Hengyi Zhu

Activation of the transcription factor nuclear factor kappa B (NF-kappaB) is controlled by sequential phosphorylation, ubiquitination, and degradation of its inhibitory subunit IkappaB. A large multiprotein complex, the IkappaB kinase (IKK) signalsome, was purified from HeLa cells and found to contain a cytokine-inducible IkappaB kinase activity that phosphorylates IkappaB-alpha and IkappaB-beta. Two components of the IKK signalsome, IKK-1 and IKK-2, were identified as closely related protein serine kinases containing leucine zipper and helix-loop-helix protein interaction motifs. Mutant versions of IKK-2 had pronounced effects on RelA nuclear translocation and NF-kappaB-dependent reporter activity, consistent with a critical role for the IKK kinases in the NF-kappaB signaling pathway.

Activation of the transcription factor NF-kappaB is controlled by the sequential phosphorylation, ubiquitination, and degradation of its inhibitory subunit, IkappaB. We recently purified a large multiprotein complex, the IkappaB kinase (IKK) signalsome, which contains two regulated IkappaB kinases, IKK1 and IKK2, that can each phosphorylate IkappaBalpha and IkappaBbeta. The IKK signalsome contains several additional proteins presumably required for the regulation of the NFkappaB signal transduction cascade in vivo. In this report, we demonstrate reconstitution of IkappaB kinase activity in vitro by using purified recombinant IKK1 and IKK2. Recombinant IKK1 or IKK2 forms homo- or heterodimers, suggesting the possibility that similar IKK complexes exist in vivo. Indeed, in HeLa cells we identified two distinct IKK complexes, one containing IKK1-IKK2 heterodimers and the other containing IKK2 homodimers, which display differing levels of activation following tumor necrosis factor alpha stimulation. To better elucidate the nature of the IKK signalsome, we set out to identify IKK-associated proteins. To this end, we purified and cloned a novel component common to both complexes, named IKK-associated protein 1 (IKKAP1). In vitro, IKKAP1 associated specifically with IKK2 but not IKK1. Functional analyses revealed that binding to IKK2 requires sequences contained within the N-terminal domain of IKKAP1. Mutant versions of IKKAP1, which either lack the N-terminal IKK2-binding domain or contain only the IKK2-binding domain, disrupt the NF-kappaB signal transduction pathway. IKKAP1 therefore appears to mediate an essential step of the NF-kappaB signal transduction cascade. Heterogeneity of IKK complexes in vivo may provide a mechanism for differential regulation of NF-kappaB activation.

The importance of basic fibroblast growth factor (bFGF) in several pathophysiological processes has stimulated interest in the design of receptor antagonists to mitigate such effects. Of key importance in this connection is the characterization of the functional binding epitopes of the growth factor for its receptor. Based on peptide mapping and molecular dynamics calculations of the three-dimensional structure of basic fibroblast growth factor, we employed site-directed mutagenesis to investigate the effect of altering residues at positions 107, 109-114, and 96 on bFGF on receptor binding affinity. All muteins were cloned and expressed in Escherichia coli, purified to homogeneity employing heparin-Sepharose columns, and evaluated for receptor binding affinity. We found that replacement of residues at positions 107 and 109-114 by alanine or phenylalanine had little effect on receptor binding affinities compared with wild type bFGF, in agreement with previous evidence that bFGF residues 109-114 comprise a low affinity binding site. By contrast, substitution of Glu-96 with alanine yielded a molecule having about 0.1% of the affinity of the wild type bFGF. The affinity of the corresponding lysine and glutamine muteins was 0.3 and 10%, respectively, emphasizing the importance of a negative charge at this position. Our findings are consistent with the view that residues 106-115 on bFGF represent a low affinity binding site on bFGF. In addition, we identify Glu-96 as a crucial residue for binding to fibroblast growth factor receptor-1. The importance of basic fibroblast growth factor (bFGF) in several pathophysiological processes has stimulated interest in the design of receptor antagonists to mitigate such effects. Of key importance in this connection is the characterization of the functional binding epitopes of the growth factor for its receptor. Based on peptide mapping and molecular dynamics calculations of the three-dimensional structure of basic fibroblast growth factor, we employed site-directed mutagenesis to investigate the effect of altering residues at positions 107, 109-114, and 96 on bFGF on receptor binding affinity. All muteins were cloned and expressed in Escherichia coli, purified to homogeneity employing heparin-Sepharose columns, and evaluated for receptor binding affinity. We found that replacement of residues at positions 107 and 109-114 by alanine or phenylalanine had little effect on receptor binding affinities compared with wild type bFGF, in agreement with previous evidence that bFGF residues 109-114 comprise a low affinity binding site. By contrast, substitution of Glu-96 with alanine yielded a molecule having about 0.1% of the affinity of the wild type bFGF. The affinity of the corresponding lysine and glutamine muteins was 0.3 and 10%, respectively, emphasizing the importance of a negative charge at this position. Our findings are consistent with the view that residues 106-115 on bFGF represent a low affinity binding site on bFGF. In addition, we identify Glu-96 as a crucial residue for binding to fibroblast growth factor receptor-1.

Basic fibroblast growth factor (bFGF) is implicated in the pathogenesis of several vascular and connective diseases. A key step in the discovery of bFGF receptor antagonists to mitigate these actions is to define the functional epitope required for receptor binding of the growth factor. In previous studies, we identified Glu96 as an essential residue in this epitope using site-directed mutagenesis. Here we examined the role of solvent accessible neighboring residues of Glu96 of bFGF on receptor binding affinity. Wild-type bFGF and its muteins were cloned and expressed in Escherichia coli and evaluated for FGF receptor binding affinity. Replacement of Asn104 of bFGF by alanine reduced receptor binding affinity over 400-fold compared with wild-type bFGF. We next explored the effect of neighboring residues of Asn104 on receptor binding affinity-Muteins in which Arg97, Leu98, Glu99, Asn101, Asn102, Thr105 and Pro141 were individually replaced by alanine exhibited receptor binding similar to wild-type bFGF. By contrast, substitution of Tyr103 or Leu140 by alanine reduced receptor binding affinity about 400- and 150-fold, respectively, in accord with a previous report. We conclude that at least six solvent-accessible residues in bFGF are crucial for high-affinity receptor binding, as evidenced by at least a 10-fold diminution in the affinity of the corresponding alanine muteins. The polar residues Glu96 and Asn104 appear to form an area important for facilitating the initial contact between ligand and receptor, whereas Tyr24, Tyr103, Leu140 and Met142 form a hydrophobic patch that may stabilize the complex. The detailed structure of this functional epitope can be employed in the discovery and design of bFGF antagonists using computational methods.

Gas chromatography (GC) coupled with dual columns and dual electron capture detectors (ECD) was established to determine 23 organochlorine pesticides in 5 soil and 5 sediment samples. Microwave assisted extraction and solid phase extraction with a Florisil column and sulfur cleanup with copper powder were used in sample pre-processing. Chromatographic analysis was performed on DB-XLB and DB-1701 capillary columns by internal standard method. This method showed good extraction efficiency, high sensitivity and good reproducibility. In the same linear range of 0.005 - 0.5 mg/L of 23 pesticides, the correlation coefficients were higher than 0.997. The average spiked recoveries were 50% - 119% with the relative standard deviations (RSDs) of 0.9% - 16.1% (n = 6) in 5 soil samples, and were 52% - 120% with RSDs of 0.3% - 28.4% ( n = 6) in 5 sediment samples, respectively. The detection limits were 0.000 05 -0.000 5 mg/kg. The results indicate that this method is suitable for the determination of the 23 organochlorine pesticides in soil and sediment samples.