Search

The genes encoding the first two enzymes of the peroxisomal beta-oxidation pathway, acyl-CoA oxidase (AOx) and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (HD), contain upstream cis-acting regulatory regions termed peroxisome proliferator response elements (PPRE). Transcription of these genes is mediated through the binding of peroxisome proliferator-activated receptor alpha (PPARalpha), which binds to a PPRE as a heterodimer with the 9-cis-retinoic acid receptor (RXRalpha). Here we demonstrate that the HD-PPRE is also a target for the constitutive androstane receptor beta (CARbeta). In vitro binding analysis showed that CARbeta bound the HD-PPRE, but not the AOx-PPRE, as a heterodimer with RXRalpha. Binding of CARbeta/RXRalpha to the HD-PPRE occurred via determinants that overlap partially with those required for PPARalpha/RXRalpha binding. In vivo, CARbeta/RXRalpha activated transcription from an HD-PPRE luciferase reporter construct. Interestingly, CARbeta was shown to also modulate PPARalpha/RXRalpha-mediated transactivation in a response element-specific manner. In the presence of the peroxisome proliferator, Wy-14,643, CARbeta had no effect on PPARalpha/RXRalpha-mediated transactivation from the HD-PPRE but antagonized transactivation from the AOx-PPRE in both the presence and the absence of proliferator. Our results illustrate that transcription of the AOx and HD genes is differentially regulated by CARbeta and that the HD gene is a specific target for regulation by CARbeta. Overall, this study proposes a novel role for CARbeta in the regulation of peroxisomal beta-oxidation.

Peroxisome proliferator-activated receptor alpha (PPARalpha) heterodimerizes with the 9-cis-retinoic acid receptor (RXRalpha) to bind to peroxisome proliferator-response elements (PPRE) present in the upstream regions of a number of genes involved in metabolic homeostasis. Among these genes are those encoding fatty acyl-CoA oxidase (AOx) and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (HD), the first two enzymes of the peroxisomal beta-oxidation pathway. Here we demonstrate that the orphan nuclear hormone receptor, RevErbalpha, modulates PPARalpha/RXRalpha- dependent transactivation in a response element-specific manner. In vitro binding analysis showed that RevErbalpha bound the HD-PPRE but not the AOx-PPRE. Determinants within the HD-PPRE required for RevErbalpha binding were distinct from those required for PPARalpha/RXRalpha binding. In transient transfections, RevErbalpha antagonized transactivation by PPARalpha/RXRalpha from an HD-PPRE luciferase reporter construct, whereas no effects were observed with an AOx-PPRE reporter construct. These data identify the HD gene as a target for RevErbalpha and illustrate cross-talk between the RevErbalpha and PPARalpha signaling pathways on the HD-PPRE. Our results suggest a novel role for RevErbalpha in regulating peroxisomal beta-oxidation.

p62 is a recently identified ubiquitin-binding, cytosolic phosphoprotein that interacts with several signal transduction molecules including the tyrosine kinase p56(lck) and the protein kinase C-zeta. p62 is therefore suggested to serve an important role in signal transduction in the cell, although the physiological function of p62 remains undefined. Here we demonstrate by transient transfection assays that p62 stimulates the transcription of reporter genes linked to the simian virus 40 (SV40) enhancer. A putative p62-responsive element was localized to the B domain of the distal 72-base pair repeat of the SV40 enhancer. p62 was unable to bind this element in vitro, nor was it able to activate transcription when directly tethered to a promoter, suggesting that p62 stimulates transcription via an indirect mechanism. Stimulation of transcription mediated by p62 was dependent on its amino-terminal region, which is also necessary for interaction with cell surface signaling molecules. These findings indicate that p62 may link extracellular signals directly to transcriptional responses, and identify the SV40 enhancer as a downstream target for signal transduction pathways in which p62 participates.

We have used oligonucleotide-directed site-specific mutagenesis to convert serine codon 27 of the Escherichia coli chloramphenicol acetyltransferase (cat) gene to UAG, UAA, and UGA nonsense codons. The mutant cat genes, under transcriptional control of the Rous sarcoma virus long terminal repeat, were then introduced into mammalian cells by DNA transfection along with UAG, UAA, and UGA suppressor tRNA genes derived from a human serine tRNA. Assay for CAT enzymatic activity in extracts from such cells allowed us to detect and quantitate nonsense suppression in monkey CV-1 cells and mouse NIH3T3 cells. Using such an assay, we provide the first direct evidence that an opal suppressor tRNA gene is functional in mammalian cells. The pattern of suppression of the three cat nonsense mutations in bacteria suggests that the serine at position 27 of CAT can be replaced by a wide variety of amino acids without loss of enzymatic activity. Thus, these mutant cat genes should be generally useful for the quantitation of suppressor activity of suppressor tRNA genes introduced into cells and possibly for the detection of naturally occurring nonsense suppressors.

The yeast two-hybrid system was used to isolate novel cellular factors that interact with the mouse peroxisome proliferator-activated receptor alpha (PPARalpha). One of the interacting clones isolated encoded LXRalpha, a recently described human orphan nuclear hormone receptor. LXRalpha bound directly to PPARalpha, as well as to the common heterodimerization partner 9-cis-retinoic acid receptor (RXRalpha). LXRalpha did not form a DNA binding complex with PPARalpha on synthetic hormone response elements composed of direct repeats of the TGACCT consensus half-site or on naturally occurring peroxisome proliferator response elements (PPREs) or LXRalpha response elements. However, LXRalpha inhibited binding of PPARalpha/RXRalpha heterodimers to PPREs, and coexpression of LXRalpha in mammalian cells antagonized peroxisome proliferator signaling mediated by PPARalpha/RXRalpha in vivo. These findings identify a novel partner for PPARalpha and suggest that LXRalpha plays a role in modulating PPAR-signaling pathways in the cell.

The herpes simplex virus transactivator VP16 directs the assembly of a multicomponent protein-DNA complex that requires the participation of two cellular factors, the POU homeodomain protein Oct-1, which binds independently to response elements, and VCAF-1 (VP16 complex assembly factor; also called HCF, C1), a factor that binds directly to VP16. A number of distinct properties of VP16 have been implicated in the assembly of the VP16-induced complex (VIC). These include its independent association with VCAF-1 and, under appropriate conditions, its ability to bind to DNA or to DNA-bound Oct-1 in the absence of VCAF-1. In order to probe the requirements of these individual interactions in the functional assembly of VIC, we mutated selected charged amino acids in two subdomains of VP16 previously shown to be important in protein-DNA complex formation. Purified VP16 proteins were analyzed for their ability to direct protein-DNA complex formation and to interact directly with VCAF-1. Several classes of mutants that were differentially compromised in VCAF-1 interaction, direct DNA binding, and/or association with DNA-bound Oct-1 were obtained. Interestingly, all of the derivatives were still capable of generating the VIC complex in vitro and activating transcription in vivo. Our findings indicate that the cooperative assembly of functional VP16-containing complexes can occur by pathways that do not necessarily require the prior interaction of VP16 with VCAF-1 or the ability of VP16 to bind directly to DNA or associate with DNA-bound Oct-1.

A synthesis of a homologous series of highly radioactive photoreactive fatty acids has been developed. Oxidative cleavage of the cis double bonds of unsaturated fatty acids by NaIO4/OsO4 produced the omega-oxo fatty acids; subsequent reduction by [3H]NaBH4 afforded a series of highly radioactive [omega-3H]hydroxy fatty acids to which a variety of photoreactive probes can be readily attached. In this study, the m-diazirinophenoxy group was coupled to the omega-hydroxy groups of the fatty acids, yielding the omega-(m-diazirinophenoxy) derivatives of nonanoic, undecanoic, tridecanoic, and pentadecanoic acid. These photoreactive derivatives were incorporated by L-cells into their major phospholipids; in addition, a number of proteins were shown to be acylated by these fatty acids. The photoreactive group was shown to remain intact as indicated by the cross-linking of the resulting photoreactive phospholipids to other phospholipids as well as to a number of L-cell proteins upon photolysis. This procedure thus identifies integral membrane proteins and directly produces photoaffinity derivatives of those proteins normally acylated by fatty acids.

Chicken ovalbumin upstream promoter transcription factor II (COUP-TFII), an orphan member of the nuclear hormone receptor superfamily, acts as a transcriptional repressor by antagonizing the functions of other nuclear hormone receptors and by actively silencing transcription. However, in certain contexts, COUP-TFII stimulates transcription directly. A cellular factor, isolated by interaction cloning, bound COUP-TFII in vitro and allowed COUP-TFII to function as a transcriptional activator in mammalian cells. This factor is identical to a recently described ligand of the tyrosine kinase signaling molecule p56(lck), suggesting that it mediates cross-talk between mitogenic and nuclear hormone receptor signal transduction pathways.

In order to identify structural domains of the herpes simplex virus trans-activator Vmw65 required for protein-DNA complex formation, subfragments of Vmw65 were expressed in Escherichia coli as fusion polypeptides with protein A of Staphylococcus aureus, and the purified hybrids were used in a band shift assay. The results indicate that a region near the amino terminus of Vmw65 between amino acids 141 and 185 is necessary for complex formation.

A number of Cephalosporium acremoniummutants blocked in the synthesis of cephalosporin C were investigated for accumulation of other β-lactam compounds. The non-cephalosporin C producers were isolated after exposing the superior cephalosporin C-producing strain M8650-4 to ultraviolet light (268 nm). One of the blocked mutants, MH63, accumulated deacetylcephalosporin C (0.4 mg/ml), deacetoxycephalosporin C (1.5 mg/ml), and penicillin N (2.7 mg/ml). In contrast, the parent of MH63 produced high levels of cephalosporin C as well as deacetylcephalosporin C (2.2 mg/ml) and penicillin N (1.0 mg/ml), but only traces of deacetoxycephalosporin C (about 0.1 mg/ml). Deacetoxycephalosporin C was isolated from the mutant strain and identified by ultraviolet light, nuclear magnetic resonance, bioactivity spectrum, and co-migration with authentic standard in three chromatography systems.

Vesicular stomatitis virus grown in the presence of omega-[9-3H]diazirinophenoxy nonanoate resulted in the biosynthetic incorporation of this photoreactive fatty acid into viral phospholipids as well as into the membrane anchoring domain of the viral G glycoprotein. Photolysis of the isolated virus at 360 nm resulted in extensive labeling of the G protein but none of the other proteins by the viral phospholipids. In addition, a new product was obtained and was identified as a G-G dimer by its molecular weight and its reaction with anti-G antibody. These results demonstrate the use of photoreactive fatty acid to identify integral membrane proteins and to make photoaffinity probes of fatty acylated membrane proteins.

The genes encoding the peroxisomal beta-oxidation enzymes enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (HD) and fatty acyl-CoA oxidase (AOx) are coordinately regulated by peroxisome proliferator-activated receptor alpha (PPARalpha)/9-cis-retinoic acid receptor (RXRalpha) heterodimers that transactivate these genes in a ligand-dependent manner via upstream peroxisome proliferator response elements (PPRE). Here we demonstrate that the monomeric orphan nuclear hormone receptor, RZRalpha, modulates PPARalpha/RXRalpha-dependent transactivation in a response-element dependent manner. Electrophoretic mobility shift analysis showed that RZRalpha bound specifically as a monomer to the HD-PPRE but not the AOx-PPRE. Determinants in the HD-PPRE for binding of RZRalpha were distinct from those required for interaction with PPARalpha/RXRalpha heterodimers. In transient transfections, RZRalpha stimulated ligand-mediated transactivation by PPARalpha from an HD-PPRE luciferase reporter in the absence of exogenously added RXRalpha, but did not affect PPARalpha-dependent transactivation of an AOx-PPRE reporter gene. These data illustrate cross-talk between the RZRalpha and PPARalpha signaling pathways at the level of the HD-PPRE in the regulation of the HD gene and characterize additional factors governing the regulation of peroxisomal beta-oxidation.

Homogeneous suspensions of streptomycete spores were desirable for plating after mutagenesis. Filtration through 8-μm membrane filters (Millipore Corp.) more effectively separated hyphae and spore clumps from single spores than did filtration through cotton wefts or paper.