Dicarboxylic aciduria: deficient [1-14C]octanoate oxidation and medium-chain acyl-CoA dehydrogenase in fibroblasts.

Dicarboxylic aciduria, an inborn error of metabolism in man, is thought to be caused by defective beta-oxidation of six-carbon to ten-carbon fatty acids. Oxidation of [1-14C]octanoate was impaired in intact fibroblasts from three unrelated patients with dicarboxylic aciduria (19 percent of control), as was the activity of medium-chain (octanoyl-)acyl-CoA dehydrogenase in the supernatants of sonicated fibroblast mitochondria (5 percent of control). These data confirm that dicarboxylic aciduria is caused by an enzyme defect in the beta-oxidation cycle.

Transcription factor requirements for in vitro formation of transcriptionally competent 5S rRNA gene chromatin.

The Saccharomyces cerevisiae 5S rRNA gene was used as a model system to study the requirements for assembling transcriptionally active chromatin in vitro with purified components. When a plasmid containing yeast 5S rDNA was assembled into chromatin with purified core histones, the gene was inaccessible to the yeast class III gene transcription machinery. Preformation of a 5S rRNA gene-TFIIIA complex was not sufficient for the formation of active chromatin in this in vitro system. Instead, a complete transcription factor complex consisting of TFIIIA, TFIIIB, and TFIIIC needed to be formed before the addition of histones in order for the 5S chromatin to subsequently be transcribed by RNA polymerase III. Various 5S rRNA maxigenes were constructed and used for chromatin assembly studies. In vitro transcription from these assembled 5S maxigenes revealed that RNA polymerase III was readily able to transcribe through one, two, or four nucleosomes. However, we found that RNA polymerase III was not able to efficiently transcribe a chromatin template containing a more extended array of nucleosomes. In vivo expression experiments indicated that all in vitro-constructed maxigenes were transcriptionally competent. Analyses of protein-DNA interactions formed on these maxigenes in vivo by indirect end labeling indicated that there are extensive interactions throughout the length of these maxigenes. The patterns of protein-DNA interactions formed on these genes are consistent with these DNAs being assembled into extensive nucleosomal arrays.

A c-Fos- and E1A-interacting component of the tissue factor basal promoter complex mediates synergistic activation of transcription by transforming growth factor-beta1.

Stimulation of quiescent mouse fibroblasts with TGF-beta1 and certain other growth factors result in cooperative activation of tissue factor (TF) gene transcription, an event accompanied by the rapid entry of c-Fos into specific AP-1 DNA-binding complexes (Felts et al. (1995) Biochemistry 34, 12355-12362). Here, we demonstrate that the ability of TGF-beta1 to synergistically activate TF transcription in serum-stimulated fibroblasts is dependent upon both c-Fos and a promoter-specific factor with functional properties characteristic of transcriptional coactivators. Inhibition of TF promoter activity by an adenovirus E1A mutant deleted in an essential CREB binding protein (CBP) interaction domain suggests that this factor is distinct from the CBP/p300 family of transcriptional coactivators. Importantly, the ability of this factor to mediate molecular interactions with c-Fos required for transcriptional synergism is directly linked to TGF-beta1 signaling. These data suggest a model in which a component of the TF basal transcription complex functions to integrate multiple signaling pathways required for full transcriptional activation of TF in fibroblasts.

Interaction of radicicol with members of the heat shock protein 90 family of molecular chaperones.

The Hsp90 family of proteins in mammalian cells consists of Hsp90 alpha and beta, Grp94, and Trap-1 (Hsp75). Radicicol, an antifungal antibiotic that inhibits various signal transduction proteins such as v-src, ras, Raf-1, and mos, was found to bind to Hsp90, thus making it the prototype of a second class of Hsp90 inhibitors, distinct from the chemically unrelated benzoquinone ansamycins. We have used two novel methods to immobilize radicicol, allowing for detailed analyses of drug-protein interactions. Using these two approaches, we have studied binding of the drug to N-terminal Hsp90 point mutants expressed by in vitro translation. The results point to important drug contacts with amino acids inside the N-terminal ATP/ADP-binding pocket region and show subtle differences when compared with geldanamycin binding. Radicicol binds more strongly to Hsp90 than to Grp94, the Hsp90 homolog that resides in the endoplasmic reticulum. In contrast to Hsp90, binding of radicicol to Grp94 requires both the N-terminal ATP/ADP-binding domain as well as the adjacent negatively charged region. Radicicol also specifically binds to yeast Hsp90, Escherichia coli HtpG, and a newly described tumor necrosis factor receptor-interacting protein, Trap-1, with greater homology to bacterial HtpG than to Hsp90. Thus, the radicicol-binding site appears to be specific to and is conserved in all members of the Hsp90 family of molecular chaperones from bacteria to mammals, but is not present in other molecular chaperones with nucleotide-binding domains.

Characterization of a TPA-response element in the 5'-flanking region of the androgen receptor gene.

The androgen receptor (AR) protein is an important transacting factor that is necessary for mediating gene expression of androgen-responsive genes. The expression of the AR gene is regulated by androgens and agents that utilize the calcium, protein kinase A, and protein kinase C pathways. Although the role of the calcium and protein kinase A pathways in the regulation of the AR gene has been investigated, the mechanism of regulation of AR through the protein kinase C pathway is not known. We have isolated the 5'-flanking region of the mouse AR gene and identified a consensus TPA (12-O-tetradecanoylphorbol 13-acetate)-response element (TRE). Transient transfection assays indicate that the TRE sequence is sufficient to confer TPA responsiveness to cells treated with TPA. Gel retardation assays and DNA footprint analysis demonstrated specific binding of the TRE and protection of the TRE sequence. Thus, these results describe a TRE in the 5'-flanking region of the AR gene and demonstrate that the TRE is responsive to TPA treatment.

A small molecule cell-impermeant Hsp90 antagonist inhibits tumor cell motility and invasion.

Heat shock protein 90 (Hsp90) is a molecular chaperone that maintains function of numerous intracellular signaling nodes utilized by cancer cells for proliferation and survival. Hsp90 is also detected on the plasma membrane of tumor cells and its expression has been suggested to correlate with metastatic potential. Given the abundance and diverse functions of the intracellular pool of this protein, the precise contribution of cell surface Hsp90 to cell motility and tumor metastasis remains to be determined. In this study we utilized the small molecule DMAG-N-oxide, a novel cell-impermeable Hsp90 inhibitor, to specifically examine the role of cell surface Hsp90 in cell motility. We observed that, while not affecting intracellular Hsp90 function, DMAG-N-oxide significantly retarded tumor cell migration and integrin/extracellular matrix-dependent cytoskeletal reorganization. Concomitant with these findings, targeting cell surface Hsp90 significantly inhibited tumor cell motility and invasion in vitro, and had a dramatic impact on melanoma cell lung colonization in vivo. These data indicate that cell surface Hsp90 plays an important role in modulating cancer cell migration that is independent of the function of the intracellular Hsp90 pool, and that small molecule inhibitors of surface Hsp90 may provide a new approach to targeting the metastatic phenotype.

Novobiocin inhibits interactions required for yeast TFIIIB sequestration during stable transcription complex formation in vitro.

Novobiocin concentrations normally used to inhibit a putative eukaryotic DNA gyrase have been found to inhibit transcription of a yeast 5S rRNA gene using an in vitro yeast transcription system. Purified RNA polymerase III and three yeast transcription factors (chromatographically separated, partially purified and free of any detectable gyrase activity) were used. Novobiocin prevents specific transcription if added to the in vitro system immediately prior to the addition of transcription factors and RNA polymerase. If a stable transcription factor complex is allowed to form prior to the addition of novobiocin, concentrations of novobiocin as high as 1000 micrograms/ml have no effect on in vitro transcription. Transcription factors TFIIIA and TFIIIC are able to be stably sequestered onto 5SrDNA-cellulose, but factor TFIIIB is not able to associate with the 5SrDNA-TFIIIA-TFIIIC complex in the presence of novobiocin. Although novobiocin is able to precipitate other basic proteins, it does not appear to precipitate any of these class III gene transcription factors, but instead appears to act by disrupting specific factor-factor interactions.

Tissue factor gene transcription in serum-stimulated fibroblasts is mediated by recruitment of c-Fos into specific AP-1 DNA-binding complexes.

Serum stimulation of quiescent mouse fibroblasts results in transcriptional activation of tissue factor (TF), the cellular initiator of the protease cascade leading to blood coagulation. In this study, we demonstrate that two AP-1 DNA-binding elements located 200-220 bp upstream of the transcription start site are both necessary and sufficient to confer serum inducibility to the TF gene promoter in fibroblasts. Analysis of AP-1 DNA-binding complexes indicates that the predominant form of AP-1 activity in quiescent cells consists of an unidentified Fos-related protein and JunD. While c-Fos is notably absent from these preexisting complexes, serum stimulation results in the rapid entry of c-Fos into the TF AP-1 DNA-binding complexes. A similar induction of c-Fos DNA-binding activity occurs in cells treated with recombinant growth factors such as platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF). Importantly, overexpression of JunD and c-Fos abrogates the requirement for serum in the stimulation of TF promoter activity in fibroblasts. Together, these data indicate that the entry of c-Fos into heterodimeric AP-1 DNA-binding complexes with JunD is a key event underlying serum-stimulated transcription of the TF gene in fibroblasts.

The p23 molecular chaperones act at a late step in intracellular receptor action to differentially affect ligand efficacies.

Multiple molecular chaperones, including Hsp90 and p23, interact with members of the intracellular receptor (IR) family. To investigate p23 function, we compared the effects of three p23 proteins on IR activities, yeast p23 (sba1p) and the two human p23 homologs, p23 and tsp23. We found that Sba1p was indistinguishable from human p23 in assays of seven IR activities in both animal cells and in yeast; in contrast, certain effects of tsp23 were specific to that homolog. Transcriptional activation by two IRs was increased by expression of any of the p23 species, whereas activation by five other IRs was decreased by Sba1p or p23, and unaffected by tsp23. p23 was expressed in all tissues examined except striated and cardiac muscle, whereas tsp23 accumulated in a complementary pattern; hence, p23 proteins might contribute to tissue-specific differences in IR activities. Unlike Hsp90, which acts on IR aporeceptors to stimulate ligand potency (i.e., hormone-binding affinity), p23 proteins acted on IR holoreceptors to alter ligand efficiencies (i.e., transcriptional activation activity). Moreover, the p23 effects developed slowly, requiring prolonged exposure to hormone. In vitro, p23 interacted preferentially with hormone-receptor-response element ternary complexes, and stimulated receptor-DNA dissociation. The dissociation was reversed by addition of a fragment of the GRIP1 coactivator, suggesting that the two reactions may be in competition in vivo. Our findings suggest that p23 functions at one or more late steps in IR-mediated signal transduction, perhaps including receptor recycling and/or reversal of the response.

Serum factors and the reticuloendothelial uptake of Staphylococcus aureus. II. Role of a zymosan-adsorbable serum opsonin.

A heat-stable factor in the serum of normal rabbits adsorbed by zymosan at 16 C in the presence of ethylenediaminetetraacetic acid has been found to enhance the uptake of an encapsulated strain of Staphylococcus aureus (Smith diffuse) by the isolated perfused rabbit liver. This opsonin does not appear to be a rate-limiting component of the hemolytic complement system, properdin, immunoglobulins G or M. It does, however, require the presence of a heat-labile cofactor(s) for expression of its activity.

Hsp90 chaperone activity requires the full-length protein and interaction among its multiple domains.

Hsp90 is an abundant and ubiquitous protein involved in a diverse array of cellular processes. Mechanistically we understand little of the apparently complex interactions of this molecular chaperone. Recently, progress has been made in assigning some of the known functions of hsp90, such as nucleotide binding and peptide binding, to particular domains within the protein. We used fragments of hsp90 and chimeric proteins containing functional domains from hsp90 or its mitochondrial homolog, TRAP1, to study the requirements for this protein in the folding of firefly luciferase as well as in the prevention of citrate synthase aggregation. In agreement with others who have found peptide binding and limited chaperone ability in fragments of hsp90, we see that multiple fragments from hsp90 can prevent the aggregation of thermally denatured citrate synthase, a measure of passive chaperoning activity. However, in contrast to these results, the luciferase folding assay was found to be much more demanding. Here, folding is mediated by hsp70 and hsp40, requires ATP, and thus is a measure of active chaperoning. Hsp90 and the co-chaperone, Hop, enhance this process. This hsp90 activity was only observed using full-length hsp90 indicating that the cooperation of multiple functional domains is essential for active, chaperone-mediated folding.

Crystal structure and activity of human p23, a heat shock protein 90 co-chaperone.

p23 is a co-chaperone for the heat shock protein, hsp90. This protein binds hsp90 and participates in the folding of a number of cell regulatory proteins, but its activities are still unclear. We have solved a crystal structure of human p23 lacking 35 residues at the COOH terminus. The structure reveals a disulfide-linked dimer with each subunit containing eight beta-strands in a compact antiparallel beta-sandwich fold. In solution, however, p23 is primarily monomeric and the dimer appears to be a minor component. Conserved residues are clustered on one face of the monomer and define a putative surface region and binding pocket for interaction(s) with hsp90 or protein substrates. p23 contains a COOH-terminal tail that is apparently less structured and is unresolved in the crystal structure. This tail is not needed for the binding of p23 to hsp90 or to complexes with the progesterone receptor. However, the tail is necessary for optimum active chaperoning of the progesterone receptor, as well as the passive chaperoning activity of p23 in assays measuring inhibition of heat-induced protein aggregation.

The hsp90-related protein TRAP1 is a mitochondrial protein with distinct functional properties.

The hsp90 family of molecular chaperones was expanded recently due to the cloning of TRAP1 and hsp75 by yeast two-hybrid screens. Careful analysis of the human TRAP1 and hsp75 sequences revealed that they are identical, and we have cloned a similar protein from Drosophila. Immunofluorescence data show that human TRAP1 is localized to mitochondria. This mitochondrial localization is supported by the existence of mitochondrial localization sequences in the amino termini of both the human and Drosophila proteins. Due to the striking homology of TRAP1 to hsp90, we tested the ability of TRAP1 to function as an hsp90-like chaperone. TRAP1 did not form stable complexes with the classic hsp90 co-chaperones p23 and Hop (p60). Consistent with these observations, TRAP1 had no effect on the hsp90-dependent reconstitution of hormone binding to the progesterone receptor in vitro, nor could it substitute for hsp90 to promote maturation of the receptor to its hormone-binding state. However, TRAP1 is sufficiently conserved with hsp90 such that it bound ATP, and this binding was sensitive to the hsp90 inhibitor geldanamycin. In addition, TRAP1 exhibited ATPase activity that was inhibited by both geldanamycin and radicicol. Thus, TRAP1 has functions that are distinct from those of hsp90.