Serine/threonine protein kinase PrkA of the human pathogen Listeria monocytogenes: biochemical characterization and identification of interacting partners through proteomic approaches

Listeria monocytogenes is the causative agent of listeriosis, a very serious food-borne human disease. The analysis of the proteins coded by the L. monocytogenes genome reveals the presence of two eukaryotic-type Ser/Thr-kinases (lmo1820 and lmo0618) and a Ser/Thr-phosphatase (lmo1821). Protein phosphorylation regulates enzyme activities and protein interactions participating in physiological and pathophysiological processes in bacterial diseases. However in the case of L. monocytogenes there is scarce information about biochemical properties of these enzymes, as well as the physiological processes that they modulate. In the present work the catalytic domain of the protein coded by lmo1820 was produced as a functional His(6)-tagged Ser/Thr-kinase, and was denominated PrkA. PrkA was able to autophosphorylate specific Thr residues within its activation loop sequence. A similar autophosphorylation pattern was previously reported for Ser/Thr-kinases from related prokaryotes, whose role in kinase activity and substrate recruitment was demonstrated. We studied the kinase interactome using affinity chromatography and proteomic approaches. We identified 62 proteins that interact, either directly or indirectly, with the catalytic domain of PrkA, including proteins that participate in carbohydrates metabolism, cell wall metabolism and protein synthesis. Our results suggest that PrkA could be involved in the regulation of a variety of fundamental biological processes.     

Levothyroxine monotherapy cannot guarantee euthyroidism in all athyreotic patients

Context

Levothyroxine monotherapy is the treatment of choice for hypothyroid patients because peripheral T4 to T3 conversion is believed to account for the overall tissue requirement for thyroid hormones. However, there are indirect evidences that this may not be the case in all patients.

Objective

To evaluate in a large series of athyreotic patients whether levothyroxine monotherapy can normalize serum thyroid hormones and thyroid-pituitary feedback.

Design

Retrospective study.

Setting

Academic hospital.

Patients

1,811 athyreotic patients with normal TSH levels under levothyroxine monotherapy and 3,875 euthyroid controls.

Measurements

TSH, FT4 and FT3 concentrations by immunoassays.

Results

FT4 levels were significantly higher and FT3 levels were significantly lower (p<0.001 in both cases) in levothyroxine-treated athyreotic patients than in matched euthyroid controls. Among the levothyroxine-treated patients 15.2% had lower serum FT3 and 7.2% had higher serum FT4 compared to euthyroid controls. A wide range of FT3/FT4 ratios indicated a major heterogeneity in the peripheral T3 production capacity in different individuals. The correlation between thyroid hormones and serum TSH levels indicated an abnormal feedback mechanism in levothyroxine-treated patients.

Conclusions

Athyreotic patients have a highly heterogeneous T3 production capacity from orally administered levothyroxine. More than 20% of these patients, despite normal TSH levels, do not maintain FT3 or FT4 values in the reference range, reflecting the inadequacy of peripheral deiodination to compensate for the absent T3 secretion. The long-term effects of chronic tissue exposure to abnormal T3/T4 ratio are unknown but a sensitive marker of target organ response to thyroid hormones (serum TSH) suggests that this condition causes an abnormal pituitary response. A more physiological treatment than levothyroxine monotherapy may be required in some hypothyroid patients.     

Modulation of histone deposition by the karyopherin kap114

The nuclear import of histones is a prerequisite for the downstream deposition of histones to form chromatin. However, the coordinate regulation of these processes remains poorly understood. Here we demonstrate that Kap114p, the primary karyopherin/importin responsible for the nuclear import of histones H2A and H2B, modulates the deposition of histones H2A and H2B by the histone chaperone Nap1p. We show that a complex comprising Kap114p, histones H2A and H2B, and Nap1p is present in the nucleus and that the presence of this complex is specifically promoted by Nap1p. This places Kap114p in a position to modulate Nap1p function, and we demonstrate by the use of two different assay systems that Kap114p inhibits Nap1p-mediated chromatin assembly. The inhibition of H2A and H2B deposition by Kap114p results in the concomitant inhibition of RCC1 loading onto chromatin. Biochemical evidence suggests that the mechanism by which Kap114p modulates histone deposition primarily involves direct histone binding, while the interaction between Kap114p and Nap1p plays a secondary role. Furthermore, we found that the inhibition of histone deposition by Kap114p is partially reversed by RanGTP. Our results indicate a novel mechanism by which cells can regulate histone deposition and establish a coordinate link between histone nuclear import and chromatin assembly.     

Quantification of Enterococcus faecalis and Enterococcus faecium in different foods using rRNA-targeted oligonucleotide probes

The abundance of Enterococcus faecalis and Enterococcus faecium in different Spanish foods was evaluated by using taxon-specific oligonucleotide probes targeted against extracted rRNA. Two satisfactory methods were developed for RNA extraction. Although the yield and purity of total RNA obtained largely depended on the type of food, method 1 should be recommended. The quantitative results obtained with the oligonucleotide probes DB6 for E. faecium and DB8 for E. faecalis showed that these two species accounted for less than 0.5% of the active microflora in all the food samples tested. These results suggest that enterococci form only a minor portion of the microflora of these products.     

Diversification and Independent Evolution of Troponin C Genes in Insects

Troponin C (TpnC), the calcium-binding subunit of the troponin regulatory complex in the muscle thin filament, is encoded by multiple genes in insects. To understand how TpnC genes have evolved, we characterized the gene number and structure in a number of insect species. The TpnC gene complement is five genes in Drosophilidae as previously reported for D. melanogaster. Gene structures are almost identical in D. pseudoobscura, D. suboboscura, and D. virilis. Developmental patterns of expression are also conserved in Drosophila subobscura and D. virilis. Similar, but not completely equivalent, TpnC gene repertoires have been identified in the Anopheles gambiae and Apis mellifera genomes. Insect TpnC sequences can be divided into three groups, allowing a systematic classification of newly identified genes. The pattern of expression of the Apis mellifera genes essentially agrees with the pattern in Drosophilidae, providing further functional support to the classification. A model for the evolution of the TpnC genes is proposed including the most likely pathway of insect TpnC diversification. Our results suggest that the rapid increase in number and sequence specialization of the adult Type III isoforms can be correlated with the evolution of the holometabolous mode of development and the acquisition of asynchronous indirect flight muscle function in insects. This evolutionarily specialization has probably been achieved independently in different insect orders.Reviewing Editor: Dr. Rüdiger Cerff     

Electron transfer between cytochrome c and p66Shc generates reactive oxygen species that trigger mitochondrial apoptosis

Reactive oxygen species (ROS) are potent inducers of oxidative damage and have been implicated in the regulation of specific cellular functions, including apoptosis. Mitochondrial ROS increase markedly after proapoptotic signals, though the biological significance and the underlying molecular mechanisms remain undetermined. P66Shc is a genetic determinant of life span in mammals, which regulates ROS metabolism and apoptosis. We report here that p66Shc is a redox enzyme that generates mitochondrial ROS (hydrogen peroxide) as signaling molecules for apoptosis. For this function, p66Shc utilizes reducing equivalents of the mitochondrial electron transfer chain through the oxidation of cytochrome c. Redox-defective mutants of p66Shc are unable to induce mitochondrial ROS generation and swelling in vitro or to mediate mitochondrial apoptosis in vivo. These data demonstrate the existence of alternative redox reactions of the mitochondrial electron transfer chain, which evolved to generate proapoptotic ROS in response to specific stress signals.     

Agreement between theory and measurement in quantification of ammonia-oxidizing bacteria

Autotrophic ammonia-oxidizing bacteria (AOB) are of vital importance to wastewater treatment plants (WWTP), as well as being an intriguing group of microorganisms in their own right. To date, corroboration of quantitative measurements of AOB by fluorescence in situ hybridization (FISH) has relied on assessment of the ammonia oxidation rate per cell, relative to published values for cultured AOB. Validation of cell counts on the basis of substrate transformation rates is problematic, however, because published cell-specific ammonia oxidation rates vary by over two orders of magnitude. We present a method that uses FISH in conjunction with confocal scanning laser microscopy to quantify AOB in WWTP, where AOB are typically observed as microcolonies. The method is comparatively simple, requiring neither detailed cell counts or image analysis, and yet it can give estimates of either cell numbers or biomass. Microcolony volume and diameter were found to have a log-normal distribution. We were able to show that virtually all (>96%) of the AOB biomass occurred as microcolonies. Counts of microcolony abundance and measurement of their diameter coupled with a calibration of microcolony dimensions against cell numbers or AOB biomass were used to determine AOB cell numbers and biomass in WWTP. Cell-specific ammonia oxidation rates varied between plants by over three orders of magnitude, suggesting that cell-specific ammonia oxidation is an important process variable. Moreover, when measured AOB biomass was compared with process-based estimates of AOB biomass, the two values were in agreement.     

Cleavage of the plasma membrane Na+/Ca2+ exchanger in excitotoxicity

In brain ischemia, gating of postsynaptic glutamate receptors and other membrane channels triggers intracellular Ca2+ overload and cell death. In excitotoxic settings, the initial Ca2+ influx through glutamate receptors is followed by a second uncontrolled Ca2+ increase that leads to neuronal demise. Here we report that the major plasma membrane Ca2+ extruding system, the Na+/Ca2+ exchanger (NCX), is cleaved during brain ischemia and in neurons undergoing excitotoxicity. Inhibition of Ca2+-activated proteases (calpains) by overexpressing their endogenous inhibitor protein, calpastatin or the expression of an NCX isoform not cleaved by calpains, prevented Ca2+ overload and rescued neurons from excitotoxic death. Conversely, down-regulation of NCX by siRNA compromised neuronal Ca2+ handling, transforming the Ca2+ transient elicited by non-excitotoxic glutamate concentrations into a lethal Ca2+overload. Thus, proteolytic inactivation of NCX-driven neuronal Ca2+ extrusion is responsible for the delayed excitotoxic Ca2+ deregulation and neuronal death.     

Enhanced expressions of endometrial tumour necrosis factor alpha and its receptors during early pregnancy in bonnet monkeys

Tumour necrosis factor alpha (TNF-alpha), a pro-inflammatory cytokine may play an active role in stimulating inflammatory reactions during pregnancy. However, the expression of endometrial TNF-alpha has not been investigated especially during early pregnancy, a phenomenon invariably accompanied by inflammatory reaction. In the present study, the endometrial expressions of TNF-alpha and its receptors (TNFR1 and TNFR2) during early pregnancy, when the embryo lies free in the zona hatched state in the uterine lumen, were analyzed by immunohistochemistry. The endometrial expressions of TNF-alpha, TNFR1 and TNFR2 were found to be significantly up-regulated (p < 0.05) in the glandular epithelium on day 6 post-ovulation in pregnant animals. The alteration in the expression of these molecules may contribute to the induction of local inflammatory reactions during implantation.     

Basal activation of the P2X7 ATP receptor elevates mitochondrial calcium and potential, increases cellular ATP levels, and promotes serum-independent growth

P2X7 is a bifunctional receptor (P2X7R) for extracellular ATP that, depending on the level of activation, forms a cation-selective channel or a large conductance nonselective pore. The P2X7R has a strong proapoptotic activity but can also support growth. Here, we describe the mechanism involved in growth stimulation. Transfection of P2X7R increases resting mitochondrial potential (delta psi(mt)), basal mitochondrial Ca2+ ([Ca2+]mt), intracellular ATP content, and confers ability to grow in the absence of serum. These changes require a full pore-forming function, because they are abolished in cells transfected with a mutated P2X7R that retains channel activity but cannot form the nonselective pore, and depend on an autocrine/paracrine tonic stimulation by secreted ATP. On the other hand, sustained stimulation of P2X7R causes a delta psi(mt) drop, a large increase in [Ca2+]mt, mitochondrial fragmentation, and cell death. These findings reveal a hitherto undescribed mechanism for growth stimulation by a plasma membrane pore.