Redox-dependent activation of CO dehydrogenase from Rhodospirillum rubrum

Studies of initial activities of carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum show that CODH is mostly inactive at redox potentials higher than −300 mV. Initial activities measured at a wide range of redox potentials (0–500 mV) fit a function corresponding to the Nernst equation with a midpoint potential of −316 mV. Previously, extensive EPR studies of CODH have suggested that CODH has three distinct redox states: (i) a spin-coupled state at −60 to −300 mV that gives rise to an EPR signal termed Cred1; (ii) uncoupled states at <−320 mV in the absence of CO2 referred to as Cunc; and (iii) another spin-coupled state at <−320 mV in the presence of CO2 that gives rise to an EPR signal termed Cred2B. Because there is no initial CODH activity at potentials that give rise to Cred1, the state (Cred1) is not involved in the catalytic mechanism of this enzyme. At potentials more positive than −380 mV, CODH recovers its full activity over time when incubated with CO. This reductant-dependent conversion of CODH from an inactive to an active form is referred to hereafter as “autocatalysis.” Analyses of the autocatalytic activation process of CODH suggest that the autocatalysis is initiated by a small fraction of activated CODH; the small fraction of active CODH catalyzes CO oxidation and consequently lowers the redox potential of the assay system. This process is accelerated with time because of accumulation of the active enzyme.

Light-induced phase-delay of the chicken pineal circadian clock is associated with the induction of cE4bp4, a potential transcriptional repressor of cPer2 gene

The chicken pineal gland contains the autonomous circadian oscillator together with the photic-input pathway. We searched for chicken pineal genes that are induced by light in a time-of-day-dependent manner, and isolated chicken homolog of bZIP transcription factor E4bp4 (cE4bp4) showing high similarity to vrille, one of the Drosophila clock genes. cE4bp4 was expressed rhythmically in the pineal gland with a peak at very early (subjective) night under both 12-h light/12-h dark cycle and constant dark conditions, and the phase was nearly opposite to the expression rhythm of cPer2, a chicken pineal clock gene. Luciferase reporter gene assays showed that cE4BP4 represses cPer2 promoter through a E4BP4-recognition sequence present in the 5′-flanking region, indicating that cE4BP4 can down-regulate the chick pineal cPer2 expression. In vivo light-perturbation studies showed that the prolongation of the light period to early subjective night maintained the high level expression of the pineal cE4bp4, and presumably as a consequence delayed the onset of the induction of the pineal cPer2 expression in the next morning. These light-dependent changes in the mRNA levels of the pineal cE4bp4 and cPer2 were followed by a phase-delay of the subsequent cycles of cE4bp4/cPer2 expression, suggesting that cE4BP4 plays an important role in the phase-delaying process as a light-dependent suppressor of cPer2 gene.

Two recombination-dependent DNA replication pathways of bacteriophage T4, and their roles in mutagenesis and horizontal gene transfer

Two major pathways of recombination-dependent DNA replication, “join-copy” and “join-cut-copy,” can be distinguished in phage T4: join-copy requires only early and middle genes, but two late proteins, endonuclease VII and terminase, are uniquely important in the join-cut-copy pathway. In wild-type T4, timing of these pathways is integrated with the developmental program and related to transcription and packaging of DNA. In primase mutants, which are defective in origin-dependent lagging-strand DNA synthesis, the late pathway can bypass the lack of primers for lagging-strand DNA synthesis. The exquisitely regulated synthesis of endo VII, and of two proteins from its gene, explains the delay of recombination-dependent DNA replication in primase (as well as topoisomerase) mutants, and the temperature-dependence of the delay. Other proteins (e.g., the single-stranded DNA binding protein and the products of genes 46 and 47) are important in all recombination pathways, but they interact differently with other proteins in different pathways. These homologous recombination pathways contribute to evolution because they facilitate acquisition of any foreign DNA with limited sequence homology during horizontal gene transfer, without requiring transposition or site-specific recombination functions. Partial heteroduplex repair can generate what appears to be multiple mutations from a single recombinational intermediate. The resulting sequence divergence generates barriers to formation of viable recombinants. The multiple sequence changes can also lead to erroneous estimates in phylogenetic analyses.

Structural studies of p21Waf1/Cip1/Sdi1 in the free and Cdk2-bound state: conformational disorder mediates binding diversity.

The cyclin-dependent kinase (Cdk) inhibitor p21Waf1/Cip1/Sdi1, important for p53-dependent cell cycle control, mediates G1/S arrest through inhibition of Cdks and possibly through inhibition of DNA replication. Cdk inhibition requires a sequence of approximately 60 amino acids within the p21 NH2 terminus. We show, using proteolytic mapping, circular dichroism spectropolarimetry, and nuclear magnetic resonance spectroscopy, that p21 and NH2-terminal fragments that are active as Cdk inhibitors lack stable secondary or tertiary structure in the free solution state. In sharp contrast to the disordered free state, however, the p21 NH2 terminus adopts an ordered stable conformation when bound to Cdk2, as shown directly by NMR spectroscopy. We have, thus, identified a striking disorder-order transition for p21 upon binding to one of its biological targets, Cdk2. This structural transition has profound implications in light of the ability of p21 to bind and inhibit a diverse family of cyclin-Cdk complexes, including cyclin A-Cdk2, cyclin E-Cdk2, and cyclin D-Cdk4. Our findings suggest that the flexibility, or disorder, of free p21 is associated with binding diversity and offer insights into the role for structural disorder in mediating binding specificity in biological systems. Further, these observations challenge the generally accepted view of proteins that stable secondary and tertiary structure are prerequisites for biological activity and suggest that a broader view of protein structure should be considered in the context of structure-activity relationships.

The cytokine-activated tyrosine kinase JAK2 activates Raf-1 in a p21ras-dependent manner.

JAK2, a member of the Janus kinase superfamily was found to interact functionally with Raf-1, a central component of the ras/mitogen-activated protein kinase signal transduction pathway. Interferon-gamma and several other cytokines that are known to activate JAK2 kinase were also found to stimulate Raf-1 kinase activity toward MEK-1 in mammalian cells. In the baculovirus coexpression system, Raf-1 was activated by JAK2 in the presence of p21ras. Under these conditions, a ternary complex of p21ras, JAK2, and Raf-1 was observed. In contrast, in the absence of p21ras, coexpression of JAK2 and Raf-1 resulted in an overall decrease in the Raf-1 kinase activity. In addition, JAK2 phosphorylated Raf-1 at sites different from those phosphorylated by pp60v-src. In mammalian cells treated with either erythropoietin or interferon-gamma, a small fraction of Raf-1 coimmunoprecipitated with JAK2 in lysates of cells in which JAK2 was activated as judged by its state of tyrosine phosphorylation. Taken together, these data suggest that JAK2 and p21ras cooperate to activate Raf-1.

Reactive oxygen species are downstream mediators of p53-dependent apoptosis.

Reactive oxygen species (ROS) have been implicated as potential modulators of apoptosis. Conversely, experiments under hypoxic conditions have suggested that apoptosis could occur in the absence of ROS. We sought to determine whether a central modulator of apoptosis, p53, regulates the levels of intracellular ROS and whether a rise in ROS levels is required for the induction of p53-dependent apoptosis. We transiently overexpressed wild-type p53, using adenoviral gene transfer, and identified cell types that were sensitive or resistant to p53-mediated apoptosis. Cells sensitive to p53-mediated apoptosis produced ROS concomitantly with p53 overexpression, whereas cells resistant to p53 failed to produce ROS. In sensitive cells, both ROS production and apoptosis were inhibited by antioxidant treatment. These results suggest that p53 acts to regulate the intracellular redox state and induces apoptosis by a pathway that is dependent on ROS production.

p53-dependent cell cycle arrest induced by N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal in platelet-derived growth factor-stimulated human fibroblasts.

Proteases are known to play important roles in cell growth control, although the underlying mechanisms are still poorly understood. Here we show that the protease inhibitor N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal induced cell cycle arrest in platelet-derived growth factor-stimulated human fibroblasts at the G1/S boundary of the cell cycle by inhibiting the proteasome. Inhibition of the proteasome resulted in accumulation of the tumor suppressor p53, which was followed by an increase in the amount of the cyclin-dependent kinase-inhibitor p21. As a consequence, both phosphorylation and activity of the cyclin-dependent kinase 2/cyclin E complex were inhibited. We further observed that the retinoblastoma gene product, pRb, remained in the hypophosphorylated state, thus preventing cells from progression into the S-phase. These studies strongly support the hypothesis that the proteasome is a key regulator in the G1-phase of cell cycle progression.

Interleukin 2 production, not the pattern of early T-cell antigen receptor-dependent tyrosine phosphorylation, controls anergy induction by both agonists and partial agonists.

Full activation of T cells requires signaling through the T-cell antigen receptor (TCR) and additional surface molecules interacting with ligands on the antigen-presenting cell. TCR recognition of agonist ligands in the absence of accessory signals frequently results in the induction of a state of unresponsiveness termed anergy. However, even in the presence of costimulation, anergy can be induced by TCR partial agonists. The unique pattern of early receptor-induced tyrosine phosphorylation events induced by partial agonists has led to the hypothesis that altered TCR signaling is directly responsible for the development of anergy. Here we show that anergy induction is neither correlated with nor irreversibly determined by the pattern of early TCR-induced phosphorylation. Rather, it appears to result from the absence of downstream events related to interleukin 2 receptor occupancy and/or cell division. This implies that the anergic state can be manipulated independently of the precise pattern of early biochemical changes following TCR occupancy, a finding with implications for understanding the induction of self-tolerance and the use of partial agonist ligands in the treatment of autoimmune diseases.

Glutamate is required to maintain the steady-state potassium pool in Salmonella typhimurium.

In many bacteria, accumulation of K+ at high external osmolalities is accompanied by accumulation of glutamate. To determine whether there is an obligatory relationship between glutamate and K+ pools, we studied mutant strains of Salmonella typhimurium with defects in glutamate synthesis. Enteric bacteria synthesize glutamate by the combined action of glutamine synthetase and glutamate synthase (GS/GOGAT cycle) or the action of biosynthetic glutamate dehydrogenase (GDH). Activity of the GS/GOGAT cycle is required under nitrogen-limiting conditions and is decreased at high external ammonium/ammonia ((NH4)+) concentrations by lowered synthesis of GS and a decrease in its catalytic activity due to covalent modification (adenylylation by GS adenylyltransferase). By contrast, GDH functions efficiently only at high external (NH4)+ concentrations, because it has a low affinity for (NH4)+. When grown at low concentrations of (NH4)+ (< or = 2 mM), mutant strains of S. typhimurium that lack GOGAT and therefore are dependent on GDH have a low glutamate pool and grow slowly; we now demonstrate that they have a low K+ pool. When subjected to a sudden (NH4)+ upshift, strains lacking GS adenylyltransferase drain their glutamate pool into glutamine and grow very slowly; we now find that they also drain their K+ pool. Restoration of the glutamate pool in these strains at late times after shift was accompanied by restoration of the K+ pool and a normal growth rate. Taken together, the results indicate that glutamate is required to maintain the steady-state K+ pool -- apparently no other anion can substitute as a counter-ion for free K+ -- and that K+ glutamate is required for optimal growth.

High copy number I-Ab transgenes induce production of IgE through an interluekin 4-dependent mechanism.

To better understand the role of class II major histocompatibility complex molecules in both normal and autoimmune responses, we have produced a series of I-Ab transgenic mice. One of these transgenic constructs, designated NOD.PD, has the sequence of the NOD beta chain (Abeta(g7)) except at positions 56 and 57, where Pro-Asp replaces His-Ser. Several NOD.PD transgenic lines have been produced. One line of these mice carried a very high number of copies (>50) of the NOD.PD transgene. As has been described in other mice carrying high copy numbers of I-Ab transgenes, B-cell development was abnormal. The steady state numbers of mature B cells (IgM+/IgD(hi)) in the periphery were greatly reduced in transgenic mice compared to nontransgenic littermates. Surprisingly, rather than being accompanied by a generalized hypogammaglobulinemia, this B-cell deficiency was accompanied by elevated concentrations of IgG1 and IgE in the serum. Conversely, the levels of IgG2a were reduced in transgenic mice compared to nontransgenic littermates. Because this isotype pattern was characteristic of interleukin (IL)-4-induced class-switching, we then investigated the role of IL-4 in causing the observed phenotype. We crossed the high copy number transgenic mice with an IL-4-deficient strain of mice. As expected, the elevated levels of IgE in high copy number transgenic mice were eliminated when the IL-4 gene was inactivated. However, the reduction in the number of B cells was not ameliorated. These data indicate that the primary defect caused by the transgene was to reduce the number of B cells in these mice. This reduction was accompanied by a secondary increase in IL-4 production, which drove the remaining B cells toward the production of IgGl and IgE.

Regulation of phosducin phosphorylation in retinal rods by Ca2+/calmodulin-dependent adenylyl cyclase.

The phosphoprotein phosducin (Pd) regulates many guanine nucleotide binding protein (G protein)-linked signaling pathways. In visual signal transduction, unphosphorylated Pd blocks the interaction of light-activated rhodopsin with its G protein (Gt) by binding to the beta gamma subunits of Gt and preventing their association with the Gt alpha subunit. When Pd is phosphorylated by cAMP-dependent protein kinase, it no longer inhibits Gt subunit interactions. Thus, factors that determine the phosphorylation state of Pd in rod outer segments are important in controlling the number of Gts available for activation by rhodopsin. The cyclic nucleotide dependencies of the rate of Pd phosphorylation by endogenous cAMP-dependent protein kinase suggest that cAMP, and not cGMP, controls Pd phosphorylation. The synthesis of cAMP by adenylyl cyclase in rod outer segment preparations was found to be dependent on Ca2+ and calmodulin. The Ca2+ dependence was within the physiological range of Ca2+ concentrations in rods (K1/2 = 230 +/- 9 nM) and was highly cooperative (n app = 3.6 +/- 0.5). Through its effect on adenylyl cyclase and cAMP-dependent protein kinase, physiologically high Ca2+ (1100 nM) was found to increase the rate of Pd phosphorylation 3-fold compared to the rate of phosphorylation at physiologically low Ca2+ (8 nM). No evidence for Pd phosphorylation by other (Ca2+)-dependent kinases was found. These results suggest that Ca2+ can regulate the light response at the level of Gt activation through its effect on the phosphorylation state of Pd.

Oral tolerance in myelin basic protein T-cell receptor transgenic mice: suppression of autoimmune encephalomyelitis and dose-dependent induction of regulatory cells.

Orally administered antigens induce a state of immunologic hyporesponsiveness termed oral tolerance. Different mechanisms are involved in mediating oral tolerance depending on the dose fed. Low doses of antigen generate cytokine-secreting regulatory cells, whereas high doses induce anergy or deletion. We used mice transgenic for a T-cell receptor (TCR) derived from an encephalitogenic T-cell clone specific for the acetylated N-terminal peptide of myelin basic protein (MBP) Ac-1-11 plus I-Au to test whether a regulatory T cell could be generated from the same precursor cell as that of an encephalitogenic Th1 cell and whether the induction was dose dependent. The MBP TCR transgenic mice primarily have T cells of a precursor phenotype that produce interleukin 2 (IL-2) with little interferon gamma (IFN-gamma), IL-4, or transforming growth factor beta (TGF-beta). We fed transgenic animals a low-dose (1 mg x 5) or high-dose (25 mg x 1) regimen of mouse MBP and without further immunization spleen cells were tested for cytokine production. Low-dose feeding induced prominent secretion of IL-4, IL-10, and TGF-beta, whereas minimal secretion of these cytokines was observed with high-dose feeding. Little or no change was seen in proliferation or IL-2/IFN-gamma secretion in fed animals irrespective of the dose. To demonstrate in vivo functional activity of the cytokine-secreting cells generated by oral antigen, spleen cells from low-dose-fed animals were adoptively transferred into naive (PLJ x SJL)F1 mice that were then immunized for the development of experimental autoimmune encephalomyelitis (EAE). Marked suppression of EAE was observed when T cells were transferred from MBP-fed transgenic animals but not from animals that were not fed. In contrast to oral tolerization, s.c. immunization of transgenic animals with MBP in complete Freund's adjuvant induced IFN-gamma-secreting Th1 cells in vitro and experimental encephalomyelitis in vivo. Despite the large number of cells reactive to MBP in the transgenic animals, EAE was also suppressed by low-dose feeding of MBP prior to immunization. These results demonstrate that MBP-specific T cells can differentiate in vivo into encephalitogenic or regulatory T cells depending upon the context by which they are exposed to antigen.

Apolipoprotein E competitively inhibits receptor-dependent low density lipoprotein uptake by the liver but has no effect on cholesterol absorption or synthesis in the mouse.

This study examines the question of whether apolipoprotein E (apoE) alters steady-state concentrations of plasma cholesterol carried in low density lipoproteins (LDL-C) by acting as a competitive inhibitor of hepatic LDL uptake or by altering the rate of net cholesterol delivery from the intestinal lumen to the liver. To differentiate between these two possibilities, rates of cholesterol absorption and synthesis and the kinetics of hepatic LDL-C transport were measured in vivo in mice with either normal (apoE+/+) or zero (apoE-/-) levels of circulating apoE. Rates of cholesterol absorption were essentially identical in both genotypes and equaled approximately 44% of the daily dietary load of cholesterol. This finding was consistent with the further observation that the rates of cholesterol synthesis in the liver (approximately 2,000 nmol/h) and extrahepatic tissues (approximately 3,000 nmol/h) were also essentially identical in the two groups of mice. However, the apparent Michaelis constant for receptor-dependent hepatic LDL-C uptake was markedly lower in the apoE-/- mice (44 +/- 4 mg/dl) than in the apoE+/+ animals (329 +/- 77 mg/dl) even though the maximal transport velocity for this uptake process was essentially the same (approximately 400 micrograms/h per g) in the two groups of mice. These studies, therefore, demonstrate that apoE-containing lipoproteins can act as potent competitive inhibitors of hepatic LDL-C transport and so can significantly increase steady-state plasma LDL-C levels. This apolipoprotein plays no role, however, in the regulation of cholesterol absorption, sterol biosynthesis, or hepatic LDL receptor number, at least in the mouse.

Coupling the phosphotransferase system and the methyl-accepting chemotaxis protein-dependent chemotaxis signaling pathways of Escherichia coli.

Chemotactic responses in Escherichia coli are typically mediated by transmembrane receptors that monitor chemoeffector levels with periplasmic binding domains and communicate with the flagellar motors through two cytoplasmic proteins, CheA and CheY. CheA autophosphorylates and then donates its phosphate to CheY, which in turn controls flagellar rotation. E. coli also exhibits chemotactic responses to substrates that are transported by the phosphoenolpyruvate (PEP)-dependent carbohydrate phosphotransferase system (PTS). Unlike conventional chemoreception, PTS substrates are sensed during their uptake and concomitant phosphorylation by the cell. The phosphoryl groups are transferred from PEP to the carbohydrates through two common intermediates, enzyme I (EI) and phosphohistidine carrier protein (HPr), and then to sugar-specific enzymes II. We found that in mutant strains HPr-like proteins could substitute for HPr in transport but did not mediate chemotactic signaling. In in vitro assays, these proteins exhibited reduced phosphotransfer rates from EI, indicating that the phosphorylation state of EI might link the PTS phospho-relay to the flagellar signaling pathway. Tests with purified proteins revealed that unphosphorylated EI inhibited CheA autophosphorylation, whereas phosphorylated EI did not. These findings suggest the following model for signal transduction in PTS-dependent chemotaxis. During uptake of a PTS carbohydrate, EI is dephosphorylated more rapidly by HPr than it is phosphorylated at the expense of PEP. Consequently, unphosphorylated EI builds up and inhibits CheA autophosphorylation. This slows the flow of phosphates to CheY, eliciting an up-gradient swimming response by the cell.

Light intensity regulation of cab gene transcription is signaled by the redox state of the plastoquinone pool.

The eukaryotic green alga Dunaliella tertiolecta acclimates to decreased growth irradiance by increasing cellular levels of light-harvesting chlorophyll protein complex apoproteins associated with photosystem II (LHCIIs), whereas increased growth irradiance elicits the opposite response. Nuclear run-on transcription assays and measurements of cab mRNA stability established that light intensity-dependent changes in LHCII are controlled at the level of transcription. cab gene transcription in high-intensity light was partially enhanced by reducing plastoquinone with 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU), whereas it was repressed in low-intensity light by partially inhibiting the oxidation of plastoquinol with 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). Uncouplers of photosynthetic electron transport and inhibition of water splitting had no effect on LHCII levels. These results strongly implicate the redox state of the plastoquinone pool in the chloroplast as a photon-sensing system that is coupled to the light-intensity regulation of nuclear-encoded cab gene transcription. The accumulation of cellular chlorophyll at low-intensity light can be blocked with cytoplasmically directed phosphatase inhibitors, such as okadaic acid, microcystin L-R, and tautomycin. Gel mobility-shift assays revealed that cells grown in high-intensity light contained proteins that bind to the promoter region of a cab gene carrying sequences homologous to higher plant light-responsive elements. On the basis of these experimental results, we propose a model for a light intensity signaling system where cab gene expression is reversibly repressed by a phosphorylated factor coupled to the redox status of plastoquinone through a chloroplast protein kinase.