Altered peptide ligands act as partial agonists by inhibiting phospholipase C activity induced by myasthenogenic T cell epitopes

Myasthenia gravis (MG) is a T cell-regulated, antibody-mediated autoimmune disease. Two peptides representing sequences of the human acetylcholine receptor α-subunit, p195–212 and p259–271, previously were shown to stimulate the proliferation of peripheral blood lymphocytes of patients with MG and were found to be immunodominant T cell epitopes in SJL and BALB/c mice, respectively. Single amino acid-substituted analogs of p195–212 and p259–271, as well as a dual analog composed of the tandemly arranged two single analogs, were shown to inhibit, in vitro and in vivo, MG-associated autoimmune responses. Stimulation of T cells through the antigen-specific T cell receptor activates tyrosine kinases and phospholipase C (PLC). Therefore, in attempts to understand the mechanism of action of the analogs, we first examined whether the myasthenogenic peptides trigger tyrosine phosphorylation and activation of phospholipase C. For that purpose, we measured generation of inositol phosphates and tyrosine phosphorylation of PLC after stimulation of the p195–212- and p259–271-specific T cell lines with these myasthenogenic peptides. Both myasthenogenic peptides stimulated generation of inositol phosphates as well as tyrosine phosphorylation of PLC. However, the single and dual analogs, although inducing tyrosine phosphorylation of PLC, could not induce PLC activity. Furthermore, the single and dual analogs inhibited the induced PLC activity whereas they could not inhibit tyrosine phosphorylation of PLC that was caused by the myasthenogenic peptides. Thus, the altered peptides and the dual analog act as partial agonists. The down-regulation of PLC activity by the analogs may account for their capacity to inhibit in vitro MG-associated T cell responses.

Identification of a structural determinant for resistance to β-lactam antibiotics in Gram-positive bacteria

Streptococcus pneumoniae is the main causal agent of pathologies that are increasingly resistant to antibiotic treatment. Clinical resistance of S. pneumoniae to β-lactam antibiotics is linked to multiple mutations of high molecular mass penicillin-binding proteins (H-PBPs), essential enzymes involved in the final steps of bacterial cell wall synthesis. H-PBPs from resistant bacteria have a reduced affinity for β-lactam and a decreased hydrolytic activity on substrate analogues. In S. pneumoniae, the gene coding for one of these H-PBPs, PBP2x, is located in the cell division cluster (DCW). We present here structural evidence linking multiple β-lactam resistance to amino acid substitutions in PBP2x within a buried cavity near the catalytic site that contains a structural water molecule. Site-directed mutation of amino acids in contact with this water molecule in the “sensitive” form of PBP2x produces mutants similar, in terms of β-lactam affinity and substrate hydrolysis, to altered PBP2x produced in resistant clinical isolates. A reverse mutation in a PBP2x variant from a clinically important resistant clone increases the acylation efficiency for β-lactams and substrate analogues. Furthermore, amino acid residues in contact with the structural water molecule are conserved in the equivalent H-PBPs of pathogenic Gram-positive cocci. We suggest that, probably via a local structural modification, the partial or complete loss of this water molecule reduces the acylation efficiency of PBP2x substrates to a point at which cell wall synthesis still occurs, but the sensitivity to therapeutic concentrations of β-lactam antibiotics is lost.

Human fatty acid synthase: Assembling recombinant halves of the fatty acid synthase subunit protein reconstitutes enzyme activity

Our model of the native fatty acid synthase (FAS) depicts it as a dimer of two identical multifunctional proteins (Mr ≈ 272,000) arranged in an antiparallel configuration so that the active Cys-SH of the β-ketoacyl synthase of one subunit (where the acyl group is attached) is juxtaposed within 2 Å of the pantetheinyl-SH of the second subunit (where the malonyl group is bound). This arrangement generates two active centers for fatty acid synthesis and predicts that if we have two appropriate halves of the monomer, we should be able to reconstitute an active fatty acid-synthesizing site. We cloned, expressed, and purified catalytically active thioredoxin (TRX) fusion proteins of the NH2-terminal half of the human FAS subunit protein (TRX-hFAS-dI; residues 1–1,297; Mr ≈ 166) and of the C-terminal half (TRX-hFAS-dII-III; residues 1,296–2,504; Mr ≈ 155). Adding equivalent amounts of TRX-hFAS-dI and TRX-hFAS-dII-III to a reaction mixture containing acetyl-CoA, malonyl-CoA, and NADPH resulted in the synthesis of long-chain fatty acids. The rate of synthesis was dependent upon the presence of both recombinant proteins and reached a constant level when they were present in equivalent amounts, indicating that the reconstitution of an active fatty acid-synthesizing site required the presence of every partial activity associated with the subunit protein. Analyses of the product acids revealed myristate to be the most abundant with small amounts of palmitate and stearate, possibly because of the way the fused recombinant proteins interacted with each other so that the thioesterase hydrolyzed the acyl group in its myristoyl state. The successful reconstitution of the human FAS activity from its domain I and domains II and III fully supports our model for the structure–function relationship of FAS in animal tissues.

Sequential Actions of Phospholipase D and Phosphatidic Acid Phosphohydrolase 2b Generate Diglyceride in Mammalian Cells

Phosphatidylcholine (PC) is a major source of lipid-derived second messenger molecules that function as both intracellular and extracellular signals. PC-specific phospholipase D (PLD) and phosphatidic acid phosphohydrolase (PAP) are two pivotal enzymes in this signaling system, and they act in series to generate the biologically active lipids phosphatidic acid (PA) and diglyceride. The identity of the PAP enzyme involved in PLD-mediated signal transduction is unclear. We provide the first evidence for a functional role of a type 2 PAP, PAP2b, in the metabolism of PLD-generated PA. Our data indicate that PAP2b localizes to regions of the cell in which PC hydrolysis by PLD is taking place. Using a newly developed PAP2b-specific antibody, we have characterized the expression, posttranslational modification, and localization of endogenous PAP2b. Glycosylation and localization of PAP2b appear to be cell type and tissue specific. Biochemical fractionation and immunoprecipitation analyses revealed that PAP2b and PLD2 activities are present in caveolin-1–enriched detergent-resistant membrane microdomains. We found that PLD2 and PAP2b act sequentially to generate diglyceride within this specialized membrane compartment. The unique lipid composition of these membranes may provide a selective environment for the regulation and actions of enzymes involved in signaling through PC hydrolysis.

Cloning and expression of the multifunctional human fatty acid synthase and its subdomains in Escherichia coli

We engineered a full-length (8.3-kbp) cDNA coding for fatty acid synthase (FAS; EC 2.3.1.85) from the human brain FAS cDNA clones we characterized previously. In the process of accomplishing this task, we developed a novel PCR procedure, recombinant PCR, which is very useful in joining two overlapping DNA fragments that do not have a common or unique restriction site. The full-length cDNA was cloned in pMAL-c2 for heterologous expression in Escherichia coli as a maltose-binding protein fusion. The recombinant protein was purified by using amylose-resin affinity and hydroxylapatite chromatography. As expected from the coding capacity of the cDNA expressed, the chimeric recombinant protein has a molecular weight of 310,000 and reacts with antibodies against both human FAS and maltose-binding protein. The maltose-binding protein-human FAS (MBP-hFAS) catalyzed palmitate synthesis from acetyl-CoA, malonyl-CoA, and NADPH and exhibited all of the partial activities of FAS at levels comparable with those of the native human enzyme purified from HepG2 cells. Like the native HepG2 FAS, the products of MBP-hFAS are mainly palmitic acid (>90%) and minimal amounts of stearic and arachidic acids. Similarly, a human FAS cDNA encoding domain I (β-ketoacyl synthase, acetyl-CoA and malonyl-CoA transacylases, and β-hydroxyacyl dehydratase) was cloned and expressed in E. coli using pMAL-c2. The expressed fusion protein, MBP-hFAS domain I, was purified to apparent homogeneity (Mr 190,000) and exhibited the activities of the acetyl/malonyl transacylases and the β-hydroxyacyl dehydratase. In addition, a human FAS cDNA encoding domains II and III (enoyl and β-ketoacyl reductases, acyl carrier protein, and thioesterase) was cloned in pET-32b(+) and expressed in E. coli as a fusion protein with thioredoxin and six in-frame histidine residues. The recombinant fusion protein, thioredoxin-human FAS domains II and III, that was purified from E. coli had a molecular weight of 159,000 and exhibited the activities of the enoyl and β-ketoacyl reductases and the thioesterase. Both the MBP and the thioredoxin-His-tags do not appear to interfere with the catalytic activity of human FAS or its partial activities.

A Novel Alkaline α-Galactosidase from Melon Fruit with a Substrate Preference for Raffinose1

The cucurbits translocate the galactosyl-sucrose oligosaccharides raffinose and stachyose, therefore, α-galactosidase (α-d-galactoside galactohydrolase, EC 3.2.1.22) is expected to function as the initial enzyme of photoassimilate catabolism. However, the previously described alkaline α-galactosidase is specific for the tetrasaccharide stachyose, leaving raffinose catabolism in these tissues as an enigma. In this paper we report the partial purification and characterization of three α-galactosidases, including a novel alkaline α-galactosidase (form I) from melon (Cucumis melo) fruit tissue. The form I enzyme showed preferred activity with raffinose and significant activity with stachyose. Other unique characteristics of this enzyme, such as weak product inhibition by galactose (in contrast to the other α-galactosidases, which show stronger product inhibition), also impart physiological significance. Using raffinose and stachyose as substrates in the assays, the activities of the three α-galactosidases (alkaline form I, alkaline form II, and the acid form) were measured at different stages of fruit development. The form I enzyme activity increased during the early stages of ovary development and fruit set, in contrast to the other α-galactosidase enzymes, both of which declined in activity during this period. In the mature, sucrose-accumulating mesocarp, the alkaline form I enzyme was the major α-galactosidase present. We also observed hydrolysis of raffinose at alkaline conditions in enzyme extracts from other cucurbit sink tissues, as well as from young Coleus blumei leaves. Our results suggest different physiological roles for the α-galactosidase forms in the developing cucurbit fruit, and show that the newly discovered enzyme plays a physiologically significant role in photoassimilate partitioning in cucurbit sink tissue.

Methyl Jasmonate Induces Lauric Acid ω-Hydroxylase Activity and Accumulation of CYP94A1 Transcripts but Does Not Affect Epoxide Hydrolase Activities in Vicia sativa Seedlings1

Treatment of etiolated Vicia sativa seedlings by the plant hormone methyl jasmonate (MetJA) led to an increase of cytochrome P450 content. Seedlings that were treated for 48 h in a 1 mm solution of MetJA stimulated ω-hydroxylation of 12:0 (lauric acid) 14-fold compared with the control (153 versus 11 pmol min−1 mg−1 protein, respectively). Induction was dose dependent. The increase of activity (2.7-fold) was already detectable after 3 h of treatment. Activity increased as a function of time and reached a steady level after 24 h. Northern-blot analysis revealed that the transcripts coding for CYP94A1, a fatty acid ω-hydroxylase, had already accumulated after 1 h of exposure to MetJA and was maximal between 3 and 6 h. Under the same conditions, a study of the enzymatic hydrolysis of 9,10-epoxystearic acid showed that both microsomal and soluble epoxide hydrolase activities were not affected by MetJA treatment.

Long-lasting reduction of inhibitory function and gamma-aminobutyric acid type A receptor subunit mRNA expression in a model of temporal lobe epilepsy.

This study evaluated hippocampal inhibitory function and the level of expression of gamma-aminobutyric acid type A (GABAA) receptor mRNA in an in vivo model of epilepsy. Chronic recurrent limbic seizures were induced in rats using injections of pilocarpine. Electrophysiological studies performed on hippocampal slices prepared from control and epileptic animals 1 to 2 months after pilocarpine injections demonstrated a significant hyperexcitability in the epileptic animals. Reduced levels of mRNA expression for the alpha 2 and alpha 5 subunits of the GABAA receptors were evident in the CA1, CA2, and CA3 regions of the hippocampus of epileptic animals. No decrease in mRNA encoding alpha 1, beta 2, or gamma 2 GABAA receptor subunits was observed. In addition, no change in the mRNA levels of alpha CaM kinase II was seen. Selective decreases in mRNA expression did not correlate with neuronal cell loss. The results indicate that selective, long-lasting reduction of GABAA subunit mRNA expression and increased excitability, possibly reflecting loss of GABAergic inhibition, occur in an in vivo model of partial complex epilepsy.

Binding of thalidomide to alpha1-acid glycoprotein may be involved in its inhibition of tumor necrosis factor alpha production.

In addition to its well known sedative and teratogenic effects, thalidomide also possesses potent immunomodulatory and antiinflammatory activities, being most effective against leprosy and chronic graft-versus-host disease. The immunomodulatory activity of thalidomide has been ascribed to the selective inhibition of tumor necrosis factor alpha from monocytes. The molecular mechanism for the immunomodulatory effect of thalidomide remains unknown. To elucidate this mechanism, we synthesized an active photoaffinity label of thalidomide as a probe to identify the molecular target of the drug. Using the probe, we specifically labeled a pair of proteins of 43-45 kDa with high acidity from bovine thymus extract. Purification of these proteins and partial peptide sequence determination revealed them to be alpha1-acid glycoprotein (AGP). We show that the binding of thalidomide photoaffinity label to authentic human AGP is competed with both thalidomide and the nonradioactive photoaffinity label at concentrations comparable to those required for inhibition of production of tumor necrosis factor alpha from human monocytes, suggesting that AGP may be involved in the immunomodulatory activity of thalidomide.

Prostate-specific membrane antigen is a hydrolase with substrate and pharmacologic characteristics of a neuropeptidase.

This report demonstrates that the investigational prostatic carcinoma marker known as the prostate-specific membrane antigen (PSM) possesses hydrolytic activity with the substrate and pharmacologic properties of the N-acetylated alpha-linked acidic dipeptidase (NAALADase). NAALADase is a membrane hydrolase that has been characterized in the mammalian nervous system on the basis of its catabolism of the neuropeptide N-acetylaspartylglutamate (NAAG) to yield glutamate and N-acetylaspartate and that has been hypothesized to influence glutamatergic signaling processes. The immunoscreening of a rat brain cDNA expression library with anti-NAALADase antisera identified a 1428-base partial cDNA that shares 86% sequence identity with 1428 bases of the human PSM cDNA [Israeli, R. S., Powell, C. T., Fair, W. R. & Heston, W.D.W. (1993) Cancer Res. 53, 227-230]. A cDNA containing the entire PSM open reading frame was subsequently isolated by reverse transcription-PCR from the PSM-positive prostate carcinoma cell line LNCaP. Transient transfection of this cDNA into two NAALADase-negative cell lines conferred NAAG-hydrolyzing activity that was inhibited by the NAALADase inhibitors quisqualic acid and beta-NAAG. Thus we demonstrate a PSM-encoded function and identify a NAALADase-encoding cDNA. Northern analyses identify at least six transcripts that are variably expressed in NAALADase-positive but not in NAALADase-negative rat tissues and human cell lines; therefore, PSM and/or related molecular species appear to account for NAAG hydrolysis in the nervous system. These results also raise questions about the role of PSM in both normal and pathologic prostate epithelial-cell function.

Phosphinate analogs of D-, D-dipeptides: slow-binding inhibition and proteolysis protection of VanX, a D-, D-dipeptidase required for vancomycin resistance in Enterococcus faecium.

VanX is a D-Ala-D-Ala dipeptidase that is essential for vancomycin resistance in Enterococcus faecium. Contrary to most proteases and peptidases, it prefers to hydrolyze the amino substrate but not the related kinetically and thermodynamically more favorable ester substrate D-Ala-D-lactate. The enzymatic activity of VanX was previously found to be inhibited by the phosphinate analogs of the proposed tetrahedral intermediate for hydrolysis of D-Ala-D-Ala. Here we report that such phosphinates are slow-binding inhibitors. D-3-[(1-Aminoethyl)phosphinyl]-D-2-methylpropionic acid I showed a time-dependent onset of inhibition of VanX and a time-dependent return to uninhibited steady-state rates upon dilution of the enzyme/inhibitor mixture. The initial inhibition constant Ki after immediate addition of VanX to phosphinate I to form the E-I complex is 1.5 microM but is then lowered by a relatively slow isomerization step to a second complex, E-I*, with a final K*i of 0.47 microM. This slow-binding inhibition reflects a Km/K*i ratio of 2900:1. The rate constant for the slow dissociation of complex E-I* is 0.24 min-1. A phosphinate analog with an ethyl group replacing what would be the side chain of the second D-alanyl residue in the normal tetrahedral adduct gives a K*i value of 90 nM. Partial proteolysis of VanX reveals two protease-sensitive loop regions that are protected by the intermediate analog phosphinate, indicating that they may be part of the VanX active site.

Human fatty acid synthase: properties and molecular cloning.

Fatty acid synthase (FAS; EC 2.3.1.85) was purified to near homogeneity from a human hepatoma cell line, HepG2. The HepG2 FAS has a specific activity of 600 nmol of NADPH oxidized per min per mg, which is about half that of chicken liver FAS. All the partial activities of human FAS are comparable to those of other animal FASs, except for the beta-ketoacyl synthase, whose significantly lower activity is attributable to the low 4'-phosphopantetheine content of HepG2 FAS. We cloned the human brain FAS cDNA. The cDNA sequence has an open reading frame of 7512 bp that encodes 2504 amino acids (M(r), 272,516). The amino acid sequence of the human FAS has 79% and 63% identity, respectively, with the sequences of the rat and chicken enzymes. Northern analysis revealed that human FAS mRNA was about 9.3 kb in size and that its level varied among human tissues, with brain, lung, and liver tissues showing prominent expression. The nucleotide sequence of a segment of the HepG2 FAS cDNA (bases 2327-3964) was identical to that of the cDNA from normal human liver and brain tissues, except for a 53-bp sequence (bases 3892-3944) that does not alter the reading frame. This altered sequence is also present in HepG2 genomic DNA. The origin and significance of this sequence variance in the HepG2 FAS gene are unclear, but the variance apparently does not contribute to the lower activity of HepG2 FAS.

DNA-strand exchange promoted by RecA protein in the absence of ATP: implications for the mechanism of energy transduction in protein-promoted nucleic acid transactions.

DNA-strand exchange promoted by Escherichia coli RecA protein normally requires the presence of ATP and is accompanied by ATP hydrolysis, thereby implying a need for ATP hydrolysis. Previously, ATP hydrolysis was shown not to be required; here we demonstrate furthermore that a nucleoside triphosphate cofactor is not required for DNA-strand exchange. A gratuitous allosteric effector consisting of the noncovalent complex of ADP and aluminum fluoride, ADP.AIF4-, can both induce the high-affinity DNA-binding state of RecA protein and support the homologous pairing and exchange of up to 800-900 bp of DNA. These results demonstrate that induction of the functionally active, high-affinity DNA-binding state of RecA protein is needed for RecA protein-promoted DNA-strand exchange and that there is no requirement for a high-energy nucleotide cofactor for the exchange of DNA strands. Consequently, the free energy needed to activate the DNA substrates for DNA-strand exchange is not derived from ATP hydrolysis. Instead, the needed free energy is derived from ligand binding and is transduced to the DNA via the associated ligand-induced structural transitions of the RecA protein-DNA complex; ATP hydrolysis simply destroys the effector ligand. This concept has general applicability to the mechanism of energy transduction by proteins.

Kinetics and mechanism of polyamide ("peptide") nucleic acid binding to duplex DNA.

To elucidate the mechanism of recognition of double-stranded DNA (dsDNA) by homopyrimidine polyamide ("peptide") nucleic acid (PNA) leading to the strand-displacement, the kinetics of the sequence-specific PNA/DNA binding have been studied. The binding was monitored with time by the gel retardation and nuclease S1 cleavage assays. The experimental kinetic curves obey pseudo-first-order kinetics and the dependence of the pseudo-first-order rate constant, kps, on PNA concentration, P, obeys a power law kps approximately P gamma with 2 < gamma < 3. The kps values for binding of decamer PNA to dsDNA target sites with one mismatch are hundreds of times slower than for the correct site. A detailed kinetic scheme for PNA/DNA binding is proposed that includes two major steps of the reaction of strand invasion: (i) a transient partial opening of the PNA binding site on dsDNA and incorporation of one PNA molecule with the formation of an intermediate PNA/DNA duplex and (ii) formation of a very stable PNA2/DNA triplex. A simple theoretical treatment of the proposed kinetic scheme is performed. The interpretation of our experimental data in the framework of the proposed kinetic scheme leads to the following conclusions. The sequence specificity of the recognition is essentially provided at the "search" step of the process, which consists in the highly reversible transient formation of duplex between one PNA molecule and the complementary strand of duplex DNA while the other DNA strand is displaced. This search step is followed by virtually irreversible "locking" step via PNA2/DNA triplex formation. The proposed mechanism explains how the binding of homopyrimidine PNA to dsDNA meets two apparently mutually contradictory features: high sequence specificity of binding and remarkable stability of both correct and mismatched PNA/DNA complexes.

Hydrothermal Carbons from Hemicellulose-Derived Aqueous Hydrolysis Products as Electrode Materials for Supercapacitors

Acid pretreatment of lignocellulosic biomass, required for bioethanol production, generates large amounts of by-products, such as lignin and hydrolyzed hemicellulose fractions, which have found so far very limited applications. In this work, we demonstrate how the recovered hemicellulose hydrolysis products can be effectively utilized as a precursor for the synthesis of functional carbon materials through hydrothermal carbonization (HTC). The morphology and chemical structure of the synthesized HTC carbons are thoroughly characterized to highlight their similarities with glucose-derived HTC carbons. Furthermore, two routes for introducing porosity within the HTC carbon structure are presented: i) silica nanoparticle hard-templating, which is shown to be a viable method for the synthesis of carbonaceous hollow spheres; and ii) KOH chemical activation. The synthesized activated carbons (ACs) show an extremely high porosity (pore volume≈1.0 cm3 g−1) mostly composed of micropores (90 % of total pore volume). Because of their favorable textural properties, the ACs are further tested as electrodes for supercapacitors, yielding very promising results (300 F g−1 at 250 mA g−1) and confirming the high suitability of KOH-activated HTC carbons derived from spruce and corncob hydrolysis products as materials for electric double layer supercapacitors.