L

L-NAME

L-NAME (N-nitro-L-arginine methyl ester), likeL-NMMA, is a structural analogue of L-arginine andcompetes with L-arginine for NO-synthase, whichuses L-arginine as a substrate for the formation ofNO. L-NMMA and L-NAME are very effectiveNO-synthesis inhibitors, both in vitro and in vivo.

▶NO Synthase

b-Lactam Antibiotics

J EAN-M AR IE G HUYS EN , JEAN-M ARIE F RERECentre d’ Ingenierie des Protéines, University of Liège,Liège, Belgium

SynonymsWall peptidoglycan inhibitors

Definition▶β-Lactam antibiotics are bicyclic or monocyclicazetidinone ring-containing compounds ( Fig. 1). Theykill bacteria by preventing the assembly of (4 –3)peptidoglycans. These covalently closed net-like poly-mers form the matrix of the cell wall by which thebacteria can divide and multiply, despite their highinternal osmotic pressure.

Mechanism of Action▶Peptidoglycans of the (4 –3) and (3–3) types ( Fig. 2)are comprised of glycan chains made of alternatingβ-1,4-linked N-acetylglucosamine and N-acetylmura-mic acid residues [1]. The D -lactyl groups on carbon C3

of the muramic acids are substituted by L-alanyl- γ - D -glutamyl- L -diaminoacyl- D -alanine stem tetrapeptides.In the (4 –3) peptidoglycans, peptides borne by adjacentglycan chains are cross-linked through direct linkagesor cross bridges (comprising one or several intervening

amino acid residues) that extend from the D-alanineresidue at position 4 of a stem peptide to the ω-aminogroup at position 3 of another stem peptide. Lipid II( Fig. 3) is the immediate biosynthetic precursor.A disaccharide bearing an L-alanyl- γ - D-glutamyl- L-diaminoacyl- D -alanyl- D-alanine stem pentapeptide (thediamino acid residue of which can be either free, i.e.,unsubstituted, or substituted by one or several aminoacid residues, i.e., branched) is exposed on the outer faceof the plasma membrane, linked to a C55 -undecaprenylvia a pyrophosphate. From this precursor, the formationof polymeric (4–3) peptidoglycans relies on glycosyltransferases ensuring glycan chain elongation andacyl transferases ensuring peptide cross linking. Acyltransferases of the SXXK superfamily (▶ SXXK AcylTransferases, also called DD-transpeptidases, with Xdenoting a variable amino acid residue) are implicated incross linking and they exhibit a specific bar code in theform of three motifs, SXXK, SXN (or analogue) andKTG (or analogue), occurring at equivalent places androughly with the same spacing along the polypeptidechains [2]. As a result of the polypeptide folding, themotifs are brought close to each other at the immediateboundary of the catalytic centre between an all-α domainand an α/β domain. SXXK acyl transferases identifyN-acyl-D-alanyl-D-alanine sequences as carbonyl donors,produce a N-acyl-D-alanyl moiety linked as an esterto the serine residue of the invariant SXXK motifand transfer the peptidyl moiety onto an amino group(transpeptidation) or a water molecule (carboxypeptida-tion). SXXK acyl transferases identify penicillin (usedas a generic term for β-lactam antibiotics) as a suicidecarbonyl donor. Because the serine-ester linked peni-cilloyl enzyme that SXXK acyl transferases produce israther stable, they are immobilized, at least for a longtime, in the form of penicillin-binding proteins, in shortPBPs. The inactivation reaction can be written

where E-OH is the enzyme, with –OH the hydroxylgroup of its active-site serine residue. Kinetically, theinteraction is described by a 3-step model

b-Lactam Antibiotics. Figure 1 Bicyclic (penams, 3-cephems, oxacephems and carbapenems) and monocyclic(aztreonam) β-lactam antibiotics. Rupture of the scissile amide bond of the azetidinone ring (arrow) by the SXXK acyltransferases implicated in (4–3) peptidoglycan synthesis results in the formation of long-lived, serine-linked acyl esterderivatives. The inactivated enzymes behave as penicillin-binding proteins or PBPs.

680 b-Lactam Antibiotics

where I is the β-lactam, P(s) the product(s) resultingfrom the very slow decay of the EI* penicilloyl enzyme.K is the dissociation constant of the EI, a non-covalentcomplex and k2 and k3 first-order rate constants. In allcases, k3 has been found to be very small and K isgenerally large so that the sensitivity of a PBP to aβ-lactam is best characterized by the k2/K ratio [3]. Thisratio varies from 1,000,000 M −1s− 1 for the mostsensitive PBP to less than 1 for the most resistant.

Lethal Target ProteinsA constellation of genes code for PBPs of varyingamino acid sequences and functionalities. PBPs occuras free-standing polypeptides and as protein fusions.This combinatorial system of structural modules resultsin a large increase in diversity.

▶SXXK PBP fusions of classes A and B are the lethalta rg et s o f β-lactam antibiotics. The PBP fusions of class Aare comprised of an SXXK acyl transferase module ofclass A, linked to the carboxy end of a glycosyltransferase module having its own five motif-bar code,itself linked to the carboxy end of a membrane anchor [2].They convert the disaccharide–pentapeptide units borneby lipid II precursormolecules into nascent polymeric (4–3) peptidoglycans. Glycan chain elongation is catalysedby the transglycosylase module. Peptide cross linkingbetween elongated glycanchains is then carried out by theassociated SXXK acyl transferase module. The PBPfusions of class B are comprised of an SXXK acyltransferase module of class B, bound to the carboxy endof a linker module having its own three-motif bar code,itself linked to a membrane anchor. They are componentsofmorphogenetic apparatus that controls wall expansion,ensures cell-shape maintenance and carries out septumformation. Likely, the linker modules ensure that the

b-Lactam Antibiotics. Figure 2 (4–3) Peptidoglycan (the synthesis of which is susceptible to β-lactam antibiotics)and (3–3) peptidoglycan (the synthesis of which is resistant to antibiotics) in Escherichia coli and Mycobacteriumtuberculosis. G:N-acetylglucosamine. M:N-acetylmuramic acid (i.e.,N-acetylglucosamine with a D-lactyl substituenton carbon C3). A2pm: meso-diaminopimelic acid. In E. coli and M. tuberculosis, the stem peptides are unbranched.COX: COOH (in E. coli) or CONH2 (in M. tuberculosis).

b-Lactam Antibiotics 681

L

associated SXXK acyl transferase modules are positionedin an active conformation within the morphogeneticapparatus where they need to be. Binding of β-lactamantibiotics to the SXXK acyl transferase modules of thePBP fusions leads to the formation of Henri–Michaeliscomplexes that exhibit ligand- and enzyme-specific hy-drogen bonding networks. Es ch er ic hi a c ol i is killed in anumber of ways; via cell lysis as a result of the selectiveinactivation of the class A PBP1a and PBP1b (whichcan substitute for each other) by cephaloridine andcefsulodin; via transformation of the cells intoround bodies as a result of the selective inactivation oft he c el l- cy cl e s ub cl as s B 2 P B P 2 b y m ec il li na m a ndthienamycin; via cell filamentation as a result of theselective inactivation of the cell-cycle subclass B3 PBP3by mezlocillin, cefaperazone, cefotaxime, cefuroxime,cephalothin and aztreonam; or via different combinationsof these morphological alterations by ampicillin, benzyl-penicillin, carbenicillin and cefoxitin.

b-Lactamase-Mediated Resistance▶SXXK free-standing PBPs are peptidoglycan-hydro-lases of one kind or another. Loss of these auxilliarycell-cycle proteins causes varying morphological aber-rations, but is not fatal at least in the laboratoryenvironment. Similarly, conversion of free-standingPBPs into β-lactam antibiotic-hydrolysing enzymes,with loss of peptidase activity and conservation of thepolypeptide fold, gives rise to the SXXK β- lactamases .There are three classes of SXXK β-lactamases A, C andD, which are easily distinguished on the basis of theirprimary structures, although the tertiary structures are

clearly similar and also related to those of the acyltransferase modules of PBPs. In particular, in additionto the characteristic SXXK, motifs reminiscent of thebar code SXN and KTG groups are found in nearlysuperimposable positions. The catalytic pathway ofβ-lactamases follows the 3-step model shown above butwith good substrates, the k3 value can be higher than1000 s −1 [4].

The class A enzymes have Mr values around 30,000.Their substrate specificities are quite variable and alarge number of enzymes have emerged in response tothe selective pressure exerted by the sometimes abusiveutilization of antibiotics. Some of these “new enzymes”are variants of previously known enzymes, with only alimited number of mutations (1–4) but a significantlybroadened substrate spectrum while others exhibitsignificantly different sequences. The first category isexemplified by the numerous TEM variants whoseactivity can be extended to third and fourth generationcephalosporins and the second by the NMCA andSME enzymes which, in contrast to all other SXXKβ-lactamases, hydrolyse carbapenems with high effi-ciency. Despite these specificity differences, the tertiarystructures of all class A β-lactamases are nearlysuperimposable.

The class C enzymes have Mr values of 39,000 andexhibit more uniform properties. They hydrolysebenzyl- and phenoxymethyl penicillin relatively well(turn-over numbers of 20–70 s−1), ampicillin andamoxicillin 10- to 20-fold less rapidly and extremelypoorly the other penicillins (generally due to lowk3 values). The early cephalosporins (cephalothin,

b-Lactam Antibiotics. Figure 3 Lipid II precursor (bottom) and polymeric (4–3) peptidoglycan of Escherichia coliand Mycobacterium tuberculosis. Glycosyl transferase and acyl transferase-catalysed reactions. G: N-acetylglucosamine. M: N-acetylmuramic acid. A2pm: meso-diaminopimelic acid. (3–3) Peptidoglycan cross linking(Fig. 2) may proceed via the formation, in a penicillin-resistant manner, of a N-acyl-L-diaminoacyl moiety linked as anester to the serine residue and the transfer of the peptidyl moiety to theω-amino group of the diamino acid residue ofanother peptide.

682 b-Lactam Antibiotics

cephalexin, cefazolin) are well hydrolysed but the latergeneration compounds also exhibit low to very lowk3 values, as do imipenem and aztreonam. A fewmutations are known to extend the spectrum of someenzymes but the most currently utilized bacterialstrategy is overproduction by deregulation of thebiosynthetic control mechanism, so that the MICs ofpoor or even very poor substrates can increasesignificantly [5].

Class D enzymes (Mr of about 27,000) exhibit a highactivity versus isoxazolyl penicillins, such as oxacillinand are referred to as the OXA-family. Surprisingly,the amino group of the SXXK lysine residues iscarboxylated in the most active forms of the enzymes.Penicillins are generally better substrates thancephalosporins but mutations have been found whichconfer extended activity spectra to these enzymes.

Inactivators of class A β-lactamases (clavulanate,sulbactam, tazobactam) are themselves β-lactams andact as suicide substrates. They can be used in

combination with β-lactamase sensitive compounds(ampicillin, amoxicillin) but are not clinically usefulagainst class C and class D producers. Some TEMmutants have also been selected that exhibit reducedsensitivity to these inactivators.The class B metallo-β-lactamases have emerged

more recently as a clinical problem but they areparticularly dangerous since many of them hydrolyseall know β-lactams, with the exception of monobac-tams. In particular, they hydrolyse the suicide substratesmentioned above, as well as carbapenems that usuallyescape the activity of all the SXXK enzymes, with theexception of the NMCA group.Three subclasses B1, B2 and B3 can be distin-

guished on the basis of the sequences. B1 and B3enzymes are optimally active with 2 Zn2+ions, while theB2 enzymes are inhibited by the second Zn2+. These B2enzymes also exhibit a very narrow activity spectrumand only hydrolyse carbapenems. The 3D structures ofrepresentative members of each subfamily have been

b-Lactamases 683

solved and they highlight a typical αββα fold,completely unrelated to that of the SXXK enzymes.The production of β-lactamases can be inducible orconstitutive and the genes carried by the chromosomeor plasmids, some genes being parts of integrons.Several clinical strains produce up to three distinctenzymes.

L

PBP-Mediated ResistanceThere are at least two modes of intrinsic resistance toβ-lactam antibiotics. Determinants conferring a decreasedsusceptibility to β-lactam antibiotics evolve by theaccumulation of point mutations in genes that codefor essential PBP fusions of classes A and/or B. Theshuffling and capture of DNA sequences from commen-sal Streptococci having a reduced susceptibility to thedrug, give rise to S tre pt oc oc cu s p ne um on ia e pathogens inwhich mosaic genes code for mosaic PBP fusions ofclasses A and/or B of decreased affinity for the drug.Mosaic and wildtype PBP fusions of the same class (andthe same subclass) differ by up to 15% amino acidresidues. Mosaic PBPs occur also in Neisseria meningi-tidis and N e is se ri a g on or rh o e ae strains.

Other strategies leading to an increased resistanceare the transfer of a complete gene encoding a resistantPBP from a non-pathogenic related species to yield theMethicillin Resistant Staphylococcus aureus (MRSA)and the overproduction by Enterococci of a pre-existingbut minor resistant PBP, which can further mutate toeven more resistant forms. Some bacteria can alsomanufacture a (3 –3) peptidoglycan ( Fig. 2) with thehelp of a penicillin-resistant LD -transpeptidase, whichis not an SXXK enzyme. Indeed, a low proportion of(3–3) cross links have been found in the walls ofEnterococcus faecium , Mycobacterium tuberculosisand Mycobacterium leprae . The quantitative influenceof these cross links on the resistance level remains to bedetermined but the synthesis of (3–3) peptidoglycan,combined with that of a set of relatively resistant SXXKPBPs and the limited permeability of the mycolic acidlayer might explain the lack of efficiency of β-lactamantibiotics as therapeutic agents against tuberculosisand leprosy [6].

Outer Membrane Permeability and Active

Efflux Systems In Gram-negative bacteria, diffusion of β-lactamantibiotics into the periplasm (where the activity ofPBPs takes place) occurs via the channels that porinscreate in the outer membrane. The number andproperties of the porin molecules are such that diffusionis relatively rapid in E. coli but much slower inEnterobacter and Pseudomonas. Mutants can beselected after the permeability of porin channels ortheir number has been decreased. A slow diffusion into

the periplasm becomes a particularly important factorwhen it is combined with the presence of a β-lactamase(even in low concentration or when poorly active) in thesame cellular compartment. Note that inMycobacteria,the mycolic acid layer plays a role similar to that of theouter membrane in Gram-negatives. Finally, β-lactamsbearing a hydrophobic side-chain can be ejectedfrom the periplasm by the active efflux systemsAcrAB-TolC in E. coli and Salmonella typhimuriumand MexAB-OprM in Pseudomonas. Overexpressionof these proteins can significantly increase the levelof resistance.

Clinical UseBecause the SXXK PBPs are specific to the prokar-yotes, the β-lactam antibiotics have a high selectivetoxicity without marked side effects except for possibleallergic reactions. Resistance is a problem of greatconcern. The use of antibiotics fuels the continuingemergence and spreading of novel β-lactamasesand intrinsic resistance determinants among bacterialpathogens.

▶Microbial Resistance to Drugs▶Quinolones▶Ribosomal Protein Synthesis Inhibitors

References1. van Heijenoort J (1996) Murein synthesis. In: Neidhart FC

et al (ed) Escherichia coli and Salmonella: cellular andmolecular biology, 2nd edn. ASM Press, Washington, DC,pp 1025–1035.

2. Goffin C, Ghuysen JM (1998) The multimodularpenicillin-binding proteins: an enigmatic family of orthologsand paralogs. Microbiol Mol Biol Rev 62:1079–1093

3. Frére JM et al (1975) Kinetics of interactions between theexocellular DD-carboxypeptidase-transpeptidase fromStreptomyces R61 and β-lactam antibiotics. Eur JBiochem 57:343–351

4. Matagne A et al (1999) The β-lactamase cycle: a tale ofselective pressure and bacterial ingenuity. Nat Prod Rep16:1–19

5. Lakaye B et al (1999) When drug inactivation renders thetarget irrelevant to antibiotic resistance: a case story withβ-lactams. Mol Microbiol 31:89–101

6. Lepage S et al (1997) Dual multimodular class Apenicillin-binding proteins in Mycobacterium leprae.J Bacteriol 179:4627–4630

b-Lactamases

▶β-Lactam Antibiotics.

684 Lateral Hypothalamic Area

Lateral Hypothalamic Area

The lateral hypothalamic area has been identified as afeeding centre by studies involving electric stimulationand discrete lesions. Neurons in the lateral hypothalam-ic area and the neighbouring perifornical area expressneuropeptides that stimulate feeding when injected intocerebral ventricles (orexins 1 and 2, melanin-concen-trating hormone (MCH)).

▶Appetite Control

Laxatives

Laxatives, also called purgatives or cathartics, aresubstances used to hasten the transit of food through theintestine. Laxatives function by different mechanisms.Bulk laxatives, like methylcellulose or bran, containagents like polysaccharide polymers, which are notfermented by the normal processes of digestion. Theyretain water in the gut lumen and promote bowelmovements. Osmotic laxatives consist of poorlyabsorbable solutes such as salines containing magne-sium cations or phosphate anions or non-digestablesugars and alcohols (glycerin, lactulose, sorbitol ormannitol). Osmotic laxatives retain an increasedvolume of fluid in the lumen of the bowel by osmosis,which accelerates the transfer of the bowel contents.Faecal softeners alter the consistency of the faeces.They are also called emollients and contain surface-active compounds similar to detergents, like docusatesalts. Stimulant laxatives/purgatives increase the motil-ity of the gut (peristalsis) and stimulate water andelectrolyte secretion by the mucosa. Stimulant laxa-tives, which can cause abdominal cramps and deterio-ration of intestinal function, include diphenylmethanederivatives (bisacodyl, phenolphthalein), anthraqui-none laxatives (derivatives of plants such as aloe,cascara or senna) and ricinoleic acid.

LCAT

▶Lecithin-Cholesterol Acyltransferase

LD50

▶Lethal Dose

LDL-Cholesterol

▶Low-Density-Lipoprotein

LDL-Receptors

LDL-receptors are receptors on the cell surface thatremove LDL (and some other forms of) lipoproteinsfrom the plasma into the cell.

▶HMG-CoA-Reductase Inhibitors (Statins)▶Lipoprotein Metabolism▶Low-Density Lipo-protein Receptor Gene Family

Lead

A lead is a hit compound that displays specificity andpotency against a target in a library screen and continuesto show the initial positive dose-dependent response inmore complex models such as cells and animals.

Lead Discovery by NMR

▶SAR-by-NMR

Lecithin-Cholesterol Acyltransferase

Enzyme that converts free cholesterol to cholesterylester on HDL.

▶Lipoprotein Metabolism

Leukotrienes 685

Leonardo

▶14-3-3 Proteins

Leptin

L

The cytokine leptin is secreted by adipocytes (fat cells) inproportion to the size of the adipose depot and circulatesvia the bloodstream to the brain, where it ultimatelyaffects feeding behavior, endocrine systems includingreproductive function and, at least in rodents, energyexpenditure. The major effect of Leptin is on the hy-pothalamous,where it suppresses appetite andhence foodintake. Leptin exerts its effects via binding to the leptinreceptor in the brain (specifically in the hypothalamus),which activates the JAK-STAT Pathway.

▶Appetite Control▶Adipokines

Lethal Dose

The lethal dose (LD50) is a measure of the toxicity of acompound. In an LD50 toxicity test, various doses of adrug are administered to groups of animals, and themortality in each group is determined. The lethal dose 50is the dose,which is lethal for 50%of a group of animals.

Leucin Zipper

A leucine zipper is a structural motif present in a largeclass of transcription factors. These dimeric proteinscontain two extended alpha helices that “grip” the DNAmolecule much like a pair of scissors at adjacent majorgrooves. The coiled-coil dimerization domain containsprecisely spaced leucine residues which are required forthe interaction of the two monomers. Some DNA-binding proteins with this general motif contain otherhydrophobic amino acids in these positions; hence, thisstructural motif is generally called a basic zipper.

Leukotrienes

ALAN R. LEFF, NILDA M. MUNOZ

Department of Medicine, University of Chicago,IL, USA

Synonyms▶Cysteinyl leukotrienes; B-leukotriene

DefinitionLeukotrienes belong to a large family of lipidmediators, termed eicosanoids, which are derived fromarachidonic acid (AA) and released from the cellmembrane by phospholipases. AA is subsequentlyconverted by the enzyme, 5-lipoxygenase (5-LO) and aprotein cofactor, ▶5-lipoxygense activating protein(FLAP), into two bioactive classes of leukotriene(LT): (i) LTB4 and (ii) ▶cysteinyl leukotrienes(cysLTs), LTC4, D4, and E4. Leukotrienes do not existin the state preformed in cells. These compounds play asignificant role in allergic responses and contributevariably to the bronchoconstrictor response in human▶bronchial asthma, and possibly, chronic obstructivepulmonary disease.

Basic CharacteristicsSynthesis and MetabolismThe site of synthesis of leukotrienes is largely orcompletely in the nuclear membrane. LTs are producedfromcellmembranephospholipid-associated arachidonicacid (AA), which is liberated by 85 kDa cytosolic groupIVAPLA2 (cPLA2) (Fig. 1) or phospholipase C (notshown) in response to cell activation. The secretoryphospholipase group V PLA2 (gVPLA2) also is capableof inducing leukotriene synthesis, probably from phos-phatidylcholine-rich membrane hydrolysis at both plas-ma or perinuclear membrane [1]. AA is converted to 5-HETE and subsequently to an unstable epoxide LTA4

([5(s)-trans-5,6-oxido-7,9-trans11,14-cis-eicosatetra-nonic acid]), a central intermediary in LT biosynthesis.LTs are synthesized in the cells by 5-LO which acts inconcert with FLAP, a protein essential for 5-LO tofunction enzymatically in intact cells. In neutrophils,LTA4 is transformed by LTA hydrolase to dihydroxyLTB4,whereas in▶eosinophils,macrophages, basophils,and mast cells that express LTC4 synthase, the terminalenzyme involved in cysLT synthesis, LTA4 is conjugatedwith the tripeptide glutathione to form LTC4. Outside ofthe cell, LTC4 is rapidly converted by γ-glutamyltran-speptidase into LTD4. LTD4 is converted more slowlyinto LTE4 by a dipeptidase, which is secreted into theurine and is sometimes used (controversially) as amarkerfor cysLT synthesis. Leukotriene B4, a noncysteine

Leukotrienes. Figure 1 Schema representing cellular biosynthesis of cysteinyl leukotrienes (cysLTs) and BLTat thenuclear membrane. In this schema, cPLA2 has migrated from the cytosol to the nuclear envelope and 5-LO issecreted from the nucleus to bind with FLAP and produce the metabolite, LTA4, which is synthesized by 5-LO from5-HPETE (not shown) in two steps. LTA4 is converted by LTC4 synthase which after LTC4, is converted rapidly toLTD4 outside of the inflammatory cell. Note that a specific hydrolase (LTA4 hydrolase) also catalyzes the formation ofLTB4, a non-cysLT, which has its own receptor. See text for details.

686 Leukotrienes

containing dihydroxy leukotriene, binds to its own BLTreceptor. The cysLTs are characterized by a side chaincontaining 3, 2, or 1 amino acid(s) (cysteine is alwayspresent); there are twoormore specific cysLTreceptors intissues at various sites including airway smooth muscle,vascular smooth muscle, and inflammatory cells them-selves. Both cysLT receptors have been characterized andcloned. LTD4 binds to high affinity CysLT1 receptorwhile CysLT2 receptor is most preferred target for LTC4.

Recent investigations have suggested that cysLTmayalso be synthesized in the cytosol in lipid bodies that areformed during cellular activation in eosinophils [2].It has been suggested that all enzyme and transportsystems essential to cysLT synthesis exist in theselipid bodies, and hence synthesis could occur withouttranslocation of cPLA2 to the nuclear envelope.Likewise, it has been suggested that 5-LO is not storedwithin the nucleus, but rather is an enzyme dispersedthroughout the cytosol, which migrates to the peri-nuclear membrane and cytosolic lipid bodies uponcellular activation. Recently, the role of 14 kDasecretory PLA2 in leukotriene synthesis has beenexplored in human granulocytes [1]. Studies demon-strated that gVPLA2 is capable of causing AA synthesisand subsequent LT secretion by both cPLA2-dependentand -independent pathways. Among the secretoryenzymes, gVPLA2 is unique because it can bind cellmembranes by two distinct mechanisms: (i) cell surfaceproteoglycan through a cluster of cationic residues atthe carboxy terminus of the enzyme and (ii) directinterfacial binding to membrane phosphatidylcholine.In eosinophils, gVPLA2 is internalized and binds toperinuclear membrane to generate AA and subsequent

translocation of cytosolic 5-LO to perinuclear mem-brane. Accordingly, LTC4 secretion caused by gVPLA2

in eosinophils does not involve ERK-1/2 mediatedcPLA2 phosphorylation. By contrast, LTB4 secretioncaused by gVPLA2 in neutrophils is mediated throughactivation of the cPLA2 pathway. These novel pathwaysfor gVPLA2 are fully established in granulocytes;however, the role of other 14 kDa sPLA2 isoforms in LTsynthesis in vitro remains an area of considerableinterest. sPLA2s have been identified in the bronch-oalveolar lavage fluid of asthmatic subjects.

Leukotriene ReceptorsThere are two distinct receptor types for LTB4 (BLT1

and BLT2 receptors) and two for cysLTs (CysLT1 andCysLT2 receptors). Classification is based mainly onfunctional data as derived from recent cloning andbiochemical characterization. For LTB4, high affinityBLT1-receptor shows high degree of specificity forLTB4 (Kd 0.15–1 nM) and is only expressed ininflammatory cells. Recently identified low affinityBLT2-receptor has a higher Kd value for LTB4 (23 nM)and is expressed ubiquitously, in contrast to BLT1,in leukocytes.The cysLTs are a family of potent bioactive lipids that

act through two structurally divergent G proteincoupled CysLT1 and CysLT2 receptors. mRNA expres-sion demonstrates the CysLT1 gene, which is located onthe X chromosome and is identified in macrophages,smooth muscle cells, leukocytes, lung, and spleen. TheCysLT1 receptor was originally identified on the basisof its contractile properties for bronchial smoothmuscle. The agonist for the CysLT1 receptor are

Leukotrienes 687

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LT D4 > LTC4 > LTE4. The second receptor, CysLT2,binds equally to LTD4 and LTC 4; mRNA is expressed inheart, leukocytes, spleen, brain, placenta, and lymphnodes. To date, the functional role of CysLT2 receptorremains unclear. The current nomenclature is summar-ized in Table 2, together with order of potency of theLTs, names of some selective antagonist drugs andexpression in human and mouse tissues/cells.

Role of Leukotrienes in Inflammatory DiseasesLT B4, a B- leukotr ie ne, is a p ot ent c hemotax in forneutr ophils and T-cells and a wea k ch em otaxin for humaneosin ophils. In human ne ut roph ils , LTB4 caus es t ranslo -cation o f i ntrace llu lar calc iu m th at in itiates an au tocrinepatter n of stimulated cellula r ac tivity. It promo tes theadhe si on of n eutrop hils to the v ascu lar en dothe li um andenha nces th e m igration a cross t h e endo thelial w all i nto t hesurro unding tissues . LTB4 also causes the release of toxicoxyg en r adica ls , l yso somal e nzymes , an d ch emokine s/cytok ines from pro in fl ammatory ce ll s. Some stud ies haveshown tha t LTB4 ma y cause contra ction of b ot h humanbron chus and guin ea pig p arenc hymal strips in vitro ;however, blockade of LTB4 rece pt or has little o r nothera peutic a ction i n h um an asth ma .

The cysLTs were formerly known as SRS-A (slowreacting substance of anaphylaxis) because of the slow,sustained contraction of airway smooth muscle thatresulted from the secretion of this substance(s) upon mastcell activation. Human bronchi may have a homogenouspopulation of CysLT1 receptors, whereas guinea pigtrachea have both CysLT1/2 receptors. Although original-ly identified in tissue mast cells, eosinophils, and mastcells, both have substantial cysLT synthetic capacity.Because the eosinophil is ubiquitous in asthma andallergic disease and because it is capable of cysLTsynthesis, this cell has generally been regarded as primarymechanism for sustained cysLT secretion in allergic states(Ta b l e 1 ). However, recent publications have suggestedthe eosinophil secretion is relatively minimal compared tomast cell secretory capacity for cysLTs.

Leukotrienes. Table 1 Pharmacologic actions of the leukotr

LTB4

PMN aggregration

PMN chemotaxis

Nuclear transcription

Exudation of plasma

Translocation of calcium

Stimulation of PLA2 (guinea pig)

Contraction of human bronchus (?)

Contraction of guinea pig parenchyma (?)

No effect with LTRA

Reprinted with permission from [3].

Asthmatic exacerbations are associated with chemo-taxis of eosinophils from the peripheral blood acrossendothelial membranes into the parenchyma and lumenof the conducting airways of the lung. This process isfacilitated by the upregulation of β1- and β2-integrins,which bind to counterligands of the immunoglobulinsupergene family on the endothelial surface. Diapedesisis facilitated by the presence of these ligands on bothsides of the endothelium (lumenal and parencymal).The migration of eosinophils from the peripheral bloodto human airways provides a continuous reservoir forleukotriene synthesis in allergic and asthma reactions.The 85 kDa cPLA2 that catalyzes the first steps ofleukotriene synthesis also serves as a messenger proteinfor cellular adhesion, likely by the synthesis oflysophospholipid, which is the by-product of mem-brane hydrolysis in which AA is also synthesized.

Leukotrienes play a role in both allergy and asthma[5]. While cysLTs are highly efficacious bronchocon-stricting agents when administered exogenously tohuman asthmatics (Fig. 2) (1000-fold the potency ofhistamine), a substantial role for cysLT in asthmaticbronchoconstriction has not been established. Amelio-ration of inflammatory response by corticosteroids inboth asthma and allergic reactions is highly efficaciousand does not appear to result from blockade ofleukotriene synthesis. No other chronic inflammatorydisease has been linked directly to cysLT secretiondespite the capacity for these compounds to causeedema and inflammatory cell migration.

DrugsThe effects of leukotrienes can be blocked at severallevels. Inhibitors of FLAP or 5-LO inhibit LTsynthesis atall levels. However, FLAP antagonists developed to datehave been too hepatotoxic for human use. Zileuton, a5-LO synthase inhibiting drug, also demonstrated somehepatotoxicity in a small percentage of patients, whichwas nonetheless entirely reversible. However, the shorthalf-life of this compound requires four times daily

ienes

Cysteinyl LTs (LTC4, LTD4, LTE4)

Airway smooth muscle contraction

Constriction of conducting airways

Contraction of guinea pig parenchyma

Secretion of mucus

Fluid leakage from venules

Edema formation

Chemoattraction of eosinophils

Autocrine activation of PLA2 (guinea pig)

Blocked by LTRA

688 Leukotrienes

administration, and accordingly, this compound is rarelyprescribed. The most widely used antiLT drugs are the▶leukotriene receptor antagonists (LTRAs; see Table 2),which were synthesized before the cloning of the CysLT1

receptor. These include pranlukast, which is widely

Leukotrienes. Table 2 Leukotriene receptor type [4]

Nomenclature BLT1 receptor CysLT1 rec

Relative po-tency of ago-nists

LTB4 > 12(R)-HETE (cysLTsare inactive)

LTD4 = LTC

Chromosome Human: 14q11.2–q12 Human: Xq1

Mouse: 14 Mouse: X-D

Selective an-tagonists(IC50)

LY-255883 (>10 nM) Montelukas

CP-195543 (570 nM) ICI198615

U-75302 (1 μM) Pobilukast (

CP-105696 (58 nM) Zafirlukast (

ZK-158252 (260 nM) Pranlukast (

Expression(Human)

Tissues: Leukocytes, thymus,spleen, liver, ovary Cells:PBLs, neutrophils,T-cells,dendritic cells, mast cells, eo-sinophils, macrophages, leu-kocytes

Tissues: splplacenta, luCells: broncCD34+ hemcells, monocmast cells, ephils, PBLs,endothelial

Expression(Mouse)

Tissues: lungs, Cells: myeloidleukocytes, neutrophils, T-cells, macrophages, mastcells, eosinophils

Tissues: lunCells: macroleukocytes

aPartial agonist in some tissues.

Leukotrienes. Figure 2 Relative airway responses ofasthmatic patients to inhaled cysteinyl leukotrinesversus histamine. CysLTs have up to 1,000-fold greaterpotency in causing bronchoconstriction than histamine.Reprinted with permission from (2).

used in Japan, and zafirlukast andmontelukast, which areused in the USA, Europe, and Asia. In controlled studiesin human asthmatics, these compounds cause 5–8%improvement in bronchoconstriction as measured by theforced expiratory volume in 1 s (FEV1). LTRAs havebeen shown to reduce the need for inhaled corticosteroids(ICS) and β-adrenoceptor agonists in human asthma.Additional studies have suggested that addition ofLTRA to ICS or β- adrenoceptor agonists augments theimprovement in FEV1. Nonetheless, LTRAs are fairlyexpensive and substantially less efficacious thaneither long-acting β-adrenergic agonists (LABA) orICS, particularly when LABA and ICS are used incombination. Accordingly, the use of LTRAs is oftenrelegated to either mild asthma or as supplementaltherapy for patients failing to respond to other drugs.There are few definitive data to substantiate the

efficacy of LTRA therapy in refractory asthma, exceptfor patients with “aspirin-sensitive asthma.” This is afairly uncommon form of asthma that occurs generallyin adults who often have no prior (i.e., childhood)history of asthma or▶atopy, may have nasal polyposis,and who often are dependent upon oral corticosteroidsfor control of their asthma. This syndrome is notspecific to aspirin but is provoked by any inhibitors ofthe cycloxygenase-1 (COX-1) pathway. These patientshave been shown to have a genetic defect that causes

eptor (LTD4 receptor) CysLT2 receptor (LTC4 receptor)

4 > LTE4a LTC4 > LTD4 > LTE4

a

3.2–21.1 Human: 13q14.12–q21.1

Mouse: 14-D1

t (MK476) none

SFK104353)

ICI204219)

ONO1078)

een, small intestine,ng smooth muscle,hial smooth muscle,apoietic progenitorytes, macrophages,osinophils, neutro-human umbilical veincells

Tissues: heart, skeletal muscle,spleen, brain, lymp node, adrenalmedulla, lung, human pumonary/saphenous vein Cells: monocytes,macrophages, mast cells, eosino-phils, cardiac muscle, coronaryartery, PBLs

g, skin, small intestinephages, fibroblasts,

Tissues: lung, skin, brain, smallintestine, spleen Cells: macro-phages, fibroblasts, endothelialcells, leukocytes

Lewy Bodies 689

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overexpression of the enzyme LTC4 synthase. Howev-er, the mechanism by which bronchoconstriction isprovoked by COX-1 inhibition remains unexplained. Insuch patients, LTRAs are specifically indicated. Thereis limited evidence suggesting that patients with aspirin-sensitive asthma can use selective COX-2 inhibitorssafely; however, COX-2-specific analgesics still are notrecommended for use in aspirin-sensitive asthma by theFDA in the USA and may themselves be cardiotoxic.

Although some studies have shown no change in theexcretion of LTE4 in the urine of patients treated withcorticosteroids, other investigations indicated that corti-costeroids inhibit cPLA2 translocation to the nuclearmembrane in inflammatory cells, thus attenuatingstimulated synthesis of cysLTs. This would suggestthat oral and possibly inhaled corticosteroids may alsoinhibit production of cysLTs both directly (see above)and indirectly by causing necrosis and apoptosis ofeosinophils.

In the USA, LTRAs have largely replaced theophyl-line as the incremental drug for the treatment ofmoderate and severe asthma, where LABA plus ICSalone do not provide adequate control. For patients withmild persistent asthma, LTRAs have been designated asa suitable substitute for low dose ICS by the NationalAsthma Education Panel Program (NAEPP) of theNational Heart and Lung Institute (National Institutes ofHealth). However, inhaled ICS are more efficacious.

Therapeutic Response to Antileukotriene DrugsWhen given acutely to patients who have no priorexposure to LTRA therapy, FEV1 increases measurablywithin 30 min and results in a maximal improvement ofabout 8% in 1 h. For short acting drugs (e.g., zileuton),this response returns rapidly to baseline, and the drugmust initially be administered in four doses daily. In thisregard, the initial effects of 5-LO inhibition are morelike bronchodilators than disease modifying agents.However, with prolonged use (>60 days), there is littledifference between peak and trough response in FEV1,even if zileuton administration is decreased to twicedaily. This acquired, longer acting effect has beensuggested to imply a disease modifying effect ofantileukotriene therapy. In some studies, even acuteadministration of LTRAs has caused a modest decreasein eosinophil migration into asthmatic airways andthe presence of the cysLT receptor, which has beenrecently identified on eosinophils, suggests a possiblemechanism for this action. Other recent studies indicatethat the LTRA, montelukast, if infused intravenouslycauses rapid incremental increase in FEV1 in patientsseeking treatment for acute asthma. The reason forthe improved efficacy by intravenous infusion isunclear, but further investigations are examining thefuture role of LTRAs as potential rescue drugs in acuteasthma.

LTRAs are extremely safe for patient use. However,the present generation of LTRAs is only modestlyefficacious. Many patients show no clinically meaning-ful response, and current recommendations suggest a 1month trial period to determine if patients will benefitfrom these drugs. With the exception of aspirin-sensitive asthmatics, there is currently no means forpredicting which patients or under what circumstancesantileukotrine therapies will be effective.

References1. Munoz NM, Kim YJ, Meliton AY et al (2003) Human

gVPLA2 induces gIVA-PLA2 independent cysteinylleukotriene synthesis in human eosinophils. J Biol Chem278:38813–38820

2. Leff AR (2001) Regulation of leukotrienes in themanagement of asthma: biology and clinical therapy.Annu Rev Med 52:1–14

3. Leff AR (2001) Discovery of leukotrienes and develop-ment of antileukotriene agents. Ann Allergy Immunol 86(Suppl):864–868

4. Brink C, Dahlen S, Drazen J et al (2003) InternationUnion of Pharmacology XXXVII:Nomenclature forLeukotriene and Lipoxin receptors. Pharmacol Review55:195–227

5. Fisher AR, Drazen JM (1997) Leukotrienes. In: Barnes P,Grunstein MM, Leff AR, Wool cock A (eds) Asthma,vol 1. Lippincott-Raven, Philadelphia, pp 547–558

Levodopa

▶L-DOPA / Levodopa▶Dopamine System▶Anti-Parkinson Drugs

Lewy Bodies

Lewy bodies are typical in neuronal degeneration,which is accompanied by the presence of theseeosinophilic intracellular inclusions of 5–25 μmdiameter in a proportion of still surviving neurons.Lewy bodies contain neurofilament, tubulin, microtu-bule-associated proteins 1 and 2, and gelsolin, an actin-modulating protein.

▶Anti-Parkinson Drugs

690 LH-RH Agonist

LH-RH Agonist

LH-RH (Luteinizing Hormone-Releasing Hormone)agonists are drugs that inhibit the secretion of sexhormones. In men, LH-RH agonists cause testosteronelevels to fall. In women, LH-RH agonists cause thelevels of estrogen and other sex hormones to fall.

▶Contraceptives▶Targeted Cancer Therapy

Liddle’s Syndrome

Liddle's syndrome is an autosomal dominant disorderthat is caused by persistent hyperactivity of theepithelial Na channel. Its symptoms mimic aldosteroneexcess, but plasma aldosterone levels are actuallyreduced (pseudoaldosteronism). The disease is char-acterized by early onset arterial hypertension, hypoka-lemia, and metabolic alkalosis.

Disease-causing mutations are found in the cytoplas-mic regulatory region of the β and γ subunits of theepithelial sodium channel (ENaC) genes. In general,patients with Liddle's syndrome can be treatedsuccessfully with the ENaC inhibitor amiloride.

▶Diuretics▶Epithelial Na+ Channels

Ligand

A ligand can be an antagonist or agonist that binds to areceptor.

Ligand-Gated Ion Channels

Ion channels that are opened by binding of aneurotransmitter (or drug) to a receptor domain onextracellular sites of the channel protein(s) are defined

as ligand-gated. Nicotinic acetylcholine, glutamate,γ-aminobutyric acidA (GABAA) and glycine receptorsare examples of this type of receptor-linked ion channel.

▶Benzodiazepines▶Ca2+ Channels▶Ionotropic Glutamate Receptors▶Nicotinic Receptors▶Non-selective Cation Channels▶K+ Channels▶Serotoninergic System▶Purinergic System▶Table Appendix: Receptor Proteins▶Transmembrane Signaling▶Glyciu Receptor▶GABAergic System

Limbic System

Collection of interconnected subcortical and corticalbrain structures (including hypothalamus, amygdala,and hippocampus) integrating multimodal intero- andexteroceptive information to produce coherent neuro-endocrine and behavioral output, and to supportmemory functions.

▶Orexins

Linkage Disequilibrium (LD)

Polymorphisms in the human genome are often notindependently transmitted; i.e., a polymorphism isassociated with particular variants present on the samechromosome. Recombination erodes this association,but for physically close polymorphisms (e.g., within agene), the correlation, known as LD, persists over time.

▶Pharmacogenomics

Lipid-lowering Drugs

Lipid-lowering drugs are drugs that affect the lipopro-teinmetabolism and that used in therapy to lower plasmalipids (cholesterol, triglycerides). The main classes of

Lipid Modifications 691

drugs used clinically are statins (HMG-CoA reductaseinhibitors), anion exchange resins (e.g. cholestyramineand cholestipol), fibrates (bezafibrate, gemfibrozil orclofibrate) and other drugs like nicotinic acid.

▶HMG-CoA-Reductase-Inhibitors▶Fibrates▶Anion Exchange Resins

Lipid Metabolism

▶Lipoprotein Metabolism

Lipid Modifications

L

PATRICK J. CASEY

Duke University Medical Center, Durham, NC, USA

SynonymsLipidation; S-acylation; N-myristoylation; Myristoy-lation; S-prenylation; Prenylation; Palmitoylation;Isoprenylation; ▶GPI anchors; Glypiation

Lipid Modifications. Figure 1 Major classes of lipid-modi

DefinitionCovalent attachment of lipid moieties to proteins playsimportant roles in the cellular localization and functionof a broad spectrum of proteins in all eukaryotic cells.Such proteins, commonly referred to as lipidatedproteins, are classified based on the identity of theattached lipid (Fig. 1). Each specific type of lipidhas unique properties that confer distinct functionalattributes to its protein host. S-acylated proteins,commonly referred to as palmitoylated proteins,generally contain the 16-carbon saturated acyl grouppalmitoyl attached via a labile thioester bond to cysteineresidue(s), although other fatty acyl chains maysubstitute for palmitoyl group.N-myristoylated proteinscontain the saturated 14-carbon myristoyl groupattached via amide bond formation to amino-terminalglycine residues. S-prenylated proteins contain one oftwo▶isoprenoid lipids, either the 15-carbon farnesyl or20-carbon geranylgeranyl. The fourth major class oflipidated proteins is those containing the glycosylpho-sphatidylinositol (GPI) moiety, a large and complexstructure of which the lipid component is an entirephospholipid.

Basic MechanismsS-acylated proteins include many GTP-binding regu-latory proteins (G proteins), including most α subunitsof ▶heterotrimeric G-proteins and also many membersof the Ras superfamily of monomeric G proteins, anumber of G protein-coupled receptors, several non-receptor ▶tyrosine kinases, and a number of othersignaling molecules. S-acylation is posttranslationaland reversible, a property that allows the cell to control

fied proteins.

692 Lipid Modifications

the modification state, and hence the localization andbiological activity, of the protein [1]. The lipidsubstrates for S-acylation are ▶acyl-CoA mole-cules. The molecular mechanism of S-acylation isonly recently being elucidated; the first identifiedacyltransferase specifically involved in the processbeing the product of the Skinny Hedgehog (Ski)gene in Drosophila. Ski orthologs have also beenidentified in mammalian genomes. This acyltransferaseappears to specifically process secreted proteins, inparticular a molecule important in development termedHedgehog.

The process by which cytoplasmic proteins aresubject to S-acylation is also becoming clarified. Thebreakthrough in this arena came from genetic screens inyeast, in which two related S-palmitoyltransferases wereuncovered that are termed Erf2 and Akr1. These twogene products share a common sequence referred to as aDHHC (aspartate-histidine-histidine-cysteine) motif,and a large number of genes encoding DHHC-motifproteins can be identified in all eukaryotes for whichgenome information is available. Although biochemicaldetails of the enzymatic function of DHHC-motifproteins are still rather limited, the available evidenceis consistent with the DHHC domain playing a directrole in the protein acyltransferase reaction.

In addition, nonenzymatic acylation of cysteinethiols on proteins incubated in the presence of acyl-CoA has been described, although the biologicalimportance of this process is still unclear.

N-myristoylated proteins include select α subunits ofheterotrimeric G proteins, a number of nonreceptortyrosine kinases, a few monomeric G-proteins, andseveral other proteins important in biological regulation[2]. There is some overlap between S-acylated andN-myristoylated proteins such that many contain bothlipid modifications. Such “dual lipidation” can haveimportant consequences, most notably the localizationof the dually modified species to distinct membranesubdomains termed ▶lipid rafts or ▶caveolae.N-myristoylation is a stable modification of proteins.The myristoyl moiety is attached to the proteincotranslationally by the enzyme myristoyl CoA: proteinN-myristoyltransferase (NMT); the lipid substrate ismyristoyl-CoA. NMT has been extensively studied inregard to substrate utilization and kinetic properties,and crystal structures of fungal enzymes are available.There are two distinct but closely related genesencoding NMTs in mammalian cells, NMT1 andNMT2, although differences between the two isoformshave not yet been described.

S-prenylation is the most recent of the four majortypes of lipid modifications to be described. As withS-acylation, S-prenylation is posttranslational. The lipidsubstrates for these modifications are farnesyl diphos-phate and geranylgeranyl diphosphate. The mechanism

involves attachment of the isoprenoid lipid to cysteineresidues at or near the carboxyl terminus through astable thioether bond [3]. Two distinct classes ofS-prenylated proteins exist in eukaryotic organisms.Proteins containing a cysteine residue fourth fromthe carboxyl terminus (the so-called CaaX-motif) canbe modified by either the 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenoid by one of two closelyrelated termed protein farnesyltransferase (FTase) andprotein geranylgeranyltransferase type 1 (GGTase-1);these enzymes are collectively referred to as CaaXprenyltransferases [4]. FTase and GGTase-1 are αβheterodimers; the α subunits of the enzymes areidentical while the β subunits are the products ofdistinct, but related, genes. The identity of the “X”residue of the CaaX motif dictates which of the twoenzymes recognize the substrate protein. Followingprenylation, most CaaX-type proteins are furtherprocessed by proteolytic removal of the three carbox-yl-terminal residues (i.e., the -aaX) by the Rce1 CaaXprotease and methylation of the now-exposed carboxylgroup of the prenylcysteine by the Icmt methyltransfer-ase. A number of S-prenylated proteins are also subjectto S-acylation at a nearby cysteine residue to produce adually lipidated molecule, although this type of dualmodification does not apparently target the protein tothe same type of membrane subdomain as the dualacylation noted above.The second class of S-prenylated proteins is a

member of the Rab family of proteins, which isinvolved in membrane trafficking in cells. Theseproteins are geranylgeranylated at two cysteine residuesat or very near to their carboxyl-terminus by proteingeranylgeranyltransferase type 2 (GGTase-2), alsoknown as Rab GGTase [5]. GGTase-2 is also a αβheterodimer and its subunits show significant se-quence similarity to the corresponding subunits of theCaaX prenyltransferases [6]. However, unlike the CaaXprenyltransferases, monomeric Rab proteins are notsubstrates for GGTase-2. In order to be processed by theenzyme, newly synthesized Rab proteins first bind andform a stable complex with a protein termed Rep, and itis the Rab–Rep complex that is recognized by GGTase-II. The enzyme acts in a processive fashion, attachingboth geranylgeranyl groups to closely spaced cysteineresidues at or near the carboxyl-terminus of the Rabproteins. All three of the protein prenyltransferases(FTase, GGTase-1, GGTase-2) have been extensivelycharacterized in regard to substrate recognition, mecha-nism, and structure.GPI-anchored proteins constitute a quite diverse

family of cell-surface molecules that participate insuch processes as nutrient uptake, cell adhesion, andmembrane signaling events [3]. All GPI-linked pro-teins are destined for the cell surface via traffickingthrough the secretory pathway, where they acquire the

Lipid Phosphate Phosphohydrolases 693

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preassembled GPI moiety. The entire procedure, whichinvolves assembly of the GPI moiety from ▶phospha-tidylinositol and sugars and proteolytic processing ofthe target protein to expose the GPI addition site at thecarboxyl-terminus of the protein, involves a number ofgene products. Many of the genes associated with GPIbiosynthesis have been cloned by complementation ofGPI-deficient mammalian cell lines and temperature-sensitive yeast GPI mutants. The precise mechanismsthrough which these enzymes work in concert toproduce a GPI-anchored protein are just now beginningto be elucidated.

Pharmacological InterventionSince the mechanisms of protein S-acylation are sopoorly understood, there is little in the way of goodpharmacological agents that target this process. Onecompound that has been used with modest success iscerulenin, which apparently mimics the acyl-CoAsubstrate in the reaction catalyzed by the putativeS-acyltransferase that acts on intracellular proteins. Incontrast, there has been substantial effort to identify andcharacterize specific inhibitors of N-myristoylation [2].Genetic and biochemical studies have established NMTas a target for development of anti-fungal drugs. Theenzyme is also a potential target for the development ofantiviral and antineoplastic agents. Both peptidic andnonpeptidic inhibitors of NMTs, particularly fungalNMTs, have been described.

There has been enormous effort in the past 10 years todevelop pharmacological agents targeting the S-pre-nylation by CaaX prenyltransferases, especially FTase[4]. This is primarily due to the interest in one subsetof S-prenylated proteins, the Ras proteins, due to theimportant role of Ras in▶oncogenesis. Ras proteins aremodified by the 15-carbon farnesyl isoprenoid, andfarnesylation of these proteins is indispensable for bothnormal biological activity and oncogenic transforma-tion. Selective inhibitors of FTase, termed FTIs, canreverse Ras-mediated oncogenic transformation ofcells, and several are in clinical development asanticancer therapeutics. Literally hundreds of potentinhibitors of FTase, and many of GGTase-1, have beenidentified using several strategies, including design ofanalogs of the CaaX peptide and isoprenoid substratesand by high-throughput screening of natural productand compound libraries. These compounds can beplaced into four distinct categories: mimics of CaaXtetrapeptides, mimics of FPP,▶bisubstrate analogs, andorganic compounds selected from natural productand chemical libraries. There has been relatively littledevelopment of pharmacological agents targetingGGTase-2. It is likely, however, that some of theisoprenoid analogs that show activity against GGTase-1will also have inhibitory activity on GGTase-2 giventhat both enzymes use the same isoprenoid substrate.

There is also increasing interest in developingpharmacological agents targeting the biosynthesis ofGPI-anchored proteins [3]. Such proteins are particu-larly abundant on the surface of a number of protozoanorganisms. Several devastating tropical diseases suchas African sleeping sickness and Chagas disease arecaused by protozoan parasites that rely heavily oncell-surface GPI-anchored proteins for both inhabitingtheir host and escaping immune detection. In studiesprimarily involving gene disruption approaches, sev-eral of the enzymes involved in GPI biosynthesishave been identified as attractive targets for develop-ment of antiparasitics. To date, there is very littlepublically available information on specific inhibitorsof the enzymes involved in GPI biosynthesis andattachment, but it is likely that such information will beforthcoming.

References1. Linder ME, Deschenes RJ (2007) Palmitoylation:

policing protein stability and traffic. Nat Rev Mol CellBiol 8:74–84

2. Farazi TA,Waksman G, Gordon JI (2001) The biology andenzymology of protein N-myristoylation. J Biol Chem276:39501–39504

3. Ferguson, MAJ (2000) Glycosylphosphatidylinositolbiosynthesis validated as a drug target for African sleepingsickness. Proc Natl Acad Sci USA 97:10673–10675

4. Lane KT, Beese LS (2006) Structural biology ofprotein farnesyltransferase and geranylgeranyltransferasetype I. J Lipid Res 47:681–699

5. Leung KF, Baron R, Seabra MC (2006) Geranylgeranyla-tion of Rab GTPases. J Lipid Res 47:467–475

Lipid Phosphate Phosphohydrolases

Lipid phosphate phosphohydrolases (LPPs), formerlycalled type 2 phosphatidate phosphohydrolases (PAP-2),catalyse the dephosphorylation of bioactive phospholi-pids (phosphatidic acid, ceramide-1-phosphate) andlysophospholipids (lysophosphatidic acid, sphingosine-1-phosphate). The substrate selectivity of individualLPPs is broad in contrast to the related sphingosine-1-phosphate phosphatase. LPPs are characterized by alack of requirement for Mg2+ and insensitivity toN-ethylmaleimide. Three subtypes (LPP-1, LPP-2,LPP-3) have been identified inmammals. These enzymeshave six putative transmembrane domains and threehighly conserved domains that are characteristic of aphosphatase superfamily. Whether LPPs cleave extracel-lularmediators or rather have an influence on intracellularlipid phosphate concentrations is still a matter of debate.

694 Lipid Rafts

Lipid Rafts

Lipid rafts are specific subdomains of the plasmamembrane that are enriched in cholesterol and sphin-golipids; many signaling molecules are apparentlyconcentrated in these subdomains.

▶Lipid Modifications

Lipid Transfer Proteins

WIM A. VAN DER STEEG1, ARIE VAN TOL2,

JOHN J.P. KASTELEIN1

1Department of Vascular Medicine, Academic MedicalCenter, University of Amsterdam, Amsterdam, TheNetherlands2Department of Cell Biology & Genetics, ErasmusUniversity Medical Center, Rotterdam, TheNetherlands

SynonymsPlasma lipid transfer proteins

DefinitionPlasma lipid transfer proteins, which include thecholesteryl-ester-transfer-protein (CETP; previouslyknown as lipid transfer protein I, LTP-I) and thephospholipid-transfer-protein (PLTP; previouslyknown as lipid transfer protein II, LTP-II) mediate thetransfer of lipids (cholesteryl esters, triglycerides andphospholipids) between lipoproteins present in humanplasma. These proteins significantly affect plasmalipoprotein concentration and composition.

Basic CharacteristicsGene and Protein CharacteristicsThe genes coding for CETP and PLTP belong to onegene family, which also includes lipopolysaccharide-binding-protein (LBP) and bactericidal/permeability-increasing-protein (BPI). This common descent not onlybecomes clear from a considerable sequence similarity(45–65% homology at the cDNA level), but also fromsubstantial conservation of exon/intron transitions.

The gene encoding CETP is located on the long armof chromosome 16 (16q12-21), spans approximately25kbp and harbors 16 exons and 15 introns. Themolecule mass of its mature protein product is 74 kDa.Major sites of CETP gene expression in humans are the

liver, spleen and adipose tissue, with lower levels ofexpression in the small intestine, adrenals, kidney andheart. CETP present in human plasma seems to originatepredominantly from the liver and adipose tissue,although data that have assessed this directly in man arelacking. The majority of human plasma CETP (�90%)is bound to ▶high-density lipoprotein (HDL) particles.An important regulatory factor of CETP gene expres-sion is hypercholesterolemia, which induces genetranscription. Sequences necessary for this cholesterol-responsiveness are located in the natural flankingregions of the human CETP gene, upstream of thetranscription start site. This regulatory sequence con-cerns an element of two direct repeats separated by fournucleotides (DR4 element). This element enhancesCETP gene expression following activation through aheterodimer, containing the transcription factors LiverXReceptor (LXR) and Retinoid X Receptor (RXR). Inorder to enhance gene transcription, these transcriptionfactors require activation through ligands,which includecholesterol and its oxysterols.The gene coding for PLTP has been mapped to

chromosome 20 (20q12-13.1), where also the genes forLBP and BPI are situated. The PLTP gene contains 13.3kbp, with similar organization as compared to CETP(16 exons-15 introns). Purified PLTP has a moleculemass of 81 kDa. PLTP is expressed in a variety of tissuesincluding placenta, pancreas, lung, kidney, heart, liver,adipose tissue, skeletal muscle and brain. Macrophagesalso express PLTP and may contribute significantlyto plasma PLTP activity levels. PLTP gene expressioncan be upregulated by cholesterol in a similar way asdescribed for CETP. In addition, PLTP gene expressionis subject to regulation via response elements forthe peroxisome proliferator activated receptor (PPAR)alpha and the farnesoid X-activated receptor (FXR).Detailed protein structures have been reported for

BPI and CETP. Given the aforementioned similaritieswithin this gene family, these protein structures serve asa likely model for the protein structure of PLTP. CETPand BPI are elongated molecules, shaped like aboomerang. There are two domains with similar folds,and a central beta-sheet domain between these twodomains. The molecules contain two lipid-bindingsites, one in each domain near the interface of thebarrels and the central beta-sheet.

FunctionIn humans, CETP and PLTP are directly involved inthe transfer of lipids between different ▶lipoproteinclasses. Through their action, these lipid transferproteins have major effects on the concentration andcomposition of HDL. This section further describes thephysiological function of CETP and PLTP in humans.CETP mediates the exchange of cholesteryl esters and

triglycerides between HDL and the proatherogenic,

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apoB-containing lipoprotein fractions, predominantly▶very-low density lipoprotein (VLDL). Specifically,triglycerides are transported from VLDL to HDL, withcholesteryl esters transferred in the opposite direction. Itis generally acknowledged that CETP itself has nopreference for the type of lipoprotein in its substratespecificity. The VLDL–HDL axis is likely to result fromthe presence of plasma lipid transfer inhibitor protein(LTIP), which is associated almost exclusively with▶low-density lipoprotein (LDL) and inhibits the CETP-mediated transfer of cholesteryl esters and triglycerides.LTIP suppresses the involvement of LDL, thereby direct-ing the CETP activity towards the remaining lipoproteinfractions, VLDL and HDL. The CETP-mediated lipidexchange is an equimolar and energyneutral process. Theexchange seems to occur by a shuttle mechanism, ratherthen by the formation of a complex between CETP anddonor and acceptor lipoproteins. In addition to LTIP,composition and concentration of lipoprotein substratesare important regulatory factors of the CETP-mediatedexchange activity, and under physiological conditions,the net mass of VLDL-triglycerides may be rate limiting.The relevance of CETP for human lipid metabolismbecame evident after the identification of humans withpartial or near complete loss of CETP activity due to thepresence of a mutation in one or both alleles of the CETPgene. In these subjects, who predominantly live in Japan,(partial) loss of plasma CETP reduces the ability ofcholesteryl esters to be transported out of HDL. Thisresults in a net increase in plasmaHDLcholesterol (HDL-C) levels, with the appearance of HDL particles that haveincreased in size. This change is paralleled by increasedconcentrations of ▶apolipoprotein A-I (apoA-I), whichresults from delayed catabolism of these large sizedHDLs. In some cases of severe CETP deficiency, thereduced transfer of cholesteryl esters fromHDL toVLDLfurthermore gives also rise to mildly decreased levels ofLDL cholesterol (LDL-C), which originates fromVLDL.Recently, the first Caucasian family was described withpartial CETP deficiency due to a novel splice sitemutation in the CETP gene.

PLTP is responsible for the majority of phospholipidtransfer activity in human plasma. Specifically, ittransfers surface phospholipids from VLDL to HDLupon lipolysis of triglycerides present in VLDL. Theexact mechanism by which PLTP exerts its activity isyet unknown. The best indications for an important rolein lipid metabolism have been gained from knockoutexperiments in mice, which show severe reduction ofplasma levels of HDL-C and apoA-I. This is most likelythe result of increased catabolism of HDL particles thatare small in size as a result of phospholipid depletion. Inaddition to the maintenance of normal plasma HDL-Cand apoA-I concentration, PLTP is also involved in aprocess called HDL conversion. Shortly summarized,this cascade of processes leads to fusion of HDL

particles into larger particles, with a concomitantrelease of lipid-poor apoA-I. This newly formed lipid-poor apoA-I is a pivotal particle in the uptake of freecholesterol from peripheral cells via efflux n throughABCA1. This is a potentially antiatherogenic functionof PLTP, but – surprisingly – overexpression of humanPLTP in mice results in increased susceptibility for diet-induced atherosclerosis and a reduction of plasmaHDL-C, again due to hypercatabolism of apoA-I. Atpresent, no reports exist on genetic PLTP deficiency inhumans. Therefore, any hypothesis on the relevance ofPLTP activity for human lipid metabolism remains to beconfirmed. When it comes to humans, plasma PLTPactivity is increased in insulin-resistant individuals withhigh plasma triglycerides and low HDL-C, as well as inpatients with diabetes mellitus. In patients with type2 diabetes, PLTP activity is positively associated withintima-media thickness, which is a surrogate measurefor the risk of atherosclerotic disease. In addition,elevated PLTP activity is decreased by statin treatment.

DrugsPlasma HDL-C concentration is negatively associatedwith occurrence of cardiovascular disease, independentof age, gender, LDL-C and other established riskfactors. Consequently, there is great interest forpharmacological interventions to raise HDL-C. Amongthe drugs that are currently available, statins, fibratesand nicotinic acid exert such an increasing effect onplasma HDL-C concentrations. These drugs, however,are limited in their HDL-C raising efficacy (statins:+3% to +9%, fibrates: +0% to +11%, nicotinic acid:+7% to +23%) or exhibit significant side effects(nicotinic acid: flushes). The high levels of HDL-Cobserved in case of CETP deficiency have led to thedevelopment of strategies to raise HDL-C by inhibitionof CETP activity in humans. Two such strategies arecurrently in advanced stage of development, i.e.,vaccine-induced inhibition and inhibition of CETPactivity through small-molecule compounds. Amongthese, the latter has attracted most attention. The effectsof CETP inhibition through small-molecule compoundsparallel those observed in genetic CETP deficientsubjects to a large extent: increase of plasma HDL-Cand apoA-I concentration, with the occurrence of largesized HDL particles due to cholesteryl ester enrichment.In case of potent CETP inhibition, plasma levels ofLDL-C and apoB are also decreased. With respect to theeffects on cardiovascular disease exerted by theselipoprotein changes, data from epidemiological studiesmight justify optimism. However, clinical trials asses-sing the consequences of CETP inhibition on surrogateand clinical cardiovascular endpoints are required.One such trial has recently been terminated due to anincreased number of lethal cardiovascular eventsoccurring in the CETP-inhibition group. At themoment,

696 Lipidation

it remains to be established whether this relates tointrinsic characteristics of the study drug or to themechanism of CETP-inhibition in general.

Due to the absence of human models for PLTPdeficiency, our knowledge about the relevance ofplasma PLTP activity for human lipid metabolism isstill incomplete. No investigational drugs are availablethat specifically target the activity of this protein.

References1. Tall A (1995) Plasma lipid transfer proteins. Annu Rev

Biochem 64:235–2572. van Tol A (2002) Phospholipid transfer protein. Curr Opin

Lipidol 13:135–1393. Jiang XC, Zhou HW (2006) Plasma lipid transfer proteins.

Curr Opin Lipidol 17:302–3084. Beamer LJ, Carroll SF, Eisenberg D (1997) Crystal

structure of human BPI and two bound phospholipids at2.4 angstrom resolution. Science 276:1861–1864

5. Qiu X, Mistry A, Ammirati MJ et al (2007) Crystalstructure of cholesteryl ester transfer protein reveals a longtunnel and four bound lipid molecules. Nat Struct Mol Biol14:106–113

Lipidation

▶Lipid Modifications

Lipopolysaccharide

A lipopolysaccharide (LPS) is any compound consist-ing of covalently linked lipids and polysaccharides. Theterm is used more frequently to denote a cell wallcomponent from Gram-negative bacteria. LPS hasendotoxin activities and is a polyclonal stimulator ofB-lymphocytes.

▶Neutrophils▶Inflammation

Lipoprotein Lipase

Endothelial anchored enzyme in muscle and adiposetissue primarily responsible for hydrolysis of chylomi-cron and VLDL triglycerides.

▶Lipoprotein Metabolism

Lipoprotein Metabolism

DANIEL J. RADER

Institute for Translational Medicine and Therapeutics,University of Pennsylvania School of Medicine,Philadelphia, PA, USA

SynonymsLipid metabolism; Cholesterol metabolism

Definition▶Lipoprotein metabolism is the process by which hy-drophobic lipids, namely triglycerides and cholesterol,are transported within the interstitial fluid and plasma. Itincludes the transport of energy in the form oftriglycerides from intestine and liver to muscles andadipose, as well as the transport of cholesterol both fromintestine and liver to peripheral tissues, as well as fromperipheral tissues back to the liver.

Basic MechanismsLipoproteins are largemacromolecular complexes,whichtransport triglycerides and cholesterol within the blood.Triglycerides are a key component of energy transportand metabolism, and cholesterol is an essential com-ponent of all cells and required for steroidogenesis.The structure of lipoproteins is a hydrophobic neutrallipid core consisting of triglycerides and cholesterylesters, surrounded by amphipathic phospholipids andspecialized proteins known as ▶apolipoproteins. Phos-pholipids serve to permit interaction with the aqueousenvironment, and apolipoproteins are required for thestructural integrity of lipoproteins and direct their meta-bolic interactions with enzymes, lipid transport proteins,and cell surface receptors. The five major families oflipoproteins are ▶chylomicrons, ▶very-low-densitylipoproteins (VLDL), ▶intermediate-density lipopro-teins (IDL), low-density lipoproteins (LDL), and▶high-density lipoproteins (HDL). Chylomicrons arethe largest andmost lipid-rich lipoproteins, whereasHDLare the smallest lipoproteins and contain the least amountof lipid. In the exogenous pathway of lipid transport(Fig. 1), dietary fat and cholesterol are absorbed byintestinal enterocytes and incorporated into chylomi-crons, which contain the major structural apolipoproteinB-48.Chylomicrons are initially secreted into lymph thusbypassing the hepatic first pass effect. Once they gainentry into the systemic circulation they bind to▶lipopro-tein lipase (LPL) on the luminal surface of the capillaryendothelium of tissues (▶endothelial lipase), especiallymuscle and adipose tissue. The LPL hydrolyzes thetriglycerides (apolipoprotein C-II on the chylomicronsurface is a required cofactor for LPL). The free fatty

Lipoprotein Metabolism. Figure 1 Exogenous pathway of lipoprotein metabolism.

Lipoprotein Metabolism. Figure 2 Endogenouspathway of lipoprotein metabolism.

Lipoprotein Metabolism 697

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acids enter the tissue to be used for energy (muscle) orstorage (adipose) and the triglyceride-depleted chylomi-cron remnant (CMR) is released. CMRs are taken up bythe liver by binding of apolipoprotein E to the LDLreceptor and the LDL receptor-related protein (LRP). Inthe endogenous pathway of lipid transport (Fig. 2), theliver synthesizes triglycerides and cholesteryl esters andpackages them into VLDL, which contain the majorstructural apolipoprotein B-100. VLDLs are hydrolyzedby LPL to form intermediate density lipoproteins (IDL).IDL can be taken up by the liver via binding ofapolipoprotein E to the LDL receptor or LRP. Alterna-tively, the triglyceride and phospholipid in IDL can behydrolyzed by ▶hepatic lipase (HL) within the hepaticsinusoids to form LDL. LDL can be taken up byperipheral cells or by the liver by the binding ofapolipoprotein B-100 to the LDL receptor.

The pathways of HDL metabolism and reversecholesterol transport are complex (Fig. 3, [1]). HDLand its major apolipoprotein apoA-I are synthesizedby both the intestine and the liver. A second major

HDL protein apoA-II is made only by the liver. NascentHDL interacts with peripheral cells to facilitate theremoval of excess free cholesterol through a processthat is facilitated by the cellular protein ATP-bindingcassette protein A1 (ABCA1). Some of the acquiredfree cholesterol is esterified to cholesteryl ester on theHDL particle by the action of the enzyme ▶lecithin-cholesterol acyltransferase (LCAT) and the nascentHDL particle becomes the larger HDL3. HDL acquiresfurther cholesteryl ester by continued LCAT action andeventually becomes the even larger HDL2. HDL2 canselectively transfer both cholesteryl ester and freecholesterol to the liver via an HDL receptor in the livercalled scavenger receptor BI (SR-BI). Cholesterylesters can also be transferred from HDL2 to apoB-containing lipoproteins such as VLDL and LDL via theaction of the▶cholesteryl ester transfer protein (CETP)and then returned to the liver by hepatic uptake of LDL.HDL2 triglycerides and phospholipids can be hydro-lyzed by HL and endothelial lipase (EL) to remodel it toHDL3. Cholesterol derived fromHDL contributes to thehepatic cholesterol pool used for bile acid synthesis andthe cholesterol is eventually excreted into the bileand feces as bile acid or free cholesterol.

Disorders of lipoprotein metabolism involve pertur-bations which cause elevation of triglycerides and/orcholesterol, reduction of HDL-C, or alteration of pro-perties of lipoproteins, such as their size or composi-tion. These perturbations can be genetic (primary) oroccur as a result of other diseases, conditions, or drugs(secondary). Some of the most important secondarydisorders include hypothyroidism, diabetes mellitus,renal disease, and alcohol use. Hypothyroidism causeselevated LDL-C levels due primarily to downregulationof the LDL receptor. Insulin-resistance and type2 diabetes mellitus result in impaired capacity tocatabolize chylomicrons and VLDL, as well as excesshepatic triglyceride and VLDL production. Chronickidney disease, including but not limited to end-stage

Lipoprotein Metabolism. Figure 3 HDL metabolism and reverse cholesterol transport.

698 Lipoprotein Metabolism

renal disease, is associated with moderate hypertrigly-ceridemia due to a defect in triglyceride lipolysis andremnant clearance. Nephrotic syndrome is associatedwith a more pronounced hyperlipidemia involving bothelevated triglycerides and cholesterol due to hepaticoverproduction of VLDL. Alcohol consumption inhi-bits oxidation of free fatty acids by the liver, whichstimulates hepatic triglyceride synthesis and secretionof VLDL; the usual lipoprotein pattern associated withalcohol consumption is moderate hypertriglyceridemia.

Inherited disorders of lipoprotein metabolism haveprovided important insights into the molecular regulationof lipoprotein metabolism in humans. The familialhyperchylomicronemia syndrome (FCS) (also known asType I hyperlipoproteinemia), characterized by severelyelevated triglycerides, is caused by genetic deficiency ofeither ▶LPL or apoC-II, demonstrating the essentialnature of each of these protein for the hydrolysis ofchylomicron triglycerides. Familial dysbetalipoprotei-nemia (FD) (also known as Type III hyperlipoproteine-mia), characterized by elevation in apoB-containinglipoprotein remnant particles containing both triglycer-ides and cholesterol, is caused by specific mutations inthe gene for apolipoprotein E (apoE), most commonlyhomozygosity for the relatively common apoE2 variant.This proved the essential nature of apoE for normalclearance of lipoprotein remnants by the liver. Familialhypercholesterolemia (FH) (also known as Type IIahyperlipoproteinemia), characterized by elevation inLDL cholesterol, is caused by loss-of-function muta-tions in the LDL receptor. Homozygotes are moreseverely affected than heterozygotes, demonstrating thegene dose effect of the LDL receptor on LDL-C levels.This disorder established the key role of the LDLreceptor in regulating plasma levels of LDL-C throughmediating uptake of LDLby the liver.Mutations in othergenes have been subsequently discovered to regulatethe hepatic expression of the LDL receptor andthus influence plasma LDL-C levels. These include

loss-of-function mutations in the autosomal recessivehypercholesterolemia (ARH) gene, a recessive trait, andgain-of-function mutations in the proprotein convertasesubtilisin/kexin type 9 (PCSK9) gene, an autosomaldominant trait. Sitosterolemia is caused by mutations inone of two members of the adenosine triphosphate(ATP)-binding cassette (ABC) transporter family,ABCG5 and ABCG8, resulting in increased cholesterolin the liver and secondary downregulation of the LDLreceptor. Finally, mutations in the receptor bindingregion of apoB-100, the ligand for the LDL receptor, canimpair its binding to the LDL receptor and delay theclearance of LDL, a condition known as familialdefective apoB-100 (FDB).Genetic conditions causing low cholesterol have also

provided important insights and novel pharmacologictargets. Abetalipoproteinemia is characterized by ab-sence of plasma apoB-containing lipoproteins and iscaused by mutations in the gene encoding microsomaltriglyceride transfer protein (MTP), a protein thattransfers lipids to nascent chylomicrons and VLDL inthe intestine and liver, respectively. Familial hypobeta-lipoproteinemia is characterized by markedly reducedLDL-C and apoB levels and is caused by mutations inapoB that generally result in premature truncation thateither reduce secretion and/or accelerate catabolism.Finally, loss-of-function mutations in PCSK9 have beenshown to cause low LDL-C levels and substantiallifetime protection from CHD. All three of these geneproducts are targets for the development of newtherapies for reducing LDL-C levels.Genetic disorders of HDL metabolism have also

resulted in greater understanding of the molecularregulation of HDL metabolism. Nonsense or missensemutations in apoA-I can result in substantially reducedHDL-C levels due to rapid catabolism of structurallyabnormal or truncated apoA-I proteins. Tangier diseaseis a rare autosomal codominant disorder characterizedby markedly low HDL-C and apoA-I levels and caused

Lipoprotein Metabolism 699

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by mutations in the gene ABCA1. ABCA1 facilitatesefflux of cholesterol from cells to lipid-poor apoA-I,and in its absence apoA-I is not appropriately lipidatedand is rapidly cleared from the circulation. LCATdeficiency causes markedly low levels of HDL-C,establishing the importance of cholesterol esterificationby LCAT in the maintenance of normal HDL metabo-lism. Finally, CETP deficiency causes markedlyelevated levels of HDL-C, establishing the importanceof cholesteryl ester by CETP in the maintenance ofnormal HDL metabolism. Again, all of these geneproducts are targets of new HDL-based therapies.

Pharmacologic RelevanceLDL Cholesterol ReductionDisorders of lipoprotein metabolism are important riskfactors for atherosclerotic vascular disease (ASCVD).Thus the field of lipoprotein metabolism has been afertile area for the development of drugs that modulatethe levels of plasma lipoproteins. Intervention withdrugs to reduce ▶LDL cholesterol has been proven todecrease the risk of cardiovascular events [2]. Interven-tion with drugs to reduce triglycerides or raise HDL-Chas not yet been definitively proven to reducecardiovascular events. The early clinical trials forLDL-C reduction utilized niacin, bile acid sequestrants,and even the surgical approach of partial ileal bypass toreduce serum cholesterol levels. However, the bulk ofthe data supporting LDL-C reduction comes from theclinical outcome trials with ▶HMG CoA-reductaseinhibitors (statins). Early studies focused on patientswith preexisting CHD, including the ScandinavianSimvastatin Survival Study (4S), the Cholesterol andRecurrent Events (CARE) study, and the Long-TermIntervention with Pravastatin in Ischemic Disease(LIPID) trial and established the efficacy of statintherapy in reducing cardiovascular events in patientswith CHD. Early primary prevention trials included theWest of Scotland Coronary Prevention Study (WOS-COPS) and the Air Force/Texas Coronary Atheroscle-rosis Prevention Study (AFCAPS/TexCAPS) andconfirmed that the benefits of LDL cholesterolreduction extend to the primary prevention setting.Subsequent studies have expanded downward the rangeof baseline LDL-C levels for which statin therapy isbeneficial as well as the target LDL that is effective [2],and include the Heart Protection Study (HPS), theAnglo-Scandinavian Cardiac Outcomes Trial Lipid-lowering Arm (ASCOT-LLA), the Collaborative Ator-vastatin Diabetes Study (CARDS), the Treat to NewTargets (TNT) trial, and the Pravastatin or AtorvastatinEvaluation and Infection Therapy (PROVE-IT) study.These studies strongly support the “lower is better”approach to reducing LDL-C as applied to high-riskpatients.

Thus drug therapy for LDL-C reduction is widelyused. Statins are the cornerstone of LDL-C reducingdrug therapy. By inhibiting cholesterol biosynthesis inthe liver, statins lead to increased hepatic LDL receptorexpression. There are six statins currently available:lovastatin, pravastatin, simvastatin, fluvastatin, ator-vastatin, and rosuvastatin. There is wide interindividualvariation in the initial response to a statin, but once apatient is on a statin, the doubling of the statin doseproduces a very predictable 6% further reduction inLDL-C. Some patients treated with statins developmuscle fatigue or pain, and severe myopathy andrhabdomyolysis have been reported. The risk of statin-associated myopathy is increased by the administrationof drugs that interfere with the cytochrome P450metabolism of statins [3]. Due to both inadequatereduction of LDL-C in some patients as well as statinintolerance in others, there is a clinical need for LDL-reducing drug therapy beyond statins. The mostcommonly used class is that of cholesterol absorptioninhibitors; currently ezetimibe is the only drug inthis class clinically available. Ezetimibe binds to andinhibits the function of NPC1L1, a major cholesteroltransporter in the intestinal enterocyte responsible forabsorption of luminal cholesterol derived both from dietas a well as from bile. The inhibition of intestinalcholesterol absorption reduces hepatic cholesterolcontent and results in upregulation of hepatic LDLreceptor expression. Ezetimibe at the 10 mg dosereduces cholesterol absorption by about 60% andreduces LDL-C levels by about 18% on average asmonotherapy, and to a similar extent when used incombination with a statin. Ezetimibe is used in combi-nation with statins to further reduce LDL-C levels andin patients who are statin-intolerant. In some cases, athird class of LDL-lowering drugs, bile acid seques-trants, is needed. Bile acid sequestrants bind bile acidsin the intestine, prevent their reabsorption, and accele-rate their loss in the feces. In order to maintain anadequate bile acid pool, the liver diverts cholesterol tobile acid synthesis, resulting in decreased hepaticcholesterol and upregulation of hepatic LDL receptorexpression. Cholestyramine and colestipol are insolubleresins that must be suspended in liquid and colesevalamis available as large tablets, and relatively large amountsof bile acid sequestrants must be used to achieveeffective LDL reduction.

Inability to achieve LDL-C goals with existing drugtherapy remains an important unmet medical need.Advances in our understanding of the molecularregulation of LDL metabolism have generated newpharmacologic targets. These include the following: (i)inhibition of squalene synthase, another importantenzyme in the cholesterol biosynthetic pathway; (ii)inhibition of MTP, which reduces VLDL production

700 Lipoproteins

and has been shown to reduce LDL-C levels; (iii)inhibition of apoB production using an antisenseoligonucleotide approach; (iv) inhibition of PCSK9based on the persuasive genetic data reviewed above.

The data supporting reduction in triglycerides orraising of HDL-C are much less abundant [4]. Fibratesand nicotinic acid are the most effective drugs inlowering TGs and raising HDL-C. Fibrates are agonistsof PPARα and aremost effective as triglyceride loweringdrugs, with modest effects in raising HDL-C. Fibrateslower triglycerides by activating PPARα to stimulate▶LPL (enhancing triglyceride hydrolysis) and reducehepatic apoC-III synthesis (enhancing clearance of TG-rich lipoproteins). A limited number of clinical trialswith fibrates support the concept that they may reducecardiovascular risk, particularly in patients with thephenotype of insulin-resistance. Increasingly, fibratesare being used in combination with statins. Nicotinicacid, or niacin, is a B-complex vitamin that in high doseslowers triglycerides and raises HDL-C levels. Niacinacutely reduces free fatty acid levels through inhibitionof adipocyte triglyceride lipolysis, an effect nowknown to be mediated by the niacin receptor GPR109A,a G protein coupled receptor primarily expressed onadipocytes. Whether this mechanism accounts for thetriglyceride lowering andHDL-C raising associatedwithniacin therapy is uncertain. The clinical use of niacin islimited by prostaglandin-mediated cutaneous flushingwhich is also mediated by GPR109A expressed ondermal macrophages. Data that niacin reduces cardio-vascular risk is limited, but are consistent with acardioprotective effect. Patients with combined hyper-lipidemia may achieve their LDL-C goal with a statinalone, but frequently have persistently elevated trigly-cerides and often do not achieve their non-HDL-C goal.In this situation, the addition of either niacin or a fibrateto the statin can be highly effective in reducing thetriglycerides and non-HDL-C levels. The therapy of lowHDL-C is amajor unmetmedical need andnew therapiesdesigned to raise HDL-C levels or promote HDLfunction are under active development [1].

References1. Rader DJ (2006)Molecular regulation of HDLmetabolism

and function: implications for novel therapies. J ClinInvest 116:3090–3100

2. Grundy SM, Cleeman JI, Merz CN et al (2004)Implications of recent clinical trials for the NationalCholesterol Education Program Adult Treatment Panel IIIguidelines. Circulation 110:227–239

3. Pasternak RC, Smith Jr, Bairey-Merz CN et al (2002)ACC/AHA/NHLBI clinical advisory on the use and safetyof statins. J Am Coll Cardiol 40:567–572

4. Szapary PO, Rader DJ (2004) The triglyceride-high-density lipoprotein axis: an important target of therapy?Am Heart J 148:211–221

Lipoproteins

The main transport form of lipids in the circulation. Theyare spherical macromolecules of 10–1200 nm diametercomposed of a core of neutral lipids (mostly cholesterolester and triglycerides) surrounded by an amphipathicshell of polar phospholipids and cholesterol. Embedded inthe shell of lipoproteins are apolipoproteins that areessential for assemblyof theparticles in tissues that secretelipoproteins, and for their recognition by target cells.

▶Lipoprotein Metabolism▶Lipid Transfer Proteins▶HMG-CoA-Reductase Inhibitors▶Low-Density Lipoprotein Receptor Gene Family

Liposomes

Phospholipid vesicles, uncoated or polyethylenglycol-coated. They can be used to vehicle drugs, antibodies ornucleic acids to target cells.

5-Lipoxygenase

5-lipoxygenase is the enzyme causing catalysis ofarachidonic acid into leukotriene A4.

▶Leukotrienes

Lipoxygenases

▶Leukotrienes

Lithocholic Acid

3α-Hydroxy-5β-cholanoic acid, a hepatotoxic andcholestatic secondary bile acid, formed by bacterialdehydroxylation of primary bile acids in the intestine.

▶Nuclear Receptor Regulation of Hepatic CytochromeP450 Enzymes

Local Anaesthetics 701

Local Anaesthetics

Local Anaesthetics. Figure 1 Common structure oflocal anaesthetics. A lipophilic moiety on the left, analiphatic spacer containing an ester or amide bond in themiddle and an amine group on the right are the typicalstructural elements for local anaesthetic drugs.

L

MICHAEL BRAU

Abteilung Anaesthesiologie und OperativeIntensivmedizin, Universitätsklinikum,Justus-Liebig-Universität, Gießen, Germany

DefinitionLocal anaesthetics are drugs that reversibly interruptimpulse propagation in peripheral nerves thus lead-ing to autonomic nervous system blockade, analgesia,anaesthesia and motor blockade in a desired area ofthe organism.

Mechanism of ActionImpulse propagation in the▶peripheral nervous systemdepends on the interplay between ion channels selectiveto potassium and sodium. Briefly, few voltage insensi-tive potassium channels allow diffusion of positivelycharged potassium ions from the internal side of theaxon to the exterior. This leaves a negative charge atthe internal side that is called resting potential andamounts to approximately −80 mV over the ▶axonalmembrane. Upon a small depolarisation of themembrane, ▶voltage–gated Na+ channels open (acti-vate) and conduct positively charged sodium ions fromthe exterior to the interior of the axon that depolarisesthe membrane to about +60 mV (action potential).After a few milliseconds the channels close spon-taneously (inactivation) terminating the action potentialand thus giving it an impulse like character. The actionpotential spreads electrotonically toneighbouring sodiumchannels that also open to produce an action potential.In this way, the impulse is propagated along the nerveto (afferent) or from (efferent) the central nervous system.Local anaesthetics inhibit ionic current through voltagegated sodium channels in a concentration dependentand reversible manner therefore directly blocking theimpulse propagation process in either direction [1].The interaction between the local anaesthetic moleculeand the sodium channel is complex. The binding affinityis low for resting channels, but dramatically increaseswhen the channel is activated. Thus, at high stimulusfrequency sodiumcurrent block increases (use-dependentblock). Use-dependent block is not important for con-duction block during local anaesthesia since very highconcentrations are reached locally producing completeand sufficient block. However, when local anaestheticssuch as lidocaine are applied intravenously, lower sys-temic concentrations may already induce use-dependentblock and thus reduce excitability in electrically activecells. This mechanism is important for the successfuluse of lidocaine as an antiarrhythmic or analgesic drug.

The putative binding site for local anaesthetic mole-cules at the sodium channel has been identified as twoamino acids in the sixth membrane-spanning segmentof domain IV [2]. This binding site is located directlyunderneath the channel pore and can only be reachedfrom the internal side of the membrane. Because localanaesthetics are applied exterior to the nerve fibre, theyhave to penetrate the axonal membrane before they canbind to the channel.

Besides sodium channels, other ion channels suchcalcium- and potassium channels as well as certainligand-gated channels are affected by local anaesthetics.However, this plays only a minor role for nerve blockbut may have more impact on adverse effects inducedby systemical concentrations of these drugs.

Structure Activity RelationLocal anaesthetics comprise a lipophilic and a hydrophil-ic portion separated by a connecting hydrocarbon chain(Fig. 1). The hydrophilic group is mostly a tertiary aminesuch as diethylamine; the lipophilic group is usuallyan unsaturated aromatic ring such as xylidine or para-aminobenzoic acid. The lipophilic portion is essential foranaesthetic potency whereas the hydrophilic portion isrequired for water solubility. Both groups are importantfor binding the drug molecule to the sodium channel.The connecting hydrocarbon chain usually contains anester or an amide, in rare cases also an ether, and separatesthe lipophilic and hydrophilic portions of the moleculein an ideal distance for binding to the channel. Localanaesthetics are classified by the nature of this bond intoester or amide local anaesthetics. This bond is alsoimportant for metabolism of the drugs and to adversereactions such as allergic reactions. Clinically relevantlocal anaesthetics have an efficacy of 100%, i.e. at highconcentrations they completely abolish the sodiumcurrent. Their blocking potencies range from a fewmicromolar to millimolar half-maximal blocking con-centration and are highly dependent on lipid solubility.To a smaller amount potency also depends on thestructure of the molecule and on the type of bonding

702 Local Anaesthetics

(Table 1). Further determinants of blocking potency arethemembrane potential and the state inwhich the sodiumchannel is in (resting, activated, inactivated). The tertiaryamine group can be protonated giving most local

Local Anaesthetics. Table 1 Examples of clinically used lo

Name Structure

Procaine

Tetracaine

Lidocaine

Prilocaine

Mepivacaine

Ropivacaine

Bupivacaine

The ending “caine” stems from cocaine, the first clinically employed locathe others are amides. Amide bonded local anaesthetics usually contadrawings, the lipophilic portion of the molecule is depicted at the left, thstereoisomeric drugs. Lipid solubility is given as the logarithm of the w

anaesthetics a pKa of about eight so that a larger amountof the drug is in the hydrophilic form when injectedinto tissue with physiological pH. However, only theunprotonated lipophilic form is able to penetrate the

cal anaesthetics

Relativepotency

Mean blockingduration [h]

Lipid solu-bility log[P]

Molwt.[g/mol]

pKa

1 0.5–1 1.92 236 9.1

8 2–3 3.73 264 8.6

2 1–2 2.26 234 8.2

2 1–2 2.11 220 7.9

2 1,5–2 1.95 246 7.9

6 3–5 2.90 274 8.2

8 4–6 3.41 288 8.2

l anaesthetic. Procaine and tetracaine are ester-linked substances,in two i’s in their name, ester-bonded only one. In the structuree amine at the right. The asterisk marks the chiral centre of theater:octanol partition coefficient, log(P).

Locus Ceruleus 703

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axonal membrane, which is required before binding tothe sodium channel can occur. In a very acidic envi-ronment, like inflamed tissue, local anaesthetics arehighly protonated, therefore cannot penetrate the axonalmembrane and have little effect.

ToxicityLocal anaesthetics interferewith all voltage-gated sodiumchannel isoforms in an organism and thus with allelectrically excitable cells in organs such as brain, heartand muscle. The major unwanted effects of localanaesthetics are thus disturbances of brain and heartfunction occurring during systemically high concentra-tions after overdosing or after accidental intra-vascularinjection. Cerebral convulsions, general anaesthesia aswell as dysrhythmias up to asystolic heart failure orventricular fibrillation are feared as rare but most harmfulcomplications. Other adverse effects can be allergicreactions. This occurs especially with ester-bondedlocal anaesthetics because their metabolism producespara-aminobenzoic acid that may serve as a hapten.As a special case, the local anaesthetic prilocaine ismetabolised to o-toluidine, which may induce methemo-globinemia, especially in patients that have a glucose-6-phosphatase deficiency. The ▶S-stereoisomers of thepiperidine local anaesthetics bupivacaine and ropivacainehave now been introduced into clinical practise to reduceside effects. Their blocking potencies are minimallylower compared to their R-counterparts but their thera-peutic index is wider.

Clinical UseLocal anaesthetics are mainly employed to induceregional anaesthesia and analgesia to allow surgicalprocedures in a desired region of the organism. Nerveblock result in autonomic nervous system blockade,analgesia, anaesthesia and motor blockade. The patientnormally stays awake during surgical procedures underregional anaesthesia, but regional anaesthesia can alsobe combined with general anaesthesia to reduce therequirement of narcotics and analgesic drugs. Localanaesthetics have to be injected locally into the circum-ference of a peripheral nerve, which gives sufficientlyhigh concentrations to achieve conduction block. Cer-tain injection techniques and procedures such as singlenerve block as well as spinal,▶epidural, or intravenousregional anaesthesia have evolved to achieve thedesired nerve block. It is also possible to place acatheter adjacent to a nerve and continuously applylocal anaesthetics to receive long-term analgesia. Lowconcentrations of lipophilic local anaesthetics may beused for differential nerve block, i.e. only block ofsympathetic and nociceptive fibres whereas somatosen-sory and motor fibres are less affected. Differential nerveblock for example is useful in labour analgesia or foranalgesia after surgical procedures of the extremities.

After local anaesthetic injection, onset of nerve blockand duration depends mainly on lipid solubility andon the region in where the drug is injected. In someformulations adrenaline is added to prolong the block-ing action by inducing regional vasoconstriction andhereby reduce absorption and metabolisation.

The amide local anaesthetic lidocainemay also be usedas an antiarrhythmic for ventricular tachycardia and exra-systoles after injection into the blood circulation. Drugswith high lipid solubility such as bupivacaine cannot beused for these purposes because their prolonged bindingto the channelmay induce dysrhythmias or asystolic heartfailure [3]. Systemically applied lidocaine has also beenused successfully in some cases of ▶neuropathic painsyndromes [4]. Here, electrical activity in the peripheralnervous system is reduced by used-dependent butincomplete sodium channel blockade.

▶General Anaesthetics▶Voltage-dependent Na+ Channels

References1. Butterworth JF, Strichartz GR (1990) Molecular mechan-

isms of local anesthesia: a review. Anesthesiology 72:722–734

2. Ragsdale DS, McPhee JC, Scheuer T et al (1994)Molecular determinants of state-dependent block of Na +channels by local anesthetics. Science 265:1724–1728

3. Clarkson CW, Hondeghem LM (1985) Mechanism forbupivacaine depression of cardiac conduction: fast blockof sodium channels during the action potential with slowrecovery from block during diastole. Anesthesiology62:396–405

4. Tanelian DL, Brose WG (1991) Neuropathic pain can berelieved by drugs that are use-dependent sodium channelblockers: lidocaine, carbamazepine, and mexiletine.Anesthesiology 74:949–951

Locus

A specific region within a DNA sequence.

▶Pharmacogenomics

Locus Ceruleus

The locus ceruleus is a structure located on the floor ofthe fourth ventricle in the rostral pons. It contains morethan 50% of all noradrenergic neurons in the brain, andprojects to almost all areas of the central nervous system.

704 Long-Chain Fatty Acids

The locus ceruleus is important for the regulation ofattentional states and autonomic nervous system acti-vity. It has also been implicated in the autonomic andstress-like effects of opiate withdrawal. A noradrenergicpathway originating from the locus ceruleus whichdescends into the spinal cord is part of the descendinginhibitory control system, which has an inhibitory effecton nociceptive transmission in the dorsal horn.

Long-Chain Fatty Acids

Long-chain fatty acids (LCFAs) are aliphatic com-pounds with a terminal carboxyl group and with a chainlength greater than 12 carbon atoms (e.g., lauric acid).Very long-chain fatty acids are fatty acids with morethan 18 carbon atoms (e.g., stearic acid).

▶Fatty Acid Transport Proteins

Long-QT Syndromes

Long-QT syndromes (LQTS) are potentially fatalinherited cardiac arrhythmias characterized by pro-longed or delayed ventricular repolarization, manifestedon the electrocardiogram as a prolongation of the QTinterval. LQTS can be caused bymutations of at least sixgenes including KCNQ1 for LQT1, KCNH2 for LQT2,SCN5A encoding a cardiac sodium channel for LQT3,KCNE1 for LQT5 and MiRP1 for LQT6. LQT4 islinked to the mutations located in chromosome 4q25–27. Blockade of HERG channels is the most commonlyidentified drug-induced LQT.

▶K+ Channels▶Voltage-dependent Na+ Channels

Long-Term Depression

Long-term depression (LTD) is a synaptic plasticityphenomenon that corresponds to a decrease in the syn-aptic strength (decrease in the post-synaptic responseobserved for the same stimulation of the pre-synaptic

terminals) observed after a specific stimulation of theafferent fibres. This decreased response is still observedhours after its induction.

▶Metabotrophic Glutamate Receptors

Long-Term Potentiation

SynonymsLTP

DefinitionLong-term potentiation (LTP) is a synaptic plasticityphenomenon that corresponds to an increase in thesynaptic strength (increase in the post-synapticresponse observed for the same stimulation of thepresynaptic terminals) observed after a high frequencystimulation (tetanus) of the afferent fibres. Thisincreased response is still observed hours and evendays after the tetanus. The phenomenon is oftenobserved at glutamatergic synapses and involves, inmost cases, the activation of the N-methyl D-aspartate(NMDA) subtype of ionotropic glutamate receptors.

▶Metabotropic Glutamate Receptors▶Nicotinic Receptors▶Ionotropic Glutamate Receptors

Loop Diuretics

▶Diuretics

Low-density-lipoprotein-cholesterol

LDL is the major carrier of cholesterol to the peripheryand supplies the cholesterol essential for the integrity ofnerve tissue, steroid hormone synthesis, and cellmembranes. The association between elevated plasmacholesterol carried in LDL and the risk of coronary heartdisease has been well established. LDL is alsosometimes called the “bad” cholesterol.

▶Lipoprotein Metabolism

Low-density Lipoprotein Receptor Gene Family 705

Low-density Lipoprotein ReceptorGene Family

L

T HOMAS E. WILLNOW

Max-Delbrueck-Center for Molecular MedicineBerlin, Germany

SynonymsLow-density lipoprotein receptor-related proteins; LRP

DefinitionThe low-density lipoprotein ( ▶LDL ) receptor genefamily encompasses a group of structurally relatedendocytic receptors that are expressed in manymammalian and nonmammalian cell types. The recep-tors mediate cellular binding and uptake of a variety ofdiverse macromolecules including lipoproteins andlipid-carrier-complexes, proteases and protease inhibi-tors, signaling factors, as well as viruses and toxins.Gene family members play crucial roles in manybiological processes ranging from lipid homeostasis, tosignal transduction, to cell migration.

Basic CharacteristicsReceptor-Mediated EndocytosisEndocytic receptors are cell surface proteins that transportmacromolecules into cells through a process known asreceptor-mediated ▶endocytosis. I n t hi s p ro ce ss , areceptor on the cell surface binds a specific ligandfrom the extracellular space, internalizes via specializedin-tucked regions of the plasma membrane calledclathrin-coated pits, and moves to an intracellular vesicle(▶endosome) to discharge its cargo. From endosomalcompartments, ligands are further directed to lysosomesfor catabolism, while the unliganded receptor returns tothe cell surface to initiate the next round of endocytosis.Endocytic receptors serve to regulate the concentration ofligands in the extracellular space and to deliver them tocells in need of these metabolites. Binding of someligands to endocytic receptor may also trigger intracellu-lar signaling cascades involved in regulation of importantphysiological processes such as embryonic patterning,neuronal cell migration, or synaptic plasticity.

Structural Organization of the LDL Receptor

Gene Family Much of our knowledge of the structure and functionof endocytic receptors is based on the analysis of theLDL receptor gene family. Members of this extendedgene family can be found in a variety of species rangingfrom roundworms to insects, to vertebrates. Ten recep-tors exist in mammalian organisms, all of which sharecommon structural motifs required for receptor-mediated

endocytosis (Fi g. 1). Among other modules, theirextracellular domains are composed of clusters of▶complement-type repeats, the site of ligand binding,as well as β-propellers, essential for pH-dependent releaseof ligands in endosomes. The cytoplasmic tails harborrecognition sites for cytosolic adaptor proteins involvedin protein trafficking and signal transduction. All ecto-domains share significant sequence similarity in linewiththe ability of the receptors to bind an overlappingspectrum of ligands. In contrast, their cytoplasmicdomains are unique, indicating distinct cellular fates formacromolecules internalized by individual receptors [1].

Functions of the LDL Receptor Gene FamilyTransgenic animal models with spontaneous or inducedreceptor gene defects have been instrumental inelucidating the physiological roles of the LDL receptorgene family. In addition, a number of human diseaseshave been identified that are caused by sporadic orinherited forms of receptor deficiency (Table 1).

LDL ReceptorThe LDL receptor is the archetype receptor of the genefamily. It is the major endocytic receptor responsible forcellular uptake of cholesterol-rich ▶lipoproteins fromthe circulation and extracellular body fluids. It bindslipoprotein particles that contain the ▶apolipoproteinsB-100 (APOB-100) or APOE, and mediates theirendocytic uptake. Particles internalized via the LDLreceptor are delivered to lysosomal degradation wherethe apolipoproteins are broken down into amino acidswhile the lipids are released into the cytosol for furthermetabolism. The uptake of lipoproteins by the LDLreceptor has a dual role in lipid homeostasis. It deliverscholesterol required for maintenance of cellular functions(e.g., formation of membranes, synthesis of steroidhormones, and bile acids), and it regulates the concentra-tion of this lipid in the circulation. The importance of thelatter function is underscored by pathological abnormal-ities observed in patientswith LDL receptor gene defects;a syndrome designated familial hypercholesterolemia(▶FH). In humanswith FH, lack of LDL receptor activityresults in massive increase in circulating LDL. The meanplasma cholesterol levels are elevated approximatelytwofold in individuals with heterozygous and fourfold inpatients with homozygous gene defects. As a conse-quence of fulminate▶hypercholesterolemia, FH patientssuffer frompremature atherosclerosis and coronary arterydisease. To date more than 150 different mutations havebeen identified in the human LDL receptor gene.

VLDLR and APOER2Very low-density lipoprotein receptor (▶VLDLR) andAPOE receptor-2 (▶APOER2) are two gene familymembers with redundant functions. They are expressedin neurons of the developing brain and act as cell

Low-density Lipoprotein Receptor Gene Family. Figure 1 The LDL receptor gene family. The figure depictsthe structural organization of mammalian members of the LDL receptor gene family. APOER2 (apolipoproteinE receptor 2); LDLR (low-density lipoprotein receptor); LRP (LDL receptor-related protein); MEGF7(multiple epidermal growth factor repeat-containing protein 7); sorLA (sorting protein-related receptor); VLDLR(very low-density lipoprotein receptor). Designations in brackets indicate alternative names for the respectivereceptors.

Low-density Lipoprotein Receptor Gene Family. Table 1 Human diseases of the LDL receptor gene family

Receptor Type of mutation Disease

LDL re-ceptor

Loss-of-function (familial, autosomaldominant)

Familial hypercholesterolemia (impaired clearance of LDL)

VLDL re-ceptor

Loss-of-function (familial, autosomalrecessive)

Autosomal recessive cerebellar hypoplasia (ataxia, mentalretardation)

LRP5 Loss-of-function (familial, autosomalrecessive)

Osteoporosis-pseudoglioma-syndrome (reduced bone mass)

Gain-of-function (familial, autosomaldominant)

High-bone-mass trait (increased osteogenic activity)

LRP6 Missense mutation (familial,autosomal dominant)

Autosomal dominant early coronary artery disease (hyperlipidemia,hypertension, diabetes)

LRP1B Loss-of-function (sporadic) Esophageal squamous cell carcinoma, nonsmall-cell lung cancer

LRP2/megalin

Loss-of-function (familial, autosomalrecessive)

Donnai-Barrow syndrome (brain malformation, renal tubulardeficiency, diaphragmatic hernia)

SorLA Polymorphisms (sporadic) Late-onset Alzheimer’s disease

706 Low-density Lipoprotein Receptor Gene Family

Low-density Lipoprotein Receptor Gene Family 707

surface receptor for ▶reelin , a secreted guidance factorfor newborn neurons that migrate to their properposition in the laminating neocortex and cerebellum.Binding of Reelin to VLDLR and APOER2 initiates anintracellular signaling cascade that involves the cyto-plasmic adaptor disabled-1 (▶ DAB1) bound to thereceptor tails, and that ultimately leads to reorganizationof the cytoskeleton and regulation of cell migration( Fig. 2a ) [2]. Receptor gene defects in humans (Table 1)and in animal models result in abnormal layering ofneurons in the brain, and in severe neuronal dysfunction(cerebellar dysplasia, ataxia).

LRP5/6These receptors are integral components of the ▶wi ng -less (Wnt) signaling cascade by acting as coreceptorsto ▶frizzled. Simultaneous binding of Wnt ligands to

Low-density Lipoprotein Receptor Gene Family. Figuregene family. (a) The reelin signaling pathway in migrating ncognate receptors VLDLR and APOER2. Binding induces activating SRC family tyrosine kinases (SFKs) that in turn pactivates phosphatidylinositol-3-kinase (PI3K) and protein synthase kinase 3β (GSK3β), resulting in reduced phosphoLissencephaly 1 (LIS1) is another interaction partner of phmultimeric protein complex that also regulates microtubuleWnt signaling pathway, Wnt ligands bind to LRP5/6 and Frmembrane-associated casein kinase 1γ (CK1γ). Phosphordomain, preventing Axin-induced destruction of β-catenin.high-mobility group protein TCF to induce target gene tran

LRP5/6 and frizzled results in signal transduction throughthe canonical Wnt signaling pathway (Fi g. 2 b) [3]. Thefunction of LRP6 seems most relevant for earlyembryonic patterning events, as loss of receptor functionin fruit fly, Xe no pu s, and mouse causes aberrant patternformation and early embryonic lethality. In contrast,LRP5 activity is mainly required for regulation of boneformation as judged from loss-of-function and gain-of-function syndromes in humans that result in low or high-bone-mass traits, respectively (Ta b l e 1 ).

LRP4LRP4 is another receptor of the LDL receptor gene familyinvolved in regulation of embryonic patterning, mainlycontrolling formation of limb structures. Loss of receptoractivity in gene targeted mice or spontaneous mutationin bovine cause abnormal limb development and

2 Signal transduction by members of the LDL receptoreurons is initiated by binding of the guidance factor to itsclustering of DAB1 on the cytoplasmic receptor tails,hosphorylate the adaptor. Phosphorylated DAB1kinase B (PKB). PKB inhibits the activity of glycogenrylation of Tau (τ) and stabilization of microtubules.

osphorylated DAB1. It associates with α-subunits to adynamics (modified from [2]). (b) As part of the canonicalizzled resulting in phosphorylation of the LRP5/6 tail byylation recruits scaffold protein Axin to the LRP tailStabilized β-catenin enters the nucleus to interact withscription.

L

708 Low-density Lipoprotein Receptor-related Proteins

polysyndactyly. The molecular mechanisms of LRP4actions remain unclear at present, but may also involvesignaling through embryonic morphogen pathways.

LRP1 and LRP1BIn hepatocytes, LRP1 serves as backup pathway to theLDL receptor in hepatic clearance of ▶chylomicronremnants , lipoproteins that transport dietary lipids fromthe intestine to the liver. In vascular smooth musclecells, LRP1 likely acts as signaling receptor thatsuppresses the activity of the platelet-derived growthfactor ( ▶PDGF) receptor. Loss of local LRP1 expres-sion in mouse models causes enhanced activation of thePDGF receptor pathway, resulting in smooth musclecell proliferation and in marked susceptibility toatherosclerotic lesion formation [4]. LRP1B is alsoknown as LRP-DIT (deleted-in-tumors) because of itsoriginal description as a gene frequently inactivatedin lung cancer cell lines. A possible role as tumorsuppressor gene remains yet to be established. LRP1Bmay share some functional redundancy with LRP1 assuggested from the close sequence similarity andoverlap in expression pattern of both receptors.

LRP2LRP2 (megalin, gp330) is the largest member of theLDL receptor gene family. Inherited Lrp2 gene defectsare the cause of Donnai-Barrow syndrome, character-ized by brain malformation, renal dysfunction, anddiaphragmatic hernia. Rather than acting as lipoproteinreceptor such as the LDL receptor and LRP1, LRP2functions as endocytic receptor for cellular uptake oflipophilic vitamins and steroid hormones bound toplasma carrier proteins [1]. Most notably, a role for thereceptor in retrieval of filtered 25-OH vitamin D3 boundto the vitamin D binding protein ( ▶DBP ) from theglomerular filtrate is appreciated. Renal uptake ofvitamin/DBP complexes prevents uncontrolled urinaryloss of this essential metabolite and it delivers to renalproximal tubular cells the precursor for conversion into1,25-(OH)2 vitamin D3, the active hormone that controlssystemic calcium and bone metabolism. Loss of receptorfunction results in excessive urinary excretion ofvitamin D3 metabolites, and consequently, in vitaminD deficiency and osteomalacia (softening of the bones).

SorLASorLA is a chimeric receptor that harbors proteinmodules not present in other members of the LDLreceptor gene family ( Fig. 1). As well as acting asendocytic receptor for extracellular ligands (such asPDGF), sorLA also regulates the intracellular traffick-ing of target proteins between secretory and endocyticcompartments. In particular, the receptor acts as sortingreceptor for the amyloid precursor protein (▶ APP), theetiologic agent in ▶Alzheimer ’s disease. In neurons,SorLA directs APP into intracellular compartments less

favorable for proteolytic processing, thereby impairingbreakdown of APP to the amyloid- β peptide, theprinciple component of senile plaques (a hallmark ofAlzheimer ’s disease). Polymorphisms in the humanSorLA gene are among the most important genetic riskfactors of late-onset Alzheimer ’s disease, likely bydetermining the expression levels of this protectivefactor in the brain of individuals [5].

DrugsCurrently no drugs directly modulating the LDLreceptor family are known. The possible use of drugstargeting the LDL receptor family or downstreamsignaling proteins may be derived from Table 1.

References1. Nykjaer A, Willnow TE (2002) The low-density lipopro-

tein receptor gene family: a cellular Swiss army knife?Trends Cell Biol 12:273–280

2. Herz J, Chen Y (2006) Reelin, lipoprotein receptors andsynaptic plasticity. Nat Rev 7:850–859

3. Niehrs C (2006) Function and biological roles ofthe Dickkopf family of Wnt modulators. Oncogene25:7469–7481

4. Herz J, Strickland DK (2001) LRP: a multifunctionalscavenger and signaling receptor. J Clin Invest 108:779–784

5. Andersen OM, Willnow TE (2006) Lipoprotein receptorsin Alzheimer’s disease. Trends Neurosci 29:687–694

Low-density LipoproteinReceptor-related Proteins

▶Low-Density Lipoprotein Receptor Gene Family

Low Molecular Mass GTPases

▶Small GTPases

Low Voltage-activated (LVA) Ca2+

Channels

▶Voltage-dependent Ca2+ Channels

Lymphokines 709

LPL

▶Lipoprotein Lipase

LPS

▶Lipopolysaccharide

LRP

L

▶Low-Density Lipoprotein Receptor Gene Family

LRP5 and LRP6

LRP5 and 6 are closely related genes in humans andmice that are structurally distinct from other LRP familymembers. A single Drosophila ortholog exists and iscalled arrow. The proteins are single-pass transmem-brane receptors with an extracellular domain compris-ing four amino-terminal epidermal growth factor(EGF)-like repeats and three low density lipoprotein(LDL) receptor type A repeats. They have a relativelyshort proline-rich intracellular domain. Genetic evi-dence in Drosophila as well as loss-of-function andoverexpression evidence in vertebrates support theconclusion that arrow and LRP5/6 are essentialcoreceptors with the Fz proteins for Wnt/β-catenin-dependent signaling.

▶Wnt Signaling▶Low-Density Lipoprotein Receptos Gene Family

LTD

▶Long Term Depression

LTP

▶Long Term Potentiation

Luteinizing Hormone

▶Contraceptives

Lymphangiogenesis

Lymphangiogenesis is the growth of lymphatic vessels,which is critically controlled by the interaction ofVEGF-C and VEGF-D with the receptor VEGF-R3 onlymphatic endothelial cells.

▶Angiogenesis and Vascular Morphogenesis

Lymphocytes

Lymphocytes are specialized white blood cells that playa crucial role in an immune response.They can be Tlymphocytes, which can directly target and destroydefective cells, or B lymphocytes, which produceantibodies directed against specific antigens. Both Tand B lymphocytes produce a variety of cytokines toaugment and amplify the immune response.

▶Immune Defense

Lymphokines

▶Cytokines▶Growth Factors

710 Lysine Acetylation of Histones

Lysine Acetylation of Histones

▶Histone Acetylation

Lysipressin

▶Vasopressin/Oxytocin

Lysolipid Mediators

▶Lysophospholipids

Lysophosphatidic Acid

Lysophosphatidic acid (LPA) is the prototype of a groupof bioactive lysophospholipids that act on specificG-protein-coupled receptors to mediate a wide varietyof cellular functions.

▶Lysophospholipids

Lysophosphatidylcholine

SynonymLPC

DefinitionPotential signaling phospholipid derived from phos-phatidylcholine by phospholipase action. No receptorsknown.

▶Proton-Sensing GPCRs▶Lysophospholipids

Lysophospholipids

DAGMAR MEYER ZU HERINGDORF

Institut für Pharmakologie, Universität Duisburg-Essen, Germany

SynonymsLysosphingolipids and lysoglycerophospholipids;Lysolipid mediators

DefinitionLysophospholipids are small bioactive lipid moleculescharacterized by a single carbon chain and a polar headgroup. Two subgroups can be distinguished: moleculescontaining the sphingoid base backbone (lysosphingo-lipids) and molecules containing the glycerol backbone(lysoglycerophospholipids). The lysolipid structurerenders these lipids more hydrophilic and versatilethan their corresponding phospholipids. Lysophospho-lipids act as extracellular mediators activating specific▶G-protein-coupled receptors (GPCRs), although someof them additionally play a role in intracellular signaltransduction. ▶Sphingosine-1-phosphate (S1P) and▶lysophosphatidic acid (LPA) have been characterizedin greatest detail so far, and the concept of lysopho-spholipids as a group of mediators is supported by thehigh homology between S1P- andLPA-specific GPCRs.Other, less well characterized lysophospholipids aresphingosylphosphorylcholine (SPC), lysophosphatidyl-choline (LPC), psychosine (galactosylsphingosine) andglucopsychosine (glucosylsphingosine); the latter twoare lacking a phosphate group. Lysophospholipids areauto- or paracrine regulators of cell growth and survival,migration and chemotaxis, cytoskeletal architecture,cell–cell-contacts and adhesion, Ca2+ homoeostasis andCa2+-dependent functions. By regulating these cellularfunctions, lysophospholipids play a role in angiogene-sis, lymphocyte trafficking, development of the nervoussystem, cancer growth andmetastasis, inflammation andarteriosclerosis. Relatives of the lysophospholipidfamily are platelet-activating factor and the ▶endocan-nabinoids, arachidonyl ethanolamide and 2-arachidonylglycerol.

Basic CharacteristicsMetabolism and OccurrenceS1P is formed from sphingosine by ▶sphingosinekinases (SphKs). Degradation of S1P occurs eitherreversibly by lipid phosphate phosphohydrolases(LPPs) and S1P phosphatases (SPPs), or irreversiblyby S1P lyase (SPL) (Fig. 1). The localization of S1Pproduction is highly important since S1P plays a roleboth as extracellular mediator and as intracellular

Lysophospholip

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Lysophospholipids 711

L

712 Lysophospholipids

second messenger (Fig. 1). Intracellular formation ofS1P by SphKs is regulated by many diverse stimuli,among them GPCR agonists, ▶growth factors, ▶cyto-kines or depolarization. Some stimuli induce atranslocation of SphK1 from the cytosol to the plasmamembrane, which appears to enable S1P secretion andso-called “inside-out signalling”. Many cell typesincluding mast cells and tumour cells can release S1P.ATP-binding cassette (ABC) transporters have shownto be involved in this process. S1P is furthermore storedin platelets and released upon platelet activation.Accordingly, it has been found in serum in higherconcentrations (�0.5–0.8 μM) than in plasma (�0.2–0.4 μM). Plasma S1P is bound to albumin and▶lipoproteins, mainly high-density lipoproteins(HDL). Tissue S1P levels are significantly lower, andthus there is a plasma/tissue S1P gradient.

LPA, i.e. monoacyl-glycerol-3-phosphate, can beformed and degraded by multiple metabolic pathways(Fig. 1). Depending on the precursor molecule andrespective pathway, the fatty acid chain in LPA differs inlength, degree of saturation and position (sn-1 or sn-2),which has an influence on biological activity. LPA

Lysophospholipids. Table 1 G-protein-coupled lysophosph

+Receptor Endogenousligands

Main tissuedistribution

Signallin

S1P1 S1P (nM)SPC (μM)

Ubiquitous Gi/o

S1P2 Ubiquitous Gi/o, Gq,G12/13

S1P3 Ubiquitous Gi/o, Gq,G12/13

S1P4 Lymphoid andhaematopoietic tissue

Gi/o, G12

S1P5 Brain, white mattertracts

Gi/o, G12

LPA1 LPA Ubiquitous Gi/o, Gq,G12/13

LPA2 Ubiquitous Gi/o, Gq,G12/13

LPA3 Ubiquitous Gi/o, Gq

Receptors Activators

P2Y9/GPR23(LPA4)

LPA

GPR63 (LPA5) Stress fibre forma

GPR3, GPR6,GPR12

High constitutiveactivity S1P or SPC

Meiotic arrest of roneurons

OGR1 SPC Proton Inhibition of prolife

GPR4 SPC, LPC Migration, angioge

G2A LPC, SPC Migration, apoptos

TDAG8 Psychosine Formation of mult

analogs with ether-bound alkyl or alkenyl chains arequantitatively less abundant than those with ester-bound acyl chains. Extracellular LPA is generated to alarge part by a lysophospholipase D named▶autotaxin.Autotaxin is an extracellular enzyme that acts asautocrine motility factor of tumour cells. LPA produc-tion by autotaxin is essential for embryonic develop-ment in mice and apparently contributes most of LPAin plasma. Alternatively, LPA can be produced fromsurface-exposed phosphatidic acid by phosphatidicacid-selective PLA1 or secretory type-II PLA2. LikeS1P, LPA occurs in plasma (�200 nM), is formedduring coagulation, and present in serum in micromolarconcentrations. Differences in the fatty acid composi-tion of plasma- and serum-LPA suggest that the sourcesof these LPA pools were different. LPA is producedby many cell types, e.g. fibroblasts, adipocytes andtumour cells.

Lysophospholipid ReceptorsAn overview of lysophospholipid GPCR is presentedin Table 1. Presently best characterized are the recep-tors of the endothelial differentiation gene (EDG)

olipid receptors

g Main biological effects

Migration ↑, proliferation, survival, cell–cell-contacts,angiogenesis, lymphocyte trafficking

Migration ↓, vascular development, differentiation ofvascular smooth muscle cells

Heart rate ↓, vascular development, NO-dependentvasorelaxation

/13 Proliferation and cytokine secretion in T-cells ↓

/13 Proliferation, cell rounding

Proliferation, survival, neurite retraction, brain devel-opment

Proliferation, survival

Implantation and embryo spacing in mice

Main biological effects

tion, neurite retraction

dent oocytes, increase in synaptic contacts in rat cortical

ration, osteoclastogenesis

nesis, impairment of endothelial barrier function

is, suppression of autoimmunity

inuclear cells, apoptosis

Lysophospholipids 713

L

family, S1P1–5 and LPA1–3. They had originally beennamed after EDG-1, now S1P1, which was detected in1990 as an orphan GPCR upregulated during thedifferentiation of endothelial cells. According to anIUPHAR committee, these receptors are now namedafter the primary natural ligand and numbered in orderof identification. S1P1–3 and LPA1–3 are widelyexpressed and often found co-expressed within a singlecell type. S1P4 is predominantly expressed in lymphaticand haematopoietic tissues and S1P5 in brain whitematter and skin. The EDG family receptors couple viaGi/o, Gq/11 and G12/13 proteins to inhibition of▶adenylyl cyclase, stimulation of phospholipase Cand increase in [Ca2+]i, stimulation or inhibition of▶mitogen-activated protein kinases (ERK, JNK andp38) and cell growth, activation of Akt and survival,activation or inhibition of Rho and Rac and rearrange-ment of the cytoskeleton, and finally stimulation orinhibition of cell migration (for details, see BiologicalActions).

Much less is known about other lysophospholipidreceptors. LPA4 and LPA5 couple via Gs to activationof adenylyl cyclase. GPR3, GPR6 and GPR12 have ahigh constitutive activity, thereby stimulating adenylylcyclase, but appear to be further activated by S1P and/or SPC. GPR6 and GPR12 are strongly expressed inmouse brain, and SPC increased synaptic contacts inGPR12-expressing embryonic cortical neurons. GPR3and GPR12 are expressed in mouse oocytes andmediate a signal that maintains the oocytes in meioticarrest, a process that is dependent on high cAMPlevels. A role for lipid ligands in meiotic arrest is likely

Lysophospholipids. Figure 2 Functional roles of S1P an

since spontaneous in vitromaturation of rodent oocyteswas delayed by SPC and S1P.

Another group of putative lysophospholipid GPCR ishighly controversial. OGR1, GPR4, G2A and TDAG8form a cluster of homologous GPCRs that are candidatehigh-affinity receptors for SPC, LPC and psychosine(Table 1). SPC and LPC are the phosphocholine-containing analogs of S1P and LPA, respectively. Bothlipids are normal constituents of plasma and serum.SPC acts also as a low-affinity or partial agonist atS1P1–5. Contradictory studies have shown that thereceptors of the OGR1 cluster can be regulated bylysophospholipids and/or by protons. Recent studiesrevealed that OGR1 was strongly induced duringdifferentiation of bone marrow mononuclear cells intoosteoclasts, and knockdown of OGR1 attenuatedosteoclastogenesis. Endogenous GPR4 was requiredfor SPC-stimulated migration of endothelial cells andendothelial tube formation. G2A and TDAG8 appear toplay a functional role in migration and apoptosis ofimmune cells.

Finally, it has to be mentioned that LPA also has anintracellular target site, which is the nuclear transcrip-tion factor, peroxisome proliferator-activated receptor-γ(PPARγ). LPA competes for thiazolidinedione bindingand activates PPARγ-dependent gene transcription.Thereby, LPA induced neointima formation in a ratcarotid artery model.

Biological ActionsCellular responses to S1P and LPA can be classifiedas growth-related (stimulation of cell proliferation and

d LPA.

714 Lysophospholipids

survival, protection from apoptosis), cytoskeleton-dependent (shape change, migration, adhesion, che-motaxis) and Ca2+-dependent (contraction, secretion).Some cellular responses are regulated in an oppositemanner by different receptors. For example, the S1P1receptor mediates cell migration and chemotaxis,thereby regulating for example angiogenesis andlymphocyte trafficking. Growth factors such as plate-let-derived growth factor need the S1P1 receptor forsignalling migration. On the contrary, this must be “andinhibits migration”. S1P2 promotes cellular stress fibreformation and inhibits migration. Furthermore, theS1P1 receptor does not increase [Ca2+]i but ratherinhibits [Ca2+]i increases by other agonists, while S1P2and S1P3 mediate [Ca2+]i increases in response to S1P.

S1P is an important mediator in the cardiovascularsystem (Fig. 2). S1P stimulates proliferation, survivalandmigration of endothelial cells and induces formationof adherens junction assembly, decrease in vascularpermeability and differentiation of endothelial cells intocapillary-like networks. Expression of the S1P1 receptorwithin endothelial cells is required for vasculogensis.Mice lacking this receptor die during embryonicdevelopment from haemorrhages caused by a failureof pericytes to migrate around newly formed capillaries.S1P andLPA induce smoothmuscle cell contraction andcan cause vasoconstriction. S1P, via endothelial S1P3receptors, can also cause NO-dependent vasorelaxation.A lack of the S1P2 receptor results in profound deafnessin mice, apparently because the cochlear spiral arterydoes not appropriately contract in the absence of S1P2.The S1P3 receptor mediates IK(ACh) activation in atrialmyocytes, leading to bradycardia. However, systemiccardiovascular parameters such as blood pressure orheart rate are marginally affected by intravenous S1P,consistent with a role of S1P as a local rather thansystemic mediator. The importance of LPA in vasculardevelopment is illustrated by the phenotype of autotaxindeficiency, which is characterized by vascular defectsearly in development. LPA promotes surface expressionof leukocyte adhesion molecules in endothelial cells. Incontrast to S1P, LPA enhances vascular permeability. Ofthe two platelet-derived lysophospholipids, LPA but notS1P stimulates platelet aggregation. S1P, LPA and otherlysolipids have been detected in lipoproteins and play arole in atherosclerosis. The major part of lipoprotein-bound S1P is associated with HDL, and many of HDL’sbeneficial vascular effects are mediated by S1P andS1P-GPCR. In contrast, LPA is produced duringmild oxidation of low-density lipoproteins (LDL) andaccumulates in atherosclerotic plaques where it actsproinflammatory and promotes thrombus formation.

The S1P1 receptor and the gradient between lym-phatic tissue (low S1P levels) and plasma (highS1P levels) are essential for lymphocyte traffick-ing. Deletion of S1P1, as well as pharmacological

downregulation of S1P1 (see Drugs), inhibit lympho-cyte emigration from lymphatic tissues. Inhibition ofS1P lyase leads to elevated S1P tissue levels and also tolymphopenia. It appears likely that the S1P1 receptor onthe lymphocyte surface senses the S1P gradient andmediates lymphocyte trafficking.LPA receptors play an important role in the nervous

system (Fig. 2). The LPA1 receptor was originallycloned from cerebral cortical neuroblasts and is highlyexpressed in the neurogenic ventricular zone of theembryonic cerebral cortex. In the adult nervous system,it is predominantly found in myelinating cells, i.e.oligodendrocytes and Schwann cells. Schwann cellmorphology, adhesion and survival are regulated byLPA. LPA1 and LPA2 receptors mediate neuronal cellrounding and neurite retraction, while LPA3 appears tostimulate neurite outgrowth. Deletion of the LPA1

receptor in mice caused �50% neonatal lethality. Thesurviving pups had a reduced size, craniofacial dy-smorphism and impaired suckling behaviour. The defectin suckling behaviour caused malnutrition of the pups,leading to growth retardation and death, and wasattributed to a defective olfaction. Deletion of LPA2 inmice did not lead to obvious defects, while LPA3

deficiency was accompanied by �50% reduced littersizes. This was caused by downregulation of cyclooxy-genase-2 (▶cyclooxygenases) in LPA3-deficient uteriduring preimplantation, which delayed implantationand disturbed embryo spacing. The mice that were bornwere grossly normal.Both S1P and LPA stimulate proliferation and

migration of fibroblasts, while they inhibit proliferationand induce migration and differentiation of keratino-cytes. LPA promotes wound healing in vivo. Thus, S1Pand LPA interact in tissue repair. Many other tissues areresponsive to S1P or LPA, including the respiratorytract (contraction of airway smooth muscle cells andcytokine release by S1P), kidney (diuresis by S1P),adipose tissue (preadipocyte proliferation by LPA), andothers. Finally, S1P and LPA promote tumour cellgrowth, chemoresistance and metastasis (Fig. 2). Thetwo mediators are produced by several tumour cells andoccur for example in malignant ascites. The antiapop-totic action of S1P and LPA may protect the cells fromchemotherapy or radiation. Furthermore, S1P and LPAstimulate tumour cell motility and invasiveness,although it has to be noted that motility of certaincancer cells (e.g. melanoma cells) is inhibited by S1P.S1P might also play a role in tumour angiogenesis. Itsimportance for tumour progression is further illustratedby the antitumour activity of SphK inhibitors and ananti-S1P antibody (see Drugs).In summary, the lysophospholipids are local media-

tors that regulate development, tissue regeneration andhomoeostasis, but also play a role in inflammation,arteriosclerosis and cancer.

Lysophospholipids 715

L

DrugsIn the field of S1P pharmacology, there is currently anovel immunosuppressive (▶immunosuppressiveagents) in clinical development for multiple sclerosis.FTY720 is a prodrug that, after being phosphorylated,acts as an agonist with low nanomolar affinity on S1P1,S1P3, S1P4 and S1P5, but not on S1P2 receptors. SphK2is required for effective phosphorylation of FTY720in vivo. The immunosuppressive action of FTY720 iscaused basically by activation and subsequent internali-zation of the S1P1 receptor, which needs to be expressedon the lymphocyte surface to enable sensing of theplasma/tissue S1P gradient and to promote emigrationof lymphocytes from lymphatic tissues. Since FTY720does not generally impair T- and B-cell proliferationand functions, it presents a novel mode of immunosup-pressive action, which might be useful in multiplesclerosis as well as transplantation or autoimmunediabetes. Activation of S1P receptor subtypes otherthan S1P1 causes further, desired and undesired effects.For example, the bradycardia that occurs during thefirst days of FTY720 treatment is mediated by activa-tion of S1P3. Animal models show further effects ofFTY720: it reduced the damage caused by ischemia-reperfusion in liver and kidney, inhibited angiogenesis,impeded tumour growth in amousemodel of melanoma,induced endothelial NO synthase activation and vaso-dilatation (via S1P3) and reduced vascular permeabilityin vivo.

Other compounds acting on S1P-GPCR are only in theexperimental stage. Several specific S1P1 agonists havebeen described. SEW2871, that has been identified byhigh-throughput screening and lacks a phosphate group,induces lymphopenia in mice (in agreement with the roleof S1P1 in lymphocyte trafficking) but no bradycardia (inagreement with a lack of an effect on S1P3). KRP-203needs to be phosphorylated, like FTY720, but thenactivates specifically S1P1 and produces effects similar tothose of SEW2871. Interestingly, the S1P1 receptor has ahigh constitutive activity which was reduced by aninverse agonist for S1P1 (SB649146). A recentlydescribed S1P1 antagonist did not induce lymphopeniabut reversed immunosuppression induced by S1P1agonist, and furthermore enhanced capillary leakage. Aputative S1P3 receptor antagonist, BML-241, wasidentified by searching a three-dimensional compounddatabase with a pharmacophore model of S1P, however,BML-241 has appears to have other, GPCR-independenteffects.A specific S1P2 receptor antagonist, JTE-013, haswidely been used to analyse S1P2 signalling pathways.This compound prevented for example S1P2-mediatedinhibition of cell migration and contraction of smoothmuscle cells. Recently, a series of aryl amide compoundswas presented which were more or less receptor subtype-selective. The lead compound, VPC23019, was acompetitive antagonist at S1P1 and S1P3. Ki values for

VPC23019 in radioligand binding assays at S1P1 andS1P3 were in the low nanomolar range, and VPC23019inhibited S1P-induced migration and Ca2+ mobilization.Small structural changes converted the molecule into anagonist. Furthermore, VPC23019 as all compounds ofthis series behaved as agonist at S1P4 and S1P5, but hadno activity at S1P2. Recently, an anti-S1P antibody wasdescribed. This antibody retarded the proliferation ofvarious transplanted mouse tumours. Furthermore, itreduced tumour-associated angiogenesis and the abilityof S1P to protect tumour cells from apoptosis. Anantitumour activity was also observed with SphKinhibitors.

LPA–GPCR pharmacology is at the experimentalstage.Dioctylglycerol pyrophosphate (DGPP8:0) acts asa competitive antagonist preferentially at human LPA3

receptors and inhibits at higher concentrations LPA1, butnot LPA2. DGPP 8:0 specifically inhibited LPA-inducedplatelet activation. Furthermore, it blocked plateletactivation by mildly oxidized low density lipoproteinsand by homogenates of lipid-rich core isolated from softarteriosclerotic plaques, illustrating the importance ofLPA as a component of arteriosclerotic plaques. Fattyalcohol phosphates (FAP) with carbon chain lengthsbetween 10 and 14 activate LPA2 and inhibit LPA3 in acompetitive manner. Another LPA–GPCR antagonist isKi16425, which was identified by high-throughputscreening of 150,000 compounds for inhibition ofLPA-induced [Ca2+]i increases. This compound inhib-ited LPA1 andLPA3 at slightly lower concentrations thanLPA2, but was highly specific for LPA-GPCR. A similarpreference for LPA1 and LPA3 was observed withVPC12249. O-methylphosphothionate (OMPT), aLPA3-selective agonist, aggravated ischemia-reperfu-sion injury inmouse kidney,while theLPA1/3 antagonist,VPC12249, reduced the kidney damage. Since mostcompounds that interact with LPA–GPCRs, exceptKi16425, have a phosphate group that can be subject todephosphorylation, a novel strategy focuses onmetabol-ically stabilized LPA analogues such as phosphonates,phosphorothioates, phosphonothioates and fluoropho-sphonates. These compounds affect LPA–GPCRs aswell as LPA metabolizing enzymes.

References1. Brinkmann V, Cyster JG, Hla T (2004) FTY720:

sphingosine 1-phosphate receptor-1 in the control oflymphocyte egress and endothelial barrier function. AmJ Transplant 4:1019–1025

2. Ishii I, Fukushima N, Ye X et al (2004) Lysophospholipidreceptors: signaling and biology. Annu Rev Biochem73:321–354

3. Meyer zu Heringdorf D, Jakobs KH (2007) Lysopho-spholipid receptors: signalling, pharmacology and regula-tion by lysophospholipid metabolism. Biochim BiophysActa 1768:923–940

716 Lysosphingolipids and Lysoglycerophospholipids

4. Moolenaar WH, Meeteren LA, van Giepmans BN (2004)The ins and outs of lysophosphatidic acid signaling.Bioessays 26:870–881

5. Parrill AL, Sardar VM, Yuan H (2004) Sphingosine1-phosphate and lysophosphatidic acid receptors: agonistand antagonist binding and progress toward developmentof receptor-specific ligands. Semin Cell Dev Biol 15:467–476

Lysosphingolipidsand Lysoglycerophospholipids

▶Lysophospholipids