Four signalling domains in the hybrid histidine protein kinase RodK of Myxococcus xanthus are required for activity

In prokaryotes, the principal signal transduction systems operating at the level of protein phosphorylation are the two-component systems. A number of hybrid histidine protein kinases in these systems contain several receiver domains, however, the function of these receiver domains is unknown. The RodK kinase in Myxococcus xanthus has an unconventional domain composition with a putative N-terminal sensor domain followed by a histidine kinase domain and three receiver domains. RodK is essential for the spatial coupling of the two morphogenetic events underlying fruiting body formation in M. xanthus, aggregation of cells into nascent fruiting bodies and the subsequent sporulation of these cells. RodK kinase activity is indispensable for RodK activity. By systematically substituting the conserved, phosphorylatable aspartate residues in the three receiver domains, genetic evidence is provided that each receiver domain is important for RodK function and that each receiver domain has a distinct function, which depends on phosphorylation. Biochemical analyses provided indirect evidence for phosphotransfer from the RodK kinase domain to the third receiver domain. This is the first example of a hybrid histidine protein kinase in which four signalling domains have been shown to be required for full activity.     

Glucose-dependent expansion of pancreatic beta-cells by the protein p8 in vitro and in vivo

p8 protein expression is known to be upregulated in the exocrine pancreas during acute pancreatitis. Own previous work revealed glucose-dependent p8 expression also in endocrine pancreatic beta-cells. Here we demonstrate that glucose-induced INS-1 beta-cell expansion is preceded by p8 protein expression. Moreover, isopropylthiogalactoside (IPTG)-induced p8 overexpression in INS-1 beta-cells (p8-INS-1) enhances cell proliferation and expansion in the presence of glucose only. Although beta-cell-related gene expression (PDX-1, proinsulin I, GLUT2, glucokinase, amylin) and function (insulin content and secretion) are slightly reduced during p8 overexpression, removal of IPTG reverses beta-cell function within 24 h to normal levels. In addition, insulin secretion of p8-INS-1 beta-cells in response to 0-25 mM glucose is not altered by preceding p8-induced beta-cell expansion. Adenovirally transduced p8 overexpression in primary human pancreatic islets increases proliferation, expansion, and cumulative insulin secretion in vitro. Transplantation of mock-transduced control islets under the kidney capsule of immunosuppressed streptozotocin-diabetic mice reduces blood glucose and increases human C-peptide serum concentrations to stable levels after 3 days. In contrast, transplantation of equal numbers of p8-transduced islets results in a continuous decrease of blood glucose and increase of human C-peptide beyond 3 days, indicating p8-induced expansion of transplanted human beta-cells in vivo. This is underlined by a doubling of insulin content in kidneys containing p8-transduced islet grafts explanted on day 9. These results establish p8 as a novel molecular mediator of glucose-induced pancreatic beta-cell expansion in vitro and in vivo and support the notion of existing beta-cell replication in the adult organism.     

Induction of specific T cell immunity in patients with prostate cancer by vaccination with PSA146–154 peptide

T cell immunotherapy of prostate cancer (CaP) offers the potential for less toxic, more effective outcomes. A clinical trial was conducted in 28 patients with locally advanced or metastatic CaP to determine whether an HLA-A2 binding epitope of prostate-specific antigen, PSA146–154 (PSA-peptide), can induce specific T cell immunity. Patients were vaccinated either by intradermal injection of PSA-peptide and GM-CSF or by intravenous administration of autologous dendritic cells pulsed with PSA-peptide at weeks 1, 4 and 10. Delayed-type hypersensitivity (DTH) skin testing was performed at weeks 4, 14, 26 and 52. Fifty percent of the patients developed positive DTH responses to PSA-peptide. The size of the DTH induration progressively increased over time in the majority of responding patients. Skin biopsies from seven DTH-positive patients were available and T cells that developed in situ were also characterized. The phenotype of recovered T cells demonstrated variable proportions of CD4⁺CD8⁻, CD4⁻CD8⁺ and CD4⁺CD8⁺ T cell populations. Cytokine analysis of PSA-peptide stimulated T cells per bead array assay exhibited specific IFN-γ and TNF-α response in six of seven patients. Specific IL-4 response was observed in five patients, while IL-10 response was detected in one patient. Purified CD4⁻CD8⁺ T cells isolated from four patients demonstrated specific cytolytic activity per chromium release assay. In conclusion, immunization with PSA-peptide induced specific T cell immunity in one-half of the patients with locally advanced and hormone-sensitive, metastatic CaP. DTH-derived T cells exhibited PSA-peptide-specific cytolytic activity and predominantly expressed a type-1 cytokine profile.     

Plant peroxisomes respire in the light: some gaps of the photorespiratory C2 cycle have become filled--others remain

The most prominent role of peroxisomes in photosynthetic plant tissues is their participation in photorespiration, a process also known as the oxidative C2 cycle or the oxidative photosynthetic carbon cycle. Photorespiration is an essential process in land plants, as evident from the conditionally lethal phenotype of mutants deficient in enzymes or transport proteins involved in this pathway. The oxidative C2 cycle is a salvage pathway for phosphoglycolate, the product of the oxygenase activity of ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO), to the Calvin cycle intermediate phosphoglycerate. The pathway is highly compartmentalized and involves reactions in chloroplasts, peroxisomes, and mitochondria. The H2O2-producing enzyme glycolate oxidase, catalase, and several aminotransferases of the photorespiratory cycle are located in peroxisomes, with catalase representing the major constituent of the peroxisomal matrix in photosynthetic tissues. Although photorespiration is of major importance for photosynthesis, the identification of the enzymes involved in this process has only recently been completed. Only little is known about the metabolite transporters for the exchange of photorespiratory intermediates between peroxisomes and the other organelles involved, and about the regulation of the photorespiratory pathway. This review highlights recent developments in understanding photorespiration and identifies remaining gaps in our knowledge of this important metabolic pathway.     

Superhelical DNA as a preferential binding target of 14-3-3γ protein

The 14-3-3 protein family is a highly conserved and widely distributed group of proteins consisting of multiple isoforms in eukaryotes. Ubiquitously expressed, 14-3-3 proteins play key roles in DNA replication, cell cycle regulation, and apoptosis. The function of 14-3-3 proteins is mediated by interaction with a large number of other proteins and with DNA. It has been demonstrated that 14-3-3γ protein binds strongly to cruciform structures and is crucial for initiating replication. In this study, we analyzed DNA binding properties of the 14-3-3γ isoform to linear and supercoiled DNA. We demonstrate that 14-3-3γ protein binds strongly to long DNA targets, as evidenced by electrophoretic mobility shift assay on agarose gels. Binding of 14-3-3γ to DNA target results in the appearance of blurry, retarded DNA bands. Competition experiments with linear and supercoiled DNA on magnetic beads show very strong preference for supercoiled DNA. We also show by confocal microscopy that 14-3-3 protein in the HCT-116 cell line is co-localized with DNA cruciforms. This implies a role for the 14-3-3γ protein in its binding to local DNA structures which are stabilized by DNA supercoiling.     

Binding of complement inhibitor C4b-binding protein contributes to serum resistance of Porphyromonas gingivalis

The periodontal pathogen Porphyromonas gingivalis is highly resistant to the bactericidal activity of human complement, which is present in the gingival crevicular fluid at 70% of serum concentration. All thirteen clinical and laboratory P. gingivalis strains tested were able to capture the human complement inhibitor C4b-binding protein (C4BP), which may contribute to their serum resistance. Accordingly, in serum deficient of C4BP, it was found that significantly more terminal complement component C9 was deposited on P. gingivalis. Moreover, using purified proteins and various isogenic mutants, we found that the cysteine protease high molecular weight arginine-gingipain A (HRgpA) is a crucial C4BP ligand on the bacterial surface. Binding of C4BP to P. gingivalis appears to be localized to two binding sites: on the complement control protein 1 domain and complement control protein 6 and 7 domains of the alpha-chains. Furthermore, the bacterial binding of C4BP was found to increase with time of culture and a particularly strong binding was observed for large aggregates of bacteria that formed during culture on solid blood agar medium. Taken together, gingipains appear to be a very significant virulence factor not only destroying complement due to proteolytic degradation as we have shown previously, but was also inhibiting complement activation due to their ability to bind the complement inhibitor C4BP.     

Improved prediction of peroxisomal PTS1 proteins from genome sequences based on experimental subcellular targeting analyses as exemplified for protein kinases from Arabidopsis

Due to current experimental limitations in peroxisome proteome research, the identification of low-abundance regulatory proteins such as protein kinases largely relies on computational protein prediction. To test and improve the identification of regulatory proteins by such a prediction-based approach, the Arabidopsis genome was screened for genes that encode protein kinases with predicted type 1 or type 2 peroxisome targeting signals (PTS1 or PTS2). Upon transient expression in onion epidermal cells, the predicted PTS1 domains of four of the seven protein kinases re-directed the reporter protein, enhanced yellow green fluorescent (EYFP), to peroxisomes and were thus verified as functional PTS1 domains. The full-length fusions, however, remained cytosolic, suggesting that PTS1 exposure is induced by specific signals. To investigate why peroxisome targeting of three other kinases was incorrectly predicted and ultimately to improve the prediction algorithms, selected amino acid residues located upstream of PTS1 tripeptides were mutated and the effect on subcellular targeting of the reporter protein was analysed. Acidic residues in close proximity to major PTS1 tripeptides were demonstrated to inhibit protein targeting to plant peroxisomes even in the case of the prototypical PTS1 tripeptide SKL>, whereas basic residues function as essential auxiliary targeting elements in front of weak PTS1 tripeptides such as SHL>. The functional characterization of these inhibitory and essential enhancer-targeting elements allows their consideration in predictive algorithms to improve the prediction accuracy of PTS1 proteins from genome sequences.     

BAG-1 family of cochaperones in the modulation of nuclear receptor action

BAG-1 is a family of cochaperones consisting of at least four polypeptides BAG-1L, BAG-1M/RAP46, BAG-1 and p29. These proteins are translated from the same mRNA at alternative translation initiation sites. They possess conserved carboxy-terminal sequences which enable them to bind and inhibit the action of the molecular chaperone Hsp70/Hsc70. BAG-1 was the first member in the family of the BAG-1 proteins to be isolated. It was identified as an anti-apoptotic protein because of its ability to bind and augment the activity of the anti-death protein, Bcl-2. Since then other BAG-1 proteins have been identified and shown to interact with several cellular factors including nuclear receptors. Recent findings show that the effect of the BAG-1 proteins on nuclear receptors ranges from inhibition to enhancement of the transactivation functions of the receptors. Available data on the negative regulation of glucocorticoid receptor (GR) action by the BAG-1 proteins identify two modes of action: inhibition of the hormone binding activity of the GR and a more direct nuclear action at the level of regulation of the transactivation function of the receptor. In the latter case, the BAG-1 proteins repress DNA binding by the GR in a process that requires prior binding of Hsp70/Hsc70 to the receptor. Positive regulatory action of the BAG-1 proteins on nuclear receptors has also been reported which may involve yet other mechanisms. This review puts together recent findings on the action the BAG-1 proteins and presents them as a novel group of regulators of action of nuclear receptor.