A Schistosoma mansoni fatty acid-binding protein, Sm14, is the potential basis of a dual-purpose anti-helminth vaccine.

Molecular cloning of components of protective antigenic preparations has suggested that related parasite fatty acid-binding proteins could form the basis of the protective immune crossreactivity between the parasitic trematode worms Fasciola hepatica and Schistosoma mansoni. Molecular models of the two parasite proteins showed that both molecules adopt the same basic three-dimensional structure, consisting of a barrel-shaped molecule formed by 10 antiparallel beta-pleated strands joined by short loops, and revealed the likely presence of crossreactive, discontinuous epitopes principally derived from amino acids in the C-terminal portions of the molecules. A recombinant form of the S. mansoni antigen, rSm14, protected outbred Swiss mice by up to 67% against challenge with S. mansoni cercariae in the absence of adjuvant and without provoking any observable autoimmune response. The same antigen also provided complete protection against challenge with F. hepatica metacercariae in the same animal model. The results suggest that it may be possible to produce a single vaccine that would be effective against at least two parasites, F. hepatica and S. mansoni, of veterinary and human importance, respectively.

Identification, purification, and molecular cloning of autonomously replicating sequence-binding protein 1 from fission yeast Schizosaccharomyces pombe.

Autonomously replicating sequence (ARS) elements of the fission yeast Schizosaccharomyces pombe contain multiple imperfect copies of the consensus sequence reported by Maundrell et al. [Maundrell K., Hutchison, A. & Shall, S. (1988) EMBO J. 7, 2203-2209]. When cell free extracts of S. pombe were incubated with a dimer or tetramer of an oligonucleotide containing the ARS consensus sequence, several complexes were detected using a gel mobility-shift assay. The proteins forming these complexes also bind ars3002, which is the most active origin in the ura4 region of chromosome III of S. pombe. One protein, partly responsible for the binding activity observed with crude extracts, was purified to near homogeneity. It is a 60-kDa protein and was named ARS-binding protein 1 (Abp1). Abp1 preferentially binds to multiple sites in ARS 3002 and to the DNA polymer poly[d(A.T)]. The cloning and sequence of the gene coding for Abp1 revealed that it encodes a protein of 59.8 kDa (522 amino acids). Abp1 has significant homology (25% identity, 50% similarity) to the N-terminal region (approximately 300 amino acids) of the human and mouse centromere DNA-binding protein CENP-B. Because centromeres of S. pombe contain a high density of ARS elements, Abp1 may play a role connecting DNA replication and chromosome segregation.

In vivo expression of a single viral DNA-binding protein generates systemic lupus erythematosus-related autoimmunity to double-stranded DNA and histones.

Although the origin of autoimmune antibodies to double-stranded DNA is not known, the variable-region structures of such antibodies indicate that they are produced in response to antigen-selective stimulation. In accordance with this, results from experiments using artificial complexes of DNA and DNA-binding polypeptides for immunizations have indicated that DNA may induce these antibodies. Hence, the immunogenicity of DNA in vivo may depend upon other structures or processes that may render DNA immunogenic. We report that in vivo expression of a single DNA-binding protein, the polyoma virus T antigen, is sufficient to initiate production of anti-double-stranded DNA and anti-histone antibodies but not a panel of other autoantigens. Expression of a mutant, non-DNA-binding T antigen did result in strong production of antibodies to the T antigen, but only borderline levels of antibodies to DNA and no detectable antibodies to histones. Nonexpressing plasmid DNA containing the complete cDNA sequence for T antigen did not evoke such immune responses, indicating that DNA by itself is not immunogenic in vivo. The results represent a conceptual advance in understanding a potential molecular basis for initiation of autoimmunity in systemic lupus erythematosus.

Induction of a Cryphonectria parasitica cellobiohydrolase I gene is suppressed by hypovirus infection and regulated by a GTP-binding-protein-linked signaling pathway involved in fungal pathogenesis.

Extracellular cellulase activity is readily induced when the chestnut blight fungus Cryphonectria parasitica is grown on cellulose substrate as the sole carbon source. However, an isogenic C. parasitica strain rendered hypovirulent due to hypovirus infection failed to secrete detectable cellulase activity when grown under parallel conditions. Efforts to identify C. parasitica cellulase-encoding genes resulted in the cloning of a cellobiohydrolase (exoglucanase, EC 3.2.1.91) gene designated chb-1. Northern blot analysis revealed an increase in cbh-1 transcript accumulation in a virus-free virulent C. parasitica strain concomitant with the induction of extracellular cellulase activity. In contrast, induction of cbh-1 transcript accumulation was suppressed in an isogenic hypovirus-infected strain. Significantly, virus-free C. parasitica strains rendered hypovirulent by transgenic cosuppression of a GTP-binding protein alpha subunit were also found to be deficient in the induction of cbh-1 transcript accumulation.

Assembly of regularly spaced nucleosome arrays by Drosophila chromatin assembly factor 1 and a 56-kDa histone-binding protein.

To ascertain the mechanism by which nucleosomes are assembled by factors derived from Drosophila embryos, two proteins termed Drosophila chromatin assembly factors (CAFs) 1 and 4 (dCAF-1 and dCAF-4) were fractionated and purified from a Drosophila embryo extract. The assembly of chromatin by dCAF-1, dCAF-4, purified histones, ATP, and DNA is a process that generates regularly spaced nucleosomal arrays with a repeat length that resembles that of bulk native Drosophila chromatin and is not obligatorily coupled to DNA replication. The assembly of chromatin by dCAF-1 and dCAF-4 is nearly complete within 10 min. The dCAF-1 activity copurified with the Drosophila version of chromatin assembly factor-1 (CAF-1), a factor that has been found to be required for the assembly of chromatin during large tumor (T) antigen-mediated, simian virus 40 (SV40) origin-dependent DNA replication. The dCAF-4 activity copurified with a 56-kDa core-histone-binding protein that was purified to > 90% homogeneity.

A single-stranded DNA binding protein binds the origin of replication of the duplex kinetoplast DNA.

Replication of the kinetoplast DNA (kDNA) minicircle of trypanosomatids initiates at a conserved 12-nt sequence, 5'-GGGGTTGGTGTA-3', termed the universal minicircle sequence (UMS). A sequence-specific single-stranded DNA-binding protein from Crithidia fasciculata binds the heavy strand of the 12-mer UMS. Whereas this UMS-binding protein (UMSBP) does not bind a duplex UMS dodecamer, it binds the double-stranded kDNA minicircle as well as a duplex minicircle fragment containing the origin-associated UMS. Binding of the minicircle origin region by the single-stranded DNA binding protein suggested the local unwinding of the DNA double helix at this site. Modification of thymine residues at this site by KMnO4 revealed that the UMS resides within an unwound or otherwise sharply distorted DNA at the minicircle origin region. Computer analysis predicts the sequence-directed curving of the minicircle origin region. Electrophoresis of a minicircle fragment containing the origin region in polyacrylamide gels revealed a significantly lower electrophoretic mobility than expected from its length. The fragment anomalous electrophoretic mobility is displayed only in its native conformation and is dependent on temperature and gel porosity, indicating the local curving of the DNA double helix. We suggest that binding of UMSBP at the minicircle origin of replication is possible through local unwinding of the DNA double helix at the UMS site. It is hypothesized here that this local melting is initiated through the untwisting of unstacked dinucleotide sequences at the bent origin site.

The ability to associate with activation domains in vitro is not required for the TATA box-binding protein to support activated transcription in vivo.

The TATA box-binding protein (TBP) interacts in vitro with the activation domains of many viral and cellular transcription factors and has been proposed to be a direct target for transcriptional activators. We have examined the functional relevance of activator-TBP association in vitro to transcriptional activation in vivo. We show that alanine substitution mutations in a single loop of TBP can disrupt its association in vitro with the activation domains of the herpes simplex virus activator VP16 and of the human tumor suppressor protein p53; these mutations do not, however, disrupt the transcriptional response of TBP to either activation domain in vivo. Moreover, we show that a region of VP16 distinct from its activation domain can also tightly associate with TBP in vitro, but fails to activate transcription in vivo. These data suggest that the ability of TBP to interact with activation domains in vitro is not directly relevant to its ability to support activated transcription in vivo.

VIP21/caveolin is a cholesterol-binding protein.

VIP21/caveolin is localized to both caveolae and apical transport vesicles and presumably cycles between the cell surface and the Golgi complex. We have studied the lipid interactions of this protein by reconstituting Escherichia coli-expressed VIP21/caveolin into liposomes. Surprisingly, the protein reconstituted only with cholesterol-containing lipid mixtures. We demonstrated that the protein binds at least 1 mol of cholesterol per mole of protein and that this binding promotes formation of protein oligomers. These findings suggest that VIP21/caveolin, through its cholesterol-binding properties, serves a specific function in microdomain formation during membrane trafficking.

Double-stranded-RNA-dependent protein kinase and TAR RNA-binding protein form homo- and heterodimers in vivo.

The yeast two-hybrid system and far-Western protein blot analysis were used to demonstrate dimerization of human double-stranded RNA (dsRNA)-dependent protein kinase (PKR) in vivo and in vitro. A catalytically inactive mutant of PKR with a single amino acid substitution (K296R) was found to dimerize in vivo, and a mutant with a deletion of the catalytic domain of PKR retained the ability to dimerize. In contrast, deletion of the two dsRNA-binding motifs in the N-terminal regulatory domain of PKR abolished dimerization. In vitro dimerization of the dsRNA-binding domain required the presence of dsRNA. These results suggest that the binding of dsRNA by PKR is necessary for dimerization. The mammalian dsRNA-binding protein TRBP, originally identified on the basis of its ability to bind the transactivation region (TAR) of human immunodeficiency virus RNA, also dimerized with itself and with PKR in the yeast assay. Taken together, these results suggest that complexes consisting of different combinations of dsRNA-binding proteins may exist in vivo. Such complexes could mediate differential effects on gene expression and control of cell growth.

G protein activation kinetics and spillover of gamma-aminobutyric acid may account for differences between inhibitory responses in the hippocampus and thalamus.

We have developed a model of gamma-aminobutyric acid (GABA)ergic synaptic transmission mediated by GABAA and GABAB receptors, including cooperativity in the guanine nucleotide binding protein (G protein) cascade mediating the activation of K+ channels by GABAB receptors. If the binding of several G proteins is needed to activate the K+ channels, then only a prolonged activation of GABAB receptors evoked detectable currents. This could occur if strong stimuli evoked release in adjacent terminals and the spillover resulted in prolonged activation of the receptors, leading to inhibitory responses similar to those observed in hippocampal slices. The same model also reproduced thalamic GABAB responses to high-frequency bursts of stimuli. In this case, prolonged activation of the receptors was due to high-frequency release conditions. This model provides insights into the function of GABAB receptors in normal and epileptic discharges.

Testis/brain RNA-binding protein attaches translationally repressed and transported mRNAs to microtubules.

We have previously identified a testicular phosphoprotein that binds to highly conserved sequences (Y and H elements) in the 3' untranslated regions (UTRs) of testicular mRNAs and suppresses in vitro translation of mRNA constructs that contain these sequences. This protein, testis/brain RNA-binding protein (TB-RBP) also is abundant in brain and binds to brain mRNAs whose 3' UTRs contain similar sequences. Here we show that TB-RBP binds specific mRNAs to microtubules (MTs) in vitro. When TB-RBP is added to MTs reassembled from either crude brain extracts or from purified tubulin, most of the TB-RBP binds to MTs. The association of TB-RBP with MTs requires the assembly of MTs and is diminished by colcemid, cytochalasin D, and high levels of salt. Transcripts from the 3' UTRs of three mRNAs that contain the conserved sequence elements (transcripts for protamine 2, tau protein, and myelin basic protein) are linked by TB-RBP to MTs, whereas transcripts that lack the conserved sequences do not bind TB-RBP. We conclude that TB-RBP serves as an attachment protein for the MT association of specific mRNAs. Considering its ability to arrest translation in vitro, we propose that TB-RBP functions in the storage and transportation of mRNAs to specific intracellular sites where they are translated.

3T3 fibroblasts transfected with a cDNA for mitochondrial aspartate aminotransferase express plasma membrane fatty acid-binding protein and saturable fatty acid uptake.

To explore the relationship between mitochondrial aspartate aminotransferase (mAspAT; EC 2.6.1.1) and plasma membrane fatty acid-binding protein (FABPpm) and their role in cellular fatty acid uptake, 3T3 fibroblasts were cotransfected with plasmid pMAAT2, containing a full-length mAspAT cDNA downstream of a Zn(2+)-inducible metallothionein promoter, and pFR400, which conveys methotrexate resistance. Transfectants were selected in methotrexate, cloned, and exposed to increasing methotrexate concentrations to induce gene amplification. Stably transfected clones were characterized by Southern blotting; those with highest copy numbers of pFR400 alone (pFR400) or pFR400 and pMAAT2 (pFR400/pMAAT2) were expanded for further study. [3H]Oleate uptake was measured in medium containing 500 microM bovine serum albumin and 125-1000 microM total oleate (unbound oleate, 18-420 nM) and consisted of saturable and nonsaturable components. pFR400/pMAAT2 cells exhibited no increase in the rate constant for nonsaturable oleate uptake or in the uptake rate of [14C]octanoate under any conditions. By contrast, Vmax (fmol/sec per 50,000 cells) of the saturable oleate uptake component increased 3.5-fold in pFR400/pMAAT2 cells compared to pFR400, with a further 3.2-fold increase in the presence of Zn2+. Zn2+ had no effect in pFR400 controls (P > 0.5). The overall increase in Vmax between pFR400 and pFR400/pMAAT2 in the presence of Zn2+ was 10.4-fold (P < 0.01) and was highly correlated (r = 0.99) with expression of FABPpm in plasma membranes as determined by Western blotting. Neither untransfected 3T3 nor pFR400 cells expressed cell surface FABPpm detectable by immunofluorescence. By contrast, plasma membrane immunofluorescence was detected in pFR400/pMAAT2 cells, especially if cultured in 100 microM Zn2+. The data support the dual hypotheses that mAspAT and FABPpm are identical and mediate saturable long-chain free fatty acid uptake.

Recoverin, a photoreceptor-specific calcium-binding protein, is expressed by the tumor of a patient with cancer-associated retinopathy.

Recoverin is a member of the EF-hand family of calcium-binding proteins involved in the transduction of light by vertebrate photoreceptors. Recoverin also was identified as an autoantigen in the degenerative disease of the retina known as cancer-associated retinopathy (CAR), a paraneoplastic syndrome whereby immunological events lead to the degeneration of photoreceptors in some individuals with cancer. In this study, we demonstrate that recoverin is expressed in the lung tumor of a CAR patient but not in similar tumors obtained from individuals without the associated retinopathy. Recoverin was identified intially by Western blot analysis of the CAR patient's biopsy tissue by using anti-recoverin antibodies generated against different regions of the recoverin molecule. In addition, cultured cells from the biopsy tissue expressed recoverin, as demonstrated by reverse transcription-PCR using RNA extracted from the cells. The immunodominant region of recoverin also was determined in this study by a solid-phase immunoassay employing overlapping heptapeptides encompassing the entire recoverin sequence. Two linear stretches of amino acids (residues 64-70, Lys-Ala-Tyr-Ala-Gln-His-Val; and 48-52, Gln-Phe-Gln-Ser-Ile) made up the major determinants. One of the same regions of the recoverin molecule (residues 64-70) also was uniquely immunopathogenic, causing photoreceptor degeneration upon immunization of Lewis rats with the corresponding peptide. These data demonstrate that the neural antigen recoverin more than likely is responsible for the immunological events associated with vision loss in some patients with cancer. These data also establish CAR as one of the few autoimmune-mediated diseases for which the specific self-antigen is known.

12(S)-hydroxyeicosatetraenoic acid and 13(S)-hydroxyoctadecadienoic acid regulation of protein kinase C-alpha in melanoma cells: role of receptor-mediated hydrolysis of inositol phospholipids.

Protein kinase C (PKC) isoenzymes are essential components of cell signaling. In this study, we investigated the regulation of PKC-alpha in murine B16 amelanotic melanoma (B16a) cells by the monohydroxy fatty acids 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] and 13(S)-hydroxyoctadecadienoic acid [13(S)-HODE]. 12(S)-HETE induced a translocation of PKC-alpha to the plasma membrane and focal adhesion plaques, leading to enhanced adhesion of B16a cells to the matrix protein fibronectin. However, 13(S)-HODE inhibited these 12(S)-HETE effects on PKC-alpha. A receptor-mediated mechanism of action for 12(S)-HETE and 13(S)-HODE is supported by the following findings. First, 12(S)-HETE triggered a rapid increase in cellular levels of diacylglycerol and inositol trisphosphate in B16a cells. 13(S)-HODE blocked the 12(S)-HETE-induced bursts of both second messengers. Second, the 12(S)-HETE-increased adhesion of B16a cells to fibronectin was sensitive to inhibition by a phospholipase C inhibitor and pertussis toxin. Finally, a high-affinity binding site (Kd = 1 nM) for 12(S)-HETE was detected in B16a cells, and binding of 12(S)-HETE to B16a cells was effectively inhibited by 13(S)-HODE (IC50 = 4 nM). In summary, our data provide evidence that regulation of PKC-alpha by 12(S)-HETE and 13(S)-HODE may be through a guanine nucleotide-binding protein-linked receptor-mediated hydrolysis of inositol phospholipids.

The amino-terminal domain of the prokaryotic enhancer-binding protein XylR is a specific intramolecular repressor.

The mechanism under which the signal-reception amino-terminal portion (A domain) of the prokaryotic enhancer-binding protein XylR controls the activity of the regulator has been investigated through complementation tests in vivo, in which the various protein segments were produced as independent polypeptides. Separate expression of the A domain repressed the otherwise constitutive activity of a truncated derivative of XylR deleted of its A domain (XylR delta A). Such inhibition was not released by m-xylene, the natural inducer of the system. Repression caused by the A domain was specific for XylR because it did not affect activation of the sigma 54 promoter PnifH by a derivative of its cognate regulator, NifA, deleted of its own A domain. The A domain was also unable to repress the activity of a NifA-XylR hybrid protein resulting from fusing two-thirds of the central domain of NifA to the carboxyl-terminal third of XylR, which includes its DNA-binding domain. The inhibitory effect caused by the A domain of XylR on XylR delta A seems, therefore, to result from specific interactions in trans between the two truncated proteins and not from mere hindering of an activating surface.