Kinetic mechanisms of IkappaB-related kinases (IKK) inducible IKK and TBK-1 differ from IKK-1/IKK-2 heterodimer

Nuclear factor-kappaB activation depends on phosphorylation and degradation of its inhibitor protein, IkappaB. The phosphorylation of IkappaBalpha on Ser(32) and Ser(36) is initiated by an IkappaB kinase (IKK) complex that includes a catalytic heterodimer composed of IkappaB kinase 1 (IKK-1) and IkappaB kinase 2 (IKK-2) as well as a regulatory adaptor subunit, NF-kappaB essential modulator. Recently, two related IkappaB kinases, TBK-1 and IKK-i, have been described. TBK-1 and IKK-i show sequence and structural homology to IKK-1 and IKK-2. TBK-1 and IKK-i phosphorylate Ser(36) of IkappaBalpha. We describe the kinetic mechanisms in terms of substrate and product inhibition of the recombinant human (rh) proteins, rhTBK-1, rhIKK-I, and rhIKK-1/rhIKK-2 heterodimers. The results indicate that although each of these enzymes exhibits a random sequential kinetic mechanism, the effect of the binding of one substrate on the affinity of the other substrate is significantly different. ATP has no effect on the binding of an IkappaBalpha peptide for the rhIKK-1/rhIKK-2 heterodimer (alpha = 0.99), whereas the binding of ATP decreased the affinity of the IkappaBalpha peptide for both rhTBK-1 (alpha = 10.16) and rhIKK-i (alpha = 62.28). Furthermore, the dissociation constants of ATP for rhTBK-1 and rhIKK-i are between the expected values for kinases, whereas the dissociation constants of the IkappaBalpha peptide for each IKK isoforms is unique with rhTBK-1 being the highest (K(IkappaBalpha) = 69.87 microm), followed by rhIKK-i (K(IkappaBalpha) = 5.47 microm) and rhIKK-1/rhIKK-2 heterodimers (K(IkappaBalpha) = 0.12 microm). Thus this family of IkappaB kinases has very unique kinetic properties.     

Identification of human IKK-2 inhibitors of natural origin (part I): modeling of the IKK-2 kinase domain, virtual screening and activity assays

Background

Their large scaffold diversity and properties, such as structural complexity and drug similarity, form the basis of claims that natural products are ideal starting points for drug design and development. Consequently, there has been great interest in determining whether such molecules show biological activity toward protein targets of pharmacological relevance. One target of particular interest is hIKK-2, a serine-threonine protein kinase belonging to the IKK complex that is the primary component responsible for activating NF-κB in response to various inflammatory stimuli. Indeed, this has led to the development of synthetic ATP-competitive inhibitors for hIKK-2. Therefore, the main goals of this study were (a) to use virtual screening to identify potential hIKK-2 inhibitors of natural origin that compete with ATP and (b) to evaluate the reliability of our virtual-screening protocol by experimentally testing the in vitro activity of selected natural-product hits.

Methodology/Principal Findings

We thus predicted that 1,061 out of the 89,425 natural products present in the studied database would inhibit hIKK-2 with good ADMET properties. Notably, when these 1,061 molecules were merged with the 98 synthetic hIKK-2 inhibitors used in this study and the resulting set was classified into ten clusters according to chemical similarity, there were three clusters that contained only natural products. Five molecules from these three clusters (for which no anti-inflammatory activity has been previously described) were then selected for in vitro activity testing, in which three out of the five molecules were shown to inhibit hIKK-2.

Conclusions/Significance

We demonstrated that our virtual-screening protocol was successful in identifying lead compounds for developing new inhibitors for hIKK-2, a target of great interest in medicinal chemistry. Additionally, all the tools developed during the current study (i.e., the homology model for the hIKK-2 kinase domain and the pharmacophore) will be made available to interested readers upon request.     

The primary structures of four subunits of the human, high-molecular-weight proteinase, macropain (proteasome), are distinct but homologous

Macropain (proteasome) is a high-molecular-weight proteinase complex composed of at least 13 electrophoretically distinct subunits. Previous work, including peptide mapping and limited amino acid sequencing, suggested that most of the subunits belong to an evolutionarily related group of different gene products (Lee et al. (1990) Biochim. Biophys. Acta. 1037, 178-185). In order to define the extent and pattern of subunit relatedness, and to determine the structural basis for possible similarities and differences in subunit functions, we are deducing the primary structures of macropain subunits by cDNA cloning and DNA sequence analysis. We report here the primary structures of four subunits. The data clearly demonstrate that the proteins represent different, but homologous gene products. Surprisingly, no evidence for homology with any other protein, including proteinases, was obtained. These results suggest that macropain is comprised of a previously unidentified family of evolutionarily related polypeptides. Because biochemical data indicate that macropain contains several different proteinase activities, the current results raise the possibility that the macropain complex is composed of a group of novel proteinases, distinct from those of other structurally identifiable proteinase families.     

Pyroglutamyl-peptidase I: cloning,sequencing, and characterisation of the recombinant human enzyme

Pyroglutamyl-peptidase I (EC 3.4.19.3) is well known from bacteria and archaea, but has not previously been cloned or sequenced from any vertebrate. We describe the cloning and sequencing of the human (AJ278828) and mouse (AJ278829) forms of pyroglutamyl-peptidase I. The deduced amino acid sequences each consist of 209 residues and show approximately 30% identity with bacterial forms of the enzyme. They show clear homology to the enzyme from prokaryotes and place the mammalian forms of the enzyme in peptidase family C15 of the MEROPS database. The catalytic residues Glu81, Cys144, and His166 in the enzyme from Bacillus amyloliquefaciens are all conserved in the human sequence. A simple cartoon model of the human protein was constructed on the basis of the published crystal structures of pyroglutamyl-peptidase I forms from Thermococcus litoralis and B. amyloliquefaciens. The human enzyme was expressed by use of a baculovirus vector in Spodoptera frugiperda cells. The recombinant protein was enzymatically active and had properties similar to those described for the naturally occurring mammalian enzyme. Gel-filtration chromatography of the active enzyme gave a molecular mass of about 24kDa, showing that the enzyme is active as the monomer. This contrasted with indications that the prokaryotic enzymes may be tetrameric. Recombinant human pyroglutamyl-peptidase I was active on pGlu-aminomethylcoumarin in the range pH 6-9, with maximal activity being seen at pH 7.0-8.5; it showed an absolute requirement for a thiol-reducing agent. In crude preparations, the enzyme was completely stable for 90 min at 50 degrees C. The enzyme was inhibited by transition metal ions including Ni(2+), Zn(2+), and Cu(2+), and by sulfhydryl-blocking agents. Reversible inhibition was seen with 2-pyrrolidone (K(i)=50 microM), and surprisingly, with N-ethylmaleimide (K(i)=30 microM).     

Early events of TCR signaling are distinct in human Th1 and Th2 cells

To study the requirements for activation of human Th1 and Th2 cells, soluble peptide/DR1 complexes were prepared from naturally expressed DR1 protein. When immobilized, this material induced T cell activation, as revealed by CD25 up-regulation. Unexpectedly, Th2 cells required a higher density of peptide/DR1 complexes than Th1 cells to initiate CD25 up-regulation. Similar findings were obtained with immobilized or soluble and cross-linked anti-CD3 mAb. In contrast, peptide/DR1 complexes displayed on the surface of nonprofessional APC similarly induced CD25 up-regulation in Th1 and Th2 cells. Signaling events distinguishing human Th1 and Th2 cells following TCR engagement by anti-CD3 mAb were then studied. It was observed that upon TCR triggering, the overall tyrosine phosphorylation profiles were fainter in Th2 than in Th1 clones. Similar results were obtained with Th1- and Th2-polarized polyclonal lines. Varying the dose of anti-CD3 mAb, the kinetics of activation, and coengagement of CD3 and CD28 failed to increase tyrosine phosphorylation in Th2 cells to levels reached in Th1 cells. In contrast, treatment with the tyrosine phosphatase inhibitor phenylarsine oxide resulted in similar tyrosine phosphorylation levels in Th2 and Th1 cells. These findings indicated that Th2 cells had an intrinsically lower TCR-induced tyrosine phosphorylation capacity than Th1 cells, which might be controlled by Th1- and Th2-specific phosphatase profiles. Finally, a weaker association was found between ZAP-70 and CD3zeta in Th2 than in Th1 cells after TCR engagement. Taken together, these results constituted evidence that early events in the TCR signaling cascades are distinct in human Th1 and Th2 cells.     

C-termini are essential and distinct for nucleic acid binding of human NABP1 and NABP2

BackgroundHuman Nucleic Acid Binding Protein 1 and 2 (hNABP1 and 2; also known as hSSB2 and 1, respectively) are two newly identified single-stranded (ss) DNA binding proteins (SSB). Both NABP1 and NABP2 have a conserved oligonucleotide/oligosaccharide-binding (OB)-fold domain and a divergent carboxy-terminal domain, the functional importance of which is unknown.MethodsRecombinant hNABP1/2 proteins were purified using affinity and size exclusion chromatography and their identities confirmed by mass spectrometry. Oligomerization state was checked by sucrose gradient centrifugation. Secondary structure was determined by circular dichroism spectroscopy. Nucleic acid binding ability was examined by EMSA and ITC.ResultsBoth hNABP1 and hNABP2 exist as monomers in solution; however, hNABP2 exhibits anomalous behavior. CD spectroscopy revealed that the C-terminus of hNABP2 is highly disordered. Deletion of the C-terminal tail diminishes the DNA binding ability and protein stability of hNABP2. Although both hNABP1 and hNABP2 prefer to bind ssDNA than double-stranded (ds) DNA, hNABP1 has a higher affinity for ssDNA than hNABP2. Unlike hNABP2, hNABP1 protein binds and multimerizes on ssDNA with the C-terminal tail responsible for its multimerization. Both hNABP1 and hNABP2 are able to bind single-stranded RNA, with hNABP2 having a higher affinity than hNABP1.ConclusionsBiochemical evidence suggests that the C-terminal region of NABP1 and NABP2 is essential for their functionality and may lead to different roles in DNA and RNA metabolism.General significanceThis is the first report demonstrating the regulation and functional properties of the C-terminal domain of hNABP1/2, which might be a general characteristic of OB-fold proteins.     

Hotspots of homologous recombination in the human genome: not all homologous sequences are equal

Homologous recombination between alleles or non-allelic paralogous sequences does not occur uniformly but is concentrated in 'hotspots' with high recombination rates. Recent studies of these hotspots show that they do not share common sequence motifs, but they do have other features in common.     

The properties of rabbit alpha1-macroglobulin upon activation are distinct from those of rabbit and human alpha2-macroglobulins

We have characterized native and activated forms of rabbit alpha1M and compared them to rabbit and human alpha2M. Similar to human alpha2M, rabbit alpha1M is a tetramer associated via disulfide bonds and non-covalent interactions that exhibits autolysis into two fragments when heated. Like human alpha2M, rabbit alpha1M is cleaved by trypsin at one site; however, rabbit alpha1M shares characteristics with rabbit alpha2M that are different from the properties of human alpha2M. Amine or trypsin treatment of rabbit alpha-macroglobulins does not result in a significant conformational change or cleavage of four thiolester bonds. Full thiolester cleavage is only observed for rabbit alpha1M after exposure to both trypsin and a small amine. Additionally, amine-treated rabbit alpha-macroglobulins retain trypsin inhibitory potential and do not fully shield bound proteinases. Methylamine and trypsin treatment of rabbit alpha1M results in two dissimilar conformations that display differing exposure of the receptor-recognition site. While ammonia- and methylamine-modified rabbit alpha1M bind to macrophages with similar affinity to that of human alpha2M, trypsin-treated rabbit alpha1M exhibits dramatically lower affinity. This suggests that rabbit alpha1M may not play the same proteinase-inhibiting physiological role as human alpha2M.     

Rac1 and PAK1 are upstream of IKK-epsilon and TBK-1 in the viral activation of interferon regulatory factor-3

The anti-viral type I interferon (IFN) response is initiated by the immediate induction of IFN beta, which is mainly controlled by the IFN-regulatory factor-3 (IRF-3). The signaling pathways mediating viral IRF-3 activation are only poorly defined. We show that the Rho GTPase Rac1 is activated upon virus infection and controls IRF-3 phosphorylation and activity. Inhibition of Rac1 leads to reduced IFN beta promoter activity and to enhanced virus production. As a downstream mediator of Rac signaling towards IRF-3, we have identified the kinase p21-activated kinase (PAK1). Furthermore, both Rac1 and PAK1 regulate the recently described IRF-3 activators, I kappa B kinase- and TANK-binding kinase-1, establishing a first canonical virus-induced IRF-3 activating pathway.     

Phosphomevalonate kinase: functional investigation of the recombinant human enzyme

Phosphomevalonate kinase (PMK) catalyzes a key step in isoprenoid/sterol biosynthesis, converting mevalonate 5-phosphate and ATP to mevalonate 5-diphosphate and ADP. To expedite functional and structural study of this enzyme, an expression plasmid encoding His-tagged human PMK has been constructed and recombinant enzyme isolated in an active, stable form. PMK catalyzes a reversible reaction; kinetic constants of human PMK have been determined for both forward (formation of mevalonate 5-diphosphate) and reverse (formation of mevalonate 5-phosphate) reactions. Animal and invertebrate PMKs are not orthologous to plant, fungal, or bacterial PMKs, limiting the information available from sequence alignment analysis. A homology model for the structure of human PMK has been generated. The model conforms to a nucleoside monophosphate kinase family fold. This result, together with sequence comparisons of animal and invertebrate PMKs, suggests an N-terminal basic residue rich sequence as a possible "Walker A" ATP binding motif. The functions of four basic (K17, R18, K19, K22) residues and one acidic (D23) residue in the conserved sequence have been tested by mutagenesis and characterization of isolated mutant proteins. Substrate K(m) values for K17M, R18Q, K19M, and D23N have been measured for forward and reverse reactions; in comparison with wild-type PMK values, only modest (<12-fold) changes are observed. In contrast, R18Q exhibits a V(max) decrease of 100/300-fold (forward/reverse reaction). K22M activity is too low for measurement at nonsaturating substrate concentration; specific activity is decreased by >10000-fold in both forward/reverse reactions, suggesting an active site location and an important role in phosphoryl transfer.     

Coassembly and complementation of Gag proteins from HIV-1 and HIV-2, two distinct human pathogens

Approximately one million people in the world are dually infected with both HIV-1 and HIV-2. To identify potential interactions between these two human pathogens, we examined whether HIV-1 and HIV-2 Gag proteins can coassemble and functionally complement each other. We generated HIV-1- and HIV-2-based vectors with mutations in Gag; compared with wild-type vectors, these mutants had drastically decreased viral titers. Coexpression of the mutant HIV-1 and HIV-2 Gag could generate infectious viruses; furthermore, heterologous complementation in certain combinations showed efficiency similar to homologous complementation. Additionally, we used bimolecular fluorescence complementation analysis to directly demonstrate that HIV-1 and HIV-2 Gag can interact and coassemble. Taken together, our results indicate that HIV-1 and HIV-2 Gag polyproteins can coassemble and functionally complement each other during virus replication; to our knowledge, this is the first demonstration of its kind. These studies have important implications for AIDS treatment and the evolution of primate lentiviruses.     

Purification and characterization of human plasma glutathione peroxidase: a selenoglycoprotein distinct from the known cellular enzyme

Glutathione peroxidase (GSHPx), (glutathione:H2O2 oxidoreductase, EC 1.11.1.9) was purified to homogeneity from human plasma. This resulted in a 6800-fold purification of the enzyme with a 2.8% yield. The purification process involved ammonium sulfate fractionation, DEAE-cellulose batch and column chromatographies, hydroxyapatite, and Sephadex G-200 and DEAE-Sephadex A-25 chromatographies. The major peak on DEAE-Sephadex A-25 column chromatography was found to be homogeneous on polyacrylamide gel electrophoresis in the presence or absence of sodium dodecyl sulfate (SDS). Relative mobility in nondenaturing polyacrylamide gel electrophoresis at pH 8.2 was 0.5 for the purified enzyme as detected by both protein staining and enzyme activity compared with 0.38 for erythrocyte GSHPx. The molecular weight of the plasma enzyme as determined by gel filtration was found to be approximately 100,000. SDS-gel electrophoresis of the plasma enzyme gave a subunit molecular weight of approximately 23,000. This suggests that the plasma enzyme exists as a tetramer in its native state, similar to that seen for the erythrocyte enzyme, but with slightly different mobility on SDS-gel electrophoresis. Plasma GSHPx, like the erythrocyte enzyme, was found to contain approximately four atoms of selenium per mole of protein. Utilizing iodinated concanavalin A, it was found that plasma GSHPx, but not the erythrocyte GSPx, is a glycoprotein. Purified plasma enzyme catalyzes both the reduction of tertiary butyl hydroperoxide and hydrogen peroxide. The apparent Km of plasma GSHPx for GSH is 5.3 mM and for tertiary butyl hydroperoxide it is 0.57 mM. Copper, mercury, and zinc strongly inhibit the enzyme activity of plasma GSHPx. Rabbit antibodies directed against the human erythrocyte GSHPx do not precipitate the enzyme activity of the purified plasma enzyme. Radioimmunoassay utilizing erythrocyte GSHPx and anti-erythrocyte GSHPx antibodies showed that less than 0.13% of the antigenically detectable protein is found in the purified GSHPx from plasma.     

Spectrin band 1 and 2 are antigenically distinct and are not composed of subunits.

Human erythrocyte membranes and freshly isolated spectrin were separated into their constituent peptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The peptides were electrophoresed from slices of such gels into agarose gels containing anti-spectrin antibodies and Triton X-100. In fresh preparations, precipitin arcs were observed only against peptides migrating as bands 1 and 2. It was found that bands 1 and 2 did not cross-react. There were two major arcs from band 1 and one principal arc from band 2, plus minor splitting of these arcs. None of the band 1 arcs fused with band 2 arcs. In fresh erythrocyte ghosts only bands 1 and 2 reacted with anti-spectrin; bands 2.1, 3, and 5, in particular, showed no precipitin arcs. However, in aged ghosts, arcs appeared in the band 3 region; in aged isolated spectrin, arcs appeared in the band 2.1 region; and in trypsin-degraded spectrin, reactive species occurred in all molecular weight classes. It is concluded that spectrin has no subunits smaller than 220,000 molecular weight and that bands 1 and 2 are immunochemically distinct.     

Hit-to-lead studies: the discovery of potent, orally active, thiophenecarboxamide IKK-2 inhibitors

A hit-to-lead optimisation programme was carried out on the thiophenecarboxamide high throughput screening hits 1 and 2 resulting in the discovery of the potent and orally bioavailable IKK-2 inhibitor 22.     

A catalytically active complex formed from the recombinant dI protein of Rhodospirillum rubrum transhydrogenase, and the recombinant dIII protein of the human enzyme

Transhydrogenase is a proton pump. It has three components: dI and dIII protrude from the membrane and contain the binding sites for NAD(H) and NADP(H), respectively, and dII spans the membrane. We have expressed dIII from Homo sapiens transhydrogenase (hsdIII) in Escherichia coli. The purified protein was associated with stoichiometric amounts of NADP(H) bound to the catalytic site. The NADP+ and NADPH were released only slowly from the protein, supporting the suggestion that nucleotide-binding by dIII is regulated by the membrane-spanning dII. HsdIII formed a catalytically active complex with recombinant dI from Rhodospirillum rubrum (rrdI), even in the absence of dII. The rates of forward and reverse transhydrogenation catalysed by this complex are probably limited by slow release from dIII of NADPH and NADP+, respectively. The hybrid complex also catalysed high rates of 'cyclic' transhydrogenation, indicating that hydride transfer, and exchange of nucleotides with dI, are rapid. Stopped-flow experiments revealed a rapid, monoexponential, single-turnover burst of reverse transhydrogenation in pre-steady-state. The apparent first-order rate constant of the burst increased with the concentration of rrdI. A deuterium isotope effect (kH/kD approximately 2 at 27 degrees C) was observed when [4B-1H]NADPH was replaced with [4B-2H]NADPH. The characteristics of the burst of transhydrogenation with rrdI:hsdIII differed from those previously reported for rrdI:rrdIII (J.D. Venning et al., Eur. J. Biochem. 257 (1998) 202-209), but the differences are readily explained by a greater dissociation constant of the hybrid complex. The steady-state rate of reverse transhydrogenation by the rrdI:hsdIII complex was almost independent of pH, but there was a single apparent pKa ( approximately 9.1) associated with the cyclic reaction. The reactions of the dI:dIII complex probably proceed independently of those protonation/deprotonation reactions which, in the complete enzyme, are associated with H+ translocation.