Maintenance of NF-kappa B activity is dependent on protein synthesis and the continuous presence of external stimuli.

The activation of NF-kappa B-like activities (called NF-kappa B) by tumor necrosis factor alpha (TNF alpha) and the phorbol ester phorbol 12-myristate 13-acetate (PMA) were compared. High levels of NF-kappa B activity were found 2 to 4 min after TNF alpha addition to human HL60 cells and lasted for at least 3 h, although the half-life of active NF-kappa B was less than 30 min. Inactive NF-kappa B, however, was relatively stable. NF-kappa B activation by TNF alpha was initially cycloheximide insensitive, but maintenance of NF-kappa B activity required ongoing protein synthesis and continuous stimulation by TNF alpha. Thus, the cells did not remain in an activated state without stimulation. In HL60 cells, NF-kappa B induction by PMA required 30 to 45 min and was completely dependent on de novo protein synthesis, while PMA (and interleukin-1) induced NF-kappa B activity rapidly in mouse 70Z/3 cells via a protein synthesis-independent mechanism. The NF-kappa B-like activities obtained under each condition behaved identically in methylation interference and native proteolytic fingerprinting assays. The NF-kappa B-like factors induced are thus all very similar or identical. We suggest that cell-specific differences in the protein kinase C-dependent activation of NF-kappa B may exist and that TNF alpha and PMA may induce expression of the gene(s) encoding NF-kappa B.

Cyclic AMP-independent activation of transcription factor NF-kappa B in HL60 cells by tumor necrosis factors alpha and beta.

No correlation exists in HL60 cells between NF-kappa B activation by tumor necrosis factor (TNF alpha) and TNF beta and intracellular levels of cyclic AMP. Cyclic AMP levels did not increase upon treatment of cells with each of these cytokines, although NF-kappa B was activated. Forskolin or 1-isobutyl-3-methylxanthine drastically increased intracellular levels of cyclic AMP, but neither activated NF-kappa B nor influenced TNF-induced NF-kappa B activation.

Isolation and characterization of the carotenoid biosynthesis genes of Flavobacterium sp. strain R1534.

The Gram-negative bacterium Flavobacterium sp. strain R1534 is a natural producer of zeaxanthin. A 14 kb genomic DNA fragment of this organism has been cloned and a 5.1 kb piece containing the carotenoid biosynthesis genes sequenced. The carotenoid biosynthesis cluster consists of five genes arranged in at least two operons. The five genes are necessary and sufficient for the synthesis of zeaxanthin. The encoded proteins have significant homology to the crtE, crtB, crtY, crtI and crtZ gene products of other carotenogenic organisms. Biochemical assignment of the individual gene products was done by HPLC analysis of the carotenoid accumulation in Escherichia coli host strains transformed with plasmids carrying deletions of the Flavobacterium sp. strain R1534 carotenoid biosynthesis cluster.

Regulation of riboflavin biosynthesis in Bacillus subtilis is affected by the activity of the flavokinase/flavin adenine dinucleotide synthetase encoded by ribC.

This work shows that the ribC wild-type gene product has both flavokinase and flavin adenine dinucleotide synthetase (FAD-synthetase) activities. RibC plays an essential role in the flavin metabolism of Bacillus subtilis, as growth of a ribC deletion mutant strain was dependent on exogenous supply of FMN and the presence of a heterologous FAD-synthetase gene in its chromosome. Upon cultivation with growth-limiting amounts of FMN, this ribC deletion mutant strain overproduced riboflavin, while with elevated amounts of FMN in the culture medium, no riboflavin overproduction was observed. In a B. subtilis ribC820 mutant strain, the corresponding ribC820 gene product has reduced flavokinase/FAD-synthetase activity. In this strain, riboflavin overproduction was also repressed by exogenous FMN but not by riboflavin. Thus, flavin nucleotides, but not riboflavin, have an effector function for regulation of riboflavin biosynthesis in B. subtilis, and RibC seemingly is not directly involved in the riboflavin regulatory system. The mutation ribC820 leads to deregulation of riboflavin biosynthesis in B. subtilis, most likely by preventing the accumulation of the effector molecule FMN or FAD.

Staphylococcus aureus and derived exotoxins induce nuclear factor kappa B-like activity in murine bone marrow macrophages.

Heat-killed gram-positive Staphylococcus aureus as well as S. aureus-derived exotoxins B and toxic shock syndrome toxin 1 can induce nuclear factor kappa B (NF-kappa B)-like activity in murine bone marrow macrophages. The induction of NF-kappa B-like activity in murine macrophages by S. aureus was as effective as induction by tumor necrosis factor alpha (TNF-alpha) or lipopolysaccharides (LPS) and was observed in macrophages derived from LPS-sensitive and LPS-resistant mice. Stimulation of macrophages with S. aureus but not with the exotoxins resulted in the accumulation of TNF-alpha in the culture medium. The induction of NF-kappa B-like activity by S. aureus, however, clearly preceded TNF-alpha secretion and was not inhibited by a neutralizing serum against TNF-alpha. In addition, pretreatment of macrophages with the protein synthesis inhibitor cycloheximide or dexamethasone, which prevented the secretion of TNF-alpha from macrophages, did not interfere with the induction of NF-kappa B-like activity by S. aureus. This findings reveal the existence of bacterial components other than LPS which can induce NF-kappa B-like activity in susceptible cells.

Cell-free sulfation of the contact site A glycoprotein of Dictyostelium discoideum and of a partially glycosylated precursor.

An 80-kDa glycoprotein of Dictyostelium discoideum, designated contact site A, has been implicated in EDTA-stable cell adhesion. This protein is known to be the major sulfated protein of aggregation-competent cells and has been shown to contain two types of carbohydrate, sulfated type 1 and unsulfated type 2 carbohydrate moieties. Here we investigate the cell-free sulfation of this protein. In the homogenate of developing cells, [35S]sulfate was transferred by endogenous sulfotransferase from [35S]3'-phosphoadenosine-5'-phosphosulfate to the contact site A glycoprotein and to various other endogenous proteins. The sulfate was transferred to carbohydrate rather than to tyrosine residues. After differential centrifugation of the homogenate, the capacity for sulfation of the contact site A glycoprotein was barely detected in the plasma membrane-enriched 10,000 X g pellet fraction which contained the bulk of this glycoprotein, but was largely recovered in the 100,000 X g pellet fraction which contained only a small portion of this glycoprotein. After sucrose gradient centrifugation, the membranes containing the sulfation capacity were found to have a density characteristic for Golgi membranes. In immunoblots, monoclonal antibodies raised against the contact site A glycoprotein recognized not only this 80-kDa protein, but also a sulfatable 68-kDa protein found in the 100,000 X g pellet fraction. The 68-kDa protein did not react with monoclonal antibodies against type 2 carbohydrate but was converted by endoglycosidases F and H into a 53-kDa protein, indicating that it was a partially glycosylated form of the 80-kDa glycoprotein containing only type 1 carbohydrate. Isoelectric focusing showed that a substantial portion of the 68-kDa glycoprotein was unsulfated, even after cell-free sulfation. The 68-kDa glycoprotein was not found in the plasma membrane-enriched 10,000 X g pellet fraction and did not accumulate in parallel with the 80-kDa contact site A glycoprotein during cell development. We conclude that the 68-kDa glycoprotein is a precursor that is converted by attachment of type 2 carbohydrate and sulfation of type 1 carbohydrate into the mature 80-kDa glycoprotein. The precursor nature of the 68-kDa glycoprotein was supported by results obtained with mutant HL220 which is defective in glycosylation (Murray, B. A., Wheeler, S., Jongens, T., and Loomis, W. F. (1984) Mol. Cell. Biol. 4, 514-519). This mutant specifically lacks type 2 carbohydrate and produces a 68-Kda glycoprotein instead of the 80-kDa contact site A glycoprotein (Yoshida, M., Stadler, J., Bertholdt, G., and Gerisch, G. (1984) EMBO J. 3, 2663-2670).(ABSTRACT TRUNCATED AT 400 WORDS)

Two-step glycosylation of the contact site A protein of Dictyostelium discoideum and transport of an incompletely glycosylated form to the cell surface.

Two different types of oligosaccharides, designated type 1 and 2 carbohydrate residues, are present on the contact site A molecule, an 80-kDa glycoprotein involved in the formation of EDTA-stable cell adhesion during cell aggregation in Dictyostelium discoideum. The first precursor detected by pulse-chase labeling with [35S]methionine was a 68-kDa glycoprotein carrying type 1 carbohydrate. Conversion of the precursor into the 80-kDa form occurred simultaneously with the addition of type 2 carbohydrate. Tunicamycin inhibited type 1 glycosylation more efficiently than type 2 glycosylation. The first precursor detected in tunicamycin-treated cells by pulse-chase labeling was a 53-kDa protein lacking both carbohydrates, which was converted through addition of type 2 carbohydrate into a 66-kDa final product. Labeling of intact cells indicated that this 66-kDa glycoprotein is transported to the cell surface. Prolonged treatment with tunicamycin resulted in the accumulation within the cells of the 53-kDa precursor with no detectable exposure of this protein on the cell surface. It is concluded that type 1 carbohydrate, which is cotranslationally added in N-glycosidic linkages, is neither required for transport of the protein to the Golgi apparatus nor for type 2 glycosylation or protection of the protein against proteolytic degradation. Incapability of tunicamycin-treated cells of forming EDTA-stable cell contacts suggests a role for type 1 carbohydrate in cell adhesion. Type 2 carbohydrate is added posttranslationally. It is required in the absence of type 1 glycosylation for transport of the protein to the cell surface.

Two different cell types have different major receptors for human tumor necrosis factor (TNF alpha).

The receptors for tumor necrosis factor alpha (TNF alpha) were analyzed on myeloid cells (HL60, U937, K562, and freshly isolated blood monocytes) and on cells of epithelial origin (MCF7, HEp2 and HeLa cells), by use of radiolabeled TNF alpha and cross-linking experiments. Both cell types had high but slightly different affinities for TNF alpha. The myeloid cells had major cross-linked products of 98-100 kDa, which were similar in their N-linked glycosylation, whereas the cells of epithelial origin contained a major cross-linked product of 75 kDa, a second product of 95 kDa. The major receptors of both cell types (studied mostly with HL60 and HEp2 cells) are different proteins because (a) their apparent molecular masses were different and no evidence was obtained for cell-specific proteases, which could generate the differently sized receptors from one common receptor molecule; (b) anti-receptor antibodies, which precipitated the 95- and 75-kDa products, did not precipitate the 100-kDa cross-linked complex; (c) the native TNF alpha-receptor complexes had different proteolytic fingerprints; (d) the tryptic fragments differed in their association with the cell membrane vesicles; (e) the receptors differed in their degree of N-linked glycosylation; and (f) O-linked glycosylation was found on the major receptor of HL60 but not of HEp2 cells. In addition, myeloid cells may also contain a small amount of the HEp2-type of TNF alpha receptor. We suggest that at least two different receptors for TNF alpha exist.

Tumor necrosis factors-alpha and -beta bind to the same two types of tumor necrosis factor receptors and maximally activate the transcription factor NF-kappa B at low receptor occupancy and within minutes after receptor binding.

HL60 cells have types A and B tumor necrosis factor (TNF) receptors whereas HEp2 cells have only the type B receptor (Hohmann, H.-P., Remy, R., Brockhaus, M., and van Loon, A.P.G.M. (1989) J. Biol. Chem. 264, 14927-14934). TNF-beta can be cross-linked to each of these receptors and competes with TNF-alpha for binding to both receptors. TNF-alpha and TNF-beta activate the transcription factor NF-kappa B in HL60 and HEp2 cells. Maximal activation of NF-kappa B required binding of TNF-alpha or TNF-beta to 20-25% of the total number of TNF receptors and was achieved within minutes after the addition of TNF-alpha to HL60 cells. Both TNF-alpha and TNF-beta activate NF-kappa B at 5-10-fold lower concentrations in HL60 cells compared with HEp2 cells, and this correlates well with their different affinities for binding to these cells. Thus, TNF-alpha and TNF-beta are indistinguishable with respect to the correlation between degrees of receptor binding and activation of NF-kappa B.

Protein kinases negatively affect nuclear factor-kappa B activation by tumor necrosis factor-alpha at two different stages in promyelocytic HL60 cells.

HL60 and EL4 cells incubated with tumor necrosis factor-alpha (TNF-alpha) plus staurosporin, a potent inhibitor of protein kinases, showed at least 2-fold increased levels of nuclear factor-kappa B (NF-kappa B) activity compared with TNF-alpha alone both during rapid NF-kappa B activation from the cytosolic pool and protein synthesis-dependent NF-kappa B activation. NF-kappa B activation by phorbol 12-myristate 13-acetate (PMA) and interleukin-1 was inhibited by staurosporin. Staurosporin treatment hardly affected the TNF-alpha-induced increase in mRNA for the p51 subunit of NF-kappa B but interfered with any phorbol ester (PMA)-induced increase in p51 mRNA. Thus, induction of NF-kappa B and p51 mRNA by TNF-alpha was not mediated by a staurosporin-sensitive factor, but NF-kappa B activation by TNF-alpha was even reduced by action of a staurosporin-sensitive factor. Decreased levels of phosphorylation of TNF-R alpha (TNF receptor type alpha) after staurosporin-treatment correlated with increased induction of NF-kappa B by TNF-alpha. Staurosporin-treatment did not affect TNF-R levels. Although protein kinase C stimulation by PMA inhibited NF-kappa B activation by TNF-alpha, its action mechanism may be different from that of the staurosporin-sensitive factor. PMA induced disappearance of TNF-R alpha by shedding into the surrounding medium, with kinetics similar to those of its inhibition of NF-kappa B activation by TNF-alpha. Phosphorylation may not mediate receptor shedding, since PMA treatment did not detectably affect TNF-R alpha phosphorylation.

Pyruvate formate-lyase (inactive form) and pyruvate formate-lyase activating enzyme of Escherichia coli: isolation and structural properties.

The catalytically active form (Ea) of pyruvate formate-lyase in Escherichia coli cells is generated from an inactive form of the enzyme (Ei) through a post-translational process that requires a distinct activating enzyme and is linked to the cleavage of adenosylmethionine to methionine and 5'-deoxyadenosine. Ei and the activating enzyme were purified to homogeneity and structurally characterized. Ei has an alpha 2 oligomeric structure (2 X 85 kDa) and contains no cofactor. The amino acid composition has been determined. Out of a total of six cysteinyl residues per subunit, one shows an unusually fast reaction with iodoacetate (k2 = 7 (M-1 s-1) at pH 6.8, 30 degrees C), which is accompanied by loss of the activatability of the enzyme. The 1500-fold purified activating enzyme is a monomeric protein of 30 kDa. It contains a covalently bound, as yet unidentified chromophoric factor which has an optical absorption peak at 388 nm. Further studies of the in situ state of pyruvate formate-lyase detected a reversible backconversion of the active form Ea into Ei when anaerobic cells become nutrient-depleted.

Molecular cloning and characterisation of the ribC gene from Bacillus subtilis: a point mutation in ribC results in riboflavin overproduction.

A mutation leading to roseoflavin resistance and deregulated riboflavin biosynthesis was mapped in the genome of the riboflavin-overproducing Bacillus subtilis strains RB52 and RB50 at map position 147 degrees. The chromosomal location indicates that the deregulating mutation in RB52 and RB50 is an allele of the previously identified ribC mutation. We cloned the ribC gene and found that it encodes a putative 36-kDa protein. Surprisingly, RibC has significant sequence similarity to flavin kinases and FAD synthases from various other bacterial species. By comparing the deduced amino acid sequence of RibC from the wild-type parent strain of RB50 with the RibC sequence from the riboflavin-overexpressing RB50 mutant we identified a point mutation that resulted in a Gly to Ser exchange in the C-terminal region of the product.

Physiology and metabolic fluxes of wild-type and riboflavin-producing Bacillus subtilis.

Continuous cultivation in a glucose-limited chemostat was used to determine the growth parameters of wild-type Bacillus subtilis and of a recombinant, riboflavin-producing strain. Maintenance coefficients of 0.45 and 0.66 mmol of glucose g-1 h-1 were determined for the wild-type and recombinant strains, respectively. However, the maximum molar growth yield of 82 to 85 g (cell dry weight)/mol of glucose was found to be almost identical in both strains. A nonlinear relationship between the specific riboflavin production rate and the dilution rate was observed, revealing a coupling of product formation and growth under strict substrate-limited conditions. Most prominently, riboflavin formation completely ceased at specific growth rates below 0.15 h-1. For molecular characterization of B. subtilis, the total amino acid composition of the wild type was experimentally determined and the complete building block requirements for biomass formation were derived. In particular, the murein sacculus was found to constitute approximately 9% of B. subtilis biomass, three- to fivefold more than in Escherichia coli. Estimation of intracellular metabolic fluxes by a refined mass balance approach revealed a substantial, growth rate-dependent flux through the oxidative branch of the pentose phosphate pathway. Furthermore, this flux is indicated to be increased in the strain engineered for riboflavin formation. Glucose catabolism at low growth rates with reduced biomass yields was supported mainly by the tricarboxylic acid cycle.

Expression of the types A and B tumor necrosis factor (TNF) receptors is independently regulated, and both receptors mediate activation of the transcription factor NF-kappa B. TNF alpha is not needed for induction of a biological effect via TNF receptors.

The expression and biological function of the types A and B tumor necrosis factor (TNF) receptors were studied using three cell types. SW480T, HEp2, and HL60 cells had, respectively, mainly the type A, only the type B, and roughly similar amounts of both receptors. Dibutyric cAMP treatment induced a 3-6-fold increase in the amount of the type A receptor in HL60 cells without affecting the amount of the type B receptor. Expression of both receptors can thus be regulated independently. HEp2 and human umbilical vein endothelial cells only showed the type B receptor, and expression of the type A receptor could not be induced in these cells. HL60 cells showed, upon Scatchard analysis, a single binding site for TNF alpha, and its Kd may correspond to that of the type A receptor. The approximately 7-fold lower affinity of TNF alpha binding to the type B receptor of HL60 cells was only detected after blocking all TNF alpha binding to the type A receptor. Both the types A and B receptors mediated TNF alpha-induced activation of the transcription factor NF-kappa B. The agonistic antibody htr9 to the type B receptor also activated NF-kappa B. Thus, signal transduction via the type B receptor may only require interaction with the receptor's extracellular domain.

Post-translational glycosylation of the contact site A protein of Dictyostelium discoideum is important for stability but not for its function in cell adhesion.

The functions of type 1 and 2 carbohydrates of the contact site A (csA) glycoprotein of Dictyostelium discoideum have been investigated using mutants lacking type 2 carbohydrate. In two mutant strains, HG220 and HG701, a 68-kd glycoprotein was synthesized as the final product of csA biosynthesis. This glycoprotein accumulated to a much lower extent on the surfaces of mutant cells than the mature 80-kd glycoprotein did in wild-type cells. There was also no accumulation of the 68-kd glycoprotein observed within the mutant cells nor was a precursor of lower molecular mass detected, in accordance with previous findings that indicated cotranslational linkage of type 1 carbohydrate by N-glycosylation. Pulse-chase labelling showed that a 50-kd glycopeptide was cleaved off from the mutant 68-kd glycoprotein and released into the medium, while the fully glycosylated 80-kd glycoprotein of the wild type was stable. These results assign a function to type 2 carbohydrate in protecting the cell-surface-exposed csA glycoprotein against proteolytic cleavage. HG220 cells were still capable of forming EDTA-stable contacts to a reduced extent, consistent with the low amounts of the 68-kd glycoprotein present on their surfaces. Thus type 1 rather than type 2 carbohydrate appears to be directly involved in intercellular adhesion that is mediated by the csA glycoprotein. Tunicamycin-treated wild-type and mutant cells produce a 53-kd protein that lacks both type 1 and 2 carbohydrates. While this protein is stable and not transported to the cell surface in the wild type, it is cleaved in the mutants and fragments of it are released into the extracellular medium. These results suggest that the primary defect in the two mutants studied is relief from a restriction in protein transport to the cell surface, and that the defect in type 2 glycosylation is secondary.

Cloning of the DNA-binding subunit of human nuclear factor kappa B: the level of its mRNA is strongly regulated by phorbol ester or tumor necrosis factor alpha.

The DNA binding subunit of nuclear factor kappa B (NF-kappa B), a B-cell protein that interacts with the immunoglobulin kappa light-chain gene enhancer, has been purified from nuclei of human HL-60 cells stimulated with tumor necrosis factor alpha (TNF alpha), and internal peptide sequences were obtained. Overlapping cDNA clones were isolated and sequenced. The encoded open reading frame of about 105 kDa contained at its N-terminal half all six tryptic peptide sequences, suggesting that the 51-kDa NF-kappa B protein is processed from a 105-kDa precursor. An in vitro synthesized protein containing most of the N-terminal half of the open reading frame bound specifically to an NF-kappa B binding site. This region also showed high homology to a domain shared by the Drosophila dorsal gene and the avian and mammalian rel (proto)oncogene products. The level of the 3.8-kilobase mRNA was strongly increased after stimulation with TNF alpha or phorbol ester. Thus, both factors not only activate NF-kappa B protein, as described previously, but also induce expression of the gene encoding the DNA-binding subunit of NF-kappa B.