Kurt Amrein

Microarray-Based Identification of a Novel Streptococcus pneumoniae Regulon Controlled by an Autoinduced Peptide

We have identified in the Streptococcus pneumoniae genome sequence a two-component system (TCS13, Blp [bacteriocin-like peptide]) which is closely related to quorum-sensing systems regulating cell density-dependent phenotypes such as the development of genetic competence or the production of antimicrobial peptides in lactic acid bacteria. In this study we present evidence that TCS13 is a peptide-sensing system that controls a regulon including genes encoding Blps. Downstream of the Blp TCS (BlpH R) we identified open reading frames (blpAB) that have the potential to encode an ABC transporter that is homologous to the ComA/B export system for the competence-stimulating peptide ComC. The putative translation product of blpC, a small gene located downstream of blpAB, has a leader peptide with a Gly-Gly motif. This leader peptide is typical of precursors processed by this family of transporters. Microarray-based expression profiling showed that a synthetic oligopeptide corresponding to the processed form of BlpC (BlpC*) induces a distinct set of 16 genes. The changes in the expression profile elicited by synthetic BlpC* depend on BlpH since insertional inactivation of its corresponding gene abolishes differential gene induction. Comparison of the promoter regions of the blp genes disclosed a conserved sequence element formed by two imperfect direct repeats upstream of extended -10 promoter elements. We propose that BlpH is the sensor for BlpC* and the conserved sequence element is a recognition sequence for the BlpR response regulator.

Domain organization and molecular characterization of 13 two-component systems identified by genome sequencing of Streptococcus pneumoniae

In bacteria, adaptive responses to environmental stimuli are often initiated by two-component signal transduction systems (TCS). The prototypical TCS comprises two proteins: a histidine kinase (HK, hk) and a response regulator (RR rr). Recent research has suggested that compounds that inhibit two-component systems might have good antibacterial activity. In order to identify TCS that are crucial for growth or virulence of Streptococcus pneumoniae, we have examined the genomic sequence of a virulent S. pneumoniae strain for genes that are related to known histidine kinases or response regulators. Altogether 13 histidine kinases and 13 response regulators have been identified. The protein sequences encoded by these genes were compared with sequences deposited in public databases. This analysis revealed that two of the 13 pneumococcal TCSs have been described before (ciaRH and comDE) and two are homologous to the yycFG and the phoRP genes of Bacillus subtilis. All the pneumococcal response regulators contain putative DNA binding motifs within the C-terminal output domain, implying that they are involved in transcriptional control. Two of these response regulators are obviously the first representatives of a new subfamily containing an AraC-type DNA-binding effector domain. To assess the regulatory role of these transcription factors, we disrupted each of the 13 response regulator genes by insertional mutagenesis. All the viable mutant strains with disrupted response regulator genes were further characterized with regard to growth in vitro, competence, and experimental virulence. Two response regulator genes could not be inactivated, indicating that they may regulate essential cellular functions. The possibility of using these systems as targets for the development of novel antibacterials will be discussed.

Protein-tyrosine Phosphatases PTP1B and Syp Are Modulators of Insulin-stimulated Translocation of GLUT4 in Transfected Rat Adipose Cells

The protein-tyrosine phosphatases PTP1B and Syp have both been implicated as modulators of the mitogenic actions of insulin. However, the roles of these protein-tyrosine phosphatases in the metabolic actions of insulin are not well characterized. In this study, we directly assessed the ability of PTP1B and Syp to modulate insulin-stimulated translocation of the insulin-responsive glucose transporter GLUT4 in a physiologically relevant insulin target cell. Primary cultures of rat adipose cells were transiently transfected with either wild-type PTP1B (PTP1B-WT), wild-type Syp (Syp-WT), or the catalytically inactive mutants PTP1B-C/S or Syp-C/S. The effects of overexpression of these constructs on insulin-stimulated translocation of a co-transfected epitope-tagged GLUT4 were studied. Cells overexpressing either PTP1B-C/S or Syp-WT had insulin dose-response curves similar to those obtained with control cells expressing only epitope-tagged GLUT4. In contrast, for cells overexpressing PTP1B-WT the level of GLUT4 on the cell surface at each insulin dose (ranging from 0 to 60 nM) was significantly lower than that observed in the control cells. Interestingly, cells overexpressing the dominant inhibitory mutant Syp-C/S also had a small but statistically significant impairment in insulin responsiveness. At a maximally stimulating concentration of insulin (60 nM), cell surface epitope-tagged GLUT4 was approximately 20% less than that of the control cells. It is possible that effects from high level overexpression of Syp and PTP1B constructs may not reflect what occurs under physiological conditions. Nevertheless, our data raise the possibility that PTP1B may be a negative regulator of insulin-stimulated glucose transport, while Syp may have a small role as a positive mediator of the metabolic actions of insulin.

11β-Hydroxysteroid dehydrogenase 1 reductase activity is dependent on a high ratio of NADPH/NADP+ and is stimulated by extracellular glucose

To assess the impact of the NADPH/NADP(+) ratio and the influence of extracellular glucose on 11beta-hydroxysteroid dehydrogenase 1 (11beta-HSD1) activity, we applied microsomal preparations and intact HEK-293 cells expressing 11beta-HSD1 in the presence or absence of hexose-6-phosphate dehydrogenase (H6PDH). A NADPH/NADP(+) ratio of ten or higher was required for efficient microsomal 11beta-HSD1 reductase activity. Measurements in intact cells suggested that the ER-luminal NADPH concentration is highly sensitive to fluctuating extracellular glucose levels. Lowering glucose in the culture medium dose-dependently decreased 11beta-HSD1 reductase activity and diminished the cortisol/cortisone ratio measured after 24h of incubation. Coexpression with H6PDH potentiated 11beta-HSD1 reductase activity at high glucose. This effect was significantly decreased at low glucose, with concomitantly increased 11beta-HSD1 dehydrogenase activity. In contrast, 11beta-HSD1 reductase activity in H4IIE liver cells and in 3T3-L1 adipocytes was less sensitive to changes in the medium. 11beta-HSD1 dehydrogenase activity was observed in H4IIE cells only at subphysiological glucose levels, indicating a highly efficient supply of substrate for H6PDH and NADPH generation in the ER-lumen. Our results suggest significant cell type-specific differences in ER-luminal NADPH generation that might allow a fine-tuned regulation of glucocorticoid action.

An inflammatory micro-environment promotes human adipocyte apoptosis

Obesity-associated macrophage infiltration into adipose tissue is responsible for both local and systemic inflammation. Recent findings suggest fat cell apoptosis as an initiator of macrophage recruitment. Here, we investigated the effects of an inflammatory micro-environment on fat cells using human THP-1 macrophages and SGBS adipocytes. Macrophage-secreted factors induced insulin resistance, inhibited insulin-stimulated Akt phosphorylation, and induced apoptosis of adipocytes. The apoptosis-inducing effect was even more pronounced in direct co-cultures of adipocytes and macrophages. Our data suggest a link between insulin resistance and apoptosis sensitivity. Accordingly, pharmacological and genetic inhibition of insulin signaling at the level of Akt2 sensitized adipocytes to apoptosis induction by macrophage-secreted factors. In conclusion, we describe here a novel interaction of macrophages and fat cells, i.e. induction of apoptosis. Our data suggest a feed-forward cycle in which macrophages further drive the inflammatory process by inducing insulin resistance and concomitant apoptosis of adipocytes.

THP-1 macrophages and SGBS adipocytes - a new human in vitro model system of inflamed adipose tissue

Obesity is associated with an accumulation of macrophages in adipose tissue. This inflammation of adipose tissue is a key event in the pathogenesis of several obesity-related disorders, particularly insulin resistance. Here, we summarized existing model systems that mimic the situation of inflamed adipose tissue in vitro, most of them being murine. Importantly, we introduce our newly established human model system which combines the THP-1 monocytic cell line and the preadipocyte cell strain Simpson–Golabi–Behmel syndrome (SGBS). THP-1 cells, which originate from an acute monocytic leukemia, differentiate easily into macrophages in vitro. The human preadipocyte cell strain SGBS was recently introduced as a unique tool to study human fat cell functions. SGBS cells are characterized by a high capacity for adipogenic differentiation. SGBS adipocytes are capable of fat cell-specific metabolic functions such as insulin-stimulated glucose uptake, insulin-stimulated de novo lipogenesis and β-adrenergic-stimulated lipolysis and they secrete typical adipokines including leptin, adiponectin, and RBP4. Applying either macrophage-conditioned medium or a direct co-culture of macrophages and fat cells, our model system can be used to distinguish between paracrine and cell-contact dependent effects. In conclusion, we propose this model as a useful tool to study adipose inflammation in vitro. It represents an inexpensive, highly reproducible human system. The methods described here can be easily extended for usage of primary human macrophages and fat cells.

Discovery of 4-Aryl-5,6,7,8-tetrahydroisoquinolines as Potent, Selective, and Orally Active Aldosterone Synthase (CYP11B2) Inhibitors: In Vivo Evaluation in Rodents and Cynomolgus Monkeys.

Inappropriately high levels of aldosterone are associated with many serious medical conditions, including renal and cardiac failure. A focused screen hit has been optimized into a potent and selective aldosterone synthase (CYP11B2) inhibitor with in vitro activity against rat, mouse, human, and cynomolgus monkey enzymes, showing a selectivity factor of 160 against cytochrome CYP11B1 in the last species. The novel tetrahydroisoquinoline compound (+)-(R)-6 selectively reduced aldosterone plasma levels in vivo in a dose-dependent manner in db/db mice and cynomolgus monkeys. The selectivity against CYP11B1 as predicted by cellular inhibition data and free plasma fraction translated well to Synacthen challenged cynomolgus monkeys up to a dose of 0.1 mg kg(-1). This compound, displaying good in vivo potency and selectivity in mice and monkeys, is ideally suited to perform mechanistic studies in relevant rodent models and to provide the information necessary for translation to non-human primates and ultimately to man.

Mapping of the p56lck-mediated phosphorylation of GAP and analysis of its influence on p21ras-GTPase activity in vitro

The protein tyrosine kinase p56lck and other members of the src family can transduce signals from activated cell-surface receptors. As we showed earlier the GTPase-activating protein (GAP), a regulator of p21ras, is a substrate of p56lck. Here, tryptic peptides of p56lck-phosphorylated GAP were generated and analyzed by two-dimensional thin layer chromatography and mass spectroscopy. Results revealed that p56lck phosphorylates GAP specifically on Tyr-460 in vitro and in vivo. The effect of tyrosine phosphorylation of GAP on its GTPase-activating activity versus p21ras was then tested using a p21ras-dependent GTPase assay system. Our results demonstrate that p56lck-mediated tyrosine phosphorylation of GAP is not sufficient to change directly its effect on the intrinsic GTPase activity of p21ras.

CD4: p56lck Association Studied in vivo Using Antibody-Induced Capping and Double Indirect Immunofluorescence Microscopy

Accumulating data suggest that the T-cell surface antigen CD4 transduces an independent signal during antigen-mediated T-cell activation. In vitro studies which showed that the cytoplasmic protein tyrosine kinase p56lck is present in anti-CD4 immunoprecipitates led to the model that p56lck is associated with the cytoplasmic domain of CD4. In this report we have extended these studies and examined potential CD4:p56lck associations in vivo. We show here by double immunofluorescence microscopy a specific co-distribution of p56lck with antibody-induced CD4 caps in intact cells. Murine T-cell hybridoma lines expressing mutant forms of CD4 were used to demonstrate that the 31 carboxyterminal aminoacids of its cytoplasmic domain, in particular cysteine-420 and cysteine-422, are crucial for the formation of CD4:p56lck complexes in vivo. The potential of the method applied is discussed with regard to studies of other transmembrane signalling systems involving src-like kinases.

Csk-mediated phosphorylation of substrates is regulated by substrate tyrosine phosphorylation

Csk is a cellular protein tyrosine kinase (PTK) that has been shown to specifically regulate the activity of Src kinase family members by phosphorylation of a carboxy-terminal tyrosine residue. The molecular mechanisms controlling Csk regulation and its substrate specificity have not been elucidated. Here we report a novel type of overlay kinase assay that allows to probe for Csk-mediated phosphorylation of cellular substrates separated by polyacrylamide gel electrophoresis and transferred to nitrocellulose filters. Most of the cell lines analyzed with this method revealed only a few potential Csk substrates. However, an increased number of Csk substrates was detected in NIH3T3 cells expressing a constitutively activated form of the Src kinase Lck or in PC12 and NIH3T3 cells that had been treated with pervanadate. These cells all display an increased level of cellular protein tyrosine phosphorylation which led to the conclusion that Csk preferentially phosphorylates tyrosine-phosphorylated proteins. To verify this hypothesis we analyzed Csk-mediated phosphorylation of recombinant Lck, a known Csk substrate. Results demonstrated that autophosphorylation of Lck (at Tyr394) facilitates Csk-mediated phosphorylation of Lck at its regulatory site (Tyr505). Subsequent peptide binding studies revealed that Csk can bind to a peptide corresponding to the Lck-autophosphorylation site only when it is phosphorylated. These findings suggest that autophosphorylation of Lck at Tyr394 triggers an interaction with Csk and thereby facilitates subsequent phosphorylation and inactivation of Lck. The phosphorylation of other cellular Csk substrates may be regulated by a similar mechanism.