Molecular and catalytic properties of Arabidopsis thaliana adenylyl sulfate (APS)-kinase

A cDNA clone (Atakn1) from Arabidopsis thaliana encoding APS-kinase (EC 2.7.1.25) was investigated for structural and catalytic properties of the gene product. Recombinant his10-AtAkn1 formed PAPS at a Vmax of 7.35 U x mg(-1). The Km for APS was 0.14 microM and for ATP 147 microM. APS caused a severe substrate inhibition (K(i) 4.5 microM). The type of inhibition is uncompetitive with respect to MgATP. High ionic strength and reducing thiols stabilized the enzyme activity. Plant APS-kinase is regulated in vitro by the redox charge with thioredoxin as essential activator. Mutagenesis of a serine in S182C and S182F presumed to be involved in the transfer of the phosphoryl group had no effect upon catalytic activity. Using a yeast two-hybrid system with AtAkn1 as bait, an interacting clone was detected from a cDNA library of A. thaliana cv. Columbia that codes for an APS-kinase iso-form (Atakn2). Complementation of APS-kinase-deficient Saccharomyces cerevisiae met14 showed that AtAkn2 is functionally active as APS-kinase. It was immunologically related to AtAkn1 and presumably represents a plastidal iso-form of the plant APS-kinase gene family.     

Electron transfer in the substrate-dependent suicide inactivation of lysine 5,6-aminomutase

The lysine 5,6-aminomutase (5,6-LAM) purified from Clostridium sticklandii was found to undergo rapid inactivation in the absence of the activating enzyme E(2) and ATP. In the presence of substrate, inactivation was also seen for the recombinant 5,6-LAM. This adenosylcobalamin-dependent enzyme is postulated to generate cob(II)alamin and the 5'-deoxyadenosyl radical through enzyme-induced homolytic scission of the Co-C bond. However, the products cob(III)alamin and 5'-deoxyadenosine were observed upon inactivation of 5,6-LAM. Cob(III)alamin production, as monitored by the increase in A(358), proceeds at the same rate as the loss of enzyme activity, suggesting that the activity loss is related to the adventitious generation of cob(III)alamin during enzymatic turnover. The cleavage of adenosylcobalamin to cob(III)alamin is accompanied by the formation of 5'-deoxyadenosine at the same rate, and the generation of cob(III)alamin proceeds at the same rate both aerobically and anaerobically. Suicide inactivation requires the presence of substrate, adenosylcobalamin, and PLP. We have ruled out the involvement of either the putative 5'-deoxyadenosyl radical or dioxygen in suicide inactivation. We have shown that one or more reaction intermediates derived from the substrate or/and the product, presumably a radical, participate in suicide inactivation of 5,6-LAM through electron transfer from cob(II)alamin. Moreover, L-lysine is found to be a slowly reacting substrate, and it induces inactivation at a rate similar to that of D-lysine. The alternative substrate beta-lysine induces inactivation at least 25 times faster than DL-lysine. The inactivation mechanism is compatible with the radical isomerization mechanism proposed to explain the action of 5,6-LAM.     

Oxidations of p-alkoxyacylanilides catalyzed by human cytochrome P450 1A2: structure-activity relationships and simulation of rate constants of individual steps in catalysis

Human cytochrome P450 (P450) 1A2 is involved in the oxidation of many important drugs and carcinogens. The prototype substrate phenacetin is oxidized to an acetol as well as the O-dealkylation product [Yun, C.-H., Miller, G. P., and Guengerich, F. P. (2000) Biochemistry 39, 11319-11329]. In an effort to improve rates of catalysis of P450 1A2 enzymes, we considered a set of p-alkoxyacylanilide analogues of phenacetin and found that variations in the O-alkyl and N-acyl substituents altered the rates of the two oxidation reactions and the ratio of acetol/phenol products. Moving one methylene group of phenacetin from the O-alkyl group to the N-acyl moiety increased rates of both oxidations approximately 5-fold and improved the coupling efficiency (oxidation products formed/NADPH consumed) from 6% to 38%. Noncompetitive kinetic deuterium isotope effects of 2-3 were measured for all O-dealkylation reactions examined with wild-type P450 1A2 and the E225I mutant, which has 6-fold higher activity. A trend of decreasing kinetic deuterium isotope effect for E225I > wild-type > mutant D320A was observed for O-demethylation of p-methoxyacetanilide, which follows the trend for k(cat). The set of O-dealkylation and acetol formation results for wild-type P450 1A2 and the E225I mutant with several of the protiated and deuterated substrates were fit to a model developed for the basic catalytic cycle and a set of microscopic rate constants in which the only variable was the rate of product formation (substrate oxygenation, including hydrogen abstraction). In this model, k(cat) is considerably less than any of the microscopic rate constants and is affected by several individual rate constants, including the rate of formation of the oxygenating species, the rate of substrate oxidation by the oxygenating species, and the rates of generation of reduced oxygen species (H(2)O(2), H(2)O). This analysis of the effects of the individual rate constants provides a framework for consideration of other P450 reactions and rate-limiting steps.     

Correct Expression of spec2a in the sea urchin embryo requires both Otx and other cis-regulatory elements

Strongylocentrotus purpuratus Otx (SpOtx) is required simultaneously in sea urchin development for the activation of endo16 in the vegetal plate and for the activation of spec2a in the aboral ectoderm. Because Otx binding sites alone do not appear to be responsible for the spatially restricted expression of spec2a, additional DNA elements were sought. We show here that consensus Otx binding sites fused to basal promoters are sufficient to activate CAT reporter gene expression in all cell types, although expression in endomesoderm progenitors is enhanced. On the other hand, three non-Otx elements derived from the spec2a enhancer are needed together with Otx sites for specifically aboral ectoderm expression. A DNA element termed Y/CBF, lying just downstream from an Otx site within the spec2a enhancer, mediates general activation in the ectoderm. A second element lying between the Otx and Y/CBF sites, called OER, functions to prevent expression in the oral ectoderm. A third site, called ENR, overlapping another Otx site, is required to repress endoderm expression. Three distinct DNA binding proteins interact sequence specifically at the Y/CBF, OER, and ENR elements. The spec2a enhancer thus consists of closely linked activator and repressor elements that function collectively to cause expression of the spec2a gene in the aboral ectoderm.     

A docking model of key components of the DISC complex: death domain superfamily interactions redefined

Apoptosis is mediated by a highly regulated signal transduction cascade that eventually leads to precisely directed cell death. The death-inducing signaling complex (DISC), composed of Fas, FADD, and caspase-8, is an apical signaling complex that mediates receptor-induced apoptosis. We have docked the experimentally determined structures of the Fas and FADD death domains into a model of a partial DISC signaling complex. The arrangement of Fas and FADD was determined using the interaction modes of the two heterodimer crystal structures determined to date, Pelle/Tube and Apaf-1/procaspase-9. The proposed model reveals that both interactions can be accommodated in a single multimeric complex. Importantly, the model is consistent with reported site-directed mutagenesis data indicating residues throughout the domain are critical for function. These results imply that members of the death domain superfamily have the potential for multivalent interactions, offering novel possibilities for regulation of apoptotic signaling.     

Functional reconstitution and regulation of IL-18 activity by the IL-18R beta chain

IL-18 and IL-12 are major IFN-gamma-inducing cytokines but the unique synergism of IL-18 and IL-12 remains unclear. In the human NK cell line NKO, IL-18R alpha, and IL-18R beta are expressed constitutively but IL-18 did not induce IFN-gamma unless IL-12 was present. COS-1 fibroblasts, which produce the chemokine IL-8 when stimulated by IL-1 beta or TNF-alpha, do not respond to IL-18, despite abundant expression of the IL-18R alpha chain. COS-1 cells lack expression of the IL-18R beta chain. The IL-18R beta cDNA was cloned from a human T-B lymphoblast cDNA library and COS-1 cells were transiently transfected with the IL-18R beta chain and a luciferase reporter. In transfected COS-1 cells, IL-18 induced IL-8 and luciferase in the absence of IL-12 and independently of IL-1 and TNF. Ab against the IL-18R alpha chain, however, prevented IL-18 responsiveness in COS-1 cells transfected with the IL-18R beta chain, suggesting that both chains be functional. In NKO cells and PBMC, IL-12 increased steady-state mRNA levels of IL-18R alpha and IL-18R beta; the production of IFN-gamma corresponded to IL-12-induced IL-18R alpha and IL-18R beta chains. We conclude that functional reconstitution of the IL-18R beta chain is essential for IL-12-independent proinflammatory activity of IL-18-induced IL-8 in fibroblasts. The synergism of IL-18 plus IL-12 for IFN-gamma production is, in part, due to IL-12 up-regulation of both IL-18R alpha and IL-18R beta chains, although postreceptor events likely contribute to IFN-gamma production.     

Effective treatment of established murine collagen-induced arthritis by systemic administration of dendritic cells genetically modified to express IL-4

Dendritic cells (DC) are APCs that are able to stimulate or inhibit immune responses, depending on levels of expression of MHC class I and II costimulatory molecules and cytokines. Our previous studies have suggested that the observed contralateral effect, where injection of a vector carrying certain immunomodulatory genes into one joint resulted in inhibition of arthritis in untreated joints, is mediated by in vivo modification of DC. Therefore, we have examined the ability of genetically modified DC to suppress established murine collagen-induced arthritis (CIA) after i.v. delivery. IL-4 has been shown to partially reduce the severity of CIA after repeated injection of recombinant protein or by injection of an adenoviral vector expressing IL-4. Here we demonstrate that i.v. injection of immature DC, infected with an adenoviral vector expressing IL-4, into mice with established CIA resulted in almost complete suppression of disease, with no recurrence for up to 4 wk posttreatment. Injection i.v. of fluorescently labeled DC demonstrated that the cells rapidly migrated to the liver and spleen after 6 h and to the lymph nodes by 24 h. In culture, spleen cells from DC/IL-4-treated mice produced less IFN-gamma after stimulation by collagen than did control groups. In addition, DC/IL-4 administration decreased the level of specific Abs against type II collagen, in particular the IgG2 Th1 isotype 14 days posttreatment. These results demonstrate the ability to treat effectively established murine arthritis by systemic administration of DC expressing IL-4.