Solution structure and function of a conserved protein SP14.3 encoded by an essential Streptococcus pneumoniae gene.

Streptococcus pneumoniae is a major human pathogen that causes high mortality and morbidity rates and has developed resistance to many antibiotics. The genome of S. pneumoniae has recently been completely sequenced revealing many genes encoding hypothetical proteins of unknown function. We have found that the gene encoding one such conserved protein, SP14.3, is essential for growth of S. pneumonia. Since it is essential, SP14.3 represents a potential target for drug discovery. Here, we describe the three-dimensional solution structure of SP14.3 as determined by NMR spectroscopy. The structure consists of two domains each with an alpha/beta-fold. The N-terminal domain contains two alpha-helices and a three-stranded beta-sheet, while the C-terminal domain is composed of one alpha-helix and a five-stranded beta-sheet. The N-terminal domain of the protein contains a highly negatively charged surface and resembles the fold of the N-terminal domain of Thermus thermophilus ribosomal protein S3. The C-terminal domain has a protein fold similar to human small nuclear ribonucleoprotein Sm D3 and Haloarcula marismortui ribosomal protein L21E. The two domains of the protein tumble in solution overall as a whole with an overall molecular rotational correlation time (tau(m)) of 12.9 ns at 25 degrees C. The relative orientation of the two domains is not defined by the nuclear Overhauser effect data. Indeed, residual dipolar couplings and the structure calculations indicate that the relative orientation of the two domains is not rigidly oriented with respect to one another in solution.

A novel B lymphocyte-associated adaptor protein, Bam32, regulates antigen receptor signaling downstream of phosphatidylinositol 3-kinase.

We have identified and characterized a novel src homology 2 (SH2) and pleckstrin homology (PH) domain-containing adaptor protein, designated Bam32 (for B cell adaptor molecule of 32 kD). cDNAs encoding the human and mouse Bam32 coding sequences were isolated and the human bam32 gene was mapped to chromosome 4q25-q27. Bam32 is expressed by B lymphocytes, but not T lymphocytes or nonhematopoietic cells. Human germinal center B cells show increased Bam32 expression, and resting B cells rapidly upregulate expression of Bam32 after ligation of CD40, but not immunoglobulin M. Bam32 is tyrosine-phosphorylated upon B cell antigen receptor (BCR) ligation or pervanadate stimulation and associates with phospholipase Cgamma2. After BCR ligation, Bam32 is recruited to the plasma membrane through its PH domain. Membrane recruitment requires phosphatidylinositol 3-kinase (PI3K) activity and an intact PI(3,4, 5)P(3)-binding motif, suggesting that membrane association occurs through binding to 3-phosphoinositides. Expression of Bam32 in B cells leads to a dose-dependent inhibition of BCR-induced activation of nuclear factor of activated T cells (NF-AT), which is blocked by deletion of the PH domain or mutation of the PI(3,4,5)P(3)-binding motif. Thus, Bam32 represents a novel B cell-associated adaptor that regulates BCR signaling downstream of PI3K.

Distinct requirements for C-C chemokine and IL-2 production by naive, previously activated, and anergic T cells.

Ag presented by activated APCs promote immunogenic responses whereas Ag presented by resting APCs leads to tolerance. In such a model, the regulation of cytokine release by the presence or absence of costimulation might potentially play a critical role in dictating the ultimate outcome of Ag recognition. C-C chemokines are a structurally defined family of chemoattractants that have diverse effects on inflammation. We were interested in determining the activation requirements for chemokine production by CD4+ T cells. Our data demonstrate for T cell clones and previously activated T cells from TCR-transgenic mice that stimulation with anti-TCR alone results in the production of copious amounts of macrophage-inflammatory protein-1alpha (MIP-1alpha) and other C-C chemokines, and that addition of anti-CD28 gives very little augmentation. Furthermore, MIP-1alpha production is nearly equivalent from both anergic and nonanergic cells. For naive T cells, anti-CD3 stimulation alone led to as much MIP-1alpha production as Ag + APC stimulation. The addition of costimulation gave a 3-10-fold enhancement, but this was 70-fold less than the effect of costimulation on IL-2 production. Thus, although C-C chemokines play a broad role in influencing inflammation, their production by signal 1 alone makes them unlikely to play a critical role in the decision between a tolerogenic and an immunogenic response. Furthermore, the production of MIP-1alpha by anergic T cells, as well as following signal 1 alone, raises the possibility that in vivo this chemokine serves to recruit activated T cells to become tolerant.

The -180 site of the IL-2 promoter is the target of CREB/CREM binding in T cell anergy.

Anergic T cells display a marked decrease in their ability to produce IL-2 even in the presence of optimal TCR and costimulatory signals. Using IL-2 enhancer/promoter-driven reporter constructs, we have previously identified a region that appears to be a target for cis transcriptional repression in anergy. This region of the promoter, which shares partial homology with a consensus AP-1-binding sequence, is located about -180 bp from the transcriptional start site. In the present study, we demonstrate that cAMP response element-binding protein/cAMP response element modulator (CREB/CREM), activating transcription factor-2/c-Jun, and Jun-Jun/Oct complexes bind to this site. However, the induction of anergy by prolonged stimulation through the TCR led to an increase in binding of only the CREB/CREM complex. Furthermore, the level of binding of this complex appeared to be up-regulated in both resting and restimulated anergic T cells. Finally, an IL-2 promoter-driven reporter construct that contained a mutation that specifically reduced the binding of the CREB/CREM complex displayed a decreased ability to be affected by anergy, while a construct that contained a mutation that decreased the binding of the Jun-Jun/Oct complex was still susceptible to anergy. These findings suggest that the -180 region of the IL-2 promoter is the target of a CREB/CREM transcriptional inhibitor that contributes to the repression of IL-2 production in T cell anergy.

DNA cleavage activities of Staphylococcus aureus gyrase and topoisomerase IV stimulated by quinolones and 2-pyridones.

We have cloned Staphylococcus aureus DNA gyrase and topoisomerase IV and expressed them in Escherichia coli as polyhistidine-tagged proteins to facilitate purification and eliminate contamination by host enzymes. The enzyme preparations had specific activities similar to previously reported values. Potassium glutamate (K-Glu) stimulated the drug-induced DNA cleavage activity and was optimal between 100 and 200 mM for gyrase and peaked at 100 mM for topoisomerase IV. Higher concentrations of K-Glu inhibited the cleavage activities of both enzymes. Using a common buffer system containing 100 mM K-Glu, we tested the enzyme-mediated DNA cleavage activities of both gyrase and topoisomerase IV with oxolinic acid, norfloxacin, ciprofloxacin, trovafloxacin, clinafloxacin, and the 2-pyridone ABT-719. As expected, all drugs tested demonstrated greater potency against topoisomerase IV than against gyrase. In addition, cleavage activity was found to correlate well with antibacterial activity.

Inhibition of cell cycle progression by rapamycin induces T cell clonal anergy even in the presence of costimulation.

Costimulation (signal 2) has been proposed to inhibit the induction of T cell clonal anergy by either directly antagonizing negative signals arising from TCR engagement (signal 1) or by synergizing with signal 1 to produce IL-2, which in turn leads to proliferation and dilution of negative regulatory factors. To better define the cellular events that lead to the induction of anergy, we used the immunosuppressive agent rapamycin, which blocks T cell proliferation in late G1 phase but does not affect costimulation-dependent IL-2 production. Our data demonstrate that full T cell activation (signal 1 plus 2) in the presence of rapamycin results in profound T cell anergy, despite the fact that these cells produce copious amounts of IL-2. Similar to conventional anergy (induction by signal 1 alone), the rapamycin-induced anergic cells show a decrease in mitogen-activated protein kinase activation, and these cells can be rescued by culture in IL-2. Interestingly, the rapamycin-induced anergic cells display a more profound block in IL-3 and IFN-gamma production upon rechallenge. Finally, in contrast to rapamycin, full T cell activation in the presence of hydroxyurea (which inhibits the cell cycle in early S phase) did not result in anergy. These data suggest that it is neither the direct effect of costimulation nor the subsequent T cell proliferation that prevents anergy induction, but rather the biochemical events that occur upon progression through the cell cycle from G1 into S phase.

Scintillation proximity assay for human DNA topoisomerase I using recombinant biotinyl-fusion protein produced in baculovirus-infected insect cells.

DNA topoisomerases are well-established targets of important therapeutic agents which include the antibacterial quinolones and anticancer camptothecins. Screens for new classes of topoisomerase inhibitors generally employ methods, such as gel electrophoresis, which are not readily amenable to a rapid high-throughput format. We describe here a high-throughput assay to screen for inhibitors of human DNA topoisomerase I based on the scintillation proximity assay. The assay employs recombinant biotinyl-topoisomerase I fusion protein, a hybrid protein which contains a domain that is biotinylated during in vivo expression. The hybrid topoisomerase I fusion protein is found to be biotinylated, active, and nuclear-localized when produced in insect cells using a baculovirus expression system. The biotinyl-topoisomerase I fusion protein can be captured from crude nuclear extracts by immobilization on streptavidin-coated scintillation proximity assay beads. The assay detects binding of 3H-labeled DNA to the bead-immobilized enzyme by scintillation counting. The method is also able to detect stabilization of covalent protein-DNA complexes by camptothecin, an inhibitor previously shown to stabilize covalent intermediates that form during catalysis.

Cross-species complementation of the indispensable Escherichia coli era gene highlights amino acid regions essential for activity.

Era is an essential GTP binding protein in Escherichia coli. Two homologs of this protein, Sgp from Streptococcus mutans and Era from Coxiella burnetii, can substitute for the essential function of Era in E. coli. Site-specific and randomly generated Era mutants which may indicate regions of the protein that are of functional importance are described.

Cold-sensitive conditional mutations in Era, an essential Escherichia coli GTPase, isolated by localized random polymerase chain reaction mutagenesis.

Conditional cold-sensitive mutations in Era, an essential Escherichia coli GTPase, were isolated. Localized random polymerase chain reaction (PCR) mutagenesis employing Taq and T7 DNA polymerases under error prone amplification conditions was exploited to generate mutations in the era gene. A plasmid exchange technique was used to identify conditional cold-sensitive mutations in Era that give rise to defective cell growth below 30 degrees C. Three recessive missense mutations in Era, N26S, A156D, and E200K, were isolated. All three mutations are located at residues conserved in Era homologues from Streptococcus mutans and Coxiella burnetti.

Synthesis of yeast cell wall glucan and evidence for glucan metabolism in a Saccharomyces cerevisiae whole cell system.

The synthesis and metabolism of yeast cell wall glucan were studied using a Saccharomyces cerevisiae construct in which radiolabelled galactose is metabolized to UDP-glucose and preferentially incorporated into glucan. Greater than 85% of the incorporated radiolabel was found within insoluble cell wall material. Our study also demonstrated that radiolabelled wall glucan is released from cells growing exponentially, and that the released radiolabel is reutilizable low molecular mass material. Size exclusion chromatography and enzymic analysis indicate that laminaribiose comprises approximately 50% of the released fraction. This is consistent with in vitro findings that laminaribiose is a by-product of a newly identified glucosyltransferase (R. P. Hartland, G. W. Emerson & P. A. Sullivan, 1991, Proc R Soc Lond B 246, 155-160) associated with fungal cell walls. Our results also suggest that pre-existing glucan undergoes less metabolic processes than newly synthesized material as evidenced by a decrease in released radiolabel over time. Pulse double labelling of glucan and total cellular protein indicate that glucan metabolism and protein synthesis (ps) are not tightly coupled although they do parallel each other during exponential growth. Inhibitors of glucan synthesis (gs) decrease the glucan to protein ratio. Measurement of ps allows normalization for non-specific decreases in the rate of cell wall synthesis due to general cessation of growth. Cilofungin and papulacandin B, two putative inhibitors of gs, inhibited galactose incorporation into glucan and thus showed a decrease in the glucan to protein ratio, although ps was affected.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the autophosphorylation of Era, an essential Escherichia coli GTPase.

Era is an essential protein in Escherichia coli which binds both GTP and GDP and has an intrinsic GTPase activity. Studies on the role of GTP/GDP binding and GTPase activity in an attempt to understand its function lead to the observation that Era is autophosphorylated. The autophosphorylation reaction is specific for GTP and cannot use ATP as a phosphoryl group donor. The reaction velocity is of first order with respect to protein concentration, suggesting an intramolecular mechanism. Autophosphorylation occurs at serine and threonine residues. The major phosphorylated tryptic peptide isolated after autophosphorylation has been identified as ISITSR, from residue 33 to 38. The peptide contains the site of phosphorylation and two potential sites for serine and threonine phosphorylation. Subsequently, both the threonine residue at position 36 and the serine residue at position 37 were altered to alanine. The double mutant Era, but not individual single mutants, was unable to functionally complement the growth of an E. coli strain which cannot produce wild-type Era protein at high temperature. This suggests that either threonine 36 or serine 37 has to exist for the function of Era in vivo. In vivo phosphorylation of Era was also examined by two-dimensional gel electrophoresis. Era has been previously assigned two distinct positions having two different X-Y co-ordinates: one of the spots (H032.0) was identified as phosphorylated Era, indicating that a substantial portion of Era in the cell is indeed phosphorylated. Therefore, Era autophosphorylation is likely to play an important physiological role in the cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimuli that induce production of Candida albicans extracellular aspartyl proteinase.

Several species of the opportunistic fungal pathogen Candida produce an extracellular aspartyl proteinase that may assist the organism to invade and colonize host tissues, evade the host immune response and assimilate nitrogen from proteinaceous sources. Although addition of exogenous proteins, such as bovine serum albumin (BSA), to cultures of C. albicans is known to elicit proteinase production, the precise molecular mechanisms controlling regulation of proteinase induction are unknown. We have examined the ability of a variety of macromolecules to induce proteinase production using a chemically-defined nitrogen-limited growth medium and a rapid, sensitive microtitre fluorescent assay for proteinase activity in culture supernatants. BSA and the extracellular matrix protein collagen induced proteinase production. Homopolymers of both poly-L- and poly-D-glutamate also induced proteinase activity, whereas polyglycine, heparin sulphate and dextran sulphate did not. Thus, molecular recognition of proteinase-inducing stimuli is not highly stereospecific, but apparently requires both main- and side-chain interactions. Peptides 8 or more residues in length generally induced proteinase production while most shorter peptides did not. These data reveal that internalization of small peptides with less than 7 residues by peptide transport was not the inducing signal for proteinase production, since Candida dipeptide and oligopeptide permeases do not efficiently transport peptides of more than 6-7 residues. In addition a tight-binding synthetic inhibitor of Candida proteinase (Ki = 0.17 nM) prevented growth of C. albicans on BSA as a sole nitrogen source by blocking protein degradation. Immunodetection of proteinase in these culture supernatants suggests that fully intact proteins, in addition to peptide fragments of sufficient size, are capable of inducing proteinase production.(ABSTRACT TRUNCATED AT 250 WORDS)

Cold-sensitive growth and decreased GTP-hydrolytic activity from substitution of Pro17 for Val in Era, an essential Escherichia coli GTPase.

A substitution mutation of Pro17 by Val (P17V) was constructed in the guanine nucleotide binding domain of Era, an essential protein in Escherichia coli. The mutation is analogous to the oncogenic activating allele at position 12 in the GTP-binding domain of p21ras. The phenotype of this mutant was analysed in a strain which exclusively expressed the mutant protein (Era-V17) in null allele chromosomal background (era1: :kan). The strain was found to be cold-sensitive for growth. Mutant Era-V17 purified from the strain was cold-sensitive for GTP-hydrolytic activity, suggesting that the GTPase activity of Era is required for cell growth since the P17V mutation resulted in both cold-sensitive growth of cells and cold-labile GTPase activity of the purified protein.

Application of a fluorogenic substrate in the assay of proteolytic activity and in the discovery of a potent inhibitor of Candida albicans aspartic proteinase.

A fluorescent method for monitoring the activity of the secreted Candida carboxyl (aspartic) proteinase (EC 3.4.23.6) was developed using a fluorogenic substrate based on resonance energy transfer. The fluorescent assay was used to monitor proteinase production, purification, and inhibition. The Km for the fluorogenic substrate, 4-(4-dimethylaminophenylazo)benzoyl-gamma-aminobutyryl-Ile-His-Pro - Phe-His-Leu-Val-Ile-His-Thr- [5-(2-aminoethyl)amino]naphthalene-1-sulfonic acid, was found to be 4.3 microM at the optimum pH of 4.5. Reaction products were separated by reverse-phase high-performance liquid chromatography and identified by amino acid analysis or by 252Cf plasma desorption mass spectrometry. Cleavage of the fluorogenic substrate was between the histidine-threonine residues, releasing the fluorescent product, threonine-[5-(2-aminoethyl)amino]naphthalene-1-sulfonic acid. Proteolytic activity was expressed as nanomoles of fluorescent product released at 22 degrees C/60 min, pH 4.5, and the release of 0.9 nmol product was equivalent to one hemoglobin proteolytic unit (O.D.A700 increase of 0.100) produced at 37 degrees C/60 min, pH 3.5. The aspartic proteinase inhibitor pepstatin had an IC50 of 27 nM when tested in a dose-response study with the purified enzyme. The apparent Ki for pepstatis was 2.9 nM. Several synthetic inhibitors of the enzymes were identified with IC50's in the nanomolar range. The most potent compound, A70450, was characterized as a fast, tight-binding inhibitor having an IC50 of 1.3 nM and apparent Ki of 0.17 nM.

Nucleoside diphosphate kinase from Escherichia coli; its overproduction and sequence comparison with eukaryotic enzymes.

The gene encoding nucleoside diphosphate (NDP) kinase of Escherichia coli was identified by polymerase chain reaction using oligodeoxyribonucleotide primers synthesized on the basis of consensus sequences from Myxococcus xanthus and various eukaryotic NDP kinases. The gene (ndk), mapped at 54.2 min on the E. coli chromosome, was cloned and sequenced. The E. coli NDP kinase was found to consist of 143 amino acid residues that are 57, 45, 45, 42, 43, and 43% identical to the M. xanthus, Dictyostelium discoideum, Drosophila melanogaster, mouse, rat, and human enzymes, respectively. The ndk gene appears to be in a monocistronic operon and, when cloned in a pUC vector, NDP kinase was overproduced at a level of approx. 25% of total cellular proteins. The protein could be labeled with [gamma-32P]ATP and migrated at a 16.5 kDa when electrophoresed in SDS-polyacrylamide gel, which is in good agreement with the Mr of the purified E. coli NDP kinase previously reported.

Pleiotropic changes resulting from depletion of Era, an essential GTP-binding protein in Escherichia coli.

Phenotypic analysis of a temperature-sensitive era mutant strain indicates that Escherichia coli cells depleted of Era undergo many physiological changes. At 43 degrees C, a completely non-permissive temperature, growth is arrested because of loss of the gene and depletion of the Era protein. Depletion of Era at 43 degrees C results in depressed synthesis of heat-shock proteins DnaK, GroEL/ES, D33.4 and C62.5, lack of thermal induction of ppGpp pool levels, and increased capacity for carbon source metabolism through the citric acid cycle. Thus, in addition to inhibition of cell growth and viability, loss of Era function results in pleiotropic changes including abnormal adaptation to thermal stress.

Isolation of subtilisin pro-sequence mutations that affect formation of active protease by localized random polymerase chain reaction mutagenesis.

In order to analyze the role of the pro-sequence in folding of the alkaline serine protease subtilisin, localized random mutagenesis using the polymerase chain reaction with Taq DNA polymerase was employed to obtain mutations in the pro-sequence which prevent production of active protease. The unique aspect of this procedure is that random mutations can be easily generated in vitro over large but defined regions of a specific gene. The method was applied to a 458-base pair fragment encompassing the coding region of the pro-sequence of subtilisin, a region of the protein which has been shown to be required for proper folding. Protease-deficient mutants containing a variety of amino acid substitutions were isolated with a frequency of 4.3%. From analysis of these mutants, four independent amino acid substitution mutations in the pro-sequence were identified. The present results demonstrate that polymerase chain reaction is an efficient and simple method for obtaining random mutations within a localized region of a given gene.