The signal peptide anchors apolipoprotein M in plasma lipoproteins and prevents rapid clearance of apolipoprotein M from plasma

Lipoproteins consist of lipids solubilized by apolipoproteins. The lipid-binding structural motifs of apolipoproteins include amphipathic alpha-helixes and beta-sheets. Plasma apolipoprotein (apo) M lacks an external amphipathic motif but, nevertheless, is exclusively associated with lipoproteins (mainly high density lipoprotein). Uniquely, however, apoM is secreted to plasma without cleavage of its hydrophobic NH(2)-terminal signal peptide. To test whether the signal peptide serves as a lipoprotein anchor for apoM in plasma, we generated mice expressing a mutated apoM(Q22A) cDNA in the liver (apoM(Q22A)-Tg mice (transgenic mice)) and compared them with mice expressing wild-type human apoM (apoM-Tg mice). The substitution of the amino acid glutamine 22 with alanine in apoM(Q22A) results in secretion of human apoM without a signal peptide. The human apoM mRNA level in liver and the amount of human apoM protein secretion from hepatocytes were similar in apoM-Tg and apoM(Q22A)-Tg mice. Nevertheless, human apoM was not detectable in plasma of apoM(Q22A)-Tg mice, whereas it was easily measured in the apoM-Tg mice. To examine the plasma metabolism, recombinant apoM lacking the signal peptide was produced in Escherichia coli and injected into wild-type mice. The apoM without signal peptide did not associate with lipoproteins and was rapidly cleared in the kidney. Accordingly, ligation of the kidney arteries in apoM(Q22A)-Tg mice resulted in rapid accumulation of human apoM in plasma. The data suggest that hydrophobic signal peptide sequences, if preserved upon secretion, can anchor plasma proteins in lipoproteins. In the case of apoM, this mechanism prevents rapid loss by filtration in the kidney.     

Uncleaved ApoM Signal Peptide Is Required for Formation of Large ApoM/Sphingosine 1-Phosphate (S1P)-enriched HDL Particles

Apolipoprotein M (apoM), a plasma sphingosine 1-phosphate (S1P) carrier, associates with plasma HDL via its uncleaved signal peptide. Hepatocyte-specific apoM overexpression in mice stimulates formation of both larger nascent HDL in hepatocytes and larger mature apoM/S1P-enriched HDL particles in plasma by enhancing hepatic S1P synthesis and secretion. Mutagenesis of apoM glutamine 22 to alanine (apoM^Q22A) introduces a functional signal peptidase cleavage site. Expression of apoM^Q22A in ABCA1-expressing HEK293 cells resulted in the formation of smaller nascent HDL particles compared with wild type apoM (apoM^WT). When apoM^Q22A was expressed in vivo, using recombinant adenoviruses, smaller plasma HDL particles and decreased plasma S1P and apoM were observed relative to expression of apoM^WT. Hepatocytes isolated from both apoM^WT- and apoM^Q22A-expressing mice displayed an equivalent increase in cellular levels of S1P, relative to LacZ controls; however, relative to apoM^WT, apoM^Q22A hepatocytes displayed more rapid apoM and S1P secretion but minimal apoM^Q22A bound to nascent lipoproteins. Pharmacologic inhibition of ceramide synthesis increased cellular sphingosine and S1P but not medium S1P in both apoM^WT and apoM^Q22A hepatocytes. We conclude that apoM secretion is rate-limiting for hepatocyte S1P secretion and that its uncleaved signal peptide delays apoM trafficking out of the cell, promoting formation of larger nascent apoM- and S1P-enriched HDL particles that are probably precursors of larger apoM/S1P-enriched plasma HDL.     

A novel human apolipoprotein (apoM).

A novel human apolipoprotein designated apolipoprotein M (apoM) is described. The unique N-terminal amino acid sequence of apoM was found in an approximately 26-kDa protein present in a protein extract of triglyceride-rich lipoproteins (TGRLP). The isolated apoM cDNA (734 base pairs) encoded a 188-amino acid residue-long protein, distantly related to the lipocalin family. The mRNA of apoM was detected in the liver and kidney. Western blotting demonstrated apoM to be present in high density lipoprotein (HDL) and to a lesser extent in TGRLP and low density lipoproteins (LDL). The first 20 amino acid residues of apoM constituted a hydrophobic segment with characteristic features of a signal peptide. This was retained in the mature protein because of the lack of a signal peptidase cleavage site. In vitro translation in the presence of microsomes demonstrated translocation of apoM over the membrane and glycosylation but no signal peptide cleavage. The in vitro translated product remained associated with the microsomes after treatment with carbonate at pH 11, demonstrating that apoM is an integral protein. This finding suggests that apoM is linked to the single phospholipid layer of lipoproteins with a hydrophobic signal anchor. In conclusion, a novel human apolipoprotein, the function of which remains to be determined, is described.     

Characterization of apoM in normal and genetically modified mice

A novel human apolipoprotein [apolipoprotein M (apoM)] was recently described and demonstrated to be a lipocalin. We have now examined apoM in wild-type mice and mice with genetically altered lipoprotein metabolism. Liver and kidney showed high mRNA expression, whereas spleen, heart, brain, and testis demonstrated low expression. ApoM gene expression was initiated on embryonic day 10. Western blot analysis of plasma suggested that mouse apoM, like its human counterpart, is secreted with a retained signal peptide, but unlike human apoM it is not glycosylated. Gel filtration of plasma showed apoM to be associated with HDL-sized particles in wild-type and apoA-I-deficient mice and with HDL- and LDL-sized particles in LDL receptor-deficient mice, whereas apoM was mainly found in VLDL-sized particles in high-fat, high-cholesterol-fed apoE-deficient mice. The plasma concentration of apoM was similar in wild-type, LDL receptor-deficient, and apoE-deficient mice but was reduced to 33% in apoA-I-deficient compared with wild-type mice (P = 0.007). These data suggest that apoM mainly associates with HDL in normal mice but also with the pathologically increased lipoprotein fraction in genetically modified mice. The substantially decreased apoM levels in apoA-I-deficient mice suggest a connection between apoM and apoA-I metabolism.     

Hydrophobic ligand binding properties of the human lipocalin apolipoprotein M

Apolipoprotein M (apoM) is a plasma protein associated mainly with HDL. ApoM is suggested to be important for the formation of prebeta-HDL, but its mechanism of action is unknown. Homology modeling has suggested apoM to be a lipocalin. Lipocalins share a structurally conserved beta-barrel, which in many lipocalins bind hydrophobic ligands. The aim of this study was to test the ability of apoM to bind different hydrophobic substances. ApoM was produced both in Escherichia coli and in HEK 293 cells. Characterization of both variants with electrophoretic and immunological methods suggested apoM from E. coli to be correctly folded. Intrinsic tryptophan fluorescence of both apoM variants revealed that retinol, all-trans-retinoic acid, and 9-cis-retinoic acid bound (dissociation constant = 2-3 microM), whereas other tested substances (e.g., cholesterol, vitamin K, and arachidonic acid) did not. The intrinsic fluorescence of two apoM mutants carrying single tryptophans was quenched by retinol and retinoic acid to the same extent as wild-type apoM, indicating that the environment of both tryptophans was affected by the binding. In conclusion, the binding of retinol and retinoic acid supports the hypothesis that apoM is a lipocalin. The physiological relevance of this binding has yet to be elucidated.     

Molecular architecture of acetylcholinesterase collagen-tailed forms; construction of a glycolipid-tailed tetramer.

Asymmetric forms of Torpedo acetylcholinesterase (AChE) are produced in COS cells by the simultaneous expression of collagenic subunits (Q) and catalytic T subunits (AChET). Truncated AChET delta subunits, from which most of the C-terminal peptide (TC) had been deleted by mutagenesis, did not associate with Q subunits. The TC peptide is therefore necessary for the association of the AChET and Q subunits. In order to determine the orientation of the Q subunit in the collagen-tailed forms, we have developed an antiserum against its non-collagenic C-terminal domain, expressed as a fusion protein in Escherichia coli. This antiserum, which recognized the Q subunit in Western blots, was found to react with intact asymmetric forms, but not with collagenase-treated forms, from which the distal part of the tail had been cleaved, suggesting that the N-terminal non-collogenic domain (QN) is responsible for the interaction with the AChET subunits. This was confirmed by creating a chimeric subunit (QN/HC), in which QN was linked to the C-terminal peptide of the H subunit of Torpedo AChE, which contains the glycophosphatidylinositol (GPI) cleavage/attachment signal: co-expression of AChET and QN/NC produced GPI-anchored tetramers, which were sensitive to PI-PLC and largely exposed to the external surface of the cells. We thus demonstrate that: (i) the HC peptide is sufficient to determine the addition of a glycolipid anchor and (ii) the QN domain is sufficient to bind a catalytic AChET tetramer by interacting with the TC peptide.     

Charge states rather than propensity for beta-structure determine enhanced fibrillogenesis in wild-type Alzheimer's beta-amyloid peptide compared to E22Q Dutch mutant

The activity of the Alzheimer's amyloid beta-peptide is a sensitive function of the peptide's sequence. Increased fibril elongation rate of the E22Q Dutch mutant of the Alzheimer's amyloid beta-peptide relative to that of the wild-type peptide has been observed. The increased activity has been attributed to a larger propensity for the formation of beta structure in the monomeric E22Q mutant peptide in solution relative to the WT peptide. That hypothesis is tested using four nanosecond timescale simulations of the WT and Dutch mutant forms of the Abeta(10-35)-peptide in aqueous solution. The simulation results indicate that the propensity for formation of beta-structure is no greater in the E22Q mutant peptide than in the WT peptide. A significant measure of "flickering" of helical structure in the central hydrophobic cluster region of both the WT and mutant peptides is observed. The simulation results argue against the hypothesis that the Dutch mutation leads to a higher probability of formation of beta-structure in the monomeric peptide in aqueous solution. We propose that the greater stability of the solvated WT peptide relative to the E22Q mutant peptide leads to decreased fibril elongation rate in the former. Stability difference is due to the differing charge state of the two peptides. The other proposal leads to the prediction that the fibril elongation rates for the WT and the mutant E22Q should be similar under acid conditions.     

载脂蛋白M

载脂蛋白M(apoM)是一类在血液中主要与高密度脂蛋白（HDL）结合的载脂蛋白,呈组织特异性表达且有着众多生物学功能.体内外多种因素可从转录或转录后水平对其表达进行调控:肝细胞核因子-1α,4α（HNF-1α,4α）、肝受体同系物-1（LRH-1）、叉头框转录因子a2（Foxa2）、血小板活化因子（PAF）等可上调其表达;肝X受体（LXR）、维甲酸X受体（RXR）、法尼酯X受体（FXR）、小异源二聚体-1(SHP-1)以及绝大多数细胞因子可下调其表达,具体调节机制复杂. 结构上,apoM含有一个特征性的疏水性信号肽,可结合1 磷酸鞘氨醇（S1P）等小的生物活性脂,以此介导多项生命活动. 功能上,apoM能促进preβ-HDL的生成,并提高其一系列抗动脉粥样硬化的生物活性,如胆固醇逆向转运、抗炎、以及低密度脂蛋白（LDL）的抗氧化等.在一些糖尿病病人体内,apoM的含量也显著降低,而apoM含量的提高可以降低血糖含量,增加胰岛素分泌以及改善胰岛素抵抗,不少学者将其视为该病发生发展的一项预测指标.本文就近年来对apoM的生物学特性,特别是其表达调控机制和功能的研究进展进行综述.     

Factor IXa:factor VIIIa interaction. helix 330-338 of factor ixa interacts with residues 558-565 and spatially adjacent regions of the a2 subunit of factor VIIIa

The physiologic activator of factor X consists of a complex of factor IXa, factor VIIIa, Ca(2+) and a suitable phospholipid surface. In one study, helix 330 (162 in chymotrypsin) of the protease domain of factor IXa was implicated in binding to factor VIIIa. In another study, residues 558-565 of the A2 subunit of factor VIIIa were implicated in binding to factor IXa. We now provide data, which indicate that the helix 330 of factor IXa interacts with the 558-565 region of the A2 subunit. Thus, the ability of the isolated A2 subunit was severely impaired in potentiating factor X activation by IXa(R333Q) and by a helix replacement mutant (IXa(helixVII) in which helix 330-338 is replaced by that of factor VII) but it was normal for an epidermal growth factor 1 replacement mutant (IXa(PCEGF1) in which epidermal growth factor 1 domain is replaced by that of protein C). Further, affinity of each 5-dimethylaminonaphthalene-1-sulfonyl (dansyl)-Glu-Gly-Arg-IXa (dEGR-IXa) with the A2 subunit was determined from its ability to inhibit wild-type IXa in the tenase assay and from the changes in dansyl fluorescence emission signal upon its binding to the A2 subunit. Apparent K(d(A2)) values are: dEGR-IXa(WT) or dEGR-IXa(PCEGF1) approximately 100 nm, dEGR-IXa(R333Q) approximately 1.8 micrometer, and dEGR-IXa(helixVII) >10 micrometer. In additional experiments, we measured the affinities of these factor IXa molecules for a peptide comprising residues 558-565 of the A2 subunit. Apparent K(d(peptide)) values are: dEGR-IXa(WT) or dEGR-IXa(PCEGF1) approximately 4 micrometer, and dEGR-IXa(R333Q) approximately 62 micrometer. Thus as compared with the wild-type or PCEGF1 mutant, the affinity of the R333Q mutant for the A2 subunit or the A2 558-565 peptide is similarly reduced. These data support a conclusion that the helix 330 of factor IXa interacts with the A2 558-565 sequence. This information was used to model the interface between the IXa protease domain and the A2 subunit, which is also provided herein.     

Mutational alterations affecting the export competence of a truncated but fully functional maltose-binding protein signal peptide.

The wild-type maltose-binding protein (MBP) signal peptide is 26 amino acids in length. A mutationally altered MBP signal peptide has been previously described that is missing one of the basic residues from the hydrophilic segment and seven residues from the hydrophobic core; however, it still facilitates MBP secretion to the periplasm at a rate and efficiency comparable to those of the wild-type structure. Thus, this truncated signal peptide (designated the R2 signal peptide) must retain all of the essential features required for proper export function. In this study, alterations were obtained in the R2 signal peptide that resulted in an export-defective MBP. For the first time, signal sequence mutations were obtained that resulted in the synthesis of a totally export-defective MBP. As was previously the case for the wild-type signal peptide, the introduction of either charged residues or helix-breaking proline residues adversely affected export function. Despite these similarities, the position of these alterations within the R2 signal peptide, their relative effects on MBP secretion and processing, and an analysis of the ability of various extragenic prl mutations to suppress the secretion defects provide additional insight into the minimal requirements for a functional MBP signal peptide.     

Mapping the site(s) of MgATP and MgADP interaction with the nitrogenase of Azotobacter vinelandii. Lysine 15 of the iron protein plays a major role in MgATP interaction.

Nitrogenase binds and hydrolyzes 2MgATP yielding 2MgADP and 2Pi for each electron that is transferred from the iron protein to the MoFe protein. The iron protein alone binds but does not hydrolyze 2MgATP or 2MgADP and the binding of these nucleotides is competitive. Iron protein amino acid sequences all contain a putatitive mononucleotide-binding region similar to a region found in other mononucleotide-binding proteins. To examine the role of this region in MgATP interaction, we have substituted glutamine and proline for conserved lysine 15. The amino acid substitutions, K15Q and K15P, both yielded a non-N2-fixing phenotype when the genes coding for them were substituted into the Azotobacter vinelandii chromosome in place of the wild-type gene. The iron protein from the K15Q mutant was purified to homogeneity, whereas the protein from the K15P mutant could not be purified in its native form. Unlike wild-type iron protein, the purified K15Q iron protein showed no acetylene reduction, H2 evolution, or ATP hydrolysis activities when complemented with wild-type MoFe protein. The K15Q iron protein and the normal iron protein had a similar total iron content and both proteins showed the characteristic rhombic EPR signal resulting from the reduced state of the single 4Fe-4S cluster bridging the two subunits. Unlike the wild-type iron protein, addition of MgATP to the K15Q iron protein did not result in the perturbation necessary to change the EPR signal of its 4Fe-4S center from a rhombic to an axial line shape. Also unlike the wild-type iron protein, addition of MgATP to K15Q iron protein in the presence of the iron chelator, alpha,alpha'-dipyridyl, did not result in a time-dependent transfer of iron to the chelator. Thus, even though the K15Q iron protein contains a normal 4Fe-4S center, it does not respond to MgATP like the wild-type protein. Examination of the ability of the K15Q iron protein to bind MgADP showed no change from the wild-type iron protein, but its ability to bind MgATP decreased to 35% of the wild-type protein. Thus, in A. vinelandii iron protein, lysine 15 is not needed for interaction with MgADP but is involved in the binding of ATP, presumably through charge-charge interaction with the gamma-phosphate. Based on the above data, this lysine appears to be essential for the MgATP induced conformational change of wild-type iron protein that is required for activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Serendipitous Fatty Acid Binding Reveals the Structural Determinants for Ligand Recognition in Apolipoprotein M

Apolipoprotein M (ApoM) is a 25-kDa HDL-associated apolipoprotein and a member of the lipocalin family of proteins. Mature apoM retains its signal peptide, which serves as a lipid anchor attaching apoM to the lipoproteins, thereby keeping it in the circulation. Studies in mice have suggested apoM to be antiatherogenic, but its physiological function is yet unknown. We have now determined the 1.95 Å resolution crystal structure of recombinant human apoM expressed in Escherichia coli and made the unexpected discovery that apoM, although refolded from inclusion bodies, was in complex with fatty acids containing 14, 16 or 18 carbon atoms. ApoM displays the typical lipocalin fold characterised by an eight-stranded antiparallel β-barrel that encloses an internal ligand-binding pocket. The crystal structures of two different complexes provide a detailed picture of the ligand-binding determinants of apoM. Additional fatty acid- and lipid-binding studies with apoM and the mutants apoM^W47F and apoM^W100F showed that sphingosine-1-phosphate is able to displace the bound fatty acids and efficiently quenched the intrinsic fluorescence with an IC₅₀ of 0.90 μM. Whereas the fatty acids bound in the crystal structure could be a mere consequence of recombinant protein production, the observed binding of sphingosine-1-phosphate might provide a key to a better understanding of the physiological function of apoM.     

Effect of OmpA signal peptide mutations on OmpA secretion, synthesis, and assembly.

In previous investigations, we have examined the effect of OmpA signal peptide mutations on the secretion of the two heterologous proteins TEM beta-lactamase and nuclease A. During these studies, we observed that a given signal peptide mutation could affect differentially the processing of precursor OmpA-nuclease or precursor OmpA-lactamase. This observation led us to further investigate the influence of the mature region of a precursor protein on protein export. Preexisting OmpA signal peptide mutations of known secretion phenotype when directing heterologous protein export (nuclease A or beta-lactamase) were fused to the homologous mature OmpA protein. Four signal peptide mutations that have previously been shown to prevent export of nuclease A and beta-lactamase were found to support OmpA protein export, albeit at reduced rates. This remarkable retention of export activity by severely defective precursor OmpA signal peptide mutants may be due to the ability of mature OmpA to interact with the cytoplasmic membrane. In addition, these same signal peptide mutations can affect the level of OmpA synthesis as well as its proper assembly in the outer membrane of Escherichia coli. Two signal peptide mutations dramatically stimulate the rate of precursor OmpA synthesis three- to fivefold above the level observed when a wild-type signal peptide is directing export. The complete removal of the OmpA signal peptide does not result in increased OmpA synthesis. This finding suggests that the signal peptide mutations function positively to stimulate OmpA synthesis, rather than bypass a down-regulatory mechanism effected by a wild-type signal peptide. Overproduction of wild-type precursor OmpA or precursors containing signal peptide mutations which lead to relatively minor kinetic processing defects results in accumulation of an improperly assembled OmpA species (imp-OmpA). In contrast, signal peptide mutations which cause relatively severe processing defects accumulate no or only small quantities of imp-OmpA. All mutations result in equivalent levels of properly assembled OmpA. Thus, a strong correlation between imp-OmpA accumulation and cell toxicity was observed. A mutation in the mature region of OmpA which prevents the proper outer membrane assembly of OmpA was suppressed when export was directed by a severely defective signal peptide. These findings suggest that signal peptide mutations indirectly influence OmpA assembly in the outer membrane by altering both the level and rate of OmpA secretion across the cytoplasmic membrane.     

Apolipoprotein M expression increases the size of nascent pre�� HDL formed by ATP binding cassette transporter A1

Apolipoprotein M (apoM) is a novel apolipoprotein that is reportedly necessary for preβ HDL formation; however, its detailed function remains unknown. We investigated the biogenesis and properties of apoM and its effects on the initial steps of nascent preβ HDL assembly by ABCA1 in HEK293 cells. Transiently transfected apoM was localized primarily in the endomembrane compartment. Pulse-chase analyses demonstrated that apoM is inefficiently secreted, relative to human serum albumin, and that ∼50% remains membrane-associated after extraction with sodium carbonate, pH 11.5. To investigate the role of apoM in nascent preβ HDL formation, ABCA1-expressing or control cells, transfected with empty vector, apoM, or C-terminal epitope-tagged apoM (apoM-C-FLAG), were incubated with ¹²⁵I-apoA-I for 24 h. Conditioned media were harvested and fractionated by fast-protein liquid chromatography (FPLC) to monitor HDL particle size. Preβ HDL particles were formed effectively in the absence of apoM expression; however, increased apoM expression stimulated the formation of larger-sized nascent preβ HDLs. Immunoprecipitation with anti-apoA-I antibody followed by apoM Western blot analysis revealed that little secreted apoM was physically associated with preβ HDL. Our results suggest that apoM is an atypical secretory protein that is not necessary for ABCA1-dependent preβ HDL formation but does stimulate the formation of larger-sized preβ HDL. We propose that apoM may function catalytically at an intracellular site to transfer lipid onto preβ HDL during or after their formation by ABCA1.     

Hepatic Apolipoprotein M (ApoM) Overexpression Stimulates Formation of Larger ApoM/Sphingosine 1-Phosphate-enriched Plasma High Density Lipoprotein

Apolipoprotein M (apoM), a lipocalin family member, preferentially associates with plasma HDL and binds plasma sphingosine 1-phosphate (S1P), a signaling molecule active in immune homeostasis and endothelial barrier function. ApoM overexpression in ABCA1-expressing HEK293 cells stimulated larger nascent HDL formation, compared with cells that did not express apoM; however, the in vivo role of apoM in HDL metabolism remains poorly understood. To test whether hepatic apoM overexpression increases plasma HDL size, we generated hepatocyte-specific apoM transgenic (APOM Tg) mice, which had an ∼3–5-fold increase in plasma apoM levels compared with wild-type mice. Although HDL cholesterol concentrations were similar to wild-type mice, APOM Tg mice had larger plasma HDLs enriched in apoM, cholesteryl ester, lecithin:cholesterol acyltransferase, and S1P. Despite the presence of larger plasma HDLs in APOM Tg mice, in vivo macrophage reverse cholesterol transport capacity was similar to that in wild-type mice. APOM Tg mice had an ∼5-fold increase in plasma S1P, which was predominantly associated with larger plasma HDLs. Primary hepatocytes from APOM Tg mice generated larger nascent HDLs and displayed increased sphingolipid synthesis and S1P secretion. Inhibition of ceramide synthases in hepatocytes increased cellular S1P levels but not S1P secretion, suggesting that apoM is rate-limiting in the export of hepatocyte S1P. Our data indicate that hepatocyte-specific apoM overexpression generates larger nascent HDLs and larger plasma HDLs, which preferentially bind apoM and S1P, and stimulates S1P biosynthesis for secretion. The unique apoM/S1P-enriched plasma HDL may serve to deliver S1P to extrahepatic tissues for atheroprotection and may have other as yet unidentified functions.     

The EF-hand Ca2+-binding protein p22 plays a role in microtubule and endoplasmic reticulum organization and dynamics with distinct Ca2+-binding requirements

We have reported that p22, an N-myristoylated EF-hand Ca(2+)-binding protein, associates with microtubules and plays a role in membrane trafficking. Here, we show that p22 also associates with membranes of the early secretory pathway membranes, in particular endoplasmic reticulum (ER). On binding of Ca(2+), p22's ability to associate with membranes increases in an N-myristoylation-dependent manner, which is suggestive of a nonclassical Ca(2+)-myristoyl switch mechanism. To address the intracellular functions of p22, a digitonin-based "bulk microinjection" assay was developed to load cells with anti-p22, wild-type, or mutant p22 proteins. Antibodies against a p22 peptide induce microtubule depolymerization and ER fragmentation; this antibody-mediated effect is overcome by preincubation with the respective p22 peptide. In contrast, N-myristoylated p22 induces the formation of microtubule bundles, the accumulation of ER structures along the bundles as well as an increase in ER network formation. An N-myristoylated Ca(2+)-binding p22 mutant, which is unable to undergo Ca(2+)-mediated conformational changes, induces microtubule bundling and accumulation of ER structures along the bundles but does not increase ER network formation. Together, these data strongly suggest that p22 modulates the organization and dynamics of microtubule cytoskeleton in a Ca(2+)-independent manner and affects ER network assembly in a Ca(2+)-dependent manner.     

Down-regulation of apolipoprotein M expression is mediated by phosphatidylinositol 3-kinase in HepG2 cells

Apolipoprotein M (apoM) is a novel apolipoprotein present mostly in high-density lipoprotein (HDL) in human plasma. In the present study, we demonstrate that insulin, insulin-like growth factor I (IGF-I), and IGF-I potential peptide (IGF-IPP) significantly inhibits apoM expression, in a dose- and a time-dependent manner, in the human hepatoma cell line, HepG2 cells. Insulin-induced down-regulation of apoM was blocked by AG1024 (a specific insulin receptor inhibitor) and LY294002 (a phosphatidylinositol 3-kinase (PI3K) inhibitor), which indicates that it is mediated via the activation of PI3K pathway. In contrast, PD98059 (a MAP kinase inhibitor) did not influence insulin-induced down-regulation of apoM expression, and activation of neither PPAR-alpha agonist (GW7647) nor PPAR-gamma agonist (GW1929) influences apoM expression in HepG2 cells, which indicates that regulation of apoM expression is not related to the activation of PPAR-alpha and PPAR-gamma in hepatic cells, whereas, both PPAR-alpha and PPAR-gamma agonists could inhibit apoB expression. Moreover, in the present study, we demonstrated that PPAR beta/delta agonist (GW501516) could inhibit both apoM and apoB expression in the HepG2 cells. In conclusion, this study shows that apoM expression is regulated by PI3-kinase in HepG2-cells.     

The secretin-specific, chaperone-like protein of the general secretory pathway: separation of proteolytic protection and piloting functions

The chaperone-like protein of the main terminal branch of the general secretory pathway from Klebsiella oxytoca , the outer membrane lipoprotein PulS, protects the multimeric secretin PulD from degradation and promotes its correct localization to the outer membrane. To determine whether these are separable functions, or whether resistance to proteolysis results simply from correct localization of PulD, we replaced the lipoprotein-type signal peptide of PulS by the signal peptide of periplasmic maltose-binding protein. The resulting periplasmic PulS retained its ability to protect PulD, but not its ability to localize PulD to the outer membrane and to function in pullulanase secretion. Periplasmic PulS competed with wild-type PulS to prevent pullulanase secretion, presumably again by causing mislocalization of PulD. A hybrid protein comprising the mature part of PulS fused to the C-terminus of full-length maltose-binding protein (MalE–PulS) had similar properties to the periplasmic PulS protein. Moreover, MalE–PulS was shown to associate with PulD by amylose-affinity chromatography. The MalE–PulS hybrid was rendered completely functional (i.e. it restored pullulanase secretion in a pulS mutant) by replacing its signal peptide with a lipoprotein-type signal peptide. However, this fatty-acylated hybrid protein was only functional if it also carried a lipoprotein sorting signal that targeted it to the outer membrane. Thus, the two functions of PulS are separate and fully dissociable. Incorrect localization, rather than proteolysis, of PulD in the absence of PulS was shown to be the factor that causes high-level induction of the phage shock response. The Erwinia chrysanthemi PulS homologue, OutS, can substitute for PulS, and PulS can protect the secretin OutD from proteolysis in Escherichia coli , indicating the possible existence of a family of PulS-like chaperone proteins.     

The phase property of membrane phospholipids is affected by the functionality of signal peptides from the Escherichia coli ribose-binding protein

We examined the effects of synthetic signal peptides from the wild-type, export-defective mutant and its revertant species of ribose-binding protein on the phase properties of lipid bilayers. The lateral segregation of phosphatidylglycerol (PG) in the lipid bilayer was detected through quenching between NBD-PGs upon the reconstitution of signal peptide into the liposome made with the Escherichia coli inner membrane composition. The tendency of lipid segregation was highly dependent on the export competency of signal peptides in vivo, with a decreasing order of wild-type, revertant, and mutant species. The colocalizations of pyrene-PG with BODIPY-PG were also induced by the signal peptides, confirming the phase separation of the acidic phospholipid. The wild-type and revertant signal peptides predominantly formed alpha-helical conformations with the presence of acidic phospholipid as determined by circular dichroism spectroscopy. In addition, they restricted the motion of lipid acyl chains as monitored by fluorescence anisotropy of DPH, suggesting a deep penetration of signal peptide into the lipid bilayer. However, the alpha-helical content of mutant signal peptide was only about half that of the wild-type or revertant peptide with a significantly smaller degree of penetration into the bilayer. An association of the defective signal peptides into the membrane was affected by salt extraction, whereas the functional ones were not. The aforementioned results indicate that the functionality of signal peptide is accomplished through its topologies in the membrane and also by its ability to induce lateral segregation of acidic phospholipid. We propose that the clustering of acidic phospholipid by the functional signal peptide is responsible for the formation of non-bilayer membrane structure, thereby promoting an efficient translocation of secretory proteins.     

Anionic phospholipids are essential for alpha-helix formation of the signal peptide of prePhoE upon interaction with phospholipid vesicles.

The conformational consequences of the interaction of the PhoE signal peptide with bilayers of different types of phospholipids was investigated using circular dichroism. It was found that interaction of the signal peptide with anionic phospholipid vesicles of dioleoylphosphatidylglycerol and dioleoylphosphatidylserine results in induction of high amounts of alpha-helical structure of 70% and 57%, respectively. Upon addition of the signal peptide to cardiolipin vesicles, less but still significant alpha-helical structure was induced (29%). In contrast, no alpha-helix formation was observed upon the interaction of the signal peptide with zwitterionic dioleoylphosphatidylcholine vesicles. In bilayers of dioleoylphosphatidylcholine with dioleoylphosphatidylglycerol, it was shown that in the presence of 100 mM NaCl a minimum amount of 50% of negatively charged lipid was required for induction of the maximal percentage of alpha-helix, whereas in the absence of salt a minimum amount of 35% of negatively charged lipid was necessary. Induction of alpha-helix structure appeared to be correlated with functionality, since, in a less functional analogue of the PhoE signal peptide, the PhoE-[Asp-19,20] signal peptide, less alpha-helix was induced than in the wild-type PhoE signal peptide. It is proposed that the interaction with anionic phospholipids is essential for a functional conformation of the PhoE signal sequence during protein translocation.