Shuffling the cards in signal transduction: Calcium, arachidonic acid and mechanosensitivity

Cell signaling is a very complex network of biochemical reactions triggered by a huge number of stimuli coming from the external medium. The function of any single signaling component depends not only on its own structure but also on its connections with other biomolecules. During prokaryotic-eukaryotic transition, the rearrangement of cell organization in terms of diffusional compartmentalization exerts a deep change in cell signaling functional potentiality. In this review I briefly introduce an intriguing ancient relationship between pathways involved in cell responses to chemical agonists (growth factors, nutrients, hormones) as well as to mechanical forces (stretch, osmotic changes). Some biomolecules (ion channels and enzymes) act as "hubs", thanks to their ability to be directly or indirectly chemically/mechanically co-regulated. In particular calcium signaling machinery and arachidonic acid metabolism are very ancient networks, already present before eukaryotic appearance. A number of molecular "hubs", including phospholipase A2 and some calcium channels, appear tightly interconnected in a cross regulation leading to the cellular response to chemical and mechanical stimulations.     

Acetyl Coenzyme A: Deacetylvindoline O-Acetyltransferase,A Novel Enzyme from Catharanthus

A new enzyme, Acetyl Coenzyme A: deacetylvindoline 0-acetyl transferase (EC 2.3.1. -) which catalyses the synthesis of vindoline from acetyl coenzyme A and deacetylvindoline was isolated from the soluble protein extract of Catharanthus roseus leaves and purified approximately 365-fold. The enzyme had an apparent pI of 4.6 upon chromatofocusing, an apparent molecular weight of 45,000 daltons and a pH optimum between 8.0 to 9.0. Dithiothreitol was essential to maintain enzyme activity.Substrate saturation studies of this enzyme resulted in Michaelis Menton kinetics giving Km values of 5.4 and 0.7µM respectively for acetyl coenzyme A and deacetylvindoline. Studies of the forward reaction demonstrated an absolute requirement for acetyl coenzyme A and deacetylvindoline derivatives containing a double bond at positions 6, 7, whereas the reverse reaction occurred only in the presence of free coenzyme A and vindoline derivatives containing the same double bond. The forward reaction was subject to product inhibition by coenzyme A with an apparent Ki of 8 µM, but was not inhibited by up to 2 mM vindoline. The rate of reaction could therefore be regulated by the level of free coenzyme A in the cell, unaffected by the accumulation of indole alkaloid product.It was suggested that this enzyme catalyses a late step in the biosynthesis of vindoline.     

Marek’s disease virus prolongs survival of primary chicken B-cells by inducing a senescence-like phenotype

Marek’s disease virus (MDV) is an alphaherpesvirus that causes immunosuppression and deadly lymphoma in chickens. Lymphoid organs play a central role in MDV infection in animals. B-cells in the bursa of Fabricius facilitate high levels of MDV replication and contribute to dissemination at early stages of infection. Several studies investigated host responses in bursal tissue of MDV-infected chickens; however, the cellular responses specifically in bursal B-cells has never been investigated. We took advantage of our recently established in vitro infection system to decipher the cellular responses of bursal B-cells to infection with a very virulent MDV strain. Here, we demonstrate that MDV infection extends the survival of bursal B-cells in culture. Microarray analyses revealed that most cytokine/cytokine-receptor-, cell cycle- and apoptosis-associated genes are significantly down-regulated in these cells. Further functional assays validated these strong effects of MDV infections on cell cycle progression and thus, B-cell proliferation. In addition, we confirmed that MDV infections protect B-cells from apoptosis and trigger an accumulation of the autophagy marker Lc3-II. Taken together, our data indicate that MDV-infected bursal B-cells show hallmarks of a senescence-like phenotype, leading to a prolonged B-cell survival. This study provides an in-depth analysis of bursal B-cell responses to MDV infection and important insights into how the virus extends the survival of these cells.     

Distribution of glutathione transferases in Gram-positive bacteria and Archaea

Glutathione transferases (GSTs) have been widely studied in Gram-negative bacteria and the structure and function of several representatives have been elucidated. Conversely, limited information is available about the occurrence, classification and functional features of GSTs both in Gram-positive bacteria and in Archaea. An analysis of 305 fully-sequenced Gram-positive genomes highlights the presence of 49 putative GST genes in the genera of both Firmicutes and Actinobacteria phyla. We also performed an analysis on 81 complete genomes of the Archaea domain. Eleven hits were found in the Halobacteriaceae family of the Euryarchaeota phylum and only one in the Crenarchaeota phylum. A comparison of the identified sequences with well-characterized GSTs belonging to both Gram-negative and eukaryotic GSTs sheds light on their putative function and the evolutionary relationships within the large GST superfamily. This analysis suggests that the identified sequences mainly cluster in the new Xi class, while Beta class GSTs, widely distributed in Gram-negative bacteria, are under-represented in Gram-positive bacteria and absent in Archaea.     

Structural differences in the hinge region of the glycoprotein hormone receptors: evidence from the sulfated tyrosine residues

Tyrosine sulfation is a late posttranslational modification of proteins that takes place in the Golgi network. In the past few years, this process has been identified as an important modulator of protein-protein interactions. Sulfated tyrosine residues have recently been identified in the C-terminal, so-called hinge region of the ectodomain of glycoprotein hormone receptors [TSH, LH/chorionic gonadotropin (CG), and FSH receptors] and were shown to play an important role in the interaction with their natural ligands. The position of two sulfated tyrosine residues in a Y-D/E-Y motif appears perfectly conserved in the alignment of TSH and LH receptors from different species, and site-directed mutagenesis experiments demonstrated that sulfation of the first residue of this motif was responsible for the functional effect on hormone binding. In contrast, the corresponding motif is not conserved in the FSH receptor, in which the first tyrosine residue is missing: the Y-D/E-Y motif is replaced by F(333)DY(335). We extend here our previous observation that, in this case, it is sulfation of the second sole tyrosine residue in the motif that is functionally important. An LH/CG receptor harboring an F(331)DY(333) motif (i.e. displaying decreased sensitivity to human CG) was used as a backbone in which short portions of the FSH receptor were substituted. Segments from the FSH receptor capable of restoring sensitivity to human CG were identified by transfection of the chimeras in COS-7 cells. These experiments identified key amino acid residues in the hinge region of the FSH receptor associated with the functional role of the second sulfated tyrosine residue in a Y-D/E-Y motif, allowing for efficient hormone binding. The experiments represent strong evidence that structural differences in the hinge regions of FSH and LH/CG receptors play a significant role in hormone-receptor-specific recognition.     

Gender-related effects on urine l-cystine metastability

Cystinuria is an autosomal recessive disease that causes l-cystine precipitation in urine and nephrolithiasis. Disease severity is highly variable; it is known, however, that cystinuria has a more severe course in males. The aim of this study was to compare l-cystine metastability in first-morning urine collected from 24 normal female and 24 normal male subjects. Samples were buffered at pH 5 and loaded with l-cystine (0.4 and 4 mM final concentration) to calculate the amount remaining in solution after overnight incubation at 4 °C; results were expressed as Z scores reflecting the l-cystine solubility in each sample. In addition, metabolomic analyses were performed to identify candidate compounds that influence l-cystine solubility. l-cystine solubility Z score was +0.44 ± 1.1 and ?0.44 ± 0.70 in female and male samples, respectively (p < 0.001). Further analyses showed that the l-cystine solubility was independent from urine concentration but was significantly associated with low urinary excretion of inosine (p = 0.010), vanillylmandelic acid (VMA) (p = 0.015), adenosine (p = 0.029), and guanosine (p = 0.032). In vitro l-cystine precipitation assays confirmed that these molecules induce higher rates of l-cystine precipitation in comparison with their corresponding dideoxy molecules, used as controls. In silico computational and modeling analyses confirmed higher binding energy of these compounds. These data indicate that urinary excretion of nucleosides and VMA may represent important factors that modulate l-cystine solubility and may represent new targets for therapy in cystinuria.