Expression of human beta-amyloid peptide in transgenic Caenorhabditis elegans.

Transgenic Caenorhabditis elegans nematodes have been engineered to express potentially amyloidic human proteins. These animals contain constructs in which the muscle-specific unc-54 promoter/enhancer of C. elegans drives the expression of the appropriate coding regions derived from human cDNA clones. Animals containing constructs expressing the 42-amino acid beta-amyloid peptide (derived from human amyloid precursor protein cDNA) produce muscle-specific deposits immunoreactive with anti-beta-amyloid polyclonal and monoclonal antibodies. A subset of these deposits also bind the amyloid-specific dye thioflavin S, indicating that these deposits have the tinctural characteristics of classic amyloid. Co-expression of beta-peptide and transthyretin, a protein implicated in preventing the formation of insoluble beta-amyloid, leads to a dramatic reduction in the number of dye-reactive deposits. These results suggest that this invertebrate model may be useful for in vivo investigation of factors that modulate amyloid formation.

A Saccharomyces cerevisiae homolog of the human adrenoleukodystrophy transporter is a heterodimer of two half ATP-binding cassette transporters.

The adrenoleukodystrophy protein (ALDP) and the 70-kDa peroxisomal membrane protein (PMP70) are half ATP-binding cassette (ABC) transporters in the human peroxisome membrane. ALDP and PMP70 share sequence homology and both are implicated in genetic diseases. PXA1 and YKL741 are Saccharomyces cerevisiae genes that encode homologs of ALDP and PMP70. Pxa1p, a putative ortholog of ALDP, is involved in peroxisomal beta-oxidation of fatty acids while YKL741 is an open reading frame found by the yeast genome sequencing project. Here we designate YKL741 as PXA2 and show that its protein product, Pxa2p, like Pxa1p, is associated with peroxisomes but not required for their assembly. Yeast strains carrying gene disruption of PXA1, PXA2, or both have similar and, in the case of the latter, nonadditive phenotypes. We also find that the stability of Pxa1p, but not Pxa2p, is markedly reduced in the absence of the other. Finally, we find that Pxa1p and Pxa2p coimmuno-precipitate. These genetic and physical data suggest that Pxa1p and Pxa2p heterodimerize to form a complete peroxisomal ABC transporter involved in fatty acid beta-oxidation. This result predicts the presence of similar heterodimeric ABC transporters in the mammalian peroxisome membrane.

Binding of a hairpin polyamide in the minor groove of DNA: sequence-specific enthalpic discrimination.

Hairpin polyamides are synthetic ligands for sequence-specific recognition in the minor groove of double-helical DNA. A thermodynamic characterization of the DNA-binding properties exhibited by a six-ring hairpin polyamide, ImPyPy-gamma-PyPyPy-beta-Dp (where Im = imidazole, Py = pyrrole, gamma = gamma-aminobutyric acid, beta = beta-alanine, and Dp = dimethylaminopropylamide), reveals an approximately 1-2 kcal/mol greater affinity for the designated match site, 5'-TGTTA-3', relative to the single base pair mismatch sites, 5'-TGGTA-3' and 5'-TATTA-3'. The enthalpy and entropy data at 20 degrees C reveal this sequence specificity to be entirely enthalpic in origin. Correlations between the thermodynamic driving forces underlying the sequence specificity exhibited by ImPyPy-gamma-PyPyPy-beta-Dp and the structural properties of the heterodimeric complex of PyPyPy and ImPyPy bound to the minor groove of DNA provide insight into the molecular forces that govern the affinity and specificity of pyrrole-imidazole polyamides.

Molecular basis of human mitochondrial very-long-chain acyl-CoA dehydrogenase deficiency causing cardiomyopathy and sudden death in childhood.

beta-Oxidation of long-chain fatty acids provides the major source of energy in the heart. Defects in enzymes of the beta-oxidation pathway cause sudden, unexplained death in childhood, acute hepatic encephalopathy or liver failure, skeletal myopathy, and cardiomyopathy. Very-long-chain acyl-CoA dehydrogenase [VLCAD; very-long-chain-acyl-CoA:(acceptor) 2,3-oxidoreductase, EC 1.3.99.13] catalyzes the first step in beta-oxidation. We have isolated the human VLCAD cDNA and gene and determined the complete nucleotide sequences. Polymerase chain reaction amplification of VLCAD mRNA and genomic exons defined the molecular defects in two patients with VLCAD deficiency who presented with unexplained cardiac arrest and cardiomyopathy. In one, a homozygous mutation in the consensus dinucleotide of the donor splice site (g+1-->a) was associated with universal skipping of the prior exon (exon 11). The second patient was a compound heterozygote, with a missense mutation, C1837-->T, changing the arginine at residue 613 to tryptophan on one allele and a single base deletion at the intron-exon 6 boundary as the second mutation. This initial delineation of human mutations in VLCAD suggests that VLCAD deficiency reduces myocardial fatty acid beta-oxidation and energy production and is associated with cardiomyopathy and sudden death in childhood.

Involvement of small GTP-binding proteins in defense signal-transduction pathways of higher plants.

Small GTP-binding proteins play a critical role in the regulation of a range of cellular processes--including growth, differentiation, and intracellular transportation. Previously, we isolated a gene, rgp1, encoding a small GTP-binding protein, by differential screening of a rice cDNA library with probe DNAs from rice tissues treated with or without 5-azacytidine, a powerful inhibitor of DNA methylation. To determine the physiological role of rgp1, the coding region was introduced into tobacco plants. Transformants, with rgp1 in either sense or antisense orientations, showed distinct phenotypic changes with reduced apical dominance, dwarfism, and abnormal flower development. These abnormal phenotypes appeared to be associated with the higher levels of endogenous cytokinins that were 6-fold those of wild-type plants. In addition, the transgenic plants produced salicylic acid and salicylic acid-beta-glucoside in an unusual response to wounding, thus conferring increased resistance to tobacco mosaic virus infection. In normal plants, the wound- and pathogen-induced signal-transduction pathways are considered to function independently. However, the wound induction of salicylic acid in the transgenic plants suggests that expression of rgp1 somehow interfered with the normal signaling pathways and resulted in cross-signaling between these distinct transduction systems. The results imply that the defense signal-transduction system consists of a complicated and finely tuned network of several regulatory factors, including cytokinins, salicylic acid, and small GTP-binding proteins.

Subcellular localization of gamma-aminobutyric acid type A receptors is determined by receptor beta subunits.

gamma-aminobutyric acid type A (GABAA) receptors are the major sites of fast synaptic inhibition in the brain. They are constructed from four subunit classes with multiple members: alpha (1-6), beta (1-4), gamma (1-4), and delta (1). The contribution of subunit diversity in determining receptor subcellular targeting was examined in polarized Madin-Darby canine kidney (MDCK) cells. Significant detection of cell surface homomeric receptor expression by a combination of both immunological and electrophysiological methodologies was only found for the beta 3 subunit. Expression of alpha/beta binary combinations resulted in a nonpolarized distribution for alpha 1 beta 1 complexes, but specific basolateral targeting of both alpha 1 beta 2 and alpha 1 beta 3 complexes. The polarized distribution of these alpha/beta complexes was unaffected by the presence of the gamma 2S subunit. Interestingly, delivery of receptors containing the beta 3 subunit to the basolateral domain occurs via the apical surface. These results show that beta subunits can selectively target GABAA receptors to distinct cellular locations. Changes in the spatial and temporal expression of beta-subunit isoforms may therefore provide a mechanism for relocating GABAA receptor function between distinct neuronal domains. Given the critical role of these receptors in mediating synaptic inhibition, the contribution of different beta subunits in GABAA receptor function, may have implications in neuronal development and for receptor localization/clustering.

Cell-type and promoter-context dependent retinoic acid receptor (RAR) redundancies for RAR beta 2 and Hoxa-1 activation in F9 and P19 cells can be artefactually generated by gene knockouts.

By using RAR type (alpha, beta, or gamma)-specific synthetic retinoids and a pan-retinoic X receptor (RXR)-specific ligand, we have investigated the contribution of RARs and RXRs in the activation of RA target genes and the differentiation of embryonal carcinoma cells. We demonstrate cell-type- and promoter context-dependent functional redundancies that differ between the three RAR types for mediating the induction of RARbeta2 and Hoxa-1 in wild-type, RARgamma-/- and RARalpha-/- F9 cells and in P19 cells. The extent of redundancy between RARs is further modulated by the synergistic activation of RXRs with a pan-RXR agonist. We also demonstrate that the expression of RARbeta2 is auto-inducible in RARgamma-/- but not in wild-type F9 cells, indicating that the functional redundancies observed between RARs in gene disruption studies can be artefactually generated. Thus, even though all three RARs can functionally substitute each other for inducing the expression of RA target genes and cell differentiation, one RAR can cell-specifically override the activity of the other RARs. Interestingly, only RARgamma can mediate the retinoic acid-induced differentiation of wild-type F9 cells, whereas the differentiation of P19 cells can be mediated by either RARalpha or RARgamma.

Protein crosslinking studies suggest that Rhizobium meliloti C4-dicarboxylic acid transport protein D, a sigma 54-dependent transcriptional activator, interacts with sigma 54 and the beta subunit of RNA polymerase.

Rhizobium meliloti C4-dicarboxylic acid transport protein D (DCTD) activates transcription by a form of RNA polymerase holoenzyme that has sigma 54 as its sigma factor (referred to as E sigma 54). DCTD catalyzes the ATP-dependent isomerization of closed complexes between E sigma 54 and the dctA promoter to transcriptionally productive open complexes. Transcriptional activation probably involves specific protein-protein interactions between DCTD and E sigma 54. Interactions between sigma 54-dependent activators and E sigma 54 are transient, and there has been no report of a biochemical assay for contact between E sigma 54 and any activator to date. Heterobifunctional crosslinking reagents were used to examine protein-protein interactions between the various subunits of E sigma 54 and DCTD. DCTD was crosslinked to Salmonella typhimurium sigma 54 with the crosslinking reagents succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate and N-hydroxysulfosuccinimidyl-4-azidobenzoate. Cys-307 of sigma 54 was identified by site-directed mutagenesis as the residue that was crosslinked to DCTD. DCTD was also crosslinked to the beta subunit of Escherichia coli core RNA polymerase with succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, but not with N-hydroxysulfosuccinimidyl-4-azidobenzoate. These data suggest that interactions of DCTD with sigma 54 and the beta subunit may be important for transcriptional activation and offer evidence for interactions between a sigma 54-dependent activator and sigma 54, as well as the beta subunit of RNA polymerase.

Aromatic-L-amino-acid decarboxylase, a pyridoxal phosphate-dependent enzyme, is a beta-cell autoantigen.

Different autoantigens are thought to be involved in the pathogenesis of insulin-dependent diabetes mellitus, and they may account for the variation in the clinical presentation of the disease. Sera from patients with autoimmune polyendocrine syndrome type I contain autoantibodies against the beta-cell proteins glutamate decarboxylase and an unrelated 51-kDa antigen. By screening of an expression library derived from rat insulinoma cells, we have identified the 51-kDa protein as aromatic-L-amino-acid decarboxylase (EC 4.1.1.28). In addition to the previously published full-length cDNA, forms coding for a truncated and an alternatively spliced version were identified. Aromatic L-amino acid decarboxylase catalyzes the decarboxylation of L-5-hydroxytryptophan to serotonin and that of L-3,4-dihydroxyphenylalanine to dopamine. Interestingly, pyridoxal phosphate is the cofactor of both aromatic L-amino acid decarboxylase and glutamate decarboxylase. The biological significance of the neurotransmitters produced by the two enzymes in the beta cells remains largely unknown.

Reexpression of retinoic acid receptor (RAR) gamma or overexpression of RAR alpha or RAR beta in RAR gamma-null F9 cells reveals a partial functional redundancy between the three RAR types.

Disruption of retinoic acid receptor (RAR) gamma in F9 embryonal carcinoma cells leads to aberrent differentiation and reduced activation of expression of several all-trans-retinoic acid (RA)-induced genes. We have analyzed the expression of several additional RA-responsive genes in RAR alpha- and RAR gamma-null F9 cells. The RA-induced activation of Cdx1, Gap43, Stra4, and Stra6 was specifically impaired in RAR gamma-null cells, supporting the idea that each RAR may regulate distinct subsets of target genes. To further investigate the role of RAR gamma in F9 cell differentiation, "rescue" cell lines reexpressing RAR gamma 2 or overexpressing either RAR alpha 1 or RAR beta 2 were established in RAR gamma-null cells. Reexpression of RAR gamma or overexpression of RAR alpha restored both target-gene activation and the differentiation potential. In contrast, over-expression of RAR beta only poorly restored differentiation, although it could replace RAR gamma for the activation of target genes. Functional redundancy between the various RARs is discussed.

Age-related learning deficits in transgenic mice expressing the 751-amino acid isoform of human beta-amyloid precursor protein.

The beta-amyloid precursor protein (beta-APP), from which the beta-A4 peptide is derived, is considered to be central to the pathogenesis of Alzheimer disease (AD). Transgenic mice expressing the 751-amino acid isoform of human beta-APP (beta-APP751) have been shown to develop early AD-like histopathology with diffuse deposits of beta-A4 and aberrant tau protein expression in the brain, particularly in the hippocampus, cortex, and amygdala. We now report that beta-APP751 transgenic mice exhibit age-dependent deficits in spatial learning in a water-maze task and in spontaneous alternation in a Y maze. These deficits were mild or absent in 6-month-old transgenic mice but were severe in 12-month-old transgenic mice compared to age-matched wild-type control mice. No other behavioral abnormalities were observed. These mice therefore model the progressive learning and memory impairment that is a cardinal feature of AD. These results provide evidence for a relationship between abnormal expression of beta-APP and cognitive impairments.

Development of pilus organelle subassemblies in vitro depends on chaperone uncapping of a beta zipper

The major subassemblies of virulence-associated P pili, the pilus rod (comprised of PapA) and tip fibrillum (comprised of PapE), were reconstituted from purified chaperone-subunit complexes in vitro. Subunits are held in assembly-competent conformations in chaperone-subunit complexes prior to their assembly into mature pili. The PapD chaperone binds, in part, to a conserved motif present at the C terminus of the subunits via a beta zippering interaction. Amino acid residues in this conserved motif were also found to be essential for subunit–subunit interactions necessary for the formation of pili, thus revealing a molecular mechanism whereby the PapD chaperone may prevent premature subunit–subunit interactions in the periplasm. Uncapping of the chaperone-protected C terminus of PapA and PapE was mimicked in vitro by freeze–thaw techniques and resulted in the formation of pilus rods and tip fibrillae, respectively. A mutation in the leading edge of the beta zipper of PapA produces pilus rods with an altered helical symmetry and azimuthal disorder. This change in the number of subunits per turn of the helix most likely reflects involvement of the leading edge of the beta zipper in forming a right-handed helical cylinder. Organelle development is a fundamental process in all living cells, and these studies shed new light on how immunoglobulin-like chaperones govern the formation of virulence-associated organelles in pathogenic bacteria.

Retinoic acid X receptor in the diploblast, Tripedalia cystophora

Nuclear hormone receptors comprise a characteristic family of transcription factors found in vertebrates, insects and nematodes. Here we show by cDNA and gene cloning that a Cnidarian, Tripedalia cystophora, possesses a retinoid receptor (jRXR) with remarkable homology to vertebrate retinoic acid X receptors (RXRs). Like vertebrate RXRs, jRXR binds 9-cis retinoic acid (Kd = 4 × 10−10 M) and binds to the DNA sequence, PuGGTCA as a monomer in vitro. jRXR also heterodimerizes with Xenopus TR beta on a thyroid responsive element of a direct repeat separated by 4 bp. A jRXR binding half-site capable of interacting with (His6)jRXR fusion protein was identified in the promoters of three T. cystophora crystallin genes that are expressed highly in the eye lens of this jellyfish. Because crystallin gene expression is regulated by retionoid signaling in vertebrates, the jellyfish crystallin genes are candidate in vivo targets for jRXR. Finally, an antibody prepared against (His6)jRXR showed that full-length jRXR is expressed at all developmental stages of T. cystophora except the ephydra, where a smaller form replaces is. These data show that Cnidaria, a diploblastic phylum ancestral to the triploblastic invertebrate and subsequent vertebrate lineages, already have an RXR suggesting that RXR is an early component of the regulatory mechanisms of metazoa.

An alternative pathway to β-carotene formation in plant chromoplasts discovered by map-based cloning of Beta and old-gold color mutations in tomato

Carotenoid pigments in plants fulfill indispensable functions in photosynthesis. Carotenoids that accumulate as secondary metabolites in chromoplasts provide distinct coloration to flowers and fruits. In this work we investigated the genetic mechanisms that regulate accumulation of carotenoids as secondary metabolites during ripening of tomato fruits. We analyzed two mutations that affect fruit pigmentation in tomato (Lycopersicon esculentum): Beta (B), a single dominant gene that increases β-carotene in the fruit, and old-gold (og), a recessive mutation that abolishes β-carotene and increases lycopene. Using a map-based cloning approach we cloned the genes B and og. Molecular analysis revealed that B encodes a novel type of lycopene β-cyclase, an enzyme that converts lycopene to β-carotene. The amino acid sequence of B is similar to capsanthin-capsorubin synthase, an enzyme that produces red xanthophylls in fruits of pepper (Capsicum annum). Our results prove that β-carotene is synthesized de novo during tomato fruit development by the B lycopene cyclase. In wild-type tomatoes B is expressed at low levels during the breaker stage of ripening, whereas in the Beta mutant its transcription is dramatically increased. Null mutations in the gene B are responsible for the phenotype in og, indicating that og is an allele of B. These results confirm that developmentally regulated transcription is the major mechanism that governs lycopene accumulation in ripening fruits. The cloned B genes can be used in various genetic manipulations toward altering pigmentation and enhancing nutritional value of plant foods.

Cloning of human acetyl-CoA carboxylase-beta and its unique features.

Acetyl-CoA carboxylase, which has a molecular mass of 265 kDa (ACC-alpha), catalyzes the rate-limiting step in the biosynthesis of long-chain fatty acids. In this study we report the complete amino acid sequence and unique features of an isoform of ACC with a molecular mass of 275 kDa (ACC-beta), which is primarily expressed in heart and skeletal muscles. In these tissues, ACC-beta may be involved in the regulation of fatty acid oxidation, rather than fatty acid biosynthesis. ACC-beta contains an amino acid sequence at the N terminus which is about 200 amino acids long and may be uniquely related to the role of ACC-beta in controlling carnitine palmitoyltransferase I activity and fatty acid oxidation by mitochondria. If we exclude this unique sequence at the N terminus the two forms of ACC show about 75% amino acid identity. All of the known functional domains of ACC are found in the homologous regions. Human ACC-beta cDNA has an open reading frame of 7,343 bases, encoding a protein of 2,458 amino acids, with a calculated molecular mass of 276,638 Da. The mRNA size of human ACC-beta is approximately 10 kb and is primarily expressed in heart and skeletal muscle tissues, whereas ACC-alpha mRNA is detected in all tissues tested. A fragment of ACC-beta cDNA was expressed in Escherichia coli and antibodies against the peptide were generated to establish that the cDNA sequence that we cloned is that for ACC-beta.