Duplication and functional divergence in the chalcone synthase gene family of Asteraceae: evolution with substrate change and catalytic simplification.

Plant-specific polyketide synthase genes constitute a gene superfamily, including universal chalcone synthase [CHS; malonyl-CoA:4-coumaroyl-CoA malonyltransferase (cyclizing) (EC 2.3.1.74)] genes, sporadically distributed stilbene synthase (SS) genes, and atypical, as-yet-uncharacterized CHS-like genes. We have recently isolated from Gerbera hybrida (Asteraceae) an unusual CHS-like gene, GCHS2, which codes for an enzyme with structural and enzymatic properties as well as ontogenetic distribution distinct from both CHS and SS. Here, we show that the GCHS2-like function is encoded in the Gerbera genome by a family of at least three transcriptionally active genes. Conservation within the GCHS2 family was exploited with selective PCR to study the occurrence of GCHS2-like genes in other Asteraceae. Parsimony analysis of the amplified sequences together with CHS-like genes isolated from other taxa of angiosperm subclass Asteridae suggests that GCHS2 has evolved from CHS via a gene duplication event that occurred before the diversification of the Asteraceae. Enzyme activity analysis of proteins produced in vitro indicates that the GCHS2 reaction is a non-SS variant of the CHS reaction, with both different substrate specificity (to benzoyl-CoA) and a truncated catalytic profile. Together with the recent results of Durbin et al. [Durbin, M. L., Learn, G. H., Jr., Huttley, G. A. & Clegg, M. T. (1995) Proc. Natl. Acad. Sci. USA 92, 3338-3342], our study confirms a gene duplication-based model that explains how various related functions have arisen from CHS during plant evolution.

Cleavage of lamin A by Mch2 alpha but not CPP32: multiple interleukin 1 beta-converting enzyme-related proteases with distinct substrate recognition properties are active in apoptosis.

Although proteases related to the interleukin 1 beta-converting enzyme (ICE) are known to be essential for apoptotic execution, the number of enzymes involved, their substrate specificities, and their specific roles in the characteristic biochemical and morphological changes of apoptosis are currently unknown. These questions were addressed using cloned recombinant ICE-related proteases (IRPs) and a cell-free model system for apoptosis (S/M extracts). First, we compared the substrate specificities of two recombinant human IRPs, CPP32 and Mch2 alpha. Both enzymes cleaved poly-(ADP-ribose) polymerase, albeit with different efficiencies. Mch2 alpha also cleaved recombinant and nuclear lamin A at a conserved VEID decreases NG sequence located in the middle of the coiled-coil rod domain, producing a fragment that was indistinguishable from the lamin A fragment observed in S/M extracts and in apoptotic cells. In contrast, CPP32 did not cleave lamin A. The cleavage of lamin A by Mch2 alpha and by S/M extracts was inhibited by millimolar concentrations of Zn2+, which had a minimal effect on cleavage of poly (ADP-ribose) polymerase by CPP32 and by S/M extracts. We also found that N-(acetyltyrosinylvalinyl-N epsilon-biotinyllysyl)aspartic acid [(2,6-dimethylbenzoyl)oxy]methyl ketone, which derivatizes the larger subunit of active ICE, can affinity label up to five active IRPs in S/M extracts. Together, these observations indicate that the processing of nuclear proteins in apoptosis involves multiple IRPs having distinct preferences for their apoptosis-associated substrates.

Evidence for an insulin receptor substrate 1 independent insulin signaling pathway that mediates insulin-responsive glucose transporter (GLUT4) translocation.

Interaction of the activated insulin receptor (IR) with its substrate, insulin receptor substrate 1 (IRS-1), via the phosphotyrosine binding domain of IRS-1 and the NPXY motif centered at phosphotyrosine 960 of the IR, is important for IRS-1 phosphorylation. We investigated the role of this interaction in the insulin signaling pathway that stimulates glucose transport. Utilizing microinjection of competitive inhibitory reagents in 3T3-L1 adipocytes, we have found that disruption of the IR/IRS-1 interaction has no effect upon translocation of the insulin-responsive glucose transporter (GLUT4). The activity of these reagents was demonstrated by their ability to block insulin stimulation of two distinct insulin bioeffects, membrane ruffling and mitogenesis, in 3T3-L1 adipocytes and insulin-responsive rat 1 fibroblasts. These data suggest that phosphorylated IRS-1 is not an essential component of the metabolic insulin signaling pathway that leads to GLUT4 translocation, yet it appears to be required for other insulin bioeffects.

U1 small nuclear RNA chimeric ribozymes with substrate specificity for the Rev pre-mRNA of human immunodeficiency virus.

The in vivo effectiveness of ribozymes strongly depends on the correct choice of the vector molecule. High levels of expression, stability, active conformation, and correct cellular localization are the most important features for a ribozyme vector. We have exploited the utilization of the U1 small nuclear RNA (snRNA) as a vector for specifically targeting a ribozyme into the nucleus. The Rev pre-mRNA of human immunodeficiency virus type 1 was chosen as target for testing the activity of the Ul-ribozyme. The catalytic core of the hammerhead motif, plus the recognition sequences, substituted the stem-loop III of the U1 snRNA. The resulting construct displays efficient cleavage activity in vitro. In addition, in the in vivo system of Xenopus laevis oocytes, the Ul-chimeric ribozyme accumulates in large amounts in the nucleus and produces a considerable reduction of Rev pre-mRNA levels. The Rev-specific ribozyme was also inserted in a derivative of the Ul snRNA mutated in the region of pairing with the 5' splice site, such as to match it with the suboptimal splice junction of the Rev precursor. This construct shows more efficient reduction of Rev pre-mRNA in vivo than the wild-type U1 vector.

Substrate-bound agrin induces expression of acetylcholine receptor epsilon-subunit gene in cultured mammalian muscle cells.

Expression of the epsilon-subunit gene of the acetylcholine receptor (AChR) by myonuclei located at the neuromuscular junction is precisely regulated during development. A key role in this regulation is played by the synaptic portion of the basal lamina, a structure that is also known to contain agrin, a component responsible for the formation of postsynaptic specializations. We tested whether agrin has a function in synaptic AChR gene expression. Synaptic basal lamina from native adult muscle and recombinant agrin bound to various substrates induced in cultured rat myotubes AChR clusters that were colocalized with epsilon-subunit mRNA. Estimation of transcript levels by Northern hybridization analysis of total RNA showed a significant increase when myotubes were grown on substrate impregnated with agrin, but were unchanged when agrin was applied in the medium. The effect was independent of the receptor aggregating activity of the agrin isoform used, and agrin acted, at least in part, at the level of epsilon-subunit gene transcription. These findings are consistent with a role of agrin in the regulation of AChR subunit gene expression at the neuromuscular junction, which would depend on its binding to the synaptic basal lamina.

Thiocalsin: a thioredoxin-linked, substrate-specific protease dependent on calcium.

We describe a protease, named "thiocalsin," that is activated by calcium but only after reductive activation by thioredoxin, a small protein with a redox-active disulfide group that functions widely in regulation. Thiocalsin appeared to be a 14-kDa serine protease that functions independently of calmodulin. The enzyme, purified from germinating wheat grain, specifically cleaved the major indigenous storage proteins, gliadins and glutenins, after they too had been reduced, preferentially by thioredoxin. The disulfide groups of the enzyme, as well as its protein substrates, were reduced by thioredoxin via NADPH and the associated enzyme, NADP-thioredoxin reductase. The results broaden the roles of thioredoxin and calcium and suggest a joint function in activating thiocalsin, thereby providing amino acids for germination and seedling development.

Protein-free cell culture on an artificial substrate with covalently immobilized insulin.

Insulin was immobilized on a surface-hydrolyzed poly(methyl methacrylate) film. Chinese hamster ovary cells overexpressing human insulin receptors were cultured on the film in the absence of serum or soluble proteins. Small amounts of immobilized insulin (1-10% of the required amount of free insulin) were sufficient to stimulate cell proliferation. In addition, the maximal mitogenic effect of immobilized insulin was greater than that of free insulin. Immobilized insulin activated the insulin receptor and downstream signaling proteins, and this activation persisted for longer periods than that obtained with free insulin, probably explaining the greater mitogenic effect of the immobilized insulin. Finally the immobilized-insulin film was usable repeatedly without marked loss of activity.

Multidrug resistance proteins QacA and QacB from Staphylococcus aureus: membrane topology and identification of residues involved in substrate specificity.

The closely related multidrug efflux pumps QacA and QacB, from the bacterial pathogen Staphylococcus aureus, both confer resistance to various toxic organic cations but differ in that QacB mediates lower levels of resistance to divalent cations. Cloning and nucleotide sequencing of the qacB gene revealed that qacB differs from qacA by only seven nucleotide substitutions. Random hydroxylamine mutagenesis of qacB was undertaken, selecting for variants that conferred increased resistance to divalent cations. Both QacA and the QacB mutants capable of conferring resistance to divalent cations contain an acidic residue at either amino acid 322 or 323, whereas QacB contains uncharged residues in these positions. Site-directed mutagenesis of qacA confirmed the importance of an acidic residue within this region of QacA in conferring resistance to divalent cations. Membrane topological analysis using alkaline phosphatase and beta-galactosidase fusions indicated that the QacA protein contains 14 transmembrane segments. Thus, QacA represents the first membrane transport protein shown to contain 14 transmembrane segments, and confirms that the major facilitator superfamily contains a family of proteins with 14 transmembrane segments.

Genetic evidence that the RAG1 protein directly participates in V(D)J recombination through substrate recognition.

RAG1 protein is essential for the activation of V(D)J recombination in developing lymphocytes (V, variable; D, diversity; J, joining). However, it has not been determined whether its role involves substrate recognition and catalysis. A single amino acid substitution mutation in the RAG1 gene has now been identified that renders its activity sensitive to the sequence of the coding region abutting the heptamer site in the recombination signal sequence. These results strongly imply that RAG1 interacts directly with DNA.

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.

An anatomical substrate for experience-dependent plasticity of the rat barrel field cortex.

The objective of this study was to examine the influence of sensory experience on the synaptic circuitry of the cortex. For this purpose, the quantitative distribution of the overall and of the gamma-aminobutyric acid (GABA) population of synaptic contacts was investigated in each layer of the somatosensory barrel field cortex of rats which were sensory deprived from birth by continuously removing rows of whiskers. Whereas there were no statistically significant changes in the quantitative distribution of the overall synaptic population, the number and proportion of GABA-immunopositive synaptic contacts were profoundly altered in layer IV of the somatosensory cortex of sensory-deprived animals. These changes were attributable to a specific loss of as many as two-thirds of the GABA contacts targeting dendritic spines. Thus, synaptic contacts made by GABA terminals in cortical layer IV and, in particular, those targeting dendritic spines represent a structural substrate of experience-dependent plasticity. Furthermore, since in this model of cortical plasticity the neuronal receptive-field properties are known to be affected, we propose that the inhibitory control of dendritic spines is essential for the elaboration of these functional properties.

Modification of the substrate specificity of an acyl-acyl carrier protein thioesterase by protein engineering.

The plant acyl-acyl carrier protein (ACP) thioesterases (TEs) are of biochemical interest because of their roles in fatty acid synthesis and their utilities in the bioengineering of plant seed oils. When the FatB1 cDNA encoding a 12:0-ACP TE (Uc FatB1) from California bay, Umbellularia californica (Uc) was expressed in Escherichia coli and in developing oilseeds of the plants Arabidopsis thaliana and Brassica napus, large amounts of laurate (12:0) and small amounts of myristate (14:0) were accumulated. We have isolated a TE cDNA from camphor (Cinnamomum camphorum) (Cc) seeds that shares 92% amino acid identity with Uc FatB1. This TE, Cc FatB1, mainly hydrolyzes 14:0-ACP as shown by E. coli expression. We have investigated the roles of the N- and C-terminal regions in determining substrate specificity by constructing two chimeric enzymes, in which the N-terminal portion of one protein is fused to the C-terminal portion of the other. Our results show that the C-terminal two-thirds of the protein is critical for the specificity. By site-directed mutagenesis, we have replaced several amino acids in Uc FatB1 by using the Cc FatB1 sequence as a guide. A double mutant, which changes Met-197 to an Arg and Arg-199 to a His (M197R/R199H), turns Uc FatB1 into a 12:0/14:0 TE with equal preference for both substrates. Another mutation, T231K, by itself does not effect the specificity. However, when it is combined with the double mutant to generate a triple mutant (M197R/R199H/T231K), Uc FatB1 is converted to a 14:0-ACP TE. Expression of the double-mutant cDNA in E. coli K27, a strain deficient in fatty acid degradation, results in accumulation of similar amounts of 12:0 and 14:0. Meanwhile the E. coli expressing the triple-mutant cDNA produces predominantly 14:0 with very small amounts of 12:0. Kinetic studies indicate that both wild-type Uc FatB1 and the triple mutant have similar values of Km,app with respect to 14:0-ACP. Inhibitory studies also show that 12:0-ACP is a good competitive inhibitor with respect to 14:0-ACP in both the wild type and the triple mutant. These results imply that both 12:0- and 14:0-ACP can bind to the two proteins equally well, but in the case of the triple mutant, the hydrolysis of 12:0-ACP is severely impaired. The ability to modify TE specificity should allow the production of additional "designer oils" in genetically engineered plants.

Interaction between focal adhesion kinase and Crk-associated tyrosine kinase substrate p130Cas.

The focal adhesion kinase (FAK) has been implicated in integrin-mediated signaling events and in the mechanism of cell transformation by the v-Src and v-Crk oncoproteins. To gain further insight into FAK signaling pathways, we used a two-hybrid screen to identify proteins that interact with mouse FAK. The screen identified two proteins that interact with FAK via their Src homology 3 (SH3) domains: a v-Crk-associated tyrosine kinase substrate (Cas), p130Cas, and a still uncharacterized protein, FIPSH3-2, which contains an SH3 domain closely related to that of p130Cas. These SH3 domains bind to the same proline-rich region of FAK (APPKPSR) encompassing residues 711-717. The mouse p130Cas amino acid sequence was deduced from cDNA clones, revealing an overall high degree of similarity to the recently reported rat sequence. Coimmunoprecipitation experiments confirmed that p130Cas and FAK are associated in mouse fibroblasts. The stable interaction between p130Cas and FAK emerges as a likely key element in integrin-mediated signal transduction and further represents a direct molecular link between the v-Src and v-Crk oncoproteins. The Src family kinase Fyn, whose Src homology 2 (SH2) domain binds to the major FAK autophosphorylation site (tyrosine 397), was also identified in the two-hybrid screen.

A protein-binding domain, EH, identified in the receptor tyrosine kinase substrate Eps15 and conserved in evolution.

In this report we structurally and functionally define a binding domain that is involved in protein association and that we have designated EH (for Eps15 homology domain). This domain was identified in the tyrosine kinase substrate Eps15 on the basis of regional conservation with several heterogeneous proteins of yeast and nematode. The EH domain spans about 70 amino acids and shows approximately 60% overall amino acid conservation. We demonstrated the ability of the EH domain to specifically bind cytosolic proteins in normal and malignant cells of mesenchymal, epithelial, and hematopoietic origin. These observations prompted our search for additional EH-containing proteins in mammalian cells. Using an EH domain-specific probe derived from the eps15 cDNA, we cloned and characterized a cDNA encoding an EH-containing protein with overall similarity to Eps15; we designated this protein Eps15r (for Eps15-related). Structural comparison of Eps15 and Eps15r defines a family of signal transducers possessing extensive networking abilities including EH-mediated binding and association with Src homology 3-containing proteins.

Tyrosine phosphorylation of protein kinase C-delta in response to the activation of the high-affinity receptor for immunoglobulin E modifies its substrate recognition.

The delta isoform of protein kinase C is phosphorylated on tyrosine in response to antigen activation of the high-affinity receptor for immunoglobulin E. While protein kinase C-delta associates with and phosphorylates this receptor, immunoprecipitation of the receptor revealed that little, if any, tyrosine-phosphorylated protein kinase C-delta is receptor associated. In vitro kinase assays with immunoprecipitated tyrosine-phosphorylated protein kinase C-delta showed that the modified enzyme had diminished activity toward the receptor gamma-chain peptide as a substrate but not toward histones or myelin basic protein peptide. We propose a model in which the tyrosine phosphorylation of protein kinase C-delta regulates the kinase specificity toward a given substrate. This may represent a general mechanism by which in vivo protein kinase activities are regulated in response to external stimuli.