Escherichia coli expression and processing of human interleukin-1 beta fused to signal peptides

Escherichia coli expression, processing, and secretion of human interleukin-1 beta (IL-1 beta) fused to the signal peptide of E. coli OmpA or PhoA protein were studied. With fusion to either signal sequence, high-level expression was observed and the products accumulated to about 20% of total cell protein. In the fusion to OmpA leader sequence, more than 50% of the product has the OmpA signal peptide removed precisely. The majority of the processed material is not released by osmotic shock. On the other hand, very little of the material from the fusion to PhoA has the PhoA signal peptide removed. Use of the host with a mutation in prlA or prlF, variation of temperature for cell growth, and alteration of the amino acid residues around the cleavage site do not facilitate processing of the PhoA signal peptide. These results suggest that some component in the PhoA signal peptide, interacting with the Il-1 beta sequence, is interfering with the processing of the signal peptide.     

Structural and mutagenic analysis of foot-and-mouth disease virus 3C protease reveals the role of the beta-ribbon in proteolysis

The 3C protease (3C(pro)) from foot-and-mouth disease virus (FMDV), the causative agent of a widespread and economically devastating disease of domestic livestock, is a potential target for antiviral drug design. We have determined the structure of a new crystal form of FMDV 3C(pro), a chymotrypsin-like cysteine protease, which reveals features that are important for catalytic activity. In particular, we show that a surface loop which was disordered in previous structures adopts a beta-ribbon structure that is conformationally similar to equivalent regions on other picornaviral 3C proteases and some serine proteases. This beta-ribbon folds over the peptide binding cleft and clearly contributes to substrate recognition. Replacement of Cys142 at the tip of the beta-ribbon with different amino acids has a significant impact on enzyme activity and shows that higher activity is obtained with more hydrophobic side chains. Comparison of the structure of FMDV 3C(pro) with homologous enzyme-peptide complexes suggests that this correlation arises because the side chain of Cys142 contacts the hydrophobic portions of the P2 and P4 residues in the peptide substrate. Collectively, these findings provide compelling evidence for the role of the beta-ribbon in catalytic activity and provide valuable insights for the design of FMDV 3C(pro) inhibitors.     

Bent bone dysplasia-FGFR2 type, a distinct skeletal disorder, has deficient canonical FGF signaling

Fibroblast growth factor receptor 2 (FGFR2) is a crucial regulator of bone formation during embryonic development. Both gain and loss-of-function studies in mice have shown that FGFR2 maintains a critical balance between the proliferation and differentiation of osteoprogenitor cells. We have identified de novo FGFR2 mutations in a sporadically occurring perinatal lethal skeletal dysplasia characterized by poor mineralization of the calvarium, craniosynostosis, dysmorphic facial features, prenatal teeth, hypoplastic pubis and clavicles, osteopenia, and bent long bones. Histological analysis of the long bones revealed that the growth plate contained smaller hypertrophic chondrocytes and a thickened hypercellular periosteum. Four unrelated affected individuals were found to be heterozygous for missense mutations that introduce a polar amino acid into the hydrophobic transmembrane domain of FGFR2. Using diseased chondrocytes and a cell-based assay, we determined that these mutations selectively reduced plasma-membrane levels of FGFR2 and markedly diminished the receptor's responsiveness to extracellular FGF. All together, these clinical and molecular findings are separate from previously characterized FGFR2 disorders and represent a distinct skeletal dysplasia.     

Purification, properties, and kinetic observations on the isoenzymes of NADP isocitrate dehydrogenase of maize.

A successful method for the purification of NADP isocitrate dehydrogenase from a plant source, Zea mays, is reported. Two mitochondrial isoenzymes were found and purified to homogeneity by a course of acetone fractionation, bulk exchange on DEAE-cellulose, cellulose hydroxylapatite column chromatography, and continuous elution electrophoresis. The mitochondrial isoenzymes are very similar with respect to kinetic properties, response to solvent perturbation, and temperature dependence of the pH/V relationship of isocitrate dehydrogenation. The Michaelis constant for isocitrate is identical for both isoenzymes. The enzymes have a molecular weight of 81,000 as estimated by permeation chromatography and an isoelectric point of 5.5 as extrapolated from gel-electrophoretic mobilities. Detectable differences are confined to differences in electrophoretic mobilities and heat denaturation. In D₂O the rate of the overall reaction from isocitrate to α-ketoglutarate and CO₂ was about 3.6 times slower than the same reaction in H₂O. Both the forward and reverse reactions, in which isocitrate is dehydrogenated or generated from oxalosuccinate, were observed to decrease by this amount in D₂O. The decarboxylation of oxalosuccinate was found to decrease by only about 25% in D₂O relative to the velocity of the reaction in H₂O. Thus the slow step in the overall reaction must be the initial dehydrogenation step rather than the decarboxylation of oxalosuccinate. The pK of the overall reaction did not change in D₂O as compared to H₂O.     

Mapping the polarity profiles of general anesthetic target sites using n-alkane-(alpha, omega)-diols

The effects of the homologous series of n-alkane-(alpha, omega)-diols have been studied on the inhibition of the purified firefly luciferase enzyme from Photinus pyralis, the inhibition of the purified bacterial luciferase enzyme from Vibrio harveyi, and the induction of general anesthesia in Xenopus laevis tadpoles. All but one of the diols tested were found to be reversible general anesthetics. The diols inhibited firefly luciferase by competing with its normal substrate firefly luciferin, and they inhibited bacterial luciferase by competing with the substrate n-decanal. For all but the smallest agent (1,4-butanediol), only a single diol molecule was found to be involved in the inhibition of the enzymes. Inhibition constants Ki were determined for the enzymes, and general anesthetic EC50 concentrations were determined for tadpoles. These data were then used in conjunction with previously determined n-alkane and n-alcohol data to calculate, as a function of chain length, the incremental standard Gibbs free energies delta (delta G0) for adding apolar -CH2- groups and for converting apolar terminal -CH3 groups to polar -CH2OH groups. The resulting plots of delta (delta G0) versus chain length gave a consistent mapping of the polarity profiles of the anesthetic-binding pockets. They clearly reveal the existence of two substantial and distinct polar regions in the anesthetic-binding pocket of firefly luciferase but only one such region for bacterial luciferase and for the unknown target sites underlying general anesthesia. The polarities and geometric properties of these different binding sites for straight-chain anesthetics are discussed in terms of simple models.