Identification of novel benzoylformate decarboxylases by growth selection

A growth selection system was established using Pseudomonas putida, which can grow on benzaldehyde as the sole carbon source. These bacteria presumably metabolize benzaldehyde via the beta-ketoadipate pathway and were unable to grow in benzoylformate-containing selective medium, but the growth deficiency could be restored by expression in trans of genes encoding benzoylformate decarboxylases. The selection system was used to identify three novel benzoylformate decarboxylases, two of them originating from a chromosomal library of P. putida ATCC 12633 and the third from an environmental-DNA library. The novel P. putida enzymes BfdB and BfdC exhibited 83% homology to the benzoylformate decarboxylase from P. aeruginosa and 63% to the enzyme MdlC from P. putida ATCC 12633, whereas the metagenomic BfdM exhibited 72% homology to a putative benzoylformate decarboxylase from Polaromonas naphthalenivorans. BfdC was overexpressed in Escherichia coli, and the enzymatic activity was determined to be 22 U/ml using benzoylformate as the substrate. Our results clearly demonstrate that P. putida KT2440 is an appropriate selection host strain suitable to identify novel benzoylformate decarboxylase-encoding genes. In principle, this system is also applicable to identify a broad range of different industrially important enzymes, such as benzaldehyde lyases, benzoylformate decarboxylases, and hydroxynitrile lyases, which all catalyze the formation of benzaldehyde.     

Widespread occurrence of glyceryl ether monooxygenase activity in rat tissues detected by a novel assay

An assay was set up for glyceryl ether monooxygenase activity in tissue samples using the novel substrate 1-O-pyrenedecyl-sn-glycerol and high-performance liquid chromatographic analysis of reaction mixtures with fluorescence detection, allowing robust detection of enzymatic activity in microgram amounts of tissue homogenates. The activity partially purified from rat liver strictly depended on the presence of a tetrahydropteridine. Tetrahydrobiopterin-dependent glyceryl ether monooxygenase activity was observed in all rat tissues tested except female heart, with highest activities in liver, intestine, and cerebellum. Activity was not uniformly distributed in brain: it was higher in cerebellum than in striatum or cortex. These data demonstrate that tetrahydrobiopterin-dependent glyceryl ether monooxygenase is found not only in liver and the gastrointestinal tract but also in brain and other organs of the rat and provide an additional goal for tetrahydrobiopterin biosynthesis in these organs.     

A novel inhibitor of cyclin-Cdk activity detected in transforming growth factor beta-arrested epithelial cells.

Transforming growth factor beta (TGF-beta) is a potent inhibitor of epithelial cell growth. Cyclins E and A in association with Cdk2 have been shown to play a role in the G1-to-S phase transition in mammalian cells. We have studied the effects of TGF-beta-mediated growth arrest on G1/S cyclins E and A. Inhibition of cyclin A-associated kinase by TGF-beta is primarily due to a decrease in cyclin A mRNA and protein. By contrast, while TGF-beta inhibits accumulation of cyclin E mRNA, the reduction in cyclin E protein is minimal. Instead, we find that the activation of cyclin E-associated kinase that normally accompanies the G1-to-S phase transition is inhibited. A novel inhibitor of cyclin-Cdk complexes was detected in TGF-beta-treated cell lysates. Inhibition is mediated by a heat-stable protein that targets both Cdk2 and Cdc2 kinases. In G0-arrested cells, a similar inhibitor of Cdk2 kinase was detected. These data suggest the existence of an inhibitor of cyclin-dependent kinases induced under different conditions to mediate antiproliferative responses.     

Regulation of heparan sulfate 6-O-sulfation by beta-secretase activity

The enzymes involved in glycosaminoglycan chain biosynthesis are mostly Golgi resident proteins, but some are secreted extracellularly. For example, the activities of heparan sulfate 6-O-sulfotransferase (HS6ST) and heparan sulfate 3-O-sulfotransferase are detected in the serum as well in the medium of cell lines. However, the biological significance of this is largely unknown. Here we have investigated by means of monitoring green fluorescent protein (GFP) fluorescence how C-terminally GFP-tagged HS6STs that are stably expressed in CHO-K1 cell lines are secreted/shed. Brefeldin A and monensin treatments revealed that the N-terminal hydrophobic domain of HS6ST3 is processed in the endoplasmic reticulum or cis/medial Golgi. Treatment of HS6ST3-GFP-expressing cells with various protease inhibitors revealed that the cell-permeable beta-secretase inhibitor N-benzyloxycarbonyl-Val-Leu-leucinal (Z-VLL-CHO) specifically inhibits HS6ST secretion, although this effect was specific for HS6ST3 but not for HS6ST1 and HS6ST2. However, Z-VLL-CHO treatment did not increase the molecular size of the HS6ST3-GFP that accumulated in the cell. Z-VLL-CHO treatment also induced the intracellular accumulation of SP-HS6ST3(-TMD)-GFP, a modified secretory form of HS6ST3 that has the preprotrypsin leader sequence as its N-terminal hydrophobic domain. Diminishment of beta-secretase activity by coexpressing the amyloid precursor protein of a Swedish mutant, a potent beta-secretase substrate, also induced intracellular HS6ST3-GFP accumulation. Moreover, Z-VLL-CHO treatment increased the 6-O-sulfate (6S) levels of HS, especially in the disaccharide unit of hexuronic acid-GlcNS(6S). Thus, the HS6ST3 enzyme in the Golgi apparatus and therefore the 6-O sulfation of heparan sulfates in the cell are at least partly regulated by beta-secretase via an indirect mechanism.     

Characterization of azo reduction activity in a novel ascomycete yeast strain

Several model azo dyes are reductively cleaved by growing cultures of an ascomycete yeast species, Issatchenkia occidentalis. In liquid media containing 0.2 mM dye and 2% glucose in a mineral salts base, more than 80% of the dyes are removed in 15 h, essentially under microaerophilic conditions. Under anoxic conditions, decolorization does not occur, even in the presence of pregrown cells. Kinetic assays of azo reduction activities in quasi-resting cells demonstrated the following: (i) while the optimum pH depends on dye structure, the optimum pH range was observed in the acidic range; (ii) the maximum decolorizing activity occurs in the late exponential phase; and (iii) the temperature profile approaches the typical bell-shaped curve. These results indirectly suggest the involvement of an enzyme activity in azo dye reduction. The decolorizing activity of I. occidentalis is still observed, although at a lower level, when the cells switch to aerobic respiration at the expense of ethanol after glucose exhaustion in the culture medium. Decolorization ceased when all the ethanol was consumed; this observation, along with other lines of evidence, suggests that azo dye reduction depends on cell growth. Anthraquinone-2-sulfonate, a redox mediator, enhances the reduction rates of the N,N-dimethylaniline-based dyes and reduces those of the 2-naphthol-based dyes, an effect which seems to be compatible with a thermodynamic factor. The dye reduction products were tested as carbon and nitrogen sources. 1-Amino-2-naphthol was used as a carbon and nitrogen source, and N,N-dimethyl-p-phenylenediamine was used only as a nitrogen source. Sulfanilic and metanilic acids did not support growth either as a carbon or nitrogen source.     

Regulation of cellular growth by sodium pump activity.

Cellular growth has been found to be directly related to the amount of sodium pumping activity in mouse lymphoblasts (L5178-Y) cultured in varying concentrations of the cardiac glycoside, ouabain. No short-term adaptation (within one generation) occured; i.e., neither growth rate nor (Na+ + K+)-ATPase activity increased in cells cultured for 1-2 days in ouabain. Growth inhibition commenced after two hours, occurring concomitantly with decreased 3H-leucine incorporation into protein. The time course of this inhibition of protein synthesis, measured by leucine incorporation was similar to, but slightly slower than the time course or the dissipation of the sodium gradient. On the other hand, 3H-thymidine incorporation is unaffected by ouabain treatment over the same period. The uptake of 3H-alanine, a neutral amino acid thought to be transported via a Na+-dependent carri-r, was depressed concurrently with the sodium gradient dissipation. It is suggested, therefore, that ouabain inhibition of cellular growth results primarily from the dissipation of the sodium gradient leading to decreased Na+-dependent transport of amino acids (e.g., alanine) and, therefore, decreased protein synthesis, as observed by leucine incorporation. A sensitive and rapid method for determining ouabain inhibition of cell volume regulation is also described, which may prove potentially useful for assaying Na pump activity.     

The anti-estrogen hydroxytamoxifen is a potent antagonist in a novel yeast system

The budding yeast Saccharomyces cerevisiae has been used extensively as a biological 'test tube' to study the regulation of the human estrogen receptor (ER) alpha. However, anti-estrogens, which are of great importance as therapeutic agents and research tools, fail to antagonize the activation by estrogen in yeast. Here, we have surveyed the antagonistic potential of five different anti-estrogens of diverse chemical nature. While they all act as agonists for wild-type ERalpha, we have established a novel yeast assay system for anti-estrogens, in which at least the commonly used anti-estrogen hydroxytamoxifen is a potent antagonist.     

Functionally orthologous viral and cellular microRNAs studied by a novel dual-fluorescent reporter system

Recent research raised the possibility that some viral microRNAs (miRNAs) may function as orthologs of cellular miRNAs. In the present work, to study the functional orthologous relationships of viral and cellular miRNAs, we first constructed a dual-fluorescent protein reporter vector system for the easy determination of miRNA function. By expressing the miRNAs and the indicator and internal control fluorescent proteins individually from a single vector, this simple reporter system can be used for miRNA functional assays that include visualizing miRNA activity in live cells. Sequence alignments indicated that the simian virus 40 (SV40) encoded miRNA sv40-mir-S1-5p contains a seed region identical to that of the human miRNA hsa-miR423-5p. Using the new reporter system, it was found that sv40-mir-S1-5p and hsa-miR423-5p downregulate the expression of common artificial target mRNAs and some predicted biological targets of hsa-miR423-5p, demonstrating that they are functional orthologs. The human immunodeficiency virus 1 (HIV-1) encoded hiv1-miR-N367 also contains a seed sequence identical to that of the human miRNA hsa-miR192. Functional assays showed that hiv1-miR-N367 and hsa-miR192 could downregulate common artificial and predicted biological targets, suggesting that these miRNAs may also act as functional orthologs. Thus, this study presents a simple and universal system for testing miRNA function and identifies two new pairs of functional orthologs, sv40-mir-S1-5p and hsa-miR423-5p as well as hiv-1-miR-N367 and hsa-miR192. These findings also expand upon our current knowledge of functional homology and imply that a more general phenomenon of orthologous relationships exists between viral and cellular miRNAs.     

One- plus two-hybrid system, a novel yeast genetic selection for specific missense mutations disrupting protein/protein interactions

To facilitate analysis of protein/protein interaction interfaces, we devised a novel yeast genetic screening method, named the "one- plus two-hybrid system," for the efficient selection of missense mutations that specifically disrupt known protein/protein interactions. This system modifies the standard yeast two-hybrid system to allow the operation of dual reporter systems within the same cell. The one-hybrid system is first used to select the intact interacting partner (prey), resulting in the positive selection of informative missense mutants from a large library of randomly generated mutant alleles. Then in a second screening step, interaction-defective prey mutants for a given protein are selected using the two-hybrid reporter system among the isolated missense mutants. We used this method to characterize the interactions between unliganded nuclear receptors (NRs) and the conserved motif within the bipartite NR interaction domains (IDs) of the NR corepressor (N-CoR) and identified the specific residues of N-CoR-IDs required either generally for optimal NR binding or to interact with a particular NR. This efficient and rapid method should allow us to quickly analyze a large number of interaction interfaces.     

Detergent-induced solubilization of cytochrome c oxidase as detected in a novel reconstituted system

A preparation of reconstituted cytochrome oxidase vesicles in which the enzyme is oriented facing inwards (such that it cannot interact with external cytochrome c) is described. No oxidase activity is expressed by these vesicles unless they are disrupted, allowing influx of cytochrome c or exposure of the oxidase-binding site to the external medium. We have exploited this property to follow detergent-induced solubilization of the membrane, a technique which allows membrane disruption and enzyme activity to be monitored simultaneously. This protocol can be employed to investigate the properties and mechanism of action of detergents as is illustrated for several ionic and nonionic detergents.     

Development of a novel spatiotemporal depletion system for cellular cholesterol

Cholesterol is an essential component of mammalian cell membranes whose subcellular concentration and function are tightly regulated by de novo biosynthesis, transport, and storage. Although recent reports have suggested diverse functions of cellular cholesterol in different subcellular membranes, systematic investigation of its site-specific roles has been hampered by the lack of a methodology for spatiotemporal manipulation of cellular cholesterol levels. Here, we report the development of a new cholesterol depletion system that allows for spatiotemporal manipulation of intracellular cholesterol levels. This system utilizes a genetically encoded cholesterol oxidase whose intrinsic membrane binding activity is engineered in such a way that its membrane targeting can be controlled in a spatiotemporally specific manner via chemically induced dimerization. In combination with in situ quantitative imaging of cholesterol and signaling activity measurements, this system allows for unambiguous determination of site-specific functions of cholesterol in different membranes, including the plasma membrane and the lysosomal membrane.