Delayed priming promotes CNS regeneration post-rhizotomy in Neurocan and Brevican-deficient mice

A wealth of literature has provided evidence that reactive tissue at the site of CNS injury is rich in chondroitin sulfate proteoglycans which may contribute to the non-permissive nature of the CNS. We have recently demonstrated using a murine model of human brachial plexus injury that the chondroitin sulfate proteoglycans Neurocan and Brevican are differentially expressed by two subsets of astrocytes in the spinal cord dorsal root entry zone (DREZ) following dorsal root lesion (Beggah et al., Neuroscience 133: 749-762, 2005). However, direct evidence for a growth-inhibitory role of these proteoglycans in vivo is still lacking. We therefore performed dorsal root lesion (rhizotomy) in mice deficient in both Neurocan and Brevican. Rhizotomy in these animals resulted in no significant increase in the number of sensory fibres regenerating through the DREZ compared to genetically matched controls. Likewise, a conditioning peripheral nerve lesion prior to rhizotomy, which increases the intrinsic growth capacity of sensory neurons, enhanced growth to the same extent in transgenic and control mice, indicating that absence of these proteoglycans alone is not sufficient to further promote entry into the spinal cord. In contrast, when priming of the median nerve was performed at a clinically relevant time, i.e. 7 weeks post-rhizotomy, the growth of a subpopulation of sensory axons across the DREZ was facilitated in Neurocan/Brevican-deficient, but not in control animals. This demonstrates for the first time that (i) Neurocan and/or Brevican contribute to the non-permissive environment of the DREZ several weeks after lesion and that (ii) delayed stimulation of the growth program of sensory neurons can facilitate regeneration across the DREZ provided its growth-inhibitory properties are attenuated. Post-injury enhancement of the intrinsic growth capacity of sensory neurons combined with removal of inhibitory chondroitin sulfate proteoglycans may therefore help to restore sensory function and thus attenuate the chronic pain resulting from human brachial plexus injur

Complete alanine scanning of the Escherichia coli RbsB ribose binding protein reveals residues important for chemoreceptor signaling and periplasmic abundance.

The Escherichia coli RbsB ribose binding protein has been used as a scaffold for predicting new ligand binding functions through in silico modeling, yet with limited success and reproducibility. In order to possibly improve the success of predictive modeling on RbsB, we study here the influence of individual residues on RbsB-mediated signaling in a near complete library of alanine-substituted RbsB mutants. Among a total of 232 tested mutants, we found 10 which no longer activated GFPmut2 reporter expression in E. coli from a ribose-RbsB hybrid receptor signaling chain, and 13 with significantly lower GFPmut2 induction than wild-type. Quantitative mass spectrometry abundance measurements of 25 mutants and wild-type RbsB in periplasmic space showed four categories of effects. Some (such as D89A) seem correctly produced and translocated but fail to be induced with ribose. Others (such as N190A) show lower induction probably as a result of less efficient production, folding and translocation. The third (such as N41A or K29A) have defects in both induction and abundance. The fourth category consists of semi-constitutive mutants with increased periplasmic abundance but maintenance of ribose induction. Our data show how RbsB modeling should include ligand-binding as well as folding, translocation and receptor binding

Characterisation of the Putative Effector Interaction Site of the Regulatory HbpR Protein from Pseudomonas azelaica by Site-Directed Mutagenesis

Bacterial transcription activators of the XylR/DmpR subfamily exert their expression control via σ54-dependent RNA polymerase upon stimulation by a chemical effector, typically an aromatic compound. Where the chemical effector interacts with the transcription regulator protein to achieve activation is still largely unknown. Here we focus on the HbpR protein from Pseudomonas azelaica, which is a member of the XylR/DmpR subfamily and responds to biaromatic effectors such as 2-hydroxybiphenyl. We use protein structure modeling to predict folding of the effector recognition domain of HbpR and molecular docking to identify the region where 2-hydroxybiphenyl may interact with HbpR. A large number of site-directed HbpR mutants of residues in- and outside the predicted interaction area was created and their potential to induce reporter gene expression in Escherichia coli from the cognate PC promoter upon activation with 2-hydroxybiphenyl was studied. Mutant proteins were purified to study their conformation. Critical residues for effector stimulation indeed grouped near the predicted area, some of which are conserved among XylR/DmpR subfamily members in spite of displaying different effector specificities. This suggests that they are important for the process of effector activation, but not necessarily for effector specificity recognition

V-ATPase-Mediated Granular Acidification Is Regulated by the V-ATPase Accessory Subunit Ac45 in POMC-Producing Cells

The regulation of the V-ATPase, the proton pump mediating intraorganellar acidification, is still elusive. We find that excess of the neuroendocrine V-ATPase accessory subunit Ac45 reduces the intragranular pH and consequently disturbs prohormone convertase activation and prohormone processing. Thus, Ac45 represents the first V-ATPase regulator

Role of glycosylation and disulfide bond formation in the beta subunit in the folding and functional expression of Na,K-ATPase

Initial folding is a prerequisite for subunit assembly in oligomeric proteins. In this study, we have compared the role of co-translational modifications in the acquisition of an assembly-competent conformation of the beta subunit, the assembly of which is required for the structural and functional maturation of the catalytic Na,K-ATPase alpha subunit. Cysteine or asparagine residues implicated in disulfide bond formation or N-glycosylation, respectively, in the Xenopus beta1 subunit were eliminated by site-directed mutagenesis, and the assembly efficiency of the mutants and the functional expression of Na+,K+ pumps were studied after expression in Xenopus oocytes. Our results show that lack of each one of the two most C-terminal disulfide bonds indeed permits short term but completely abolishes long term assembly of the beta subunit. On the other hand, lack of the most N-terminal disulfide bonds allows the expression of a small number of functional Na+,K+ pumps at the cell surface. Elimination of all three but not of one or two glycosylation sites produces beta subunits that remain stably expressed in the endoplasmic reticulum, in association with binding protein but not as irreversible aggregates. The assembly efficiency of nonglycosylated beta subunits is decreased but a reduced number of functional Na+,K+ pumps is expressed at the cell surface. The lack of sugars does not influence the apparent K+ or ouabain affinity of the Na+,K+ pumps. Thus, these data show that disulfide bond formation and N-glycosylation may play important but qualitatively distinct roles in the initial folding of oligomeric protein subunits. Moreover, the results suggest that an endoplasmic reticulum degradation pathway exists, which is glycosylation-dependent

Adrenergic, dopaminergic, and muscarinic receptor stimulation leads to PKA phosphorylation of Na-K-ATPase.

Na-K-adenosinetriphosphatase (Na-K-ATPase) is a potential target for phosphorylation by protein kinase A (PKA) and C (PKC). We have investigated whether the Na-K-ATPase alpha-subunit becomes phosphorylated at its PKA or PKC phosphorylation sites upon stimulation of G protein-coupled receptors primarily linked either to the PKA or the PKC pathway. COS-7 cells, transiently or stably expressing Bufo marinus Na-K-ATPase wild-type alpha- or mutant alpha-subunits affected in its PKA or PKC phosphorylation site, were transfected with recombinant DNA encoding beta 2- or alpha 1-adrenergic (AR), dopaminergic (D1A-R), or muscarinic cholinergic (M1-AChR) receptor subspecies. Agonist stimulation of beta 2-AR or D1A-R led to phosphorylation of the wild-type alpha-subunit, as well as the PKC mutant, but not of the PKA mutant, indicating that these receptors can phosphorylate the Na-K-ATPase via PKA activation. Surprisingly, stimulation of the alpha 1B-AR, alpha 1C-AR, and M1-AChR also increased the phosphorylation of the wild-type alpha-subunit and its PKC mutant but not of its PKA mutant. Thus the phosphorylation induced by these primarily phospholipase C-linked receptors seems mainly mediated by PKA activation. These data indicate that the Na-K-ATPase alpha-subunit can act as an ultimate target for PKA phosphorylation in a cascade starting with agonist-receptor interaction and leading finally to a phosphorylation-mediated regulation of the enzyme

Opioides intrathecaux dans le traitement des douleurs chroniques: mecanismes d'action et aspects cliniques. [Spinal opioids: mechanisms of action and chronic pain management]

The efficacy of spinal opioids is well known, the analgesia is potent and long lasting, due to the central localization of the opioid receptors. The analgesia is intimately related to the inhibition of the nociceptive signal in the spinal cord but side effects are mainly mediated by the activation of the mu opioid receptor in the brain and the brain stem. Only a limited number of controlled clinical studies compared systemic versus spinal administration of morphine in chronic pain patients, and the real benefit for the intrathecal route remains controversial. Implanted devices for a continuous intrathecal delivery of opioids should be prescribed only to patients with intractable chronic pain for which conventional methods were previously ineffective

Regulation of expression and function by subunits of oligomeric P-type ATPases

Na,K-ATPase activity must be finely controlled to meet the constantly changing physiological demands and to avoid destabilization of body homeostasis. Recent experimental evidence suggests that certain regulatory mechanisms are closely linked to the multisubunit structure of the Na,K-pump molecule. Na,K-ATPase is composed of a catalytic alpha and a glycoprotein beta subunit and sometimes of a third component, the gamma subunit. The beta subunit is a fundamental element of Na,K-ATPase in that its assembly in the ER is required for the structural and functional maturation of the catalytic alpha subunit and in consequence the beta subunit controls the expression of functional pumps at the cell surface. Furthermore, beta subunits influence the transport properties of the mature catalytic alpha subunits. Distinct interaction sites mediate the two functions of the beta subunit. Recently, we have started to characterize the gamma subunit, the functional role of which is yet not known. Immuno-radiolabeling of epitope-tagged gamma subunits expressed in Xenopus oocytes shows that the gamma subunits is a type I membrane protein which specifically associates only with Na,K-ATPase but not with other oligomeric P-type ATPases. The gamma peptide does not influence the formation or the cell surface expression of functional alpha-beta complexes. On the other hand, the gamma peptide itself needs association with Na,K-ATPase to be stably expressed and to be efficiently transported to the plasma membrane. Finally, the gamma subunit can modulate the K activation of Na,K-pumps. In conclusion, processes such as subunit assembly or the subunit composition of the cell surface expressed Na,K-pumps appear to cooperate with hormones in the control of the expression and the activity of Na,K-ATPase