lipids across membranes and aqueous phases and act to solubilise their cargo, protect it from chemical damage and probably also to define its destination. As such, they have been adapted to carry out a broad spectrum of biological functions in addition to their classical roles in energy metabolism and the transmission or blocking of retinoid-based signalling. The set of reviews in this issue of CMLS is designed to draw attention to some newly understood aspects and principles of their biology and structure, and concentrates on the proteins involved in transport of fatty acids and retinoids.     

Physiological importance of omega-3/omega-6 polyunsaturated fatty acids in man. An overview of still unresolved and controversial questions

The 'essentiality' of (omega-6) and (omega-3) fatty acids in mammals is well known. Nevertheless, some important points remain unclear concerning their implication in physiology. After a short discussion about the definition of essential fatty acids deficiency, this brief overview deals with some of these points, pointing out some of the unresolved questions. Different subjects are approached concerning the (omega-6) and (omega-3) fatty acids metabolism: desaturases, eicosanoids, production, as well as some of their metabolic effects on cell membranes, intestinal function, glucose and lipid metabolism, haemorheology.     

How do thermophilic proteins deal with heat?

Recent years have witnessed an explosion of sequence and structural information for proteins from hyperthermophilic and thermophilic organisms. Complete genome sequences are available for many hyperthermophilic archaeons. Here, we review some recent studies on protein thermostability along with work from our laboratory. A large number of sequence and structural factors are thought to contribute toward higher intrinsic thermal stability of proteins from these organisms. The most consistent are surface loop deletion, increased occurrence of hydrophobic residues with branched side chains and an increased proportion of charged residues at the expense of uncharged polar residues. The energetic contribution of electrostatic interactions such as salt bridges and their networks toward protein stability can be stabilizing or destabilizing. For hyperthermophilic proteins, the contribution is mostly stabilizing. Macroscopically, improvement in electrostatic interactions and strengthening of hydrophobic cores by branched apolar residues increase the enthalpy change between the folded and unfolded states of a thermophilic protein. At the same time, surface loop deletion contributes to decreased conformational entropy and decreased heat capacity change between the folded and unfolded states of the protein. Received 28 February 2001; received after revision 26 March 2001; accepted 27 March 2001     

The metabotropic GABA receptor: molecular insights and their functional consequences

Recent years have seen rapid and significant advances in our understanding of the G-protein-coupled gamma-amino butyric acid, B-type (GABA(B)) receptor, which could be a therapeutic target in conditions as diverse as epilepsy and hypertension. This progress originated with the ground-breaking work of Bernhard Bettler's team at Novartis who cloned the DNA encoding a GABA(B) receptor in 1997. Currently, the receptor is thought to be an unusual, possibly unique, example of a heterodimer composed of homologous, seven-transmembrane-domain (7TMD) subunits (named GABA(B) R1 and GABA(B) R2), neither of which is fully functional when expressed alone. The large N-terminal domain of the GABA(B) R1 subunit projects extracellularly and contains a ligand binding site. The similarity of the amino acid sequence of this region to some bacterial periplasmic amino acid-binding proteins of known structure has enabled structural and functional modelling of the N-terminal domain, and the identification of residues whose substitution modulates agonist/antagonist binding affinities. The intracellular C-terminal domains of the R1 and R2 subunits appear to constitute an important means of contact between the two subunits. Alternative splice variants, a common and functionally important feature of 7TMD proteins, have been demonstrated for the R1 subunit. Notably GABA(B) R1a differs from GABA(B) R1b by the possession of an N-terminal extension containing two complement protein modules (also called SCRs, or sushi domains) of unknown function. The levels at which each of the respective variants is expressed are not equal to one another, with variations occurring over the course of development and throughout the central nervous system. It is not yet clear, however, whether one variant is predominantly presynaptically located and the other postsynaptically located. The existence of as yet unidentified splice variants, additional receptor subtypes and alternative quaternary composition has not been ruled out as a source of receptor heterogeneity.