A C. elegans orphan nuclear receptor contributes to xenobiotic resistance.

Lipophilic endocrine signals in metazoans, including the steroid, thyroid, and retinoid hormones, alter gene expression in target cells by binding to and modulating the activity of nuclear receptor (NR) transcription factors [1]. In vertebrates, xenobiotic and pharmacologic compounds can regulate the expression of protective metabolic enzymes via specific "xenobiotic sensing" NRs [2-4]. Here, we report evidence suggesting that this activity is an ancient conserved function for the NR class containing these receptors. Specifically, we show that a Caenorhabditis elegans member of this NR class, nhr-8, is required for wild-type levels of resistance to the toxins colchicine and chloroquine. The nhr-8 promoter is active in the nematode gut, a tissue that also expresses the ABC transporter, PGP-3, which contributes to defense against these toxins [5]. In contrast to pgp-3 mutants, nhr-8 mutants are not more sensitive than wild-type to pyocyanin-dependent killing by the pathogenic bacterium Pseudomonas aeruginosa. We conclude that NHR-8 functions in the nematode xenobiotic defense system and that NHR-8 and PGP-3 have overlapping, but distinct, spectra of toxin specificity.

Evidence that dim1 associates with proteins involved in pre-mRNA splicing, and delineation of residues essential for dim1 interactions with hnRNP F and Npw38/PQBP-1.

The small evolutionarily conserved protein Dim1p/hDim1/Dib1p/DML-1 was initially defined as a factor essential for progression through the G2/M transition, and shown to be required to maintain the steady state level of a component of the fission yeast anaphase promoting complex/cyclosome. More recently, Dib1p has been defined as a component of the U4/U6.U5 tri-snRNP, required for pre-mRNA splicing. To investigate the mechanism(s) of Dim1 function, reiterative two-hybrid screening was performed to identify interacting proteins. Proteins thus identified were solely those involved in pre-mRNA splicing or related functions, and one partner induced a striking synthetic phenotype when co-expressed with hDim1 in mammalian cells. Saturating alanine scanning mutagenesis of Dim1 allowed delineation of amino acids essential for its ability to interact with its defined partners: mapping these residues on the structural coordinates of hDim1 defined an interactive sector of the protein. Finally, depletion studies have recently shown that Dim1 function is essential for pre-mRNA splicing in yeast. We find that elimination of DML-1 expression in C. elegans by RNA interference leads to embryonal lethality during gastrulation, marked by a failure to correctly express early zygotic transcripts. These results parallel the arrest phenotypes associated with global disruption of zygotic gene expression, suggesting that Dim1 proteins maintain an essential function in gene expression in higher eukaryotes.

The Caenorhabditis elegans orphan nuclear hormone receptor gene nhr-2 functions in early embryonic development.

We have identified a Caenorhabditis elegans gene, nhr-2, that is a member of the nuclear hormone receptor superfamily of transcription factors and defines a new subclass of the superfamily. nhr-2 messenger RNA is expressed in the maternal germline and during the first half of embryogenesis. Zygotic expression of nhr-2 begins by the 16-cell stage, making it one of the earliest genes known to be transcribed in the embryo. Immunolocalization detects NHR-2 protein in embryonic nuclei as early as the 2-cell stage. The protein is present in every nucleus until the 16- to 20-cell stage. Subsequently, expression continues in many, but not all, cell lineages, becoming progressively restricted to the anterior and dorsal regions of the embryo and disappearing during the initial stages of morphogenesis. Disruption of nhr-2 function with antisense RNA results in embryonic and early larval arrest, indicating that the gene has an essential function in embryonic development. nhr-2 does not correspond to known mutations mapped to the same genetic interval, and will provide an entry point for further study of a heretofore uncharacterized zygotic gene regulatory pathway.

Subunits of human alpha 2-macroglobulin produced by specific reduction of interchain disulfide bonds with thioredoxin.

Disulfide bonds in alpha 2-macroglobulin (alpha 2M) were reduced with the thioredoxin system from Escherichia coli. Under the conditions selected, 3.5-4.1 disulfide bonds were cleaved in each alpha 2M molecule, as determined by the consumption of NADPH during the reaction and by the incorporation of iodo[3H]acetate into the reaction product. This extent of disulfide bond reduction, approximately corresponding to that expected from specific cleavage of all four interchain disulfide bonds of the protein, coincided with the nearly complete dissociation of the intact alpha 2M molecule to a species migrating as an alpha 2M subunit in gel electrophoresis, under both denaturing and nondenaturing conditions. The dissociation was accompanied by only small changes of the spectroscopic properties of the subunits, which thus retain a near-native conformation. Reaction of isolated subunits with methylamine or trypsin led to the appearance of approximately 0.55 mol of thiol group/mol of subunits, indicating that the thio ester bonds are largely intact. Moreover, the rate of cleavage of these bonds by methylamine was similar to that in the whole alpha 2M molecule. Although the bait region was specifically cleaved by nonstoichiometric amounts of trypsin, the isolated subunits had minimal proteinase binding ability. Reaction of subunits with methylamine or trypsin produced changes of farultraviolet circular dichroism and near-ultraviolet absorption similar to those induced in the whole alpha 2M molecule, although in contrast with whole alpha 2M no fluorescence change was observed. The methylamine- or trypsin-treated subunits reassociated to a tetrameric species, migrating as the "fast" form of whole alpha 2M in gradient gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)

Properties of the complex between alpha 2-macroglobulin and brinase, a proteinase from Aspergillus oryzae with thrombolytic effect.

The proteinase, brinase (Mr approximately 35000), from Aspergillus oryzae, which has been used in therapeutic attempts as a thrombolytic agent in arterial thrombosis, binds to purified human alpha 2-macroglobulin (alpha 2M) with a stoichiometry of 1.7-1.9 mol of enzyme/mol inhibitor. This binding leads to quantitative cleavage of the bait region of the inhibitor and to release of 3.6 thiol groups per molecule of alpha 2M, reflecting cleavage of the thioester bonds. The reaction with brinase is accompanied by a similar conformational change of alpha 2M as the reaction with trypsin, as shown by gradient gel electrophoresis and spectroscopic analyses. Brinase thus binds to alpha 2M in a similar manner as most small proteinases. However, in the complex formed at saturation of alpha 2M with brinase, the enzyme retains considerable proteolytic activity against macromolecular substrates, corresponding to about 25% of that of the free enzyme with fibrin as substrate. This finding indicates that the trapping of brinase by alpha 2M is less efficient than that of smaller proteinases. The complex formed at equimolar concentrations of the reactants has appreciably lower, although still significant, activity, amounting to 5-10% of that of free brinase against fibrin. This proteolytic activity of alpha 2M-brinase complexes against high-molecular-weight substrates most likely accounts for the thrombolytic effect of brinase in vivo. The observations also indicate that this thrombolytic activity increases more than proportionally to the brinase concentration as the latter is increased to approach saturation of alpha 2M in plasma.

Changes of the proteinase binding properties and conformation of bovine alpha 2-macroglobulin on cleavage of the thio ester bonds by methylamine.

Cleavage of the thio ester bonds of human alpha2-macroglobulin (alpha 2M) by methylamine leads to an extensive conformational change and to inactivation of the inhibitor. In contrast, cleavage of these bonds in bovine alpha 2M only minimally perturbs the hydrodynamic volume of the protein [Dangott, L. J., & Cunningham, L. W. (1982) Biochem. Biophys. Res. Commun. 107, 1243-1251], as well as its spectroscopic properties, as analyzed by ultraviolet difference spectroscopy, circular dichroism, and fluorescence in this work. A conformational change analogous to that undergone by human alpha 2M thus does not occur in the bovine inhibitor. However, changes of several functional properties of bovine alpha 2M are induced by the amine. The apparent stoichiometry of inhibition of trypsin thus is reduced from about 1.2 to about 0.7 mol of enzyme/mol of inhibitor. In spite of this decrease, the interaction with the proteinase induces similar conformational changes in methylamine-treated alpha 2M as in intact alpha 2M, as revealed by spectroscopic analyses, indicating that the mode of binding of the proteinase to the inhibitor is essentially unperturbed by thio ester bond cleavage. The reaction with methylamine also greatly increases the sensitivity of bovine alpha 2M to proteolysis by trypsin at sites other than the "bait" region. Moreover, the second-order rate constant for the reaction with thrombin is reduced by about 10-fold. These results indicate that the thio ester bonds of bovine alpha 2M, although not required per se for the binding of proteinases, nevertheless are responsible for maintaining certain structural features of the inhibitor that are of importance for full activity.

Stoichiometry of reactions of alpha 2-macroglobulin with trypsin and chymotrypsin.

The stoichiometry of the individual steps, i.e. polypeptide chain cleavage, hydrolysis of the putative thioester bond and conformational change, of the reaction between alpha 2-macroglobulin and trypsin or chymotrypsin was analysed. The chain cleavage was monitored by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the thioester hydrolysis by both a spectroscopic and a fluorimetric technique and the conformational change by tryptophan fluorescence. A stoichiometry of close to 2:1 was obtained for all reactions. This finding indicates that the alpha 2-macroglobulin half-molecule is an independent functional unit of the inhibitor, within which co-operativity between the two subunits may occur.