Ligand binding pocket function of Drosophila USP is necessary for metamorphosis.

The widely accepted paradigm that epoxidized methyl farnesoates ("juvenile hormones," JHs) are the principal sesquiterpenoid hormones regulating insect metamorphosis was assessed in Drosophila melanogaster. GC-MS analysis of circulating methyl farnesoids during the mid to late 3rd instar showed that methyl farnesoate is predominant over methyl epoxyfarnesoate (=JH III). The circulating concentration of methyl farnesoate (reaching nearly 500 nM), was easily high enough on a kinetic basis to load the Drosophila ortholog of the nuclear hormone receptor RXR (also known as "ultraspiracle," USP), whereas the circulating concentrations of JH III and methyl bisepoxyfarnesoate (bisepoxyJH III) were not. The hypothesis that the ligand pocket of USP necessarily binds an endogenous ligand for differentiation of the immature to the adult was tested with USP mutated at residue that normally extends a side chain into the ligand binding pocket. An equilibrium binding assay confirmed that the mutation (Q288A) strongly altered methyl farnesoate interaction with USP, while a heterologous cell-line transfection assay confirmed that the mutation did not allosterically alter the transcriptional response of the ultraspiracle/ecdysone receptor heterodimer to ecdysteroid signaling. Transgenic wildtype USP driven by the cognate natural promoter rescued null animals to develop to the adult inside a normally formed puparium, while in contrast animals transgenically expressing instead the ligand pocket mutant exhibited developmental derangement at the larval to pupal transition, including failure to form a properly shaped or sclerotized puparium. Other point mutations to the pocket strongly reducing affinity for methyl farnesoate similarly disrupted the larval to pupal metamorphosis. These results suggest that normal larval to pupal maturation in this mecopteran model insect requires the involvement of a distinct endocrine axis of USP binding to its own endogenous terpenoid ligand.

In vitro inhibition of human MMP-2 collagenolytic and gelatinolytic activity by neutralizing monoclonal antibodies.

Matrix metalloproteinases (MMPs) have been implicated in the degradation of the extracellular matrix in normal and pathological tissue remodelling. Among the MMPs, MMP-2 is the most commonly studied protease that has been involved in cancer, inflammation, infective diseases, degenerative diseases of the brain and vascular diseases. In this study, monoclonal antibodies (MAbs) were generated against human MMP-2, purified, characterized and tested for their ability to inhibit the enzymatic activity of MMP-2. Out of 12 positive clones generated against MMP-2, 2 clones (F2-1-11 and G8-25-5) were selected for further characterization. The selected clones react specifically with human pro and active form of MMP-2 in enzyme linked immunosorbant assay (ELISA), dot immunobinding assay (DIA) and Western blot and do not cross react with other human metalloproteinases or MMP-2 from other species. Additionally, these MAbs (F2-1-11 and G8-25-5) selectively inhibit collagenolytic and gelatinolytic activity of APMA ((p-aminophenylmercuric acetate)-activated-pro-MMP-2 and MMP-2, respectively.

Serum levels of matrix metalloproteinase-2 and -9 and conventional tumor markers (CEA and CA 19-9) in patients with colorectal and gastric cancers.

BACKGROUND: Matrix metalloproteinases (MMPs), especially MMP-2 and MMP-9, play an important role in tumor invasion and metastasis. This study aimed to determine the serum levels of MMP-2, MMP-9, 130- and 225-kDa gelatinolytic bands and conventional tumor markers, carcinoembryonic antigen (CEA) and cancer antigen (CA) 19-9, in patients with gastrointestinal cancers. The relationship between these parameters and clinicopathological factors was also studied. METHODS: Sera from controls (n=19), and patients with colorectal (n=47) and gastric (n=34) cancer were collected prospectively. The gelatinolytic activities of MMP-2, MMP-9, 130- and 225-kDa bands were determined using gelatin zymography. CEA and CA 19-9 were determined using immunoradiometric assay (IRMA). RESULTS: Serum levels of MMP-9, 130- and 225-kDa gelatinolytic bands, CEA, and CA 19-9, but not MMP-2, in colorectal and gastric cancer were significantly higher than that of controls. No significant correlation was found between histological grade or clinical stage and levels of MMP-9, 130- and 225-kDa gelatinolytic bands, which were correlated (r=0.61-0.89, p<0.005). CONCLUSIONS: Our findings suggest that zymographic determination of MMP-9, 130- and 225-kDa gelatinolytic bands in colorectal and gastric cancer may be useful in studying these types of cancer in parallel with conventional tumor markers.

The retinoid-X receptor ortholog, ultraspiracle, binds with nanomolar affinity to an endogenous morphogenetic ligand.

The in vivo ligand-binding function and ligand-binding activity of the Drosophila melanogaster retinoid-X receptor (RXR) ortholog, ultraspiracle, toward natural farnesoid products of the ring gland were assessed. Using an equilibrium fluorescence-binding assay, farnesoid products in the juvenile hormone (JH) biosynthesis pathway, and their epoxy derivatives, were measured for their affinity constant for ultraspiracle (USP). Farnesol, farnesal, farnesoic acid and juvenile hormone III exhibited high nanomolar to low micromolar affinity, which in each case decreased upon addition of an epoxide across a double bond of the basic farnesyl structure. Similar analysis of the substitution on C1 of methyl ether, alcohol, aldehyde, and carboxylic acid showed that each conferred weaker affinity than that provided by the methyl ester. Attention was thus focused for a ring-gland farnesoid product that possesses the features of methyl ester and lack of an epoxide. A secreted product of the ring gland, methyl farnesoate, was identified possessing these features and exhibited an affinity for ultraspiracle (K(d) = 40 nm) of similar strength to that of RXR for 9-cis retinoic acid. Mutational analysis of amino acid residues with side chains extending into the ligand-binding pocket cavity (and not interacting with secondary receptor structures or extending to the receptor surface to interact with coactivators, corepressors or receptor dimer partners) showed that the mutation C472A/H475L strongly reduced USP binding to this ring gland product and to JH III, with less effect on other ring-gland farnesoids and little effect on binding by (the unnatural to Drosophila) JH I. Along with the ecdysone receptor, USP is now the second arthropod nuclear hormone receptor for which a secreted product of an endocrine gland that binds the receptor with nanomolar affinity has been identified.

Alternative farnesoid structures induce different conformational outcomes upon the Drosophila ortholog of the retinoid X receptor, ultraspiracle.

In view of recent studies that the ligand-binding pocket of the Drosophila melanogaster nuclear hormone receptor, ultraspiracle (dUSP), is a necessary component of dUSP-dependent transcriptional activation by methyl epoxyfarnesoate, we have assessed qualitative differences in the effect of farnesoid and dodecanoid compounds on receptor conformation and transcriptional activation. Farnesoids possessing terminal alcohol, aldehyde, acid, ester and/or epoxide moieties induced different changes in the local environment of the ligand-binding pocket, as monitored by the change each induced in the fluorescence of the two tryptophan residues existing in dUSP (that are situated 10 residues apart on the alpha-helix 5 that forms one lining of ligand-binding pocket). Similarly, each compound differed in the extent that it promoted an increase in anisotropy (dimerization state) of the receptor. Dodecanoid derivatives were much weaker in causing such effects. Methyl expoxyfarnesoate (insect juvenile hormone III) exhibited the greatest biological activity to increase transcription of a DR12JHECore reporter construct in transfected Sf9 cells, even though it did not exert the most suppression of USP fluorescence nor exert the greatest increase in USP anisotropy. In a comparison of farnesoid derivatives possessing the three side branches either as all methyl groups (JH III), or one of the side branches as ethyl (JH II), or two of the side branches as ethyl (JH I), the JH III and JH I were more similar to each other in the fluorescence suppression and in vivo morphogenetic activity than either was to JH II, evidencing that dUSP does not sense JH II as a structural 'intermediate' between JH III and JH I. Ligand-binding domains of vertebrate retinoid X receptors respond to agonists by repositioning alpha-helix 12 to the edge of a hydrophobic groove, and there with the groove jointly forms a coactivator binding surface. When alpha-helix 12 in dUSP was mutated to place two signaling tryptophan residues its C-terminus, fluorescence signaling indicated that upon dUSP binding of methyl epoxyfarnesoate, the alpha-helix 12 was repositioned differently than what occurred upon binding of non-JH farnesoids. These leads on alternative ligand-induced conformations that dUSP can adopt provide a foundation for commercial development of synthetic molecules that induce specific dUSP conformations, and for identification of in vivo conditions under which endogenous molecules may exert these conformational outcomes to this receptor.