The C2 domains of classical PKCs are specific PtdIns(4,5)P2-sensing domains with different affinities for membrane binding

C2 domains are conserved protein modules in many eukaryotic signaling proteins, including the protein kinase (PKCs). The C2 domains of classical PKCs bind to membranes in a Ca(2+)-dependent manner and thereby act as cellular Ca(2+) effectors. Recent findings suggest that the C2 domain of PKCalpha interacts specifically with phosphatidylinositols 4,5-bisphosphate (PtdIns(4,5)P(2)) through its lysine rich cluster, for which it shows higher affinity than for POPS. In this work, we compared the three C2 domains of classical PKCs. Isothermal titration calorimetry revealed that the C2 domains of PKCalpha and beta display a greater capacity to bind to PtdIns(4,5)P(2)-containing vesicles than the C2 domain of PKCgamma. Comparative studies using lipid vesicles containing both POPS and PtdIns(4,5)P(2) as ligands revealed that the domains behave as PtdIns(4,5)P(2)-binding modules rather than as POPS-binding modules, suggesting that the presence of the phosphoinositide in membranes increases the affinity of each domain. When the magnitude of PtdIns(4,5)P(2) binding was compared with that of other polyphosphate phosphatidylinositols, it was seen to be greater in both PKCbeta- and PKCgamma-C2 domains. The concentration of Ca(2+) required to bind to membranes was seen to be lower in the presence of PtdIns(4,5)P(2) for all C2 domains, especially PKCalpha. In vivo experiments using differentiated PC12 cells transfected with each C2 domain fused to ECFP and stimulated with ATP demonstrated that, at limiting intracellular concentration of Ca(2+), the three C2 domains translocate to the plasma membrane at very similar rates. However, the plasma membrane dissociation event differed in each case, PKCalpha persisting for the longest time in the plasma membrane, followed by PKCgamma and, finally, PKCbeta, which probably reflects the different levels of Ca(2+) needed by each domain and their different affinities for PtdIns(4,5)P(2).     

Loss of hippocampal neuronal nitric oxide synthase contributes to the stress-related deficit in learning and memory

Nitric oxide (NO) has been involved in many pathophysiological brain processes. However, the exact role of NO in the cognitive deficit associated to chronic stress exposure has not been elucidated. In this study, we investigated the participation of hippocampal NO production and their regulation by protein kinase C (PKC) in the memory impairment induced in mice subjected to chronic mild stress model (CMS). CMS mice showed a poor learning performance in both open field and passive avoidance inhibitory task respect to control mice. Histological studies showed a morphological alteration in the hippocampus of CMS mice. On the other hand, chronic stress induced a diminished NO production by neuronal nitric oxide synthase (nNOS) correlated with an increment in gamma and zeta PKC isoenzymes. Partial restoration of nNOS activity was obtained after PKC activity blockade. NO production by inducible nitric oxide synthase isoform was not detected. The magnitude of oxidative stress, evaluated by reactive oxygen species production, after excitotoxic levels of NMDA was increased in hippocampus of CMS mice. Moreover, ROS formation was higher in the presence of nNOS inhibitor in both control and CMS mice. Finally, treatment of mice with nNOS inhibitors results in behavioural alterations similar to those observed in CMS animals. These findings suggest a novel role for nNOS showing protective activity against insults that trigger tissue toxicity leading to memory impairments.     

CB1 receptors diminish both Ca(2+) influx and glutamate release through two different mechanisms active in distinct populations of cerebrocortical nerve terminals

We have investigated the mechanisms by which activation of cannabinoid receptors reduces glutamate release from cerebrocortical nerve terminals. Glutamate release evoked by depolarization of nerve terminals with high KCl (30 mmol/L) involves N and P/Q type Ca(2+)channel activation. However, this release of glutamate is independent of Na(+) or K(+) channel activation as it was unaffected by blockers of these channels (tetrodotoxin -TTX- or tetraethylammonium TEA). Under these conditions in which only Ca(2+) channels contribute to pre-synaptic activity, the activation of cannabinoid receptors with WIN55,212-2 moderately reduced glutamate release (26.4 +/- 1.2%) by a mechanism that in this in vitro model is resistant to TTX and consistent with the inhibition of Ca(2+) channels. However, when nerve terminals are stimulated with low KCl concentrations (5-10 mmol/L) glutamate release is affected by both Ca(2+) antagonists and also by TTX and TEA, indicating the participation of Na(+) and K(+) channel firing in addition to Ca(2+) channel activation. Interestingly, stimulation of nerve terminals with low KCl concentrations uncovered a mechanism that further inhibited glutamate release (81.78 +/- 4.9%) and that was fully reversed by TEA. This additional mechanism is TTX-sensitive and consistent with the activation of K(+) channels. Furthermore, Ca(2+) imaging of single boutons demonstrated that the two pre-synaptic mechanisms by which cannabinoid receptors reduce glutamate release operate in distinct populations of nerve terminals.     

uPA deficiency exacerbates muscular dystrophy in MDX mice

Duchenne muscular dystrophy (DMD) is a fatal and incurable muscle degenerative disorder. We identify a function of the protease urokinase plasminogen activator (uPA) in mdx mice, a mouse model of DMD. The expression of uPA is induced in mdx dystrophic muscle, and the genetic loss of uPA in mdx mice exacerbated muscle dystrophy and reduced muscular function. Bone marrow (BM) transplantation experiments revealed a critical function for BM-derived uPA in mdx muscle repair via three mechanisms: (1) by promoting the infiltration of BM-derived inflammatory cells; (2) by preventing the excessive deposition of fibrin; and (3) by promoting myoblast migration. Interestingly, genetic loss of the uPA receptor in mdx mice did not exacerbate muscular dystrophy in mdx mice, suggesting that uPA exerts its effects independently of its receptor. These findings underscore the importance of uPA in muscular dystrophy.     

Classical Xenopus laevis progesterone receptor associates to the plasma membrane through its ligand-binding domain

During the last decade, considerable evidence is accumulating that supports the view that the classic progesterone receptor (xPR-1) is mediating Xenopus laevis oocyte maturation through a non-genomic mechanism. Overexpression and depletion of oocyte xPR-1 have been shown to accelerate and to block progesterone-induced oocyte maturation, respectively. In addition, rapid inhibition of plasma membrane adenylyl cyclase (AC) by the steroid hormone, supports the idea that xPR-1 should be localized at the oocyte plasma membrane. To test this hypothesis, we transiently transfected xPR-1 cDNA into Cos-7 cells and analyzed its subcellular distribution. Through Western blot and immunofluorescence analysis, we were able to detect xPR-1 associated to the plasma membrane of transfected Cos-7 cells. Additionally, using Progesterone-BSA-FITC, we identified specific progesterone-binding sites at the cell surface of xPR-1 expressing cells. Finally, we found that the receptor ligand-binding domain displayed membrane localization, in contrast to the N-terminal domain, which expressed in similar levels, remained cytosolic. Overall, these results indicate that a fraction of xPR-1 expressed in Cos-7 cells, associates to the plasma membrane through its LBD.     

Phylogenetic and functional analysis of Arabidopsis RCI2 genes

Six new Arabidopsis thaliana genes (AtRCI2C-H) have been identified that show high homology to AtRCI2A and AtRCI2B. Sequence comparisons revealed that AtRCI2-related genes are widely spread among very different organisms, including other plant species, prokaryotes, fungi, and simply organized animals, and are also organized in gene families. Most RCI2 genes show a similar exon-intron organization, which indicates that they have been structurally conserved during evolution, and encode small, highly hydrophobic proteins containing two putative transmembrane domains. Consistently, the majority of AtRCI2 proteins localize in the plasma membrane. RCI2 proteins exhibit an elevated level of sequence similarity and seem to have evolved from a common ancestor. In spite of their high similarity, conserved subcellular localization, and common origin, experimental evidence is presented suggesting that different RCI2 proteins may have distinct functional roles. Thus, as previously demonstrated for AtRCI2A and AtRCI2B, the newly identified AtRCI2 genes (AtRCI2C-H) are differentially regulated in Arabidopsis organs and in response to abiotic stresses and ABA treatment. Furthermore, only the AtRCI2 proteins that do not contain the C-terminal hydrophilic tail (i.e. AtRCI2A-C and AtRCI2H) are able to complement for the loss of the yeast AtRCI2-related gene PMP3. On the basis of these results, different aspects on the evolution and roles of RCI2 genes are discussed.     

How can organellar protein N-terminal sequences be dual targeting signals? In silico analysis and mutagenesis approach

Organellar nuclear-encoded proteins can be mitochondrial, chloroplastic or localized in both mitochondria and chloroplasts. Most of the determinants for organellar targeting are localized in the N-terminal part of the proteins, which were therefore analyzed in Arabidopsis thaliana. The mitochondrial, chloroplastic and dual N-terminal sequences have an overall similar composition. However, Arg is rare in the first 20 residues of chloroplastic and dual sequences, and Ala is more frequent at position 2 of these two types of sequence as compared to mitochondrial sequences. According to these observations, mutations were performed in three dual targeted proteins and analyzed by in vitro import into isolated mitochondria and chloroplasts. First, experiments performed with wild-type proteins suggest that the binding of precursor proteins to mitochondria is highly efficient, whereas the import and processing steps are more efficient in chloroplasts. Moreover, different processing sites are recognized by the mitochondrial and chloroplastic processing peptidases. Second, the mutagenesis approach shows the positive role of Arg residues for enhancing mitochondrial import or processing, as expected by the in silico analysis. By contrast, mutations at position 2 have dramatic and unpredicted effects, either enhancing or completely abolishing import. This suggests that the nature of the second amino acid residue of the N-terminal sequence is essential for the import of dual targeted sequences.     

Generation and characterization of a recombinant vesicular stomatitis virus expressing the glycoprotein of Borna disease virus

Borna disease virus (BDV) is an enveloped virus with a nonsegmented negative-strand RNA genome whose organization is characteristic of mononegavirales. However, based on its unique genetics and biological features, BDV is considered to be the prototypic member of a new virus family, Bornaviridae, within the order Mononegavirales. BDV cell entry occurs via receptor-mediated endocytosis, a process initiated by the recognition of an as yet unidentified receptor at the cell surface by the BDV surface glycoprotein (G). The paucity of cell-free virus associated with BDV infection has hindered studies aimed at the elucidation of cellular receptors and detailed mechanisms involved in BDV cell entry. To overcome this problem, we generated and characterized a replication-competent recombinant vesicular stomatitis virus expressing BDV G (rVSVDeltaG*/BDVG). Cells infected with rVSVDeltaG*/BDVG produced high titers (10(7) PFU/ml) of cell-free virus progeny, but this virus exhibited a highly attenuated phenotype both in cell culture and in vivo. Attenuation of rVSVDeltaG*/BDVG was associated with a delayed kinetics of viral RNA replication and altered genome/N mRNA ratios compared to results for rVSVDeltaG*/VSVG. Likewise, incorporation of BDV G into virions appeared to be restricted despite its high levels of expression and efficient processing in rVSVDeltaG*/BDVG-infected cells. Notably, rVSVDeltaG*/BDVG recreated the cell tropism and entry pathway of bona fide BDV. Our results indicate that rVSVDeltaG*/BDVG represents a unique tool for the investigation of BDV G-mediated cell entry, as well as the roles of BDV G in host immune responses and pathogenesis associated with BDV infection.