Effects of Alzheimer's peptide and alpha1-antichymotrypsin on astrocyte gene expression.

We employed gene array technology to investigate the effects of alpha1-antichymotrypsin (ACT), soluble or fibrillar Alzheimer's peptide (Abeta(1-42)) alone and the combination of ACT/Abeta(1-42) on human astrocytes. Using a 1.2-fold change as significance threshold, 398 astrocyte genes showed altered expression in response to these treatments compared to controls. Of the 276 genes affected by the ACT/soluble Abeta(1-42) combination, 195 (70.6%) were suppressed. The ACT/fibrillar Abeta(1-42) combination affected expression of 64 genes of which 58 (90.5%) were up-regulated. The most prominent gene expression changes in response to the ACT/soluble Abeta(1-42), were the down-regulation of at least 60 genes involved in transcription, signal transduction, apoptosis and neurogenesis. The ACT/fibril Abeta(1-42) increased the expression of genes involved in transcription regulation and signal transduction. Surprisingly, gene expression of astrocytes exposed to soluble or fibrillar Abeta(1-42) alone was largely unaffected. Thus, the molecular forms generated by the combination of ACT/Abeta(1-42) alter expression of astrocyte genes more profoundly in breadth and magnitude than soluble or fibrillar Abeta(1-42) alone, suggesting that pathogenic effects of Abeta(1-42) may occur as a consequence of its association with other proteins.

Crystal structure of a substrate complex of Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein synthase III (FabH) with lauroyl-coenzyme A.

Beta-ketoacyl-acyl carrier protein synthase III (FabH) catalyzes a two step reaction that initiates the pathway of fatty acid biosynthesis in plants and bacteria. In Mycobacterium tuberculosis, FabH catalyzes extension of lauroyl, myristoyl and palmitoyl groups from which cell wall mycolic acids of the bacterium are formed. The first step of the reaction is an acyl group transfer from acyl-coenzyme A to the active-site cysteine of the enzyme; the second step is acyl chain extension by two carbon atoms through Claisen condensation with malonyl-acyl carrier protein. We have previously determined the crystal structure of a type II, dissociated M.tuberculosis FabH, which catalyzes extension of lauroyl, myristoyl and palmitoyl groups. Here we describe the first long-chain Michaelis substrate complex of a FabH, that of lauroyl-coenzyme A with a catalytically disabled Cys-->Ala mutant of M.tuberculosis FabH. An elongated channel extending from the mutated active-site cysteine defines the acyl group binding locus that confers unique acyl substrate specificity on M.tuberculosis FabH. CoA lies in a second channel, bound primarily through interactions of its nucleotide group at the enzyme surface. The apparent weak association of CoA in this complex may play a role in the binding and dissociation of long chain acyl-CoA substrates and products and poses questions pertinent to the mechanism of this enzyme.

Crystals of X29, a Xenopus laevis U8 snoRNA-binding protein with nuclear decapping activity.

Eukaryotic ribosome biosynthesis requires modification (methylation, pseudouridylation) and nucleolytic processing of precursor ribosomal RNAs in the nucleolus. The RNA components of the small nucleolar RNPs (snoRNAs) are essential for many of these events. One snoRNP, called U8, is necessary for maturation of 5.8S and 28S rRNA in vertebrates. In Xenopus laevis, U8 snoRNA was found to bind specifically and with high affinity to a protein called X29. X29 is a Nudix hydrolase, a nucleotide diphosphatase that removes the m(7)G and m(227)G caps from U8 and other RNAs. X29 requires an RNA as substrate and cap analogues are not substrates or inhibitors of cleavage. To study the determinants of X29 activity and its specificity for U8 RNA substrate, X29 was crystallized in an orthorhombic crystal form that diffracts to 2.1 A resolution.

Glioma cell activation by Alzheimer's peptide Abeta1-42, alpha1-antichymotrypsin, and their mixture.

We compared the effects ofAlzheimer's peptide (Abeta1-42), a,-antichymotrypsin (ACT) and an ACT/Abeta1-42 mixture on human glioma DK-MG cells. The solution of Abeta (5 microM) formed by 2-h incubation at room temperature induced tumour necrosis factor-alpha (TNF-alpha) and interleukin (IL)-6 levels by 55 and 45%, respectively, and increased gelatinase B activity by 67%, while exposure of cells to the ACT/Abeta1-42 mixture (1:10 molar ratio ACT: Abeta1-42) under the same experimental conditions showed no effect on IL-6 levels or gelatinase B activity, but strongly induced TNF-alpha (by 190%), compared to the controls. Stimulation of the cells with Abeta1-42 alone, but not with ACT, increased by about 20% low-density lipoprotein (LDL) uptake and mRNA levels for LDL receptor and HMG-CoA reductase, while the ACT/Abeta1-42 mixture significantly increased LDL uptake (by 50%), up-regulated mRNA levels for LDL receptor and HMG-CoA reductase by 48 and 63%, respectively, and increased lipid accumulation by about 20-fold. These data suggest a possible new role for Abeta in Alzheimer's disease through its interaction with the inflammatory reactant, ACT.

Alpha1-antichymotrypsin/Alzheimer's peptide Abeta(1-42) complex perturbs lipid metabolism and activates transcription factors PPARgamma and NFkappaB in human neuroblastoma (Kelly) cells.

Amyloid-beta peptide (Abeta) and the serpin proteinase inhibitor alpha1-antichymotrypsin (ACT) are components of the amyloid plaques associated with Alzheimer's disease (AD). Abeta exists in soluble monomeric and oligomeric forms and in an insoluble polymerised fibrillar form, but it is not clear which of these plays the most important role in the etiology of AD. In vitro, Abeta(1-42) interacts with ACT, and as a result of this, ACT loses its proteinase inhibitor activity and polymerisation of Abeta(1-42) is promoted. Here we provide evidence that new molecular forms resulting from incubation of ACT with Abeta(1-42) have multiple cellular level effects on neuronal cells. The mixture of soluble Abeta and an ACT/Abeta complex formed by 2 hr incubation at a 10:1 molar ratio of Abeta:ACT strongly induce cellular proliferation and expression of transcription factors peroxisome proliferator-activated receptor-gamma (PPARgamma) and NFkappaB, and also increase uptake and depress degradation of native and oxidised low-density lipoprotein (LDL) by cells. Similar but less pronounced effects are seen when cells are exposed to the Abeta peptide alone preincubated for 2 hr. Abeta(1-42) and to a lesser extent ACT/Abeta(1-42) complex mixture prepared by 2 hr incubation both inhibit association of native LDL with cells. Neither ACT alone nor the Abeta(1-42) and ACT/Abeta(1-42) forms prepared by 24-hr incubation show any significant effects in these assays. We propose that specific molecular forms of Abeta(1-42) and ACT/Abeta(1-42) complex mixture, both dependent on the abundances of Abeta(1-42) and ACT/Abeta(1-42) in vivo and on their time of exposure to each other, have cellular effects which are important for the initiation and progression of the pathologies associated with AD.

Crystal structure of the Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein synthase III.

Mycolic acids (alpha-alkyl-beta-hydroxy long chain fatty acids) cover the surface of mycobacteria, and inhibition of their biosynthesis is an established mechanism of action for several key front-line anti-tuberculosis drugs. In mycobacteria, long chain acyl-CoA products (C(14)-C(26)) generated by a type I fatty-acid synthase can be used directly for the alpha-branch of mycolic acid or can be extended by a type II fatty-acid synthase to make the meromycolic acid (C(50)-C(56)))-derived component. An unusual Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein (ACP) synthase III (mtFabH) has been identified, purified, and shown to catalyze a Claisen-type condensation between long chain acyl-CoA substrates such as myristoyl-CoA (C(14)) and malonyl-ACP. This enzyme, presumed to play a key role in initiating meromycolic acid biosynthesis, was crystallized, and its structure was determined at 2.1-A resolution. The mtFabH homodimer is closely similar in topology and active-site structure to Escherichia coli FabH (ecFabH), with a CoA/malonyl-ACP-binding channel leading from the enzyme surface to the buried active-site cysteine residue. Unlike ecFabH, mtFabH contains a second hydrophobic channel leading from the active site. In the ecFabH structure, this channel is blocked by a phenylalanine residue, which constrains specificity to acetyl-CoA, whereas in mtFabH, this residue is a threonine, which permits binding of longer acyl chains. This same channel in mtFabH is capped by an alpha-helix formed adjacent to a 4-amino acid sequence insertion, which limits bound acyl chain length to 16 carbons. These observations offer a molecular basis for understanding the unusual substrate specificity of mtFabH and its probable role in regulating the biosynthesis of the two different length acyl chains required for generation of mycolic acids. This mtFabH presents a new target for structure-based design of novel antimycobacterial agents.

Role of tyrosine 65 in the mechanism of serine hydroxymethyltransferase.

Crystal structures of human and rabbit cytosolic serine hydroxymethyltransferase have shown that Tyr65 is likely to be a key residue in the mechanism of the enzyme. In the ternary complex of Escherichia coli serine hydroxymethyltransferase with glycine and 5-formyltetrahydrofolate, the hydroxyl of Tyr65 is one of four enzyme side chains within hydrogen-bonding distance of the carboxylate group of the substrate glycine. To probe the role of Tyr65 it was changed by site-directed mutagenesis to Phe65. The three-dimensional structure of the Y65F site mutant was determined and shown to be isomorphous with the wild-type enzyme except for the missing Tyr hydroxyl group. The kinetic properties of this mutant enzyme in catalyzing reactions with serine, glycine, allothreonine, D- and L-alanine, and 5,10-methenyltetrahydrofolate substrates were determined. The properties of the enzyme with D- and L-alanine, glycine in the absence of tetrahydrofolate, and 5, 10-methenyltetrahydrofolate were not significantly changed. However, catalytic activity was greatly decreased for serine and allothreonine cleavage and for the solvent alpha-proton exchange of glycine in the presence of tetrahydrofolate. The decreased catalytic activity for these reactions could be explained by a greater than 2 orders of magnitude increase in affinity of Y65F mutant serine hydroxymethyltransferase for these amino acids bound as the external aldimine. These data are consistent with a role for the Tyr65 hydroxyl group in the conversion of a closed active site to an open structure.

Crystal structure at 2.4 A resolution of E. coli serine hydroxymethyltransferase in complex with glycine substrate and 5-formyl tetrahydrofolate.

Serine hydroxymethyltransferase (EC 2.1.2.1), a member of the alpha-class of pyridoxal phosphate enzymes, catalyzes the reversible interconversion of serine and glycine, changing the chemical bonding at the C(alpha)-C(beta) bond of the serine side-chain mediated by the pyridoxal phosphate cofactor. Scission of the C(alpha)-C(beta) bond of serine substrate produces a glycine product and most likely formaldehyde, which reacts without dissociation with tetrahydropteroylglutamate cofactor. Crystal structures of the human and rabbit cytosolic serine hydroxymethyltransferases (SHMT) confirmed their close similarity in tertiary and dimeric subunit structure to each other and to aspartate aminotransferase, the archetypal alpha-class pyridoxal 5'-phosphate enzyme. We describe here the structure at 2.4 A resolution of Escherichia coli serine hydroxymethyltransferase in ternary complex with glycine and 5-formyl tetrahydropteroylglutamate, refined to an R-factor value of 17.4 % and R(free) value of 19.6 %. This structure reveals the interactions of both cofactors and glycine substrate with the enzyme. Comparison with the E. coli aspartate aminotransferase structure shows the distinctions in sequence and structure which define the folate cofactor binding site in serine hydroxymethyltransferase and the differences in orientation of the amino terminal arm, the evolution of which was necessary for elaboration of the folate binding site. Comparison with the unliganded rabbit cytosolic serine hydroxymethyltransferase structure identifies changes in the conformation of the enzyme, similar to those observed in aspartate aminotransferase, that probably accompany the binding of substrate. The tetrameric quaternary structure of liganded E. coli serine hydroxymethyltransferase also differs in symmetry and relative disposition of the functional tight dimers from that of the unliganded eukaryotic enzymes. SHMT tetramers have surface charge distributions which suggest distinctions in folate binding between eukaryotic and E. coli enzymes. The structure of the E. coli ternary complex provides the basis for a thorough investigation of its mechanism through characterization and structure determination of site mutants.

Deamidation of asparagine residues in a recombinant serine hydroxymethyltransferase.

Serine hydroxymethyltransferase purified from rabbit liver cytosol has at least two Asn residues (Asn(5) and Asn(220)) that are 67 and 30% deamidated, respectively. Asn(5) is deamidated equally to Asp and isoAsp, while Asn(220) is deamidated only to isoAsp. To determine the effect of these Asn deamidations on enzyme activity and stability a recombinant rabbit liver cytosolic serine hydroxymethyltransferase was expressed in Escherichia coli over a 5-h period. About 90% of the recombinant enzyme could be isolated with the two Asn residues in a nondeamidated form. Compared with the enzyme isolated from liver the recombinant enzyme had a 35% increase in catalytic activity but exhibited no significant changes in either affinity for substrates or stability. Introduction of Asp residues for either Asn(5) or Asn(220) did not significantly alter activity or stability of the mutant forms. In vitro incubation of the recombinant enzyme at 37 degrees C and pH 7.3 resulted in the rapid deamidation of Asn(5) to both Asp and isoAsp with a t(1/2) of 50-70 h, which is comparable to the rate found with small flexible peptides containing the same sequence. The t(1/2) for deamidation of Asn(220) was at least 200 h. This residue may become deamidated only after some unfolding of the enzyme. The rates for deamidation of Asn(5) and Asn(220) are consistent with the structural environment of the two Asn residues in the native enzyme. There are also at least two additional deamidation events that occur during prolonged incubation of the recombinant enzyme.

Atherogenic properties of human monocytes induced by the carboxyl terminal proteolytic fragment of alpha-1-antitrypsin.

Atherosclerotic plaques contain a significant number of macrophage foam cells and are associated with an inflammatory state. Inflammation induces the secretion from monocytes and other cells of cytokines, reactive oxygen species, proteinases and proteinase inhibitors among many other molecular species. AAT is prominent among the serine proteinase inhibitors and is an important regulator of leukocyte elastase and proteinase-3. It has been shown that the stable AAT-proteinase complex can upregulate AAT biosynthesis, and we have shown that the shorter, carboxyl terminal peptide (C-36) resulting from proteinase cleavage of AAT polymerizes, and in its fibrillar form alters cellular metabolism. To test for a possible link between the inflammation-generated C-36 peptide and cellular processes associated with atherogenesis, we have studied the effects of the fibrillar form of this peptide at varying concentrations on human monocytes in culture. We have found that fibrillar C-36 at concentrations of greater than or equal to 5 micromol/l in monocyte cultures for 24 h significantly increases LDL binding and uptake, upregulates LDL receptors, induces cytokine production and glutathione reductase activity, and upregulates AAT synthesis. The expression of CD36 protein, LDL Scavenger receptor, is also upregulated by fibrillar C-36 and native LDL in the presence of C-36-activated monocytes is more oxidized than with unactivated control monocytes. The majority of monocytes cultured for 24 h in the presence of C-36 fibrils were transformed morphologically into macrophages. These data establish a direct molecular link, mediated by C-36 peptide of AAT, between inflammation and the oxidation and accumulation of lipid in monocyte-derived macrophages. This may be important for an understanding of the events conducive to atherogenesis.

Crystal structure of rabbit cytosolic serine hydroxymethyltransferase at 2.8 A resolution: mechanistic implications.

Serine hydroxymethyltransferase (SHMT) catalyzes the reversible cleavage of serine to form glycine and single carbon groups that are essential for many biosynthetic pathways. SHMT requires both pyridoxal phosphate (PLP) and tetrahydropteroylpolyglutamate (H4PteGlun) as cofactors, the latter as a carrier of the single carbon group. We describe here the crystal structure at 2.8 A resolution of rabbit cytosolic SHMT (rcSHMT) in two forms: one with the PLP covalently bound as an aldimine to the Nepsilon-amino group of the active site lysine and the other with the aldimine reduced to a secondary amine. The rcSHMT structure closely resembles the structure of human SHMT, confirming its similarity to the alpha-class of PLP enzymes. The structures reported here further permit identification of changes in the PLP group that accompany formation of the geminal diamine complex, the first intermediate in the reaction pathway. On the basis of the current mechanism derived from solution studies and the properties of site mutants, we are able to model the binding of both the serine substrate and the H4PteGlun cofactor. This model explains the properties of several site mutants of SHMT and offers testable hypotheses for a more detailed mechanism of this enzyme.

Fibrillar Alzheimer's amyloid peptide Abeta(1-42) stimulates low density lipoprotein binding and cell association, free radical production and cell cytotoxicity in PC12 cells.

Amyloid forming peptides are known to disturb vital cellular functions and induce cell death. However, the mechanisms by which fibrillogenic peptides induce cytotoxic effects in various cells has not been established. In this study the effects on low density lipoprotein binding and uptake of fibrils of the Alzheimer's amyloid beta-peptide (Abeta(1-42)), which is known to play a central role in the pathogenesis of Alzheimer's disease, were investigated in pheochromocytoma PC12 cells. Fibrillar Abeta(1-42) at micromol concentrations increased low-density lipoprotein (LDL) binding and cell association by 460% and 200% respectively, and LDL degradation by about 62%. Approximately 49% and 34% of Abeta fibril stimulated LDL cell association and degradation was inhibited by anti-LDL receptor antibodies. The soluble form of Abeta had no effect on any of these measures of LDL metabolism. The observed increased glutathione reductase activity, DNA fragmentation (TUNEL assay) and decreased DNA synthesis ([(3)H] thymidine incorporation assay) in cells treated with Abeta(1-42) fibrils alone or together with LDL relative to controls, suggests that the interaction of fibrils with LDL receptors might be one possible pathway which contributes to fibril cytotoxicity.

Crystal forms and subunit stoichiometry of serine hydroxymethyltransferase.

Serine hydroxymethyltransferase catalyzes the formation of one-carbon units essential for anabolic processes leading to the formation of such essential cellular components as purines, pyrimidines, amino acids, and lipids. Crystal structure determinations of several forms of the enzyme are under way, and to expand the comparative scope of these studies, we have crystallized the rabbit cytosolic and mitochondrial enzymes. Crystallization of serine hydroxymethyltransferase from different sources has often been problematic, and we report studies addressing these difficulties that may have more general application. The crystal lattice symmetry and the stoichiometry of the crystal asymmetric units and of the enzyme in solution suggest that serine hydroxymethyltransferase may exist as dimers, trimers, and possibly higher order complexes and that their aggregation state is affected by ionic strength.

Alzheimer's peptide Abeta1-42 binds to two beta-sheets of alpha1-antichymotrypsin and transforms it from inhibitor to substrate.

The serpin alpha1-antichymotrypsin is a major component of brain amyloid plaques in Alzheimer's disease. In vitro alpha1-antichymotrypsin interacts with the Alzheimer's amyloid peptide Abeta1-42 and stimulates both formation and disruption of neurotoxic Abeta1-42 fibrils in a concentration-dependent manner. We have constructed a new hybrid model of the complex between Abeta1-42 and alpha1-antichymotrypsin in which both amino and carboxyl sequences of Abeta1-42 insert into two different beta-sheets of alpha1-antichymotrypsin. We have tested this model and shown experimentally that full-length and amino-terminal segments of Abeta1-42 bind to alpha1-antichymotrypsin as predicted. We also show that Abeta1-42 forms both intra- and intermolecular SDS-stable complexes with alpha1-antichymotrypsin and that the binding of Abeta1-42 to alpha1-antichymotrypsin abolishes the inhibitory activity of the latter and its ability to form stable complex with chymotrypsin. The existence of both inter- as well as intramolecular complexes of Abeta1-42 explains the nonlinear concentration-dependent effects of alpha1-antichymotrypsin on Abeta1-42 fibril formation, which we have reinvestigated here over a broad range of Abeta1-42:alpha1-antichymotrypsin ratios. These data suggest a molecular basis for the distinction between amorphous and fibrillar Abeta1-42 in vivo. The reciprocal effects of Abeta1-42 and alpha1-antichymotrypsin could play a role in the etiology of Alzheimer's disease.

The C-terminal peptide of alpha-1-antitrypsin increases low density lipoprotein binding in HepG2 cells.

Hydrophobic fragments generated from various proteolytically degraded precursor proteins are known to form amyloid fibrils which have biological effects unrelated to precursor function. We examined the effects on HepG2 cells of the fibrillar and soluble C-terminal peptide [AAT-(358-394)] generated during the cleavage of the alpha-1-antitrypsin molecule by target proteinase. Soluble and fibrillar forms of AAT-(358-394)-peptide increased low-density lipoprotein (LDL) binding by 1-fold and 15-fold, respectively, an effect which was diminished by incubation or coincubation of cells with LDL, but was not affected by the serpin-enzyme complex. This effect of AAT-(358-394)-peptide appears to be on LDL receptor binding and not on the serpin-enzyme complex (SEC) or LDL receptor-related protein binding. The terminal deoxynucleotidyl transferase-biotin dUTP nick-end labeling assay for apoptosis showed increased DNA fragmentation in cells incubated with AAT-(358-394)-peptide fibrils relative to controls and LDL-incubated cells. [3H]Thymidine incorporation in cells incubated with fibrils for 4 days decreased by 60%. We conclude that interaction of AAT-(358-394)-peptide fibrils with cell surface receptor(s) disturbs intracellular cholesterol homeostasis and induces cell death.

Inflammation, antichymotrypsin, and lipid metabolism: autogenic etiology of Alzheimer's disease.

Alzheimer's disease is a multifactor pathology, some of whose causes have been inferred from genetic studies, primarily of associated early-onset cases. Much evidence implicates the A beta amyloid peptide as a neurotoxic agent, with chronic inflammation as an accompanying physiological contributor to the disease. The two central questions of how A beta kills neurons and of the autogenic basis of disease remain unanswered. We hypothesize that specific interactions of A beta with the inflammatory serpin, alpha 1-antichymotrypsin, abolish the serpin proteinase inhibitor activity and stimulate formation of the neurotoxic fibrillar form of A beta. Further, the fibrillar A beta interacts with specific cell surface receptors, prompting its own biosynthesis and disrupting cellular cholesterol metabolism. These molecular and cellular interactions autogenically sustain the processes of A beta formation, fibrillization, and receptor interaction, the last of which culminates in neuronal death through disruption of cholesterol metabolism.

The structural puzzle of how serpin serine proteinase inhibitors work.

Serine proteinase cleavage of proteins is essential to a wide variety of biological processes and is primarily regulated by protein inhibitors. Many inhibitors are conformationally rigid simulations of optimal serine proteinase substrates, which makes them highly efficient competitive inhibitors of target proteinases. In contrast, members of the serpin family of serine proteinase inhibitors display extensive flexibility and polymorphism, particularly in their reactive site segments and in beta-sheet secondary structure, which can take up and expel strands. Reactive site and beta-sheet polymorphism appear to be coupled in the serpins and may account for the extreme stability of serpin-proteinase complexes through the insertion of the reactive site strand into a beta-sheet. These unusual properties may have opened an adaptive pathway of proteinase regulation that was unavailable to the conformationally rigid proteinase inhibitors.

Structural basis for serpin inhibitor activity.

The mechanism of formation and the structures of serpin-inhibitor complexes are not completely understood, despite detailed knowledge of the structures of a number of cleaved and uncleaved inhibitor, noninhibitor, and latent serpins. It has been proposed from comparison of inhibitor and noninhibitor serpins in the cleaved and uncleaved forms that insertion of strand s4A into preexisting beta-sheet A is a requirement for serpin inhibitor activity. We have investigated the role of this strand in formation of serpin-proteinase complexes and in serpin inhibitor activity through homology modeling of wild type inhibitor, mutant substrate, and latent serpins, and of putative serpin-proteinase complexes. These models explain the high stability of the complexes and provide an understanding of substrate behavior in serpins with point mutations in s4A and of latency in plasminogen activator inhibitor I.